Epigenetics Papers

Regulation of Replication Fork Progression Through Histone Supply and Demand

2008-02-19T07:52:00.005-06:00

This work shows that the replacement of histones is coordinated with the fork progression during DNA replication. After finding that Asf1, the H3-H4 chaperone, co- purified with the putative helicase MCM2-7 in HeLa cells, the question then was if problems during replication could be the consequence of abnormal chromatin unwinding. This was confirmed by following the cell cycle progression with DNA binding markers and by silencing of Asf1 with siRNAs. Formation of ssDNA was also reduced after Asf1 depletion, i.e.,

[indicating...] that impaired replication reflects a DNA unwinding defect and implied that DNA in chromatin cannot be properly unwound by the replicative helicase. This could reflect a direct effect of Asf1 on DNA unwinding and fork progression or indirect effects, involving DNA damage at the replication fork, replisome collapse, and/or checkpoint signaling. [...] Our results show that replication fork progression is dependent on the histone H3-H4 chaperone, Asf1, and on an equilibrium between histone supply and demand. This dependency could ensure that replication only proceeds when nucleosomes are being formed behind the fork with a proper balance between new and parental histones H3-H4

Model for Asf1 function in replication as a histone acceptor and donor (Groth et al./Science)

Groth A., et al. 2007. Science 318(5858):1928-1931

epigenetics

More comments can be found in the same issue of Science, in the Research Roundup section of the Journal of Cell Biology, and the Faculty of 1000 website.

The histone deacetylase inhibitor valproic acid enhances acquisition, extinction, and reconsolidation of conditioned fear

2008-02-15T07:48:00.004-06:00

Histone modifications have been found to contribute with certain brain phenomena like memory and behavior, and in this respect, some HDAC inhibitors have been shown to enhance long- term memory, which makes them interesting candidates for treatment of cognitive disorders, particularly phobias, but also for other behavior alterations, like anxiety disorders. In this paper, authors report that sodium valproate (VPA), a HDAC inhibitor, improves the extinction of conditioned fear by enhancing long- term memory in mice, whose effects not only last but are also more pronounced while in training.

When administered prior to training, VPA enhances long- term memory for both the acquisition and extinction of conditioned fear. These findings are consistent with several studies demonstrating that HDAC inhibitors facilitate memory. [...These findings also] support the long- standing view that extinction is new learning and not simply erasure of the original fear association [...].

T. W. Bredy & M. Barad (2008) Learn Mem 15(1):39-45
epigenetics

The Obesity-Associated FTO Gene Encodes a 2-Oxoglutarate-Dependent Nucleic Acid Demethylase

2008-01-09T10:52:00.000-06:00

Studies have revealed an association between variants of the first intron of the human FTO gene and obesity. Computational analyses predict that the protein product of FTO contains a 2-oxoglutarate oxygenase (2OG) fold, and these enzymes have been found to be involved in diverse cellular processes, ranging from DNA repair to histone demethylation. Here, authors characterized a recombinant form of FTO to determine if this protein is a 2OG enzyme. To this end, they expressed a His- tagged form of FTO in E.coli, and assays specific to 2OG oxygenases showed that Fto catalyzed 2OG decarboxylation, activity which was in turn stopped by 2OG oxygenase- specific inhibitors. As the sequence of FTO resembled that of AlkB, an DNA repair oxygenase from E.coli related to methylation lesions, Gerken et al., screened for known AlkB targets and other common substrates of 2OG oxygenases (for example, me-H3K9), and found that only 1-methyl adenine "stimulated turnover of 2OG above control levels." Mass spectrometry coupled with liquid chromatography revealed that Fto catalyzed Fe++ and 2OG- dependent DNA demethylation. Fto was active also in single- stranded DNA, and YFP-Fto transfected to cell lines were located mostly at nuclei, as expected for any DNA demethylase. With many links already found between DNA methylation modifications and nutrition, it still remains to see if there is an actual causal relationship between obesity and epigenetics.

Gerken T., et al. 2007. Science 318(5855):1469-1472
epigenetics

The role of site accessibility in microRNA target recognition

2007-10-23T04:09:00.000-06:00

Currently, most microRNA predictors perform so poorly that it was easy to assume that secondary structure formation has a more important role in target recognition, instead of relying in sequence complementarity alone. To further evaluate this hypothesis, this group previously reported the development of a quantitative (luminiscent) assay for measuring repression by microRNAs, and here they employed this methodology over a selection of targets that were engineered for specific sequence mutations. These in vitro (cell culture) observations were used to train an in silico model, which they implemented into a new predictor, available from their website.

[...] Our results show that site accessibility is as important as sequence match in the seed for determining the efficacy of microRNA- mediated translational repression, and they suggest that effective microRNA binding also requires unpairing of the local region flanking the target. We introduce a parameter-free thermodynamic model that explains these effects and shows that preferential positioning of microRNA target sites in regions of high accessibility is a conserved feature in genomes. Our findings thus indicate that the thermodynamics of intra- and intermolecular base pairing account for a significant portion of the microRNA- target interaction, consistent with observations for siRNA- target interactions. However, our model does not explain the entire variance in our experiments. This may be in part due to limitations of RNA structure prediction algorithms and their inability to account for the effects that RNA binding proteins have on secondary structures. Moreover, microRNAs bind as part of the RISC complex, [... and] RISC proteins are likely to constrain base-pairing interactions between microRNA and target site and otherwise influence the mechanics of the duplex formation.

Kertesz M. et al. 2007. Nat Genet 39:1278-84

epigenetics

More comments can be found in the same issue of Nature Genetics.

Periodic epi-organization of the yeast genome revealed by the distribution of promoter sites

2007-10-11T08:42:00.000-06:00

Some studies of the chromatin structure have revealed that particular chromosome regions are compartmentalized in some areas of the nucleus, as is the case for nucleoli; other similar phenomena of regional specialization is the case of the centrosomal and nucleolar polarity, and it is very likely that these spatial arrangements must have an influence over gene expression regulation. In this paper, Kepes analyzed the transcriptional organization of Saccharomyces and found periodicity between the location of protein binding sites. He used ChIP data from several transcription factors, like Rap1p, and noticed regularities in distances between the targets from the same chromosome; for example, in the chromosome IX, Rap1p targets were spaced by ~31 kb. Data used from different ChIP experiments in different labs showed the same regularity. Other proteins analyzed showed also specificity in their preferred periods, different for each chromosome. To validate these model and to evaluate if this results have an impact on other cellular processes, Kepes analyzed the distribution of the ARS sequences -and these were distributed in a regular manner for each independent chromosome as well. Periodicity was not an artifact of the ChIP experiments, because lowering the stringency only produced loss of signal. These results not only provide a way to discriminate ChIP results, using spacing distances from the same chromosome, but also establish a link between spatial arrangement and expression regulation:

Relative positions of clusters of coregulated genes (Kepes/JMB)

[..] This concept also accounts for the optimization of gene control by several regulators, despite combinatorial intricacy, through target assignment to a cluster positioned for the correct set of regulators. It solves the paradox of the high efficiency of DNA-related processes despite extreme DNA compaction, as compaction now appears to be partly achieved by a dynamic epi-organization that actually increases transcriptional, replicational and perhaps recombinational efficiencies. Finally, it provides a framework to understand why yeast “heterochromatin barriers” that block the spread of transcriptionally silent chromatin consist of multiple binding sites for various regulators

Dr.Kepes recently gave a conference here at the MPI entitled "The solenoidal model of chromosomes", which motivated to read more about his work.

Kepes, F. 2003. J Mol Biol 329(5):859-65

epigenetics

N6-methyladenine: the other methylated base of DNA

2007-09-27T12:35:00.000-06:00

Besides of containing methyl- cytosines, it is known that DNA also includes a sixth base, N6- methyl adenine (m6A), which is essential for the viability of bacteria, but also occurs in archaea and eukaryotes. In bacteria, adenine methylation is associated with the protection of the DNA from endonucleases, via the restriction- modification system, and the enzymes involved (adenine methyltransferases), have been also found to be encoded by phage genomes. On the other hand, adenine methylation marks are also used to identify parental and nascent DNA strands during chromosome replication and DNA mismatch repair.

In eukaryotes, m6A and its endonucleases and methyl- transferases are coded in some protists like Chlorella, which might possibly protect this unicellular organism from infection. In ciliate protozoa, m6A is only found in macronuclear DNA only, and occurring with a non- random distribution. However, the assumption that m6A is not present in higher eukaryotes, which comes from experiments made in the 1970s, is somewhat evolutively surprising if we consider all its functions in bacteria, not to mention the high mutability of m5C to thymine (which is supposed to be a defense against genomic parasites such as transposons). Hence, the large number of transposons in eukaryotic genomes could have masked the small amounts of m6A, and thus this base has not been as throughly investigated as the other epigenetic mark. Indeed, there are reports of m6A in higher eukaryotes, particularly plant and mosquito DNA.

[To date of this paper], in mammals there is only indirect evidence of m6A in rodents (from sensitive restrictases assays), but the definitive physical detection is still needed. It seems that in these studied mammalian genes (mouse Myo-D1 and rat steroid reductase), m6A could be modulating gene expression, since it was showed that it interferes with protein – DNA binding. This could be a matter of concern considering that plasmids for transient expression are propagated in bacterial cells before introduced to animal cell cultures.

This controversy of the presence or not of m6A in higher eukaryotes is reminiscent of the one that arose regarding the presence of 5mC in Drosophila, which was just demonstrated in this decade. Thus, more sensitive approaches are required to clarify its presence, for example using HPLC coupled to tandem mass spectrometry, and technological limitations could be indirectly solved if the corresponding methyl- transferase is found. Authors proposed that m6A could function as a defense mechanism, as it is for bacterial chromosomes, and also to mark the “immortal” DNA strand of adult stem cells, that divides asymmetrically.

For several decades now, the importance of epigenetic mechanisms involving DNA methylation in mammals has resulted in m5C being considered as the fifth base of DNA. However, it seems likely that the relative high abundance of m5C in mammalian DNA has focused attention on the role of m5C to the detriment of investigations on m6A. The fundamental role played by m6A in bacteria raises fascinating questions on its phyletic distribution and on its possible functions in eukaryotes. Hence, determining unambiguously whether our genome is made of five or six bases, and elucidating the biological functions played by m6A in eukaryotes is now a matter of crucial concern for both basic research and drug development.

Ratel, D. et al. 2006. BioEssays 28: 309-315

epigenetics

DNA methylation during differentiation of a lower eukaryote, Physarum polycephalum

2007-09-26T03:49:00.000-06:00

Physarum polycephalum is a lower eukaryotic organism which stands out as a promising model for epigenetic studies because of its very unique life and cell cycles. Preliminary analysis of its DNA methylation levels showed similar content to that of vertebrates, and reports suggested that this epigenetic modification could be involved in its sporulation. In this short paper, authors showed that this is indeed the case, and proved that methylation targeted specifically a sporulation- related gene, spherulin-4.

After transference to the starvation medium, cultures undergo a metabolic transition which results in the formation of spherules. To see if DNA methylation regulation is involved in this transition, authors added azaC to the starvation medium, and assayed the presence of spherules after a period of time (72 hrs). They found that azaC inhibited the formation of spherules when used at concentrations over 50 uM, and this inhibition was presumed to be complete [and not only slowed down], since longer incubations (up to 168 hrs) did not produce spherules either; the inhibition also was not due to unspecific toxicity, since cultures resumed growth promptly after transfer to medium without azaC. Complete inhibition of spherule formation was seen after continuous presence during six or more hours. On the other hand, germination of spherules in absence of azaC was not influenced, which suggests that DNA methylation is involved in the formation of spherules, but not in their later germination (although authors recognized that this might require further investigations). Other methylation inhibitors assayed (5-aza- 2-deoxycytidine and L-ethionine) also produce similar effects.

To assess the DNA methylation content during spherulation, cultures were pulse- labeled with methyl- ³H-methionine, and the incorporation of ³H was measured; these experiments showed however that the methylation remained almost unchanged. Later they measured the DNA methylation by HPLC, and although the levels were not statistically significative, important differences were seen when individual culture time- points were compared. This supported the hypothesis that changes in DNA methylation patterns were occurring indeed.

To observe DNA methylation of specific sequences, several spherulation genes were analyzed by Southern blotting, using methylation- sensitive restrictases. Here, only a probe corresponding to the spherulin-4 gene showed differential patterns, which indicated changes in DNA methylation, and although several independent cultures displayed similar band patterns, some gave more complex hybridizations, suggesting that the methylation changes could be transient.

Our studies with inhibitors of DNA methylation indicated that for P.polycephalum to differentiate into spherules methylation of DNA must take place at least during the second half of spherulation [...]. Two classes of inhibitors were used: (i) nucleoside analogues [...], and (ii) L-ethionine [...]. Since these two classes of inhibitors operate by distinct mechanisms their influence on spherulation is likely to be caused via the feature they have in common, i.e. by inhibition of DNA methylation. [...] We have also shown that inhibiting methylation of DNA does not lead to significant overall growth retardation of P. polycephalum, thus indicating the specific role of DNA methylation in spherulation.

[...A previous] study [...] failed to detect any changes in the methylation pattern of several genes during differentiation of P. polycephalum. The most likely explanation of this discrepancy is the fact that not all developmentally regulated genes undergo differential methylation detectable by the method used in the study in question as well as in this work [...].

Fronk J, and R.Magiera. 1994. Biochem J 304(Pt 1):101-104

epigenetics

Epigenetic characterization of hematopoietic stem cell differentiation using miniChIP and bisulfite sequencing analysis

2007-09-12T17:51:00.000-06:00

Attema et al., have developed a ChIP variant that overcomes the limitation of standard protocols, that require a large number of cells (~5x10⁸). Their method, termed miniChIP, allows the analysis of histone modifications in as low as ~50,000 cells. They applied their assay to analyze the epigenetic marks at lineage- specific genes in hematopoietic stem cells (HSC) for lineage commitment:

[This method is] amenable to the numbers of HSC and progenitors that can be reasonably obtained to high purity from adult mouse bone marrow [...]. We established the method on 50,000 cells through the systematic adjustment of formaldehyde cross-linking, sonication, preclearing, salt concentration and antibody immunoprecipitation conditions. [... Besides,] when used in conjunction with [...] carrier ChIP and Q²ChIP assays, [...] epigenetic analysis should be possible on extremely rare cells (i.e., 100) contained within primary tissues. Therefore, these newly described ChIP methods will serve as invaluable tools for the epigenetic analysis of primary cells in numerous developmental systems and neoplastic cell populations, including cancer stem cells, where cell numbers are limiting.

Attema JL. et al. 2007. Proc Natl Acad Sci USA 104(30):12371-76
epigenetics

Diet-induced hypermethylation at agouti viable yellow is not inherited transgenerationally through the female

2007-09-10T16:39:00.000-06:00

A^vy locus in mice displays phenotypes ranging from yellow (hypomethylated A^vy) to brown (pseudoagouti, hypermethylated A^vy), and the coat color of A^vy/a mice resembles that of their A^vy/a dams, due to epigenetic inheritance. Besides, A^vy can be influenced by environment during embryonary stages -specifically, diet. Both epigenetic inheritance and environmental influence could account for a transgenerational epigenetic effect, and Cropley et al. recently reported evidence for this. Here, however, Waterland et al. said that the findings by Cropley et al. demonstrate only that maternal methyl supplementation helps to prevent the loss of DNA methylation but not prove the transgenerational inheritance of acquired characters.

[...Our] study had two potential outcomes. If the effects of maternal diet on offspring A^vy methylation are partially transmitted to the next generation, we would expect the difference in coat color between the two diet groups to increase with successive generations. If, on the other hand, the effects of methyl supplementation on A^vy methylation are not transmitted transgenerationally, we would expect the coat color difference between the two diet groups to be stable across the three generations. Our findings are clearly consistent with the latter.

As their results are in contradiction with those of Cropley et al., authors suggested that the observed differences could be due mainly for two parameters in the study design: the initial epigenetic state of the A^vyallele (pseudoagouti -hypermethylated A^vy- in the Cropley et al. experiment, and yellow -hypomethylated A^vy- here); and the diet (Cropley et al. used a 3-fold higher level of methyl donors, which authors said could have introduced a toxicity bias). Finally, authors mentioned that, although CpG methylation appears to play a central role in the phenotypic variation of A^vy, additional mechanisms (like histone modifications, for example) must be identified, to see if this loci follows an epigenetic inheritance.

Waterland, RA. et al. 2007. FASEB J [Epub ahead of print]
epigenetics

The language of covalent histone modifications

2007-09-07T00:36:00.000-06:00

This is the classic paper where the histone code hypothesis was proposed. Evidences that support this were that (1) histone modifications (acetylation, phosphorylation, methylation) may alter chromatin structure, influencing gene expression; and (2) growing data showing diversity and biological specificity associated with different histone modification patterns. Characterization of histone acetyl- transferases (HATs) and deacetylases (HDACs) provided further support, because they brought the possibility of steady- state regulation of the process. On the other hand, structural periodicities between modified residues may suggest that combinations of modified amino acids could have different meanings (e.g. a code)

Considering only electrostatic requirements for folding the chromatin polymer, histone acetylation [...] and histone phosphorylation [...] would cause decondensation of the chromatin fibre. Thus, the use of multiple marks on histone tails [...] could serve to amplify the readout of upstream signalling pathways causing greater changes in the overall charge density of tails that lead to greater changes in the chromatin structure of target genes. [...] If the function of H3 phosphorylation is to 'open' chromatin, how then can H3 phosphorylation at the same site also be involved in chromatin condensation? [...] Perhaps a single histone modification does not function alone. We will refer to the hypothesis -that multiple histone modifications, acting in a combinatorial or sequential fashion on one or multiple histone tails, specify unique downstream functions- as the histone code hypothesis.

The 'histone code' hypothesis (Strahl and Allis/Nature)

How is this code established? Authors proposed that modifications are located close enough to influence the ability of the modifying enzymes to add more covalent groups, and these modifications might in turn influence the outcome on other histone tails that are in contact. How is this code read? Relaxation of the chromatin could promote the binding of efectors, or alternatively, histone modifications could act as receptors that recruit distinct specific sets of proteins. For example, PCAF binds acetylated lysine in H3 or H4 -which might suggest the possibility of specific binding motifs recognizing histone modifications. Authors pointed that all these evidences remark the necessity for identifying:

the complete dictionary of histone modifications;
phenotypes associated with each modification;
modifying enzymes and their recruitment processes; and
the way that these modifications specify all these interactions.

Perhaps the most difficult questions are those that were not discussed in the paper: How is this code passed through generations? How this code appeared and evolved, since some histone modifications seemed to be species- specific?

Strahl DB. and Allis CD. 2000. Nature 403:41-45
epigenetics

High- resolution profiling of histone methylations in the human genome

2007-09-06T17:44:00.000-06:00

Here authors reported another ChIPSeq implementation (see Johnson DS. et al., and Mikkelsen TS. et al. for related posts), and used it to map histone modifications in human CD4+ T cells.

The flow chart of ChIPSeq (Barski A. et al./Cell)

To distinguish between this procedure [ChIP combined with Solexa sequencing] and our previous procedure, GMAT, or other similar procedures that incorporate the SAGE protocol, we have termed this direct sequencing procedure 'ChIP-Seq'. [...] Our data indicate that H3K27me1 and H4K20me1 are associated with actively transcribed regions. Moreover, H3K9me1 and H2BK5me1 are also associated with transcribed regions. [...] However, our data suggest that H3K79me1 is modestly associated with activation while H3K79me3 is associated with repression in human cells. H3K79me2 did not show any preference toward either active or silent genes. Therefore, actively transcribed regions are marked with high levels of H3K36me3, H3K27me1, H3K9me1, and H2BK5me1. H3K36me3 exhibits a more 3' distribution, whereas H3K9me1 and H2BK5me1 exhibit higher levels near the 5' end, and H3K27me1 distribute more evenly throughout the transcribed regions.

Barski A. et al. 2007. Cell 129:823-837

epigenetics

Histone modifications induced by a family of bacterial toxins

2007-09-05T12:07:00.000-06:00

In this article, authors demonstrate that the intracellular pathogen Listeria monocytogenes modulates gene expression of the host by inducing histone modifications, via secretion of the virulence factor listeriolysin O (LLO). They found that LLO provokes the dephosphorylation of H3S10 and deacetylation of H4 in HeLa cells. As H3S10 dephosphorylation occurs early in infection, they hypothesized that the effect might occur by secretion of toxins. To this end, they evaluated several virulence factor mutants, and found that the ∆hly strain (LLO defective) had no effect on phospho- H3S10 levels, compared to the wild- type; complementation of the mutation restored the wild type phenotype. Later, when HeLa cells were treated with purified LLO, it was able to induce both H3S10 dephosphorylation and H4 deacetylation. Total levels of histone did not vary, meaning that bacteria did not induce histone degradation. The same effect was seen in other cell lines. As LLO is member of the CDC family of toxins, they evaluated other members of this group (Clostridium perfringens PFO and Streptococcus pneumoniae PLY) for the same effects, and found that both PFO and PLY induced similar levels of dephosphorylation, suggesting that this could be a property of the whole CDC family. They later observed (using microarrays and real time PCR) that the expression was being modulated by the infection: ~50 repressed and ~100 induced genes. phospho- H3S10 ChIP experiments confirmed that LLO treatment modulated the expression in these genes, and western blots showed that the same subset of genes were targeted for H4 modification. Interestingly, none of the up- regulated genes has known implications in the immune response; in contrast, some down- regulated genes are indeed involved in immune processes, meaning that Listeria might counteract the host immunity before invasion. Other assays using MAPK, PKC, and NF-kB pathways inhibitors, did not block the effect of LLO on H3S10, suggesting that H3S10 dephosphorylation must be induced through a pathway not yet described. The same results were seen for inhibitors to tyrosine kinases, serine/threonine kinases, and phosphatidyl- inositol kinases.

Epigenetic regulation by LLO is thus a so far unsuspected function for this key virulence factor [...]. LLO most likely activates a [yet unknown] signaling cascade.

Hamon MA. et al. 2007. Proc Natl Acad Sci USA 104(33):13467-72
epigenetics

Molecular genetic studies of the memory of winter

2007-09-04T09:54:00.000-06:00

Vernalization is the process by which exposure to prolonged cold establishes competence to flower. This vernalized state can be stable, meaning that plants can exhibit a memory of winter, and this process acts as an epigenetic switch -changes depending on the presence or absence of an inductor. In Arabidopsis, FLC is a repressor which prevents plants from flowering unless vernalized. Expression of this gene is repressed after long exposure to cold, and this state can be maintained even after the cold ends. Therefore, the memory of winter is the stable repression of FLC.

Cold exposure triggers signalling events leading to the VIN3 expression, a protein that encodes a PHD domain, common in chromatin- remodelling complexes. After sensing cold temperatures, VRN2 is required for maintenance of the PcG- mediated FLC repression by vernalization. Involvement of a PRC2 component (VRN2), and a PHD domain containing protein (VIN3), as well as the mitotical stability of the vernalized state, motivated an examination of histone modifications at the FLC chromatin:

H3K9 appears to be more revelant because H3K27 methylation alone is not enough to maintain FLC repression. Three enzymes involved in vernalization- mediated histone modification have been described: MEA, CLF and SWN, all Arabidopsis homologs of the animal E(z), the H3K27 methyl- transferase of the PRC2 complex. However, although E(z) is able to methylate H3K9 per se, this property has not been observed in its plant homologs. Thus, some other enzymes must exist that remain to be identified.

Other epigenetic modifications such as DNA methylation, do not appear to be involved in FLC repression. Although animal PRC2 complexes are associated with DNA methyl- transferases, plant PRC2 repression does not involve DNA methylation. Besides, there are no changes in FLC DNA methylation by vernalization, which might suggest that this epigenetic mechanism would not be involved in vernalization- mediated FLC repression.

Similarity between Drosophila PRC2 repression and vernalization suggested that FLC repression may involve cis- elements similar to PRE. In this respect, a region (vernalization response element, VRE) has been identified to be required for vernalization- mediated H3K9 methylation; and it is likely that the VRN1 protein might be involved in VRE recognition, because it shares some properties with PRE binding proteins, and VRN1 is enriched at FLC chromatin during vernalization.

Few histone- modification specific binding proteins have been identified in plants. LHP1, the plant homolog for HP1, is not related to constitutive heretochromatin but to euchromatic regions. This protein is involved in the maintenance of FLC repression by vernalization, and because it is enriched in FLC chromatin, its role could be similar to Pc in polycomb repression. Also, there is a possibility that VIN3 could recognize H3K4me3 with its PHD finger domain, for the restoration of FLC activity. Another PHD finger protein required for proper vernalization response VIL1 (a VIN3 binding protein), has been recently identified.

Vernalized state is lost as repressed genes like FLC become reactivated in next generation; thus this could occur during meiosis. In support for this could be the observation that VRN1 and LHP1 expression is lower in pollen. Another possibility is that during meiosis, increased phosphorilation of H3S10 could lower LHP1 binding to FLC which in turn would lower the H3K9 methylation levels, lading to the re- establishment of heterochromatin.

In the end, it is clear from this review that much work needs to be done to explain the whole epigenetics of the flowering process.

Sung S, Amasino RM. 2006. J Exp Bot 57(13):3369-77
epigenetics

Gene-specific control of inflammation by TLR-induced chromatin modifications

2007-08-28T02:03:00.000-06:00

Toll- like receptors (TLRs) have an important role in mediating inflammation, and because this process can cause different changes in physiology, TLRs induce many genes with different functions. This makes unlikely that the regulation of these genes relies only at the signalling pathway level. Authors thus hypothesized:

[...] TLR-induced genes with different biological functions should have distinct requirements for regulation. Specifically, genes encoding pro-inflammatory mediators should be transiently inactivated in tolerant macrophages to limit tissue damage. On the other hand, genes encoding antimicrobial effectors and other proteins that do not negatively affect tissue physiology should remain inducible even after repeated stimulation of TLRs to provide continuous protection from infection.

First, using microarrays and real- time PCR, they found two classes of genes after their responsiveness to re-stimulation with LPS: tolerizable (T, genes not inducible in tolerant macrophages, pro- inflammatory), and non- tolerizable (NT, inducible in tolerant macrophages, antimicrobial) genes. These results, and other experiments detailed in the paper, confirmed that the induction of LPS tolerance inhibits the pro- inflammatory genes, while the other group (antimicrobial genes) remains inducible. This led to analyze the gene- specific regulation by chromatin modifications. ChIP experiments of acetylated H4 and trimethylated H3K4 showed that these chromatin modifications are lost in class T genes:

Acetylated H4 was found in both classes of genes, but only NT genes were re- acetylated after stimulation of tolerant cells;
H3K4 trimethylation was also found in both classes of genes, but only maintained in class NT genes following LPS stimulation;
Exposure to trichostatin A (TSA) inhibited the expression of class T genes;
Pargyline (an LSD1 inhibitor) treatment prevented the silencing of class T genes; and
Both treatments (TSA, pargyline) did not affect activations via NF-kB or MAPK pathways.

Recruitment of nucleosome remodelling complexes, Brg1 and Mi-2ß, was also inhibited in class T genes -this was observed using ChIP and REA/LM-PCR experiments. Taken together, these results mean that although both groups of genes were induced by the same receptor, their regulation is gene- specific and controlled via different chromatin modifications. Authors proposed a model for this process:

Model for gene-specific regulation of class T and NT genes
(Foster SL. et al./Nature).

[...] Collectively, these results indicate that gene products induced by LPS in naive macrophages differentially modify chromatin at class T and class NT promoters to silence the former and to prime the latter for their differential regulation by a second LPS stimulation.

Foster SL. et al. 2007 May 30. Nature [Epub ahead of print]
epigenetics

Genome-wide maps of chromatin state in pluripotent and lineage-committed cells

2007-08-27T04:50:00.000-06:00

This paper describes the development of another ChIPSeq methodology, and evaluated its usefulness by creating genome- wide maps of chromatin states that distinguished three mouse cell types: embryonic stem (ES) cells, neural progenitor cells, and embryonic fibroblasts. For each group, they used ChIP to analyze the occupancy of trimethylated histones (H3K9, H3K4, H3K27, H3K36, and H4K20) and RNA polymerase II. Later, Solexa/Illumina sequencings were performed from nanogram quantities of ChIP DNAs, and reads were computationally assembled. Validation of the methodology was both by computational analysis and by comparison to other wet lab methods. This first large- scale application of pyrosequencing in epigenetics suggests that we are getting closer to the era of disease- specific epigenomic maps, and public annotated databases of chromatin- states as well.

Mikkelsen TS. et al. 2007. Nature 448: 553-560
epigenetics

More comments can be found on the same issue of Nature [Editor's summary: Chromatin profiling; and Genomic biology].

DNA Methylation Analysis by MethyLight Technology

2007-08-18T19:26:00.000-06:00

This review discusses the advantages of MethyLight, a real- time PCR based method for DNA methylation quantitation from bisulfite- modified samples, and compares MethyLight to other related procedures like bisulfite sequencing, MSP, MS-SNuPE, and COBRA. First, as MethyLight is based on real- time PCR, there is no risk of contamination from input DNA or from handling errors. A limitation for this implementation is that MethyLight only detects a particular methylation at a time, but this can be corrected by automation, via simultaneous analysis of many samples in microtiter plates. The detection of CpG hypermethylation is better performed in this manner:

Principles of MethyLight technology (Trinh et al./Methods)

Authors discussed each step for this procedure: (1) determining the site of interest for methylation analysis; (2) methylation-specific primers and fluorogenic probes design; (3) genomic DNA isolation; (4) bisulfite conversion; (5) real- time methylation analysis; and (6) data processing. For primer design, some complications particular to the bisulfite modification should be taken into account:

All unmethylated cytosine residues in the genome are converted [...;] this reduction in genomic complexity reduces the annealing specificity of PCR primers and fluorogenic probes, which not only complicates primer and probe design, but also reduces the efficiency of each PCR cycle. Therefore, PCR amplification of bisulfite converted DNA requires more cycles, and is much less efficient than amplification of [... unmodified] DNA. Essentially, the amplification is being performed on a 3-base genome [...;] furthermore, amplification [...] is inefficient [... because] the template consists of a mixture of different DNA methylation patterns.

The amplicon typically should be 50-200 bp long, but if longer amplicons are desired, probes should be designed as close to the primer as possible. This probe is usually linked to a 5' reporter (mostly FAM) and a 3' quencher (e.g., TAMRA). Thus, a polymerase with 5' -> 3' exonuclease activity is required to process the reporter. On the other hand, respect to the sample DNA isolation, it does not need to be extensively purified as the bisulfite modification removes most contaminants -although in some cases these bisulfite chemicals are inhibitors of the PCR amplification. However, it should be kept in mind that very long bisulfite incubations produce more DNA degradation, and that even the sample storage is crucial since bisulfite- modified DNA is unstable. In conclusion, MethyLight has some drawbacks, but is very useful because it is able to process many samples in short time, can be automated, and does not require post- PCR manipulation; therefore is very practical for rapid screenings and other similar applications. The original article that introduced this method will be commented here later.

Trinh, BN. et al. 2001. Methods 25(4):456-462
epigenetics

Epigenetic Inheritance in Rice Plants

2007-08-16T20:41:00.000-06:00

In this study, authors tried to establish a causal relationship between changes in methylation patterns and acquired traits. To this end, they exposed rice seeds to azadC, and a line was selected from the surviving seedlings ('Line-2'), which was somehow phenotypically different from the wild type. From here, and although there was no conclusive evidence for a transgenerational effect so far, authors wrote:

[...] a pulse treatment of germinated seeds with azadC had induced phenotypic changes evident at maturity, and that such changes were stably inherited by the progeny.

To demonstrate that their observation had an epigenetic origin, they evaluated the methylation status of some loci using digestion with methylation- sensitive restrictases followed by blotting, but they found no significative differences. Perhaps a global methylation assay in this step could have been more informative. They later identified ~20 MSAP unique fragments in Line-2, one of them -termed 'HMF2'- predictably showing similarity to retrotransposon sequences; bisulfite analysis of HMF2 indicated that this fragment was hypomethylated in Line-2. Another fragment, HMF5, related to a pathogen- resistance gene, was also hypomethylated in the studied clone. Authors mentioned that this acquired change was directed instead of being at random, and that these sequences were methylated in wild type plants to prevent recombination, because they also contained a downstream retrotransposon sequence. Although these evidences were insufficient to prove the stable inheritance of methylation status in these plants, they concluded that:

[...] It was thus suggested that methylation/demethylation does occur under natural conditions, and that heritable epigenetic mutations play a significant role in evolution. The present finding substantiates this idea, showing that gene expression is flexibly tuned by methylation, allowing plants to gain or lose particular traits which are heritable as far as methylation patterns of corresponding genes are maintained. This is in support of the concept of Lamarckian inheritance, suggesting that acquired traits are heritable.

Clearly, a more detailed study is needed. And perhaps this next time the future authors will give a more informative title to their paper, instead of using this very generic and misleading one.

Akimoto K. et al. 2007. Ann Bot 100(2):205-217
epigenetics

Telomere length regulates the epigenetic status of mammalian telomeres and subtelomeres

2007-07-26T11:36:00.000-06:00

Recently, some research groups have reported an association between epigenetic regulation and telomere processing: For example, DNA methylation has been indicated as a repressor of telomere recombination, and some chromatin modifications (such as trimethylation of H3K9 and H4K20) are more frequently found in telomere regions. Besides, loss of these heterochromatin marks is linked to abnormal telomere elongation. In this paper, authors showed that telomere lenghts are related to the maintenance of the heterochromatic status of telomeres, using telomerase- null mouse fibroblasts as their model. First -in ChIP experiments- they found that heterochromatic marks (trimethylation of H3K9 and H4K20) decrease, global H4 acetylation increases, and the density of CBX3 (a protein involved in heterochromatin compaction) is lower, as telomeres became shorter, suggesting "an open [chromatin] state as telomere[s...] are lost." No other heterochromatin domains in the genome were associated with this telomere shortening, confirming the specificity of this effect. On the other hand, using bisulfite sequencing they found that subtelomeric regions, that are normally hypermethylated, loss most of this kind of modifications, in relation to this telomere shortening. CO-FISH experiments later showed that telomere- null cells have higher telomere recombination frequencies

In summary, telomere shortening leads to a changes in histone and DNA modifications at mammalian telomeres and subtelomeres characterized by decreased H3K9 and H4K20 trimethylation, increased histone H3 and H4 acetylation and as decreased DNA methylation at subtelomeric repeats, consistent with defective heterochromatin assembly at these regions. [...] Changes in telomeric and subtelomeric modifications in turn could provide a mechanism by which mammalian telomere repeats are counted and autoregulated.

Benetti R. et al. 2007. Nature Genet 39(2):243-250

epigenetics

Nucleosome positioning signals in genomic DNA

2007-07-24T21:41:00.000-06:00

Currently, there is great interest in developing a tool for effective prediction of nucleosome positioning. Here, authors employed data generated by a whole genome nucleosome mapping experiment by Yuan et al. (2005), to train a support vector machine (SVM) classifier. They found that their developed program was equally predictive as one previously reported by Segal et al. (2006), which established that ~50% of positioning was determined by DNA sequence (this study was already discussed here). This, authors said, demonstrates that the proposal of a nucleosome positioning code is inconsistent. In my opinion, respect to the developed tools, clearly a detailed comparison between this and previous nucleosome positioning programs is required since they all have similar predictive abilities. Their website is here.

Peckham HE. et al. 2007 July 9. Genome Res [Epub ahead of print]
epigenetics

Derivation of pluripotent epiblast stem cells from mammalian embryos

2007-07-23T23:21:00.000-06:00

In this paper, a novel type of stem cells are described, the epiblast stem cells (EpiSCs), derived from late rodent epiblast layers and discovered for its ability to grow in a chemically defined medium (CDM) containing activin A and FGF2 (CDM/AF), forming colonies with characteristics of pluripotent cells, and expressing markers of pluripotency; thus they differ from mouse ES cells not only in their tissue of origin, but also in their pluripotency pathway (activin/Nodal). Four levels of characterization confirmed the uniqueness of EpiSCs: first, a morphological analysis of cultures; second, EpiSCs were analyzed both with qualitative (PCR, immunostaining) and quantitative (q-PCR) methods, confirming that they were neither mouse ES cells nor primordial germ cells; third, microarray expression patterns of EpiSCs resembled their original layer; and finally, the pluripotency of these cells was assessed both in vivo (injected colonies were able to produce teratomas containing several types of tissues -confirming that EpiSCs have a multi- lineage pluripotency) and in vitro (EpiSCs differentiated into a wide variety of cell types). Clonal sublines produced similar results. Interestingly, EpiSCs share with human ES cells many differences with mouse ES cells that were previously attributed to species divergence, for example their epigenetic stability, that could reflect a similar epiblast origin. This was the editor's summary for this and a related paper that appeared in the same issue of Nature:

A new type of stem cell. Human embryonic stem (ES) cells are potentially important in therapy because they are pluripotent, capable of differentiating into virtually any cell type given appropriate encouragement. One obstacle to progress in research on them has been the baffling differences between human and mouse ES cells. Now two groups working independently have created a new kind of pluripotent ES cell. Derived from mouse embryos after they implant in the wall of the uterus, these EpiSCs (epiblast stem cells) are distinct from 'classic' mouse ES cells and mirror key features of human ES cells. The discovery of EpiSCs should provide an important experimental model to accelerate the use of human ES cells in research and eventually perhaps, in therapy.

Brons IGM. et al. 2007. Nature 448:191-195

epigenetics

Conformational flexibility in the chromatin remodeler RSC observed by electron microscopy and the orthogonal tilt reconstruction method

2007-07-16T23:56:00.000-06:00

Although structural characterizations of chromatin remodeling complexes would help to understand how this remodelers work, to date their electron microscopy reconstructions have been limited, because of its large macromolecular size and other impediments. In this paper, authors used a novel approach in cryo- electron microscopy, the orthogonal tilt reconstruction (OTR) method, to obtain a structure of the yeast RSC (remodel the structure of chromatin) complex, a remodeler that belongs to the SWI/SNF family. OTR consists on collecting data with a complete rectangle angle of difference, resulting on orthogonal views. With this new approach, they identified a conformational variability in the RSC structure (a region that opens and closes much like a turntable needle) that might have functional repercussions, because it could function as a entry point and it could promote the translocations of the nucleosomes. They also showed, using EMSA assays, that RSC has no preference for mono- or dinucleosomes. Perhaps what I found more interesting is that they employed optical tweezers to demonstrate, in single molecule experiments, that these translocations occur in mononucleosomes. All these results support their proposed model that fits only a mononucleosome, in both open and close conformations.

Model of nucleosome binding by RSC
(Leschziner AE et al/PNAS)

Taken together, the striking complementarity between the RSC central pocket and a nucleosome, the similar affinity of RSC for mono- and dinucleosomes, and the fact that the translocation observed with mononucleosomal substrates in single molecule assays is identical to that observed on nucleosomal arrays, strongly suggest that remodeling by RSC only requires the binding of a single nucleosome.

Besides of their conclusions respect to the RSC structure, as I am a big fan of single molecule experiments, I look forward to see more applications of optical tweezers and other biophysics tools in Epigenetics research.

Leschziner AE et al. 2007. Proc Natl Acad Sci USA 104(12):4913-4918
epigenetics

A Chip off the Old Block: A Model for the Evolution of Genomic Imprinting via Selection for Parental Similarity

2007-07-12T07:18:00.000-06:00

Here, a two- loci mathematical model for the evolution of imprinting is hypothesized, in which imprinting of the alleles depends on the alleles of another loci, a cis- acting modifier. This paramutation- based model can explain either maternal or paternal inactivation.

we [... propose] that imprinting will evolve in situations in which selection favors offspring being similar to the parent of one sex. This "chip-off-the-old-block" hypothesis points out that a direct consequence of imprinting is that offspring will phenotypically resemble one parent more than the other, independent of the offspring's sex [...]. A potential scenario for the generation of selection for parental resemblance arises because of the male-biased differential migration rate in most mammals [...]. In a heterogeneous habitat, mothers are likely to be better adapted to the local environment than fathers, because the latter are more likely to have been subject to different selection pressures elsewhere. Thus, at least early in life, before any migration, offspring of both sexes would be selected to resemble their mothers. Thus the chip-off-the-old-block hypothesis would predict that almost any locus exhibiting local adaptation, especially in juveniles [...], could be imprinted. This prediction contrasts with those of the genetic conflict and ovarian time bomb hypotheses that focus on genes active during fetal development and, in the case of genetic conflict, soon after birth.

This is an interesting conceptualization, but authors recognize that this hypothesis is unlikely to explain the evolution of imprinting because it requires high levels of recombination and migration; however, this difficulty would prove "why imprinting is not more widespread phylogenetically". The importance here is that these mathematical models would help not only to understand the origin and evolution of the mechanisms of imprinting, but also this may enable predictions.

Spencer HG, and AG Clark. 2006. Genetics 174:931-935
epigenetics

Cancer initiation and progression: an unsimplifiable complexity

2007-07-10T17:13:00.000-06:00

In this short paper, authors discuss the potential of creating mathematical models of cancer in basis of the current knowledge in genetics and epigenetics, but instead of using the traditional reductionist methodology, they support a more comprehensive systemic approach. In their opinion, in this way it would be possible to reveal tumor properties not yet considered, towards better practices in research and medical care. Some of their conclusions were:

[...] Cancer is a hierarchical system. The decisive step in carcinogenesis is the result of an irreversible qualitative change in one or more of the genetic characteristics of cancer cells. Although this modification governs the transformation of normal human cells into malignant cancer cells, it may or may not lead to visible changes [...]. This can be explained using the concept of emergence [...].
Although the alterations usually occur at a characteristic stage of tumour advancement, experimental evidence indicates that the ongoing accumulation of changes is more important than their order of occurrence in the course of cancer. In physical terms, it is true that alterations in one parameter [...] are not necessarily associated with the loss of stability of a system, and it is also true that an unstable system is more sensitive to small changes in parameters [...].
Considering cancer as a robust system [i.e. the ability of a living system to maintain performance (phenotypic stability) in the face of perturbations arising from environmental changes, stochastic events (or intracellular noise) and genetic variations] would provide us with a framework for future research strategies [...].

Perhaps a systemic modelling approach could answer some of the still elusive epigenetic questions, or reveal new ones that could never appear using a reductionist methodology.

Grizzi F. et al. 2006. Theor Biol Med Model 3:37
epigenetics

Histone replacement marks the boundaries of cis-regulatory domains

2007-07-08T23:35:00.000-06:00

One of the still unsolved questions is how the epigenetic memory is maintained in regulatory regions, and passed through generations. Previously, Mito et al. (2005), developed an strategy to identify patterns of histone replacement at a genomic scale. In this report, they used that biotin epitope tag approach to measure the histone H3.3 (that is found throughout the cell cycle) to H3 (deposited during replication) ratio in premitotic Drosophila homeotic genes. They found that the replacement of histones and nucleosome occupancy is distinctive in cis- regulatory regions; and they suggest a cyclic model for this process:

What process maintains the chromatin of cis-regulatory elements in a state of flux? Many DNA- binding and chromatin- binding proteins involved in gene regulation display short residence times on DNA, and some mouse transcription factors show dynamic behavior at their functional binding sites. A model for this process has been proposed, involving alternating cycles of nucleosome disruption by a Brahma- related SWI/SNF chromatin-remodeler and transcription factor binding. The binding of PcG and trxG proteins is also dynamic, and we propose that a similar cycle of nucleosome disruption and factor binding takes place at boundaries and PREs. Nucleosome disruption by SWI/SNF remodeling complexes would occasionally evict nucleosomes and transiently expose DNA, which would become available to other diffusible factors, including PcG proteins. The continued local presence of nucleosome remodelers would result in another cycle of remodeling, nucleosome depletion, nuclease hypersensitivity, and histone replacement at the site. […] The resulting dynamic process would allow for proteins that promote opposite epigenetic outcomes to act at common cis-regulatory sites.

Mito Y. et al. 2007. Science, Vol. 315(5817):1408-1411

epigenetics

More comments can be found in this issue of Science STKE, and at the Faculty of 1000 website. Also, a related paper from the same issue of Science by Dion et al., was previously discussed here.

Inheritance of a cancer-associated MLH1 germ-line epimutation

2007-07-05T07:52:00.001-06:00

Germ- line genetic mutations or epigenetic modifications of mismatch- repair genes, MLH1 and MSH2, have been associated to hereditary nonpolyposis colorectal cancer. In this paper, authors claimed that they have found evidence of germ- line epimutation of MLH1 in a woman with cancer and in her son, thus supporting transgenerational epigenetic inheritance in humans, something that has been previously demonstrated in other species, like mice. Although they favour their epigenetic inheritance hypothesis, they did not discard other possible explanations:

An alternative explanation for our findings is that epimutations are not inherited per se. Rather, they are erased in gametogenesis but reestablished in successive generations because of cis-acting or even trans-acting genetic factors that increase susceptibility to MLH1 epimutations. Examples of epigenetic silencing that are driven by genetic events in cis include deletion of imprint-control centers in imprinted disorders and expansion of triplet repeats within the FMR1 promoter in the fragile X syndrome. Such a mechanism may also explain the recently reported strongly heritable pattern of epimutation in MSH2, since the methylation state segregated faithfully with the genetic haplotype. In contrast, in the two families described in our study, we found no evidence of a fully penetrant in cis defect. Rather, they showed epimutations that were meiotically reversible and transmitted in a nonmendelian fashion. A simple explanation for this pattern is that epimutations can occur on any haplotype, and although they usually are cleared in the germ line, they may be retained at low but uncertain frequency.

However, their observations could account for other possibilities, for example: Why isn't this an effect of the same shared environment, rather than an inherited trait? This would be in agreement with the spontaneous reversion of the MLH1 epimutation to normality during spermatogenesis that they found in one of the patients. Besides, epigenetic marks may vary through time, so any reseach that hopes to demonstrate transgenerational epigenetic inheritance should follow up the variation of methylation and histone marks periodically. For example, Fraga et al used pairs of twins of different age groups in a paper previously discussed here. As a consequence, in my opinion their observations are inconclusive to demonstrate epigenetic inheritance in humans, as was a controversial paper by Chan et al. (2006), that was previously discussed here.

Hitchins MP. et al. 2007. N Engl J Med 356(7):697-705

epigenetics

More comments can be found in the same issue of the NEJM, in this article of Gastroenterology, and at the Faculty of 1000 website.

Epigenetic Regulation of Tumor Necrosis Factor Alpha

2007-07-04T21:13:00.000-06:00

Tumor necrosis factor alpha (TNF-) is an inflammatory cytokine that is involved in several immune responses and metabolism. TNF expression regulation is complex, and still very little is known about its epigenetic control. In this paper, Sullivan et al. evaluated the CpG methylation and histone modifications in various cell lines and primary tissues. Their study suggests that cells acquire certain epigenetic marks in the course of development, mostly at the level of transcription.

Our data appear to show an orderly series of events that accompany myeloid differentiation and that these events progressively lead to competence for the production of TNF-alpha

Epigenetic changes at TNF- during development
(Sullivan et al./MCB)

[...] The first event is DNA demethylation of the TNF-alpha locus, which seems to arise from the coding region and extend 5' into the promoter. The DNA demethylation begins to occur between the EB stage and differentiation into hematopoietic stem cells. Further demethylation continues as the cells differentiate into monocytes. A cell such as the hepatocyte, which is capable of producing TNF-alpha only after a priming stimulus, is only partially demethylated. A second step in the acquisition of transcriptional competence is the relocation of the TNF-alpha locus into euchromatin.

Sullivan KE. et al. 2007. Mol Cell Biol 27(14):5147-60
epigenetics

An RNA-dependent RNA polymerase is required for paramutation in maize

2007-07-03T22:26:00.000-06:00

Paramutation, an allele-dependent transfer of epigenetic information, which results in the heritable silencing of one allele by another, requires the mop1 (mediator of paramutation 1) gene to occur in maize. Current proposed and demonstrated models of paramutation include trans- acting RNAs, and interactions between chromatin protein complexes and encoded tandem repeats. To elucidate the mechanism of this phenomena, Alleman et al. identified by positional cloning that mop1 coded for an RNA- dependent RNA polymerase (RDRP) homolog gene, in a mutated form that has a stop codon upstream to the polymerase domain. Therefore, their results provide support for the trans- acting RNA model. This discovery is in agreement with other investigations that also found an involvement of RNAi pathways in RNA- directed DNA methylation, for example with one previously discussed here; although Alleman and colleagues failed to detect any specific siRNA for this proposed mechanism of paramutation in maize, and they provided possible explanations for this:

We propose that the level of transcription occurring in the three genotypes is not sufficient to trigger paramutation or to maintain transcriptional silencing [...] and that the RDRP encoded by mop1 is required to produce a higher threshold of RNA [...] that mediates these processes. [...] An intriguing question is why the transcribed single-copy sequence does not induce silencing. A hypothesis about the importance of tandem arrays in maintaining silencing in centromeric heterochromatin offers a possible explanation. Another idea is that the unique junction fragments created by the tandem repeats have specific properties. Because the penetrance of paramutation increases with the number of repeats, a mechanism that senses the number of repeat junctions needs to be postulated.

[...] Our results add the classical paramutation phenomenon to a growing list of processes involving the RNA-mediated formation of chromatin domains. Some examples include dosage compensation in flies, X-inactivation in mammals, RNA-directed DNA methylation in plants, meiotic silencing of unpaired chromatin in Neurospora and C. elegans, and transcriptional silencing of centromeres, transgenes and transposons in multiple species. Like paramutation, most of these phenomena share epigenetic regulation, long-range interactions and chromosome dynamics. Unique features of b1 paramutation [in maize] are its extreme penetrance and faithful transmission through meiosis.

Now that these results proposed that mop1 is a RDRP homolog, it would be interesting to characterize the activities of this yet hypothetical protein, and its structural properties as well.

Alleman M. et al. 2006. Nature 442:295-298

epigenetics

More comments can be found at the Faculty of 1000 website.

A Genomic Scanning Method for Higher Organisms Using Restriction Sites as Landmarks

2007-07-02T15:31:00.000-06:00

Restriction landmark genomic scanning (RLGS) is a method that is employed to analyze the genomes of vertebrates and plants, using restriction enzyme recognition sites as landmarks. The method consists of seven steps: (1) Blocking of the cleaved genomic DNA, to avoid high backgrounds, with nucleotide analogues; (2) Landmark cleavage, with a rare cutting restrictase; (3) Labeling of the cleavage ends, followed by digestion with a higher frequency restrictase; (4) Agarose gel electrophoresis; (5) Digestion with a third restrictase (average fragment size <10 Kb); Polyacrilamide gel electrophoresis; and (7) Autoradiography.

The RLGS Procedure (Hatada et al./PNAS)

This bidimensional electrophoretic procedure is reproducible and, in this way, whole genome profiles can be generated with mean intervals of ~150 Kb and ~1 Mb, depending on the total genome size. Besides, as each spot represents a locus and its copy number, this technique can detect the hetero- or homozygosity allelic status. Furthermore, as no probes are needed, it could be used to any organism. The authors mentioned that it can be applied to genome mapping, evolutionary distances, polymorphism, etc., but it also has been used to identify imprinted loci, using a combination of methylation- sensitive restriction enzymes. One of those protocols was previously discussed here.

Hatada I. et al. 1991. Proc Natl Acad Sci USA 88:9523-27
epigenetics

Repeat-induced epigenetic changes in intron 1 of the frataxin gene and its consequences in Friedreich ataxia

2007-06-30T02:44:00.000-06:00

Friedreich ataxia (FRDA) is the most commonly inherited ataxia. It is recessively inherited, and caused by an expansion of the GAA TTC repeat, present in the intron 1 of the frataxin FXN gene. The expanded alleles produce less mature mRNA, although the repeats could alter the expression in other ways, as occurs with the centromeric tandem repeats that are normaly silenced. Since this control is probably by means of alteration of epigenetic marks, these chromatin alterations were evaluated for the FXN gene. First, analysis of the region of intron 1 between exon 1 and the FXN repeat was found to contain three interspersed repeated elements. Bisulfite sequencing of this region showed that most CpGs were methylated both on affected and unaffected individuals, but methylation was more extensive in the first group. Patients with FRDA also had elevated levels of H3K9me2, a mark of chromatin compaction, that correlated with low levels of FXN mRNA. This data support the idea of a epigenetic mechanism responsible for the low expression of FXN.

Model for chromatin organization in FRDA alleles
(Greene et al./NAR)

How could the long FXN-repeat tracts in patient cells generate transcriptionally silent chromatin? One possibility is that this process involves small double-stranded RNAs (dsRNAs) that target the transcriptional silencing machinery to homologous regions of the genome. [...] Repeat-mediated heterochromatin formation is seen in two other Repeat Expansion diseases: Fragile X syndrome and the congenital form of Myotonic Dystrophy type 1. Thus repeat-mediated transcriptional silencing may provide a common thread linking these diseases where the repeat is transcribed but not translated.

Greene E. et al. 2007. Nucleic Acids Res 35(10):3383-90
epigenetics

Sex chromosome silencing in the marsupial male germ line

2007-06-26T22:33:00.000-06:00

In this paper, Namekawa et al., studied the X-inactivation (XCI) by imprinting in marsupials, a process not described before at the molecular level for this taxa. The complication here was that, opposite to what happens in eutheria where XCI is regulated by Xist and other components of the Xic locus, marsupials lack of a XIC homolog. Using several FISH and immuno- staining experiments in Cot-1 DNA, they found that both meiotic sex chromosome inactivation (MSCI) and postmeiotic sex chromatin (PMSC) do occur in the opossum. They also found similarities in the histone epigenetic marks between eutheria and marsupials:

In conclusion, our study [...] show that MSCI and PMSC occur in the marsupial and that the silencing initiated during the first meiotic prophase continues through meiosis II and into the postmeiotic period. Thus, spermatogenic events regulating transcriptional activity of the sex chromosomes are very well conserved in the marsupial and eutherian. This is in striking contrast to the absence of conservation in XIC elements that regulate XCI in the eutherian soma

Continuity of silencing from MSCI to PMSC in marsupial (Namekawa, SH. et al./PNAS).

[...] The state of the paternal X upon arrival in the opossum zygote requires further study. In the absence of any significant cytoplasm and the replacement of histones for protamines, how might epigenetic information be transmitted from the sperm to the zygote? [...] Our studies have highlighted three persistent marks of meiotic silencing (H3–3meK9, HP1beta, and HP1gamma) that are shared between eutherians and metatherians. Interestingly, H3–3meK9, HP1beta, and HP1gamma are also the last chromatin- associated marks to be detected before protamine- mediated compaction during mouse spermiogenesis. These marks are therefore candidates for transgenerational inheritance of epigenetic programming associated with the paternal X.

Namekawa SH. et al. 2007. Proc Natl Acad Sci U S A 104(23):9730-35
epigenetics

More comments can be found at the same issue of PNAS.

Computational prediction of methylation status in human genomic sequences

2007-06-25T22:07:00.000-06:00

Computational epigenomics is a growing field that until now has been mainly focused in two topics: identification of methylated CpG islands, and allele- specific cytosine methylation. But still there is no algorithm for methylation pattern prediction, based on sequence alone. In order to develop this kind of predictor, Zhang's group generated a training dataset from the enzymatic digestion of human brain DNA. They found three kinds of differences between the methylated (M) and unmethylated (U) fragment groups: (i) the Takai- Jones criteria for CpG islands (a larger number in the U set); (ii) the distribution of Alu elements (M sequences were richer in AluY and AluS); and (iii) hexamer abundance. These measures were integrated into a support vector machine (SVM) classifier approach. SVM correctly predicted the methylation status of non-CGIs and CGIs, with 84% and ~97% accuracies respectively, using an optimal sliding window of 800 bp. This was an important advance because, opposite to previous datasets, this came from normal human DNA. The implemented algorithm, HDMFINDER, is available from the authors.

Das R. et al. 2006. Proc Natl Acad Sci USA 103(28): 10713-16
epigenetics

Bypassing genomic imprinting allows seed development

2007-06-23T18:26:00.000-06:00

Angiosperm plants imprint some genes during endosperm development, and although some components of the FIS complex (mea, fis2 and fie; related to the Polycomb group of chromatin remodelling factors) have been implicated in mediating this phenomenon, the actual role of imprinting in seed development remains unclear. In this paper, Nowack et al. generated Arabidopsis seeds whose endosperm is uniparental (maternal: the CDKA;1 mutant tagged with a YFP reporter), and using mutants for the FIS- class genes, they showed that uniparental seeds can develop without a paternal genome contribution if they lack of any of the functional FIS- class genes, meaning that imprinting in developing seeds can be bypassed. This investigation brings new insights for the evolutionary role of the endosperm, in contrast to the gymnosperm development, where descendants are produced by a single fertilization.

Nowack MK. et al. 2007. Nature 447: 312-316
epigenetics

More comments can be found at the same issue of Nature, and at the Faculty of 1000 website.

Genome-Wide Mapping of in Vivo Protein-DNA Interactions

2007-06-22T23:40:00.000-06:00

Current methods for protein- DNA interaction identification at a genomic scale are limited because of their inability for mapping binding sites with high resolution, they are not very cost- effective and they are mostly non- high- throughput. Besides these technologies are usually based on ChIP assays, and therefore they share the same problems (low accuracy, limited coberture, poor resolution). Here the authors report a large- scale ChIP assay ("ChIPSeq"), that consists of a first step of standard ChIP, followed by size selection of the generated immuno- enriched DNA fragments (a step that increases resolution), and then a ultrahigh- throughput sequencing -the Solexa/Illumina method- is performed. The sequencing reads are computationally filtered and mapped, and because of the very large number of reads produced by the Solexa/Illumina system, a greater sensitivity and statistical certainty is achieved (which is especially helpful when the desired goal is to identify a motif). Unlike other related techniques, this involves no plasmid library construction, single copy sites are available, and is feasible for any sequenced genome, rather than only for those whose arrays have been developed. The authors evaluated the utility and power of their method by building an interactome map for the NRSF/REST transcription factor. They also suggested that other ultrahigh- throughput sequencing platforms could be used instead of the Solexa/Illumina system.

Johnson DS. et al. 2007. Science 316:1497-1501
epigenetics

More comments can be found at the same issue of Science, and at the STKE and Faculty of 1000 websites.

HIV integration site selection: Analysis by massively parallel pyrosequencing reveals association with epigenetic modifications

2007-06-21T14:12:00.000-06:00

Selection of favorable integration sites is required for efficient HIV infection. Integration shows no sequence- specificity, and although some factors that support efficient integration have been identified, cells depleted for them are still infected. Some studies have suggested that nucleosomes could play a role as a substrate for integration. Here, Wang et al. (2007) used pyrosequencing to generate more than ~40,000 unique integration sites, and with a nucleosome positioning prediction tool (discussed previously here), they identified a pattern for integration: It was "favored at phosphate backbone sites at the edges of outwardly facing major grooves" of chromatin. ENCODE annotations correlated positively the generated integration sites with active expression epigenetic marks (H3 acetylation, H4 acetylation, H3K4 methylation), and negatively with silencing modifications (H3K27 trimethylation and CpG methylation). Authors suggested that the viral integrase could interact with methylated histone tails, or with other chromatin associated proteins, or simply these epigenetic modifications could be only markers of favored integration sites, with no active role. Besides, this novel experimental design could be useful to evaluate the integration sites not only for other viruses, but also for the toxicity problems sometimes associated with gene therapy viral vectors.

Wang, GP. et al. 2007 Jun 1; Genome Res 17:1186-1194
epigenetics

The Locus of Evolution: Evo Devo and the Genetics of Adaptation

2007-06-20T14:51:00.000-06:00

This recent paper discusses the affirmation made by evolutionary developmental (evo-devo) biologists, that adaptive mutations leading to morphological changes are more likely to occur in cis- regulatory sequences rather than in protein- coding regions.

But are this claims supportable? [...] We will conclude that evo devo's enthusiasm for cis- regulatory changes is unfounded and premature. There is no evidence at present that cis- regulatory changes play a major role -much less a pre-eminent one- in adaptive evolution.

Despite their comprehensive explanation, they did not mention the now well documented impact of the epigenetic regulation of cis- acting elements on adaptive evolution, as we however have read about it here before in some papers, and overall in the book by Jablonka and Lamb (2005)

Hoekstra HE. and Coyne JA. 2007. Evolution 61(5):995-1016
epigenetics

More comments can be found at the Faculty of 1000 website.

Dynamics of Replication-Independent Histone Turnover in Budding Yeast

2007-06-19T15:25:00.000-06:00

Nucleosomes are forced out from the promoters for gene expression, and later reassembled for repression. Disagreement between studies on histone replacement dynamics in Drosophila, Saccharomyces and Physarum, lead this group to address this two questions at a genomic scale: (1) Is there evidencefor general transcription-dependent H3 turnover? and (2) Arethere additional mechanisms for histone turnover? To this end, they compared the ratios of two systems carrying histone H3 protein fusions: A constitutively Myc-H3, and an inducible Flag-H3. First, turnover rates were measured in arrested G1 cells using ChIP-on-chip of the fusions, and hybridized to an array covering 4% of the genome (the turnover rate was defined as the number of H3 replacements per unit of time). They found that nucleosomes over protein- coding regions were "coldest" (meaning that they rarely turn over), whereas promoter region nucleosomes were "hotter" (with faster replacement rate). These results were confirmed by repeating the experiment in unsynchronized yeast cells. Analysis of RNA polymerase II occupancy of coding regions was found to be correlated with the variation of histone replacement rates in protein- coding regions; although authors mentioned that it was unlikely that this turnover is only a result of the polymerase activity. Altogether, they concluded that "erasure of histone marks ... by rapid turnover delimits the spread of chromatin states"

Dion MF. et al. 2007. Science 315:1405-8
epigenetics

More comments can be found in the same issue of Science.

DNA demethylation in the Arabidopsis genome

2007-06-18T21:44:00.000-06:00

DNA methylation in Arabidopsis is found in different target sequences, besides of the CpG dinucleotide. The DNA demethylation process is very active and it is mediated by the DEMETER (DME) family if helix- hairpin- helix DNA glycosilases, that also have an additional apurinic/apyrimidinic (AP) lyase activity. This family has four known members: DME, REPRESSOR OF SILENCING1 (ROS1, a DME homolog), DEMETER- LIKE2 (DML2) and DML3; and demethylation can occur by mediation of DME but also by ROS1; the activity of the two other members of this family has not been reported. In this paper, Penterman et al. (2007) used microarrays to identify loci demethylated by ROS1, DML2 and DML3. To this end, they first confirmed the 5meC demethylase activity of DML2 and DML3. Then, they compared the genome- wide methylation pattern changes when wild- type or single- knock out mutants were screened for expression; here, they found "~179 loci were methylation and demethylation pathways converge". These results were confirmed by bisulfite sequencing. Comparison between bisulfite sequencing data from a triple mutant, and from single mutants and wild type plants showed that hypermethylation at the majority of these loci was regulated by this family of glycosilases; and also that redundancy of this regulation exists for DML enzymes at some loci (i.e., a loci can be demethylated by more than one DML enzyme). Crosses between single mutants and wild type individuals restored the demethylation levels in most loci (11 out of 14), and methylation levels were intermediate between mutant and wild type in the remaining loci, with a parental origin- specificity. Thus these were epilalleles, and hence DML demethylation "prevents the formation of stable epialleles" in some loci, a very interesting housekeeping function for this family.

Penterman J. et al. 2007. Proc Natl Acad Sci USA 104(16):6752-57
epigenetics

More comments can be found at the Faculty of 1000 website.

Methylation-Specific Oligonucleotide Microarray: A New Potential for High-Throughput Methylation Analysis

2007-06-08T10:32:00.000-06:00

Here Gitan et al. (2001) reported a microarray- based method for the assessment of the methylation status of DNA sequences. Genomic target DNA is modified by bisulfite treatment, and a PCR amplification of the CpG island is carried out. The amplification products are then labelled with fluorescent dyes, one for unmethylated and another for methylated sequences, and hybridized to the oligo probes fixed in the microarray. The authors evaluated the feasibilty of this development by assessing the methylation status of the estrogen receptor (ER) CpG island.

[...] Test DNA samples are bisulfite-modified, PCR-amplified products that contain pools of DNA fragments with altered nucleotide sequences due to their differential methylation status. As shown, the unmethylated allele of a given DNA sequence is expected to have the unmethylated cytosine of the test CpG sites converted to thymine, whereas these CpG sequences remain unchanged in the methylated allele. Target DNA is then hybridized to arrayed oligonucleotide probes specifically designed to discriminate between converted and unconverted nucleotides at these CpG sites

Schematic outline of the MSO assay (Gitan et al./Genome Res)

[...] As with other oligonucleotide microarrays, cross-hybridization between imperfect-match probes and targets was observed inour initial study using the MSO assay. To overcome this, we havefound that selecting the optimal sequence composition for eacholigonucleotide probe is critical in the assay. The specificityof a probe drops greatly when it contains more than four consecutiveT or G residues. In addition, some probes may have inherentlydiminished hybridization signals, probably due to decreased duplexstability of targets and probes. Through carefuldata analysis, we have noticed that cross-reactivity might alsoincrease when oligonucleotide probes are designed to query methylationdifferences in one single CpG site. This issue is easily overcomeby designing probes to include two or more CpG sites. This designconsideration may limit our ability to detect methylation changesin single CpG sites. Nonetheless, it is usually not necessaryto assess the overall methylation status of a given CpG islandby analyzing every CpG site within the locus [...].

Gitan RS. et al. 2001. Genome Res 12:158-164
epigenetics

Methylome profiling of cancer cells by amplification of inter-methylated sites (AIMS)

2007-05-21T23:24:00.000-06:00

Here Frigola et al. (2002) reported a novel technique for the discovery of methylome regions by fingerprinting, based on the MCA method. This new technology consists of two procedures: First, DNA is digested with the methylation- sensitive SmaI restrictase, which leaves blunt ends, and then a second cleavage (this time with the isoschizomer PspAI) is performed, leaving an overhang that will be used to ligate complementary adapters. In the second step, a PCR amplification is carried out, with primers that are homolog to the adaptor, the restriction site, and 1-4 arbitrary nucleotides. A diagram of the method can be seen in this figure. Then, for a sequence to be amplified, it requires: (1) to contain to spaced SmaI sites, and (2) to have homology with the 3' end of the primer. Sensitivity tests with tumor tissue samples showed that the technique is able to detect hypermethylation even when it is present only in less than 1% of the cells. However, interpretation of the AIMS fingerprints should take into account problems that could arise with some samples, for example with age- related methylation patterns, hypermethylations present in small subsets of cells or heterogeneous (mosaic) methylation profiles, and of course incomplete restrictase digestions. AIMS is useful because it allows the assesment of global methylation profiles, generates a high number of sequences with methylated CpGs, and reduces the complexity of the methylome by obtaining fingerprints, allowing the discovery of novel target sequences. Other important advantages are that it can process a large number of samples simultaneously, and that it does not require very high amounts of starting material.

Frigola, J. et al. 2002. Nucleic Acids Res 30(7):e28
epigenetics

CpG islands as gene markers in the vertebrate nucleus

2007-05-20T23:03:00.000-06:00

A small proportion of the unmethylated genome (1%), the HpaII tiny fragment (HTF) fraction, has been observed in a wide range of species, by cutting with the methylation- sensitive HpaII restrictase. This differs from bluk DNA by being mostly unmethylated at the CpG dinucleotides, and a high G+C content, and they are frequently found as "islands" (Although they were called as CpG islands by Gardiner-Garden and Frommer before, the name was well established after this paper). Their distinguishable features are their very high CpG density, that is partially attributed to the elevated G+C content, but it could be due because of the absence of CpG deficiency - CpGs occur here at the expected composition frequency, and in other sequences it is perhaps lower because of the mutability of the 5mC to T by deamination. This has been observed as a very probable explanation of the different compositions seen in the human alpha-globin pseudogene in comparison with the functional copy, where only four out of 70 CpGs in the expressed gene still persist in the non- expressed pseudogene, and the remaining have been replaced by TpG and CpA. Another example of this mutability phenomenon has been seen in Neurospora 5S rDNA. This suggests that CpG islands have not lost their CpGs because they are mostly unmethylated. CpG islands are closely related to genes, and they have been associated both to housekeeping and to tissue- specific genes as well, where most islands include the 5' end of the transcribed region. Froman evolutionary point of view, on the other hand, invertebrate genes are found in unmethylated regions or CpG deficient, so it is reasonable to think that in the vertebrate lineage of the methylated fraction has grown at expenses of the old invertebrate sequences. As CpG islands are clearly associated with genes, they are helpful for locating and isolating genes of interest (different cloning strategies are mentioned in the paper). Existance of the CpG islands can be explained as accidental ("an inconsequential by-product of the evolution"), or they are essential, that is a more interesting possibility, because of their association with 5' regions, that suggests a role in the DNA - transcription factors interaction. Then, they could be crucial in the regulation of gene expression, and one form could be by rendering sequences through altered chromatin structures.

Bird, A. 1987. Trends Genet 3(12): 342-347
epigenetics

Specific protection of methylated CpGs in mammalian nuclei

2007-05-18T23:19:00.000-06:00

In this historical paper, Antequera et al. (1989) demonstrated that methylated CpGs were specifically associated with MeCP, a protein that was the first characterized from the MBD family. They started with a simple -but very powerful- hypothesis: If specific proteins are bound to methylated CpG rich sequences, this DNA should be resistant to the restriction enzyme cleavage, in contrast with unmethylated CpGs that would be fully accessible. To test this, they employed two methylation sensitive (HpaII and HinPI) enzymes, and MspI, that cleaves CpGs irrespective of its methylation status. They first cleaved liver nuclear DNA with the three enzymes, and then the digested DNA was end-labelled with radioactive dCTP and the Klenow fragment of the DNA Polymerase I. Electrophoretic separation showed a similar pattern for the three enzymes, a ladder of ~200 bp fragments, that corresponded to the nucleosomal repeat. These results were surprising since the difference in sensitivity to methylation between these enzymes. They evaluated if DNA- associated proteins were responsible of this, by repeating the experiment in naked DNA, and this time MspI cut more frequently than the other enzymes. This showed that MspI was insensitive when naked DNA was used as substrate, but it became indistinguishable from methyl-CpG sensitive enzymes when chromatin is cleaved. This particularity was confirmed by digestion with enzymes that contained no CpGs in their recognition sites (HaeIII and Sau3A). Also, it was observed that this resistance to cleavage could not be overcome by the addition of more restrictase. Confirmation that MspI was cutting only in unmethylated DNA was seen by thin layer chromatography of the 5' terminal Cs, after full DNase I digestion of the 32P-ATP labelled DNA. Using another enzyme (Tth) proved that the cleavage blockage was not unique to MspI. However, artificially methylated DNA was cleaved by MspI, thus showing that the methylation alone is not enough to inhibit the cleavage, but it was a consequence of the exclusion by 5mCpG- associated proteins. As this results predicted, analysis of specific sequences (here they used a 16 Kb region of the alpha-globin gene) showed the same effect, as seen by Southern blot. They considered that MeCP, a methyl-CpG binding protein, could be responsible of the protection. To evaluate this possibility, they used PC13 cells, which have very low levels of MeCP, and measured the amount of cleaved methylated CpG by thin layer chromatography. This time, although some protection was still visible, the amount of cleavage in methylated sites was considerably greater than in brain cells. In vitro protection assays gave the same results. Taken together, their results demonstrated that, although nucleosomes protected the DNA from restriction endonucleases, as seen by the ~200 bp ladders formation, they could be associated with methylated sequences, but because the reduced protection of 5mCpGs in PC13 cells, these could not be responsible of the less efficient digestion; however, MeCP is a better candidate, as it binds CpGs in several sequence contexts, and enzymatic cleavage protection was reduced in PC13 (MeCP deficient) cells and in vitro. From this, the authors postulated that MeCP binds to the 5mCpG sequences, and could function as a transcriptional repressor.

Antequera, F. et al. 1989. Cell 58(3):509-517
epigenetics

Histone modifications and silencing prior to DNA methylation of a tumor suppressor gene

2007-05-17T23:58:00.000-06:00

Tumor suppresor genes are often silenced in cancer, and this phenomenon is usually accompanied by promoter DNA hypermethylation. In this paper, Bachman et al., used a double knockout system (termed DKO, with DNMT1 and DNMT3B genes disrupted), to evaluate the molecular events that occur during gene silencing in a temporal fashion. They first noted that colorectal HCT116 DKO cancer cells grew very slowly, and they observed that the silencing of the p16^INK4a gene (the wild type condition) was eliminated, but by passage 22 wild type was restored. Real time MSP showed discordance between expression and DNA methylation. By passage 87 the wild type allele became methylated again -then, silencing preceded methylation. Confimation that no other gene region was methylated was obtained by bisulfite sequencing. Later, they asked whether re- silencing of p16^INK4a was associated with histone modification, and with ChIP experiments they observed that the wild type allele contained methylated H3K9, but not the mutated allele; after DKO, methylated H3K9 was removed from the wt allele, but by passage 22 chromatin for this allele has become re- methylated, while the mutant remained unmethylated. Histone acetylation was changed as well, and after erasing DNA methylation H4 acetylation reappeared, but this was not fully restored until late passages (No.85). Thus, silencing occur in conjunction with H3K9 methylation and before H4 acetylation or DNA methylation. Finally, they tested if wild type p16^INK4a silencing was essential for growth. Two triple mutants (DKO and p16^INK4a disrupted, called TKO^mut/−, and other with DKO and the mutant gene disrupted, TKO^wt/−) were generated. As expected TKO^wt/− re- expressed p16^INK4a but TKO^wt/− grew slower than TKO^mut/−, and the authors thought that altogether, these results meant that:

[...] First, they prove that the growth rate of cancer cells can depend on silencing of a specific tumor suppressor gene due to epigenetic modifications [...]. Second, and most importantly, our data show not only that DNA methylation is required to maintain silencing of the wt p16^INK4a gene, but also that erasure of the methylation signature of DNA leads to histone modifications. Moreover, there appeared strong selective pressure for re-silencing of the wt p16^INK4a allele in these cells. This re-silencing was independent of DNA methylation and was associated with histone H3-K9 methylation, but not with changes in histone H4 acetylation. Only after the H3-K9 methylation associated with gene silencing was replaced did other epigenetic changes come about. These results suggest that DNA methylation and H4 deacetylation serve to lock chromatin in a specific repressed state that was originally initiated by the methylation of histone H3-K9 [...]. Our experiments demonstrate that histone H3-K9 methylation can occur in association with gene silencing and long before DNA methylation. This formally demonstrates that the enzymatic mechanisms and recognition elements required for chromatin modifications are not dependent on prior DNA methylation.

Bachman, KE. et al. 2003. Cancer Cell 3:89-95
epigenetics

Transmission of Stress-Induced Learning Impairment and Associated Brain Gene Expression from Parents to Offspring in Chickens

2007-05-16T23:46:00.000-06:00

Stress affects individuals under captivity. This could have been an important selection factor of adaptation to domestication, and it has been demonstrated that this adaptation could be acquired in few generations. The mechanism that generates stress is not clear, as well as if it could be transmissible through generations, although some advances have been made that showed that maternal behavior changes the epigenetic patterns of their descendants, producing stably inheritable alterations. In this paper, Lindqvist et al., used red junglefowl (RJF), the ancestor of domestic chickens, and domesticated white leghorn (WL) chickens, to show that the stress- induced learning is associated with a modification of gene expression in hypothalamus and pituitary. To this end, they subjected RJF (derived from a zoo population) and WL chickens to a chronic mild stressful treatment in the parental generation, and then both progenitors and descendants were selected for learning and competition tests. Parental WL birds took more tests to reach the solving criteria, while RJF and unstressed birds did it faster. Thus, the treatment affected the learning capacity. On the other hand, chicks from stressed WL parents were slower than descendants of control birds in another learning test (although no significant differences were found with RJF offspring). A modification of the weight was also observed in the WL offspring, and the data suggested to the authors that it is a consequence of the stressful environment. Later, as hypothalamus and pituitary are the centers of stress in the body, they hypothesized that this behavior could be due to differential gene expression patterns in these organs, and using microarrays they found a correlation both in WL parental and offspring, but not in RJF, and from this they concluded that:

[...] the regulatory change induced by stress was transferred to the offspring in WL, while there was no transfer between generations in RJF [...]

Comment: A difference expression is seen, but neither "transgenerational inheritance" nor any other "transfer" mechanism was measured. Alterations in gene expression could be due to many factors that Lindqvist et al., have not evaluated. In any case, these observations should motivate further investigations that hopefully will explain why stress responses could be passed through generations of poultry, as it has already been observed and discussed here before for other organisms.

Lindqvist C. et al. 2007. PLoS One 4:e364
epigenetics

Restriction landmark genome scanning

2007-05-15T23:49:00.000-06:00

Restriction landmark genomic scanning (RLGS) provides an assessment of CpG islands' status without previous knowledge of the sequence. This method consists of a two- dimensional electrophoretic separation of radiolabeled DNA; it is highly reproducible and the produced fragments can be analyzed using spot detection software. These fragments can be later cloned, or also they can be predicted if a previous dataset is available. In this paper, a detailed protocol of RLGS is described, from the isolation of genomic DNA, until the analysis of fragments and further cloning. Besides, the authors have built a profile dataset that would be helpful to compare with our in-house results; this could be useful for reproducibility, and also to keep a database of fragments -unfortunately, the site that they mentioned on the paper was not working by the time of writing (If it is temporarily down, the URL can be accessed here). Conclusion: Read this paper if you need a protocol (recipe). Else, you should read the original paper that described first this technique, by Hatada et al., 1991.

Costello, JF. et al. 2002 Methods 27:144-149
epigenetics

Intracytoplasmic Sperm Injection May Increase the Risk of Imprinting Defects

2007-05-14T18:04:00.000-06:00

Intracytoplasmic sperm injection (ICSI) was introduced as a solution to male infertility, but some investigators have found that it could be associated with an increased risk of birth defects, and also to a higher incidency of aneuploidies. In this paper, authors report two cases of unrelated children who where conceived by ICSI that were affected by Angelman syndrome (AS), a neurological disorder with genetic or epigenetic origin, caused by mutation or loss of imprinting in the UBE3A gene. The two patients studied were girls that showed abnormal methylation patterns in a Southern analysis using the SNRPN probe, and this was confirmed by MSP. This report led the authors to hypothesize that ICSI was causally related to the imprinting defect, based on these facts: (1) AS is a rare disease; (2) maternal methylation imprint on chromosome 15 is only established after fertilization; (3) ICSI bypasses many of the normal steps involved in fertilization, and besides, the oocyte undergoes mechanical stress; and (4) some investigations in sheep have shown association between fetal overgrowth and hypomethylation of the Igf2r allele. These observations were later confirmed in studies of related diseases, for example the Prader-Willi syndrome (PWS).

Cox, GF. et al. 2002. Am J Hum Genet 71(1): 162–164
epigenetics

COBRA: a sensitive and quantitative DNA methylation assay

2007-05-13T23:39:00.000-06:00

Accurate methods to quantitate the methylation levels of samples are required, mostly to analyze tumor clinical samples, for large- scale studies. The authors here report a method called Combined Bisulfite Restriction Analysis (COBRA), which fulfills the above mentioned requirements and also is compatible with paraffin samples. The method consist in (1) carrying out a bisulfite modification; (2) then a PCR amplification; (3) doing a digestion with restriction enzymes whose sites would be different between experimental and control samples; and finally (4) a hybridization with a later phosphorimager quantitation.Clearly, the method relies on the distinction of restriction sensitive sites, and the fraction that contains CpG sites will be a reflection of the percentage of methylation in the original sample. The authors tested the reliability of this development with the human estrogen receptor gene, that is increasingly methylated in some colorectal cancer forms. Finally, Xiong and Laird discussed that this method overcomes problems in using hybridizations alone (that are excellent for quantitation but not compatible with paraffin samples), in restriction digestion followed by PCR (less sensitivity), and in MSP (a technique that is only qualitative). Comment: This method could have been a great development by the time of the publication of this paper, but currently it is not widely used, perhaps because it relies on restriction enzyme digestions (that depend on the quality of the sample, and other crucial factors), and in Southern hybridizations (a non- high- throughput method with many problems). Developments in mass spectrometry, capillary electrophoresis, real time PCR and pyrosequencing should lead to better ways to quantify methylation in samples, towards more precise cancer prognosis.

Z.Xiong and P.W.Laird. 1997. Nucleic Acids Res 25(12):2532-2534
epigenetics

A genomic code for nucleosome positioning

2007-05-09T23:16:00.000-06:00

Nucleosomes contain 147 bp stretches of DNA, packed into histone octamers. It has been observed that DNA sequences differ in their ability to bend with and bind to the histone octamer, suggesting a sequence preference for nucleosome formation; this sequence constraint may, for example, limit the access to the transcription machinery, thus functioning as a system for gene expression regulation. Two models have been proposed to explain this sequence preference: trans- positional regulation of nucleosome positioning (by nucleosome remodelling complexes); and a cis- regulation model, that expects that the genome encodes sequences with high and low nucleosome affinities. In this paper, Segal et al. used both experimental and computational approaches to show that ~50% of the in vivo nucleosome organization in the yeast genome is explained by the cis- regulation model: First, they employed a genome- wide assay to isolate the DNA forming stably nucleosomes, and then with the resulting sequences, they built a stochastic model containing the information that describes the relative affinities of nucleosomes to bind to specific sequences; this approach was similar to that used in bioinformatics to describe transcription factor binding motifs and patterns. As expected, they found periodicity that resembles the DNA helical repeat, and the same results were obtained with avian sequences, which implied that these genomic positioning signals were strong and perhaps evolutionary conserved. They used several analyses to confirm that positioning was dictated by sequences, and between chromosomal regions, the highest occupancy was over centromeres, and as expected rDNA and tRNA genes presented a notorious low predicted occupancy. Other important sites with low predicted nucleosome occupancy were functional binding sites (46 transcription factor recognition sequences evaluated), and transcription start sites. However, we should keep in mind that these results are just predictions, as the authors also recognize, and they mentioned some improvements that should be taken into account in the future to better determine the nucleosome occupancy, like competition between nucleosome proteins and other DNA binding proteins (that depends on affinity and concentration), consideration of favourable nucleosome interaction thermodynamics, and of course more in vitro experimentation in these and other species. This work could be the first step towards another form of biological information: One that specifies that the nucleosome organization is encoded in the genome.

Segal E. et al. 2006. Nature 442:772-778
epigenetics

An online computational tool was built by the authors, based on the model reported on this paper, that can be found here. More comments can be found in the same issue of Nature^{1, 2}, and at the Faculty of 1000 and EurekAlert websites.

Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors

2007-05-08T18:45:00.000-06:00

Embryonic stem cells (ES) are able to grow indefinitely while maintaining pluripotency and keeping the ability to differentiate. Several transcription factors are related to the maintenance of pluripotency, and some genes that are commonly found upregulated in tumors are associated with the long- term maintenance of the ES phenotype and culture proliferation ability. In this study, investigators Takahashi and Yamanaka from Kyoto University, analyzed which factors are crucial for inducing pluripotency. To this end, they first selected 24 candidate genes, and transfected mouse embryonic fibroblasts (MEF) with these into the Fbx15 gene locus using a beta-geo cassette and a helper retrovirus vector. They found that only transfection of all 24 genes together produced G418 resistance, in contrast of using single genes alone. Then, they evaluated which of these genes were critical individually, and found that Oct3/4, Klf4, Sox2 and c-Myc were relevant to generate the induced pluripotent stem (iPS) cells. RT- PCR showed that iPS cells expressed ES markers, and global expression profiles -using microarrays- revealed that iPS cells clusters with ES cells but not with fibroblasts, although some genes seemed to be more efficiently regulated in ES cells -thus, iPS were similar but not identical to ES cells. Evaluation of pluripotency of the obtained clones was performed by subcutaneous injection to form teratomas. Histology, immuno- staining and RT- PCR confirmed that some clones differentiated into all three germ layers without expressing trophoblast markers. Pluripotency was confirmed by growing the clones in culture dishes: the embryoids attached to the surface and initiated differentiation. The experiments were repeated using adult mouse fibroblasts, and again the same four factors succeeded in producing pluripotent cells. This research results interesting for the advance that it represents for stem cell in vitro differentation, although more research is required to establish the role of each of the four factors towards the generation and control of pluripotency.

Takahashi, K., and S.Yamanaka. 2006. Cell 126:663-676
epigenetics

More comments about this paper can be found in the same issue of Cell, at the Faculty of 1000 website, and here.

The history of cancer epigenetics

2007-05-06T23:05:00.000-06:00

This timeline traces the beginnings of cancer epigenetics to 1982, when Feinberg and Vogelstein identified CpG hypomethylation in cancer cells, and here I will summarize some interesting ideas that I found in this paper. After this initial observation, other discoveries showed more facts related to DNA hypomethylation: (1) It may lead to gene activation; (2) the existence of a 'methylator phenotype', that might be linked to mismatch repair; (3) a relationship between CpG hypomethylation and chromosomal instability; and (4) some toxins, drugs, diets and viruses can provoke methylation alterations that could be found in tumor cells. The mechanism responsible for global hypomethylation in cancer remains unknown, but it has been linked to disfunctional chromatin-remodelling SNF2 family helicases. Later, hypermethylation of tumor suppressor genes was also observed in cancer -conveniently in Rb. By that time, parallel studies showed a relationship between CpG island hypermethylation, imprinting and gene inactivation, that were helpful to formulate the concept of gene silencing in cancer. Other investigations presented a classification between frequent and infrequent methylation groups, called CIMP (the 'methylator phenotype'), which is still a controversial definition. But it is not clear yet if hypermethylation is the cause or the consequence of gene silencing, and some studies point that this could help to maintain the silenced state, although it may not be the causal agent. The resistance to the idea comes from the fact that there are known disorders of the methylation machinery but these do not predispose to cancer.

"[...] the counter- argument from human genetic studies is that the many tumour- suppressor genes in cancer are not modifiers of DNA methylation, even though they are involved in virtually every other potential growth or regulatory pathway. On the other hand, many tumour-suppressor genes are involved in some aspect of chromatin structure [...]."

Another epigenetic mechanism, loss of imprinting (LOI), which may lead to a functional loss of heterozygosity, has been also associated to some tumor forms. A third epigenetic mechanism, the histone modification, is also linked to cancer development, and was first demonstrated by microinjection of in vitro methylated chromatin that would package into a silenced state. An open question in this field that remains undiscovered is whether histone replacement might be perturbed in tumor cells. Finally, solutions to these questions will help in the acceptance of the role of epigenetic disturbances in cancer: (1) explaining the epigenetic inheritance; (2) tumor suppresor genes are linked to almost every signalling pathway but not to DNA methylation (thus, epigenetic changes might be secondary causal elements); and (3) most epigenetic studies come from rare familial disorders. This review, although a little outdated, is interesting because shows the advances in Epigenetics historically, and it is not limited to the studies in cancer.

Feinberg, AP., and B.Tycko. 2004. Nat Rev Cancer 4:143-153
epigenetics

Transgenerational epigenetic imprints on mate preference

2007-05-04T23:56:00.000-06:00

Endocrine- disrupting chemical (EDC) exposure can induce an epigenetic transgenerational reprogramming, with effects that differ between sexes, and this could have an evolutionary impact, because exposed individuals would not reproduce effectively; besides, exposure to EDCs during the embryonal stages provoke changes in behavior related to reproductive functions in the late adult individual. Then, the hypothesis would be that, if individuals have special preferences for mating, then an evolutionary impact may exist. To test this assumption, the authors studied the mating behavior of an F3 generation rats (n=12), descendants from an F0 group that was previously exposed to vinclozolin. After testing the mate preference behavior, they "found that all females preferred males from the control lineage [not exposed to vinclozolin but to DMSO], whereas males from both lineages exhibited no preferences for female type". They later repeated the experiments to minimize the effect of odor discrimination on behavior, and found that the "inability to discriminate among social odors [...] cannot account for the observed differences". Although the purpose of this paper was not to find a causal agent for this, the authors mentioned that many factors can be influential in this mate preference behavior, such as the MHC (HLA) variability, the exocrine gland- secreting peptide (ESP1), ultrasound emission, and others. They concluded that "the current study demonstrates that an environmental factor can promote a transgenerational alteration in the epigenome that influences sexual selection and could impact the viability of a population and evolution of the species." In my opinion, putting aside the fact that the purpose was not, for the moment, to establish a causal agent for the change in mate preference, I cannot help to think that the behaviour was "measured" using subjetive qualitative variables, while some other markers (biochemical, genetic, epigenetic, brain scan patterns, etc.) could be more informative -besides, I don't know for sure if twelve is a statistically significative number. Questions apart, the paper suggest an interesting principle of "perception of wealthness", that was long ago assumed by neodarwinists, but here from a neolamarckian perspective.

Crews, D. et al. 2007. Proc Natl Acad Sci USA 104(14):5942-5946
epigenetics

Heritable germline epimutation of MSH2 in a family with hereditary nonpolyposis colorectal cancer

2007-05-03T23:49:00.000-06:00

Mutations and epimutations of DNA mismatch repair genes, MLH1 and MSH2, are involved in certain forms of hereditary colorectal cancer. A paper by Suter et al. (2004) found aberrant methylation in MLH1 in a small proportion of sperm cells, suggesting the possibility that this epigenetic modification could be heritable -an evidence that, although already demonstrated in other organisms, is still elusive in humans. In this work, the authors reported a family, which they considered showed a heritable germline methylation in MSH2, enough to prove the epigenetic inheritance in man. To this end, they first screened the MSH2 promoter using MSP, and found a methylation signal in both normal and cancer colon tissues of an cancer- affected individual; sequencing showed a frameshift in this gene. Their pedigree interpretation suggested a possible familial inheritance of this methylation pattern, so they looked for this change in different tissues in three generations of the family, and this time the MSP showed the same methylation band in both blood and rectal mucosae cells (also, sequencing associated this methylation pattern with a particular SNP). Results of this experiment were repeated with other somatic tissues, with the same results. Later, they quantified the proportion of methylated alleles in several tissues using pyrosequencing, but failed to obtain similar results between tissues, and they could not distinguish from those in the control group -they interpreted this likely as a sample contamination. The authors concluded that they have clearly demonstrated a "stably inherited, allele-specific and mosaic methylation of the MSH2 promoter in a family putatively affected with HNPCC". They hypothesized an explanation for this: "This epimutation could act as the first 'hit' to inactivate one allele of MSH2; the resulting scattered hemimethylated cells then acquire a somatic second hit to progress to cancer". They compared their results with those of Suter et al., that also showed mosaic methylation of the epimutated allele, for which they suggested that this phenomenon could "mask the inheritance pattern", making more difficult to diagnose familial colon cancer.

Chan, TL. et al. 2006. Nature Genetics 38:1178 - 1183
epigenetics

In my opinion, Chan et al. have rushed in their interpretations for this "epimutation inheritance explanation", because they not showed any concluding evidence of transgenerational inheritance, for example they not examined changes in germ cells. Of course, this paper brought much discussion, and these different communications have been published in the current issue of Nature Genetics. Here are some opinions:

[...] Two recent reports [by Suter et al., and Chan et al.] claim to have identified germline epimutations in humans. [...] Although the data are very interesting and compatible with the presence of a germline epimutation, they are not sufficient to prove it. First, it should be noted that mosaic hypermethylation [...] points to a somatic event that occurred after fertilization. The finding of MLH1 or MSH2 hypermethylation in cancerous cells and noncancerous cells [...] of an affected individual does not necessarily imply that the epimutation occurred in a parental germ cell [...]. It is not possible to "extract germline DNA from blood leukocytes" [as Chan et al. mentioned in their paper] . Second, it is necessary to distinguish between an epimutation that is relatively independent of the DNA sequence context and an epimutation that is a direct consequence of a cis-acting DNA mutation [...]. Sensu stricto, transgenerational epigenetic inheritance refers to the germline transmission of an epigenetic mark. Thus, the epimutation should be detectable in the gametes. Chan et al. provide no evidence for this. [...] As epigenetic marks of endogenous mammalian genes are generally erased during gametogenesis and early embryogenesis, transgenerational epigenetic inheritance is typically associated with incomplete penetrance. Therefore, it is surprising that Chan et al. observed mendelian segregation and complete penetrance of the epimutation in the family they studied. On the other hand, epigenetic marks are rather stable after the embryonic stage. Thus, the presence of mosaic hypermethylation in the reported family is difficult to reconcile with the germline transmission of an epimutation [...] It is more likely that the family described by Chan et al. shows segregation of a DNA variant that predisposes in cis to postzygotic de novo methylation in each generation, as in the fragile X mental retardation syndrome. [...] Although Chan et al. have not found a point mutation in the immediate vicinity of the MSH2 gene, the presence of a DNA sequence change further upstream or downstream of the gene is not excluded. [...] Although Chan et al. do not completely exclude a "genetic event", they claim to have found "compelling evidence" for "stably inherited...methylation". As argued above, there is little evidence for this.

(Horsthemke, Nat Genet 39:573 - 574)

Suter and Martin, referenced by Chan et al., also showed discordance with the results:

The phenomena described by us and by Chan et al. seem to share an association [...] However, we suggest that what Chan and colleagues have found is distinctly different from the germline epimutation of MLH1[...]. Our conclusion rests on two features of the MSH2 epimutation: its mosaicism with prominent tissue specificity and its strong heritability [...]. The authors propose that the MSH2 epimutation is a germline event and that somatic reversion has produced the tissue-specific pattern, but the evidence seems insufficient for this conclusion. It would be helpful to know more about the tissue distribution of the MSH2 epimutation—in particular, whether it is found in cells derived from all three germ layers. [...] It is striking that the MSH2 epimutation appears to be inherited in a mendelian pattern: all individuals carrying the at-risk haplotype also have the epimutation. This is in sharp contrast to the MLH1 epimutation [...]. We suggest that the MSH2 haplotype in this kindred predisposes in cis to somatic hypermethylation [...]. We have proposed that the MLH1 epimutation is an accident conditioned by the presence of epigenetically silent material [... but t]he MSH2 epimutation may be something quite different: a haplotype that is prone to somatic hypermethylation in certain cell types.

(Suter and Martin, Nat Genet 39: 573)

On the other hand, Chong et al., pointed out some mistaken conceptual attributions in both Suter and Chan works:

The phrases used to describe the two phenomena [the Suter and Chan experiments], 'heritable germline epimutation' and 'transgenerational epigenetic inheritance', are similar, but they have different meanings. 'Heritable germline epimutation' describes an atypical epigenetic state that occurs in all tissues of an individual [...] and that is detected in more than one generation. 'Transgenerational epigenetic inheritance' is the direct transfer of epigenetic information across generations. Heritable germline epimutation may or may not be the result of transgenerational epigenetic inheritance. [...On the other hand, ] although [Chan et al.] suggest that this is a case of transgenerational epigenetic inheritance, they are careful to add that it could be controlled by a mutation in the disease haplotype. We would support the latter explanation [...] To argue a case for transgenerational epigenetic inheritance in non-human organisms, the scientific community has generally demanded the use of inbred, genetically identical animals. In the absence of this, there is always an alternative explanation for any finding of a heritable germline epimutation. An underlying genetic change could direct the re-establishment of an atypical epigenetic state each generation; that is, a DNA variant could predispose in cis to methylation. Although Chan and colleagues have sequenced the exons and promoter of MSH2 and find no changes, cis effects cannot be ruled out.

(Chong et al., Nat Genet 39:574 - 575)

Later, Suter and Martin replied the above mentioned letters:

[...] we disagree with the criticisms of work on the MLH1 germline epimutations. Whitelaw and co-workers conflate a variety of different phenomena; Hortshemke dismisses the evidence that the MLH1 epimutation is present in germline cells.Whitelaw and co-workers have confused somatic and germline epigenetics by including somatic epigenetic events in their redefinition of 'heritable germline epimutation'. The term actually means just what it says. [...] 'Transgenerational epigenetic inheritance' is what happens when a germline epimutation remains stable for one or more generations [...]

(Suter and Martin, Nat Genet 39:575 - 576)

Finally, the Chan group replied that they agree with "the possibility of an underlying genetic change that causes the heritable methylation cannot be excluded", although they think that this could effectively represent transgenerational epigenetic inheritance (Leung et al., Nat Genet 39:576).

More opinions on Chan et al. (2006) can be found at the Faculty of 1000 website.

Transgeneration memory of stress in plants

2007-05-02T12:03:00.000-06:00

Changes in the dynamic of the plant genome have been observed as a consequence of exposure to different environmental factors, either biotic or abiotic. However, the evolutionary continuity of these well documented changes is not known because studies not considered to follow the effects in subsequent generations. In this experiment, Molinier et al. (2006) showed that stress- stimulated recombination persist for several generations, regardless of the origin of the causal agent. To this end, they employed Arabidopsis harbouring truncated versions of the glucuronidase reporter, and two sources of stress: Ultraviolet-C radiation, and flagellin, a bacterium- derived plant defensin stimulator that mimicks an infection. Both types of stress succeeded in inducing quantifiable recombination, and this was greater in plants exposed to stresses than of plants descended from untreated progenitors. They hypothesized that the transgenerational effect must be epigenetic because the entire population of descendants changed their phenotype. To test this possibility, they selfed the descendants, and found that these changes were stable up to the S4 progeny in plants exposed to UV-C only in S0; this epigenetic phenomenon was thus stable. Then, they asked whether these changes were transmitted through the male or the female progenitors, by crossing stress- exposed with untreated plants, and found that the recombination was elevated irrespective of the gamete in a dominant fashion. They concluded that "environmental influences [...] change the flexibility of the plant genome in somatic tissue of treated plants and in somatic tissue of their progeny. As this influences persist in the entire population of plants, the basis for the change is epigenetic rather than genetic". Nevertheless, they did not explained how the stress stimulation exerted such recombination activity, but they have not observed significant changes in gene expression, and discarded paramutation as a possibility because plants lacking the transgene were induced to recombination (they however did not rejected the idea that microRNAs could be involved). Based on their observations, and citing the discussions by Jablonka and Lamb (1995), the authors proposed that "the environmental influences that lead to increased genomic dynamics even in successive, untreated generations may increase the potential for adaptive evolution". This investigation suggested that changes in somatic cells can pass to the subsequent generations and can be stably inherited, although more research is needed to explain the precise mechanisms underlying these adaptive responses.

Molinier, J. et al. 2006. Nature 442:1046-1049
epigenetics
More comments can be found at the same issue of Nature, the Faculty of 1000 and at The Scientist websites.

Genome-Wide Location and Function of DNA Binding Proteins

2007-04-30T23:56:00.000-06:00

This is the original paper of the ChIP-on-chip technology. This development was tested by following the activity of two transcription factors (Gal4 and Ste12), and its interaction with the DNA molecule across the whole genome of the Saccharomyces genome. Briefly, the principle of the technique in words of the authors:

The method combines a modified romatinmmunorecipitation (ChIP) procedure, [...] with DNA microarray analysis. Briefly,cells were fixed with formaldehyde, harvested, and disrupted bysonication. The DNA fragments cross-linked to a protein of interestwere enriched by immunoprecipitation with a specific antibody.After reversal of the cross-links, the enriched DNA was amplifiedand labeled with a fluorescent dye (Cy5) with the use of ligation- mediated- polymerasechain reaction. A sample of DNA that was not enrichedby immunoprecipitation was subjected to LM-PCR in the presenceof a different fluorophore (Cy3), and both immuno- precipitation-enriched and -unenriched pools of labeled DNA were hybridizedto a single DNA microarray containing all yeast intergenic sequences(see figure)

Results with each evaluated transcription factor were confirmed by conventional ChIP analysis. The authors found novel binding sites for these proteins, some including less stringent sequences; but also that most canonical binding sites were not detected, meaning that the union of the transcription factor required additional factors might help to their specificity. Because they found connection and coordination between multiple cellular pathways, they concluded that this new development will help to uncover the global regulatory networks

Ren B., et al. 2000. Science 290(5500):2306-9
epigenetics

Capillary electrophoretic analysis of genomic DNA methylation levels

2007-04-29T20:54:00.000-06:00

At the time of the publication of this paper, a robust method for the large- scale determination of cytosine methylation in genomic DNA was required. Some labs used HPLC, but this failed because it needed large amounts of starting samples and it was hard to standardize to reproducibility. In this article, Stach et al. (2003) established a method for the detection of 5meC using capillary electrophoresis. They modified the procedure developed by Schmitz et al. (2002) where:

[...] DNA is digested to single nucleotides followedby chemical derivatization with a fluorescent marker and separationby micellar electrokinetic chromatography [see figure]. Derivatizednucleotides are detected by laser-induced fluorescence at 510nm, thus permitting a well defined experimental readout withvery little baseline fluctuation [...]

A novel method for the determination of genomic cytosine methylation levels. (A) Outline of the method. Five micrograms of genomic DNA were enzymatically hydrolyzed to single nucleotides and chemically derivatized with the fluorescent marker Bodipy FL EDA. After derivatization, the reaction mixture was separated by capillary electrophoresis and detected by laser-induced fluorescence. (B) Representative result. Electropherograms revealed five defined peaks representing adenine (dAMP), guanine (dGMP), thymine (dTMP), cytosine (dCMP) and 5-methylcytosine (5m-dCMP), respectively. The well defined baseline allows a precise determination of cytosine methylation levels (Stach et al., 2003/NAR)

In spite of important technological advance that this new method represents, the authors did not mention any difference with the previously reported capillary electrophoresis- based method by Fraga et al. (2002).

Stach, D. et al. 2003. Nucleic Acids Res 31(2):e2
epigenetics

Sex-specific, male-line transgenerational responses in humans

2007-04-28T23:16:00.000-06:00

Previously, these authors found an association between ancestral food supply and longevity and with cardiovascular and diabetic mortality, using historical data. The longevity of the probands was influenced by the paternal grandparents access to food but only during the grandfathers' slow growth period (SGP: 8-10 years for girls, 9-12 years for boys). In the present investigation, the authors used the ALSPAC group and the historical records of Överkalix (Sweden) to test the transgenerational effects of cigarrette consumption and ancestral food supply (only estimated from food availability, no actual nutrition data) on mortality, respectively. They found three periods of exposure sensitivity, and a remarkable sex- specificity in their results: Paternal grandfather's food supply was only related to the mortality of grandsons, and paternal grandmother's food supply was associated with granddaughters' mortality only. The authors suggested that the possible mechanism for this could be epigenetic inheritance involving the sex chromosomes, although their hypothesis does not deny a possible role of autosomal genes for this phenomena. On the other hand, the largest transgenerational response found in this analysis was associated to the food supply during the period of paternal grandmother's conception to age 3 years, perhaps showing a relationship with the oogenesis. Taken together, these results demonstrated that the SGP is a critical ontogenetic period, and the sex- specific pattern suggest and evolved transgenerational response mechanism, but the reason remains to be determined. More detailed studies are needed in order to establish a clear role for the early exposure to environmental agents and their transgenerational effects in disease susceptibility.

Pembrey ME et al. 2006. Eur J Hum Genet 14:159–166
epigenetics

Comment in the same issue of the European Journal of Human Genetics:

[This] fascinating article [...] suggests that the behaviour (or environment) of prepubescent boys could influence the phenotype of their sons and grandsons. Using data collected in the Avon Longitudinal Study of Parents and Children (ALSPAC), they showed that early paternal smoking was associated with greater body mass index at 9 years of age in sons, but not in daughters. This prompted them to return to the records [...] from Overkalix [...]. Reanalysis showed that the paternal grandfathers' food supply during mid childhood was linked to the mortality risk ratio of grandsons, but not granddaughters. The study suggested that in humans, a one-off environmental event could influence phenotype for more than one generation in a sex- specific way [...]. In animal models, these types of effects, termed fetal programming, can be produced by exposing offspring in utero to a manipulation such as dietary restriction of the pregnant female [...]. From a public health point of view, what makes the Pembrey study particularly interesting is that it argues that exposure in the male can affect the development and health of males for at least two generations, and this is rarely, if ever, considered (E. Whitelaw, Queensland Institute of Medical Research, Australia).

More comments can be found at the Faculty of 1000 website.

Evidence for an epigenetic mechanism by which Hsp90 acts as a capacitor for morphological evolution

2007-04-27T09:09:00.000-06:00

Drosophila Kruppel (Kr) codes for a transcription factor required for the development of thoracic segments. The authors of this paper used a mutant strain of Kr to study the observed phenotypic variation in isogenic strains, a phenomenon already described in other species such as Arabidopsis. They first selected a dominant mutation, the Kr^If-1 allele, that is responsible of ectopic expression of Kr and consequently of developmental defects. Deficiency crosses showed the expected ectopic expression but reciprocal crosses (Kr^If-1 females x Df/+ males) produced a different phenotype, perhaps due to imprinting. Mapping of the Kr^If-1 mutations showed that most were in TrxG genes, known to be related to epigenetic inheritance, and functionally opposed to the Polycomb group (PcG), that are involved in keeping the inactive chromatin state. Apart to the TrxG mutations, one was found in Hsp83, a member of the Hsp90 heat shock proteins family. This suggested that the fixation of the phenotype could be due to stress. To test this, the authors isogenized the mutant strain and used an Hsp90 inhibitor, geldanamycin. After the drug exposure, they continued breeding the strain for 13 generations in absence of the inhibitor, and found that the ectopic growth provoked by the mutation was higher in each succesive generation until it stabilized in F6. On another experiment, this phenotype was for the most part suppresed in presence of trichostatin A, and HDAC inhibitor, thus confirming the epigenetic nature of the phenomenon. Taken together this results, the authors concluded that genetic variation was not required and thus could not account for the fixation and selection of the mutant outgrowth. The authors suggested that this novel ability of trait fixation give a "refinement" of the the capacitor model of Waddington, because they proposed:

[...] that an environmental stress causes a reduction in Hsp90 levels and, through some unknown interaction with TrxG proteins, induces an immediate 'chromatin effect'. Our model allows an adaptive response to be 'fixed' epigenetically, and therefore obviates the need to wait for the selection of existing genetic variation. In other words, it predicts a more rapid evolutionary process than is required for selection of existing genetic variation. Because of the inherent instability of epigenetic inheritance, fixation of an epigenetically-determined phenotype is probably less stable than fixation through a genetic selection mechanism.

Sollars V. et al. 2002. Nat Genet 33:70 - 74

epigenetics
A comment by Rutherford and Hennikoff can be found in the same issue of Nature Genetics:

[...] Reductions in Hsp90 have previously been shown to generally uncover morphological variation [in Arabidopsis... and the] findings of Sollars et al. suggest that chromatin-based germ-line inheritance is subject to selection as well. Does the demonstration of epialleles call for a complete reinterpretation of genetic assimilation experiments? Probably not. In those experiments, assimilation did not occur instantly but gradually over several generations. Furthermore, a characteristic feature of Hsp90 buffering is that the genetic variation is pre-existing in particular strain backgrounds, as evidenced by the fact that repeated crosses to the same strains produced the same abnormality. Nevertheless, the evidence that heritable epigenetic variation is common raises questions about the contribution of epigenetic variation to quantitative traits in general.

More comments can be found on the Faculty of 1000 website.

Evolution of Duplicate Gene Expression in Polyploid and Hybrid Plants

2007-04-25T22:43:00.000-06:00

This review summarizes the effects of plant polyploidization in gene expression and genomic evolution. The abnormal gene dosage that occurs in this phenomenon can be controlled by epigenetic gene silencing -most studies have been carried out in genes involved in vernalization like Arabidopsis FLC.

[...] Polyploidy can result in chromosomal rearrangements and gene loss, interlocus concerted evolution of ribosomal DNA repeats, unequal rates of sequences evolution of duplicated genes, and changes in DNA methylation. There can be considerable consequences on the expression of genes duplicated by polyploidy, termed "homeologs." [...] What molecular mechanisms are responsible for gene silencing and reactivation in polyploids and hybrids? Hypermethylation of DNA cytosines has been shown to silence homeologs in Arabidopsis neopolyploids and in A. suecica. These experiments were done by using a methyltransferase inhibitor or by silencing a methyltransferase gene using RNA interference, causing reactivation of silenced genes. Activation of retrotransposons on allopolyploidy in wheat was shown to cause gene silencing by readout transcription into adjacent genes that were in the opposite orientation [...]. Another mechanism for gene activation on allopolyploidy was shown for the FLC gene in neopolyploids of Arabidopsis. Activation of the A. thaliana copy of FLC was associated with changes in histone modifications including demethylation and acetylation [...]. Why are some genes silenced, downregulated, or upregulated in polyploids? Several hypotheses have been proposed relating to gene dosage, altered regulatory networks, epigenetic remodeling, interactions of parental copies with each other, as well as side effects of other molecular processes [...]. Gene dosage factors include preservation of the appropriate level of gene expression despite the increased dosage caused by chromosome doubling, as well as increased variation in dosage-regulated gene expression. Altered regulatory networks include the consequences of reuniting diverged regulatory factors and their target genes. It is likely that multiple factors cause gene expression changes in polyploids and that the causes vary by gene and perhaps by organism.

Adams, K. 2007. J Hered 98(2):136-141
epigenetics

Histone Demethylation Mediated by the Nuclear Amine Oxidase Homolog LSD1

2007-04-24T20:35:00.000-06:00

The most common forms of histone modification are acetylation and methylation, both linked to active and silenced transcription, respectively. The process of histone acetylation was very well known at the time of the publication of this paper, but the turnover of histone methylation was explained with two theoretical models, one was the removal of the complete histone tail or the replacement of the methylated histone, and the other was the existence of histone demethylases. Some authors proposed that amino oxidases could be function as demethylases, and a protein that could fulfill the picture was KIAA0601, because it copurified with corepressor complexes (such as Co-REST in non- neuronal cells), and showed significant sequence similarity with FAD- dependent amino oxidases. The authors first asked whether KIAA0601 functions as a repressor. To this end they expressed a Gal4- KIAA0601 fusion, and the reporter Gal4-luciferase promoter was strongly repressed; however, a C-terminal deletion construct, that lacks the amine oxidase homologous region, was not able to repress transcription. Therefore, the transcriptional function of this protein is linked to its enzymatic activity. KIAA0601 is known to bind FAD, and computational analyses predicted that it may catalyze oxidation reactions of amines. To answer this, they expressed a His-tag- KIAA0601 fusion, and the purified protein, that copurified with FAD, was incubated with either H3K4me2 or H3K9me2, and the methylation status was determined with antibodies. They observed that KIAA0601, but not the C-terminal deleted form of the protein, reduced the methylation levels in H3K4me2 but had no effect on H3K9me2 or unmethylated H3, which indicates specificity, and that the enzymatic activity is linked to its silencing function. This experiment was repeated with proteins isolated from cell lines, obtaining similar results. Then, they evaluated if this protein would have the same activity on the mono- or trimethylated forms of H3K4, and they found that the enzyme could reduce the methylation levels on the monomethyl K4 but not on the trimethylated form of the amino acid. Taken together with previous results, Shi et al. proposed a demethylation pathway for H3K4. In other similar assays (detected by Western blotting or by mass spectrometry), no other methylated residue was altered by this protein, further confirming the specificity, and the endogenous form of KIAA0601 showed the same activity and specificity that the recombinant fused form. As their model of reaction predicted the formation of formaldehyde, they used enzymatic assays to determine the presence of this metabolite, which resulted in a confirmation of their hypothetical pathway. The specificity of this protein, now termed as LSD1, for "lysine specific demethylase", was also confirmed with these biochemical assays. The formation of formaldehyde was also observed in mass spectrometry experiments. Finally, they asked whether LSD1 regulates transcription and histone methylation in vivo. They first knocked out LSD1 using an RNAi system, and observed that the Co-REST associated promoters were derepressed. ChIP experiments showed that LSD1 occupied the Co-REST associated promoters in vivo, and in the knocked out cells, the decrease of occupancy was accompanied with an increase of H3K4 dimethylation and promoter activity. They concluded that LSD1 is an specific histone lysine demethylase associated with epigenetic repression, and this paper is valuable because it was the initial characterization of an enzyme involved in processing H3K4 demethylation.

Shi Y. et al. 2004. Cell 119:941-953
epigenetics
More comments can be found at the Faculty of 1000 website.

Non-genomic transgenerational inheritance of disease risk

2007-04-23T23:49:00.000-06:00

Some diseases are monogenic but for most, a range of different factors operates, and recent experiments have demonstrated that environmental influences affecting one generation, can have consequences on the phenotype of subsequent generations. These effects are not from a mendelian inheritance but due to epigenetic transmission or other undiscovered indirect mechanisms. Epidemiological analysis, like those from Pembrey et al. (2006), suggested that the same could be happening in humans. In this review, Gluckman et al. (2007) analyzed the familial (maternal effects and non- genomic inheritance) influences on susceptibility to type 2 diabetes as a model to explain the impact of environmental epigenetic effects in the development of the disease. Evidences of induction of disease risk by non- genomic transmission, comes from a number of experiments involving drug exposure (endocrine disruptors such as vinclozolin and methoxychlor), physical activity, or diet manipulation of females during pregnancy. This challenges resulted in broad phenotypic traits such as low birth- weight, endocrine or behavioral effects, accompanied with common alterations of DNA methylation patterns in tissues, particularly germline cells. This form of stable inheritance of acquired characters was first described by Waddington (1957), who termed the phenomenon as genetic assimilation. The molecular mechanisms involved in epigenetic inheritance are well known (DNA methylation, chromatin histones modification, and small non- coding RNA- mediated processes), but only the RNA promoted-changes have direct sequence- specific effects, and all having in common a correlated altered gene expression. In mammals, epigenetic variations generated by this type of inheritance can be random (epimutations) or induced. One form of normal control of allele expression is genomic imprinting, and it is regulated by allele- specific methylation. But epigenetic effects act on either imprinted or non- imprinted genes, because environmental exposure can have influence over later generations (although more long- term experiments are needed to explain better this mechanisms). Besides of the well studied diet or hormonal manipulation effects, some other non- genomic influences like changes in mitochondrial copy number and function, could have influences over nuclear imprinting. On the other hand, it is not clear how the epigenetic memory could be inherited; transmission of epigenetic marks through generations require sucessive steps of reprogramming, have parental allele- specificity, and display dependence on the early divergence between germline and somatic cells (thus, most examples of epigenetic inheritance could be found when environmental factors produce epialleles in the early development). Heritable changes in somatic cells with transgenerational consequences seem to be less plausible, but an experiment of heritability in C.elegans demonstrated that this is not a closed possibility. In humans, transgenerational inheritance has been suggested that is due to altered dietary (epidemiological studies of the effects of famine in populations in Sweden and the Netherlands, related to increased diabetes mortality or insulin resistance) or endocrine exposure (reproductive abnormalities due to diethylstilbestrol in descendants and in later generations), and Pembrey hypothesized that this could be a consequence of exposure during gametogenesis. Some studies have suggested that the variation of epigenetic patterns genetically identical individuals is a consequence of differences in environmental exposures, and others point to epimutations such as those in DNA repair enzymes, that have a potential for heritability. Other maternal influences, such as altered uterine development, changes in metabolism, behavioral and cultural practices, also account as non- epigenetic mechanisms of non- genomic inheritance, that in some cases can lead to alterations in the pattern of DNA methylation. The authors said that this form of phenotypic memory, represented by the non- genomic inheritance, is an evolutionary advantage because it opens the possibility to incorporate short- term changes that overcome the problems of long- term fluctuations, that rely on prediction of environmental conditions. This model has been termed as the developmental plasticity in human evolution, and it behaves as a "variable, selectable and heritable trait", as an indication that it might have been positively selected in the genome. Examples of this adaptive processes are those related the above mentioned effects of diet and drug exposure, and this model raises the possibility that deleterious messages (such as those motivated by toxins) "might also be transmitted to subsequent generations". A practical consequence that the authors mentioned, is that predicted by the hypothesis of Neel, who said that the early hominids developed tolerance to nutritional uncertainty, but those fixed traits now could promote cardiovascular and metabolic diseases in the present "energy dense (abundant nutrition and low energy expenditure)" environment. In this respect, individuals living in constantly changing societies are more vulnerable to diseases because of the bigger mismatch between prenatally predicted and actual environment. Further studies of this phenomena will help to prevent and counter the effects of epigenetic traits.

Gluckman, PD. et al. 2007. BioEssays 29(2):145-154
epigenetics

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Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome

2007-04-22T23:43:00.000-06:00

The role of CpG islands as targets for DNA methylases in epigenetic silencing is very well established. However, some problems still exist related to the dynamics of action of methylation of CpG- poor promoters, maybe because of the limited number of genes analyzed, or the origin of the samples that could have lead to inconclusive results. On the other hand, it is supposed that the genomic depletion of CpGs is attributed to the high mutability of this dinucleotides, although there are other theories explaining this fact from an evolutionary point of view. To answer this, Weber et al. (2007) generated a whole epigenomic map of DNA methylation, RNA polymerase II occupancy and histone modification status for 16,000 promoters in human somatic and germline cells. They first assessed the methylation state, using fibroblast DNAs in MeDIP combined with microarray detection, and filtered their obtained set using computational comparison with several annotation databases like RefSeq and others. The obtained dataset featured three classes of promoters, based on their CpG ratio, GC content and lenght of CpG- rich region: High CpG (HCPs), Low CpG (LCPs), and a group of intermediate CpG content (ICPs), that did not fit the previously defined criteria. MeDIP and bisulfite sequencing showed that HCPs were either free (least MeDIP- enriched) or strongly methylated (just 3% of analyzed HCPs), and this confirmed previous observations that CpG islands remain free of methylation in terminally differentiated cells. Overall methylation analysis defined that LCPs are methylated, HCPs mostly unmethylated, and ICPs have a high frequency of methylation, i.e. hypermethylated promoters belong to LCP and ICP classes. Next they used ChIP to define the RNA polymerase occupancy, and found that, as expected, most active promoters are HCPs and the least occupied by RNAP are LCPs (only 11% of active promoters). Considering the results of the previous experiment, they concluded that most LCPs are methylated in either their active or inactive state, meaning that low concentration of methylated sites does not preclude activity; however, in HCPs and ICPs, activity was negatively correlated to their methylation status, and HCPs remain unmethylated even when inactive. Later, they evaluated the association between chromatin modification and promoter class, using H3K4 dimethylation, a marker of active chromatin. They obtained two groups of inactive promoters (those with lower levels of H3K4me2), that correlated with their methylation status: inactive HCPs (that are almost permanently hypomethylated) and unmethylated ICPs (hypermethylated ICPs showed no H3K4me3 enrichment). From this, they concluded that HCPs are protected from methylation by histones, and this chromatin state can be a marker of methylation status. Taken together with their previous results, this showed that DNA methylation is sufficient but not necessary to inactivate CpG island promoters. To validate their results, they repeated the MeDIP experiments in sperm cells, showing high reproducibility, but hypermethylation of HCPs and ICPs in fibroblasts was absent in germline cells, meaning that these changes might have been acquired during differentiation. But because most inactive CpG island promoters remain unmethylated in germ cells, they argued the possibility that DNA methylation could not be a default repression mechanism for tissue- specific gene expression; instead somatic methylation was found at a significant rate. Then, tested if the CpG depletion was because of the high mutation rate of cytosines using alignment comparison between human, chimpanzee and rhesus monkey genome sequences, and found that CpG loss was higher in LCPs than in the other classes, but because these LCPs were mostly methylated in sperm cells (previous experiment), this supports the hypothesis that DNA methylation favors evolutionary CpG depletion. Annotations of methylated CpG-rich promoters in several databases linked this genes to those with testis- specific gene expression, and this results were later confirmed by PCR and bisulfite sequencing. Besides, methylation of germline- specific genes was absent in sperm cells; from this, they concluded that the methylation of these genes is established during somatic development and authors think that this could preclude abnormal activation of testis- specific meiotic genes, as it happens in tumors. Overall, this experiments showed that DNA methylation of a promoter is a function of its CpG content and its gene function. Furthermore, this paper is considered a very important approach to the future human methylome.

Weber M. et al. 2007. Nature Genetics 39(4):457-466
epigenetics
More comments can be found in the same issue of Nature Genetics

Epigenetic silencers and Notch collaborate to promote malignant tumours by Rb silencing

2007-04-20T14:33:00.000-06:00

A common occurrence in tumors is the activation of developmental signalling pathways, such as those of Wnt, Hedgehog and Notch. Being cancer a genetic and epigenetic disease, a natural step is to find links between both activation pathways, and here Ferres-Marco et al. (2006) used the eye development in Drosophila as a model to find mutations interacting with the Notch pathway that lead to tumors with alterations of epigenetic states. To this end, they generated a triple transgenic fly over- expressing the Notch ligand Delta, that induced tumor formation and metastasis, and then they identified the P-element insertional positions relevant to the phenotype, and found that they were inside the lola and psq genes, whose proteins could work as transcription factors and epigenetic silencers. From these assumption, they evaluated the possibility that Psq and Lola could lead to tumors by epigenetic processes, so they assesed the epigenetic state of mutant discs, and noticed aberrant gene silencing (reduced H3K4 trimethylation). If this was actually happening, it was possible that some cell- cycle control genes could be altered, so then they compared the expression of twelve tumor- related genes and found that Rbf (homolog of human tumor supressor gene Rb) was downregulated and later, bisulphite sequencing experiments showed that Delta over- expression was causing the hypermethylation of the Rbf promoter in this system, a confirmation of cross- talk between genetic and epigenetic pathways in cancer development. The authors propose a model for tumorigenesis that could begin with the activation of Notch pathway that would begin gene repression, and at the same time Psq and Lola would stimulate aberrant gene silencing; later the association of Notch with cellular memory could lead to the continuous signals of tumor proliferation.

Ferres-Marco D. et al. 2006. Nature 439:430-436
epigenetics
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Genome-wide non-mendelian inheritance of extra-genomic information in Arabidopsis

2007-04-18T15:47:00.000-06:00

Lolle et al. (2005) found that alleles with eleven point mutations in the HOTHEAD (HTH) locus of Arabidopsis produced wild- type descendants when homozygous plant mutants were selfed. Incomplete penetrance of the phenotype was discarded as a valid explanation because an allele- specific PCR experiment revealed that clear heterozygosity just have arosen. Contamination or outcrossing were also ruled out, and the real manifestation of the phenomenon was demonstrated when wild- type HTH alleles were observed in descendants from male homozygous hth-4, via pollen transmission. Other possible causes were ruled out, such as the involvement of transposons or repetitive elements (using sequence analysis), an specific high rate of mutation (discarded when high frequency of reversion was seen in successive generations), or gene conversion (neither other copies of hth nor related HTL genes were found that contained the sequence information needed to revert the genotype). They also observed that the loss of HTH function affected the stability of sequences distributed all across the genome, but this instability was not random. To explain this phenomena, they postulated a model for an ancestral sequence cache, as an extra- genomic memory, that could be used to restore the information of the ancestors when it is required. They further supported this hypothesis with previous observations like re- appearance of molecular markers, or transmission of markers through grafting. They hypothesized that RNA could be involved in this extra- genomic non- mendelian inheritance, supporting this with recent findings in siRNA-, microRNA- and RNA-DNA hybrid- mediated stably gene silencing through generations. Authors said that this possible model would avoid the problems coming from inbreeding or the stress originated from the absence of the HTH gene product, and it is similar to the adaptive response found in micro- organisms. They concluded that exists a non- genomic mechanism leading to a "high frequency of modification of DNA sequences in a template- directed manner". They, however, did not identified the causal agent molecule (RNA or protein), and paramutations were also not considered.

Lolle SL. et al. 2005. Nature 434:505-509
epigenetics
More comments can be found at the Faculty of 1000 website

Epigenetic programming by maternal behavior

2007-04-17T14:54:00.000-06:00

Maternal effects influence the development of defensive responses through epigenetic mechanisms, for example in rats these effects depend upon patterns of maternal behavior in the transmission of stress responses. Exist two forms of maternal behavior over the initial lactation days: licking and grooming (LG) and arched-back nursing (ABN); and these variations are associated with differences in hypothalamic- pituitary- adrenal (HPA) responses to stress in the offspring. The high-LG-ABN mothers show increased both hippocampal glucocorticoid receptor (GR) and nerve growth factor- inducible protein A (NGFI-A) expression, and GR expression can be induced in the offspring of high LG-ABN mothers or by artificial manipulations. These facts suggested that an epigenetic control mechanism must exist that is programmed by maternal behavior, and this adaptive advantage could be transmitted across generations. The question then was if maternal behavior altered the epigenetic state of the GR promoter, and additionally, if these possible changes could be stably maintained through adulthood. In this paper, Weaver et al. (2004) using bisulfite sequencing found that the CpG positions, especially those in the NGFI-A binding region of the GR promoter, were methylated in the offspring of the low- LG-ABN mothers, and unmethylated in the descendants of the high LG-ABN group; cross fostering confirmed that the maternal behavior was the effector of the CpG methylation, thus demonstrating that epigenetic patterns can be established through behavioral programming without germ line transmission. Timing experiments showed that in both groups, at birth the GR promoter was completely unmethylated, and later in postnatal day one (P1), the GR promoter was de novo methylated. The differences in the methylation of this promoter developed in the first week, when the maternal care of both groups of mothers exactly differed, and these epigenetic patterns remained through adulthood. Besides, ChIP against acetylated H3K9 and NGFI-A showed greater association of both proteins to the GR promoter in high- LG-ABN mothers, thus linking the DNA methylation with the histone modification states. It is commonly accepted that the methylation is rarely modified in adult post- mitotic cells; however, in this research Weaver et al. were also able to artificially reverse the maternally mediated epigenetic marking by pharmacological administration of TSA in the offspring of low- LG-ABN mothers, resulting in increased acetylated H3K9 and NGFI-A association to the promoter and decreasing the CpG methylation status, with concomitant elevated expression levels of GR. Finally, they observed that the TSA treatment totally suppresed the maternal effect in HPA responses to stress. This work is of great value because it has demonstrated that behavior can induce changes in the epigenetic status of the genome, and thus giving the first evidence of environmental programming of gene expression, and these changes were stably imprinted through lifespan.

Weaver ICG. et al. 2004. Nature Neuroscience 7(8):847 - 854
epigenetics
More comments can be found at the Faculty of 1000 website

Induction of DNA methylation and gene silencing by short interfering RNAs in human cells

2007-04-16T23:43:00.000-06:00

After the role of siRNAs was clear in the post-transcriptional silencing, and the existence of RNA- directed DNA methylation was already observed in plants, investigators Kawasaki and Taira (2004) wanted to see if these RNAs could be related to the epigenetic gene silencing. To this end, they designed a set of siRNAs targeted to control the E-cadherin promoter, after its aberrant methylation patterns have been observed in cancer lines. Using this strategy, they found that siRNA treatment induced DNA methylation, but controls (mutated siRNAs and non- targeted promoters) failed to provoke the same epigenetic change; also, the addition of 5-aza- deoxycitidine reduced the siRNA- mediated DNA methylation silencing, and later Northern blot experiments showed that these siRNAs have additive effects, i.e., using more siRNAs induced more DNA methylation that using a particular siRNA alone. Moreover, parallel ChIP assays against methylated H3K9 revealed that the E-cadherin promoter coprecipitated with this modified histone. Lower expression in both cases was confirmed by Northern and Western analysis. Taking together, these experiments showed that siRNAs against this promoter sequence not only promoted gene silencing via DNA methylation of CpG islands in a specific manner, but also they induced histone H3 methylation at lysine 9 in both cancer and normal cells, therefore demonstrating that RNA interference is linked to both epigenetic control pathways. Next, they evaluated the effect of using siRNAs against DNA methyl- transferases (DNMTs) in combination with the E-cadherin- specific siRNAs, and they found that levels of DNA methylation were lower in cells transfected with DNMT1 or DNMT3 but not with DNMT2, so only the first two were related to the siRNA- mediated epigenetic silencing. They confirmed these results when they observed that E-cadherin siRNAs supressed expression in cells containing DNMT2 siRNA, but not in those with DNMT1 or DNMT3 siRNAs. As a manner to test their results on a practical case, they used short hairpin RNAs gene therapy vectors against the erbB2 promoter, and succesfully supressed the expression driven from that promoter, via DNA methylation (and confirmed by Northern blotting). This work is important because it was the first evidence of a common pathway between siRNA- and epigenetic- driven gene silencing in animal cells. However, controversy arose when Sbovoda et al., (2004) and later several other different groups failed to induce siRNA- mediated DNA methylation, which ultimately led to a retraction of the published paper, although Kawasaki insisted that their results were valid (Another paper showing opposite results was already discussed in this blog). As of today, the RNA- directed DNA methylation is still an unanswered topic.

Kawasaki, H. and K.Taira. 2004. Nature 431:211-217
epigenetics
More comments can be found at the Faculty of 1000 and The Scientist websites

Epigenetic differences arise during the lifetime of monozygotic twins

2007-04-15T12:03:00.000-06:00

Monozygotic twins are genetically identical because they are originated from the same single zygote; although it is supposed that some differences (like those in placenta, vascularization, mosaicism, and others) can count on the phenotype, there is no clear evidence that these could be the cause of the variation typically showed between twins, for example in their differences in susceptibilities to neurological disorders. Here, the authors evaluated the epigenotypic differences in an heterogeneous group of monozygotic twins, using several experimental approaches (quantification of X- chromosome inactivation by PCR, 5-methyl cytosine content, histone acetylation levels, AIMS fingerprinting, bisulfite sequencing, CGH, RLGS, expression microarrays), and found distinctive epigenetic patterns between pairs of twins, and these differences become more notorious as individuals became older: Younger twins showed little changes in epigenotypical patterns (DNA methylation, H3/H4 acetylations), and older twins, with different lifestyles, and with less time of cohabitation, had uneven results. The authors think that the reason that genetically identical individuals have different epigenotypes, and accumulate differences with age, could be due to environmental factors. Another explanation to this fact could be possible errors in the transmission of the epigenotype during mitosis, but this is not yet clear.

Fraga, M.F. et al. 2005. Proc Natl Acad Sci USA 102(30): 10604
epigenetics

Comment on the same issue of PNAS:

"[...] Their general conclusion is that whereas young identical twinpairs are essentially indistinguishable in their epigeneticmarkings, older identical twin pairs show substantial variations.Moreover, and of considerable importance, were their studiesof gene expression in these aging twin pairs. Differences ingene expression among older twin pairs were some four timesgreater than those observed in young twin pairs [...]. Theauthors therefore concluded that there is indeed widespread"epigenetic drift" associated with aging. Their article citesother lines of evidence consistent with an important role ofepigenetic alterations in aging mammalian tissues [...]. The reported epigenetic shifts in these aging identical twinscould have arisen through endogenous, stochastic mechanisms,independent of environmental perturbations, or could have resultedfrom such environmental perturbations. The fact that there wasan association between the extent of environmental differencesbetween twins and thedegree of epigenetic shifts cannot definitively answer thatquestion. Moreover, one cannot know from this data the extentto which specific gene alterations were adaptive or nonadaptive.Although the classic evolutionary biological theory of why agingexists argues that senescent phenotypes are nonadaptive,compensatory changes in gene expression can continue for somedecades after the peak of reproductive activity [...]. I have one technical concern about Fraga et al.'s interpretationsof their results, however. Shifts in cellular population heterogeneitywithin mammalian tissues during aging are exceedingly well establishedand have, in fact, been one of the major stumbling blocks ofbiochemical investigations of aging for many decades [...] More definitive analyses of epigenetic shifts in aging twinswould therefore have to use such techniques as flow cytometricseparations of specific cell types; these methods are particularlywell developed for the case of human peripheral blood lymphocytes [...] Stochastic events such as retrotranspositions by LINE-1, point mutations of microRNAs, andsegmental duplications and haploinsufficiencies couldalso contribute to our observations that some identical twinsseem to age more rapidly than their siblings. My guess, however,is that epigenetic shifts will prove to be the most significantclass of altered genetic expression, despite the caveats expressedin the preceding paragraph [...]" (George Martin, University of Washington- Seattle).

More comments can be found at the Faculty of 1000, EurekAlert, The Scientist, Scientific American, and the Washington Post websites.

Histone modifications: signalling receptors and potential elements of a heritable epigenetic code

2007-04-13T23:29:00.000-06:00

The histone code hypothesis was first proposed in 1992 by Turner and until now there is still a great deal of controversy in this topic. On one side, some people (like the authors of this review) think that this is a real scientific fact with some missing points waiting to be discovered, and on the other, some researchers refer to this as something, if real, yet to be proved. In this paper, Nightingale et al. (2006) defined the histone code, discussed the strategies followed to locate the coding elements and the heritable potential of histone modifications, and then commented most of the histone marks relative to their particular functional outcomes.

In the definition of the histone code, they pointed that the code is real and supported by these three facts: (1) histones in nucleosomes suffer a variety of modifications at their N-terminal regions, that could be reversible; (2) protein binding to the histone N-tails depend upon the existance or removal of these modifications; and (3) different histone- protein(s) associations have different functional outcomes. The authors think that the controversy arises when some investigators have tried to associate roles in heritability to the histone modifications, which has not yet been proved in any way. If there is an heritable histone code, it should have an expected result, and must exist even in the absence of the function. To avoid confussion, Nightingale et al. suggested a different naming convention: the histone code concept should be used when refering to the gene expression regulation due to modifications, and the epigenetic code in cases dealing with cellular memory. In the rest of the paper, the authors give an extense compilation of data, with less discussion, of instances of histone modifications. They concluded that there is still more work needed to establish a clear role of the histone modifications in the heritability of transcriptional states.

Nightingale, KP. et al. 2006. Curr Opin Genet Dev 16:125-136
epigenetics

A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters

2007-04-12T23:26:00.000-06:00

The mutability of 5-methyl-cytosine causes a lower abundance of CpG dinucleotides in human DNA, and consequently, methylated CpGs are likely to be lost over time. The associated CpG islands are thus retained because they are hypomethylated in the germ line, but some can surpass this with other mechanisms (selection, repetitive regions). Although there is not a formal mathematical definition of a CpG island (the first attempt was discussed in a previous post), they have been extensively studied, for example to be used as a measure of association with promoters in computational gene identification. Those arbitrary definitions were based on thresholds of CpG fractions, GC content and length of the CpG island. Here, Saxonov et al. (2006) surveyed the pattern of CpGs over the whole human genome. They found and classified two classes of promoters, based on their CpG content: 72% of promoters with high CpG concentrations (HCG), and a remaining 28% of promoters with CpG content characteristic of the overall genome (low CpG concentration, LCG). They noted that the frequency of CpGs is higher near the transcriptional start site of HCGs, compared to a flat distribution of CpGs in LCGs, showing that HCG promoters contain CpG islands and the others lack them. Later, they compared the CpG mutation rates in order to find the cause of the difference in CpG content between the two classes, using human and chimpanzee SNPs; however, the mutation frequencies were not considerably different, thus the effect could not have been CpG- specific. Then, the authors compared the expression and distribution patterns of the genes of both classes, using gene ontologies and expression datasets, and did find differences: HCGs were present in genes expressed in all tissues, and only a small number of cell types were associated with LCGs. The authors proposed that this could be consistent with the supposition that these promoters are hypomethylated in the germ line. They also suggested that CpG islands were evolutively favoured because they could be epigenetically regulated, taking into account that most tissue- specific genes were in the HCG class. Ontologies confirmed the bias towards similar functional grouping in the two classes. Besides, HCG genes showed very similar expression profiles, independently of their CpG content inside the HCG class, showing that the HCG/LCG classification is specifically correlated with expression and not with a CpG content. This research is interesting because it helps to understand the mechanisms of establishment of the CpG patterns, and this would eventually enable to characterize the CpG islands properties, and identify the influence of CpG island locations and boundaries related to their associated trans- active elements.

Saxonov, S. et al. 2006. Proc Natl Acad Sci USA 103(5):1412-1417
epigenetics

Fluoxetine and Cocaine Induce the Epigenetic Factors MeCP2 and MBD1 in Adult Rat Brain

2007-04-10T12:41:00.000-06:00

MeCP2, a member of the MBD (methyl-CpG binding domain) protein family, silences genes when it is bound to methylated upstream CpG regions, by recruiting histone deacetylases (HDAC). Mutations in MeCP2 are associated to the Rett syndrome, and studies have shown that its expression is not homogeneous between different brain areas. To assess the spatio- temporal effects of MeCP2 regulation in nervous cells, Cassel et al. (2006) evaluated the influence of some serotonin (5-HT)- elevating agents, such as fluoxetine (an antidepressant, inhibitor of the serotonin- reuptake) and cocaine, using immuno- histochemistry and real- time PCR in normal adult rat brains. They found that after repeated doses of both drugs, there was an increase of the number of cells that immuno- reacted more for MeCP2 and another member of the methyl-CpG binding domain protein family, MBD1; but not concurrent HDAC1 or HDAC2 increase was seen, suggesting a different epigenetic regulation pathway for these cells. Quantitative analyses of the expression of both MBDs and HDACs in different serotonin- responsive brain regions, showed that the serotonergic system (particularly GABA-ergic neurons) was involved in this regulation pathway indeed because both drugs employed are 5-HT uptake and transport inhibitors, and this was later confirmed using other inhibitors of dopamine and noradrenaline transporters, obtaining the same pattern of MBD expression. In short, this research indicates that there is a relationship between epigenetic regulation (via the stimulus of MeCP2 and MBD1 expression) and the mode of action of these serotonin- elevating agents. This could be important not only for the control of depression but besides it gives more clues to understand the symptoms of the Rett syndrome.

Cassel S. et al. 2006. Mol Pharmacol 70(2):487-492
epigenetics
Comment on the same issue of Molecular Pharmacology

Reversal of Neurological Defects in a Mouse Model of Rett Syndrome

2007-04-09T13:35:00.000-06:00

Rett syndrome (RTT) is a severe autism disorder caused by mosaic expression of mutated X-linked MECP2 in neurons, although mutations in this gene can also be found in patients with other neurological pathologies. Phenotype of RTT includes abnormal neuron morphology but not neuronal death, i.e. Rett is a neurodevelopmental disorder (A short description can be found in Science 314:1536). Previously, transgenic neuron- specific Mecp2 mutant mice have showed that the condition is specifically due to the null expression of this gene solely in neurons, although these cells preserve their viability. This, and the possibility that MeCP2 could be necessary only during a short developmental period, motivated the authors to try to revert RTT, by creating model mice with their endogenous MeCP2 silenced by a lox-Stop cassette, but still can be activated by cassette deletion. The activation of Mecp2 was dependent of a fused Cre-ER transgene with a Mecp2^lox-Stopcopy; the Cre-ER protein product relocates to the nucleus after exposure to the estrogen- analog Tamoxifen (TM), producing deletion of the lox-Stop cassette.

Guy et al. (2007), first evaluated the effect of the TM- dependent deletion of the lox-Stop cassette in transgenic male mice, and found that it lead to more that 75% deletion in neurons, restoring the expression of Mecp2; however, some males showed toxicity with phenotype similar to those caused by Mecp2 over- expresion, and later this effect was eliminated with more gradual TM injections. Sudden activation of Mecp2 could then lead to toxicity or complete phenotypic reversal. After this, they tested if male mice with advanced RTT symptoms could be rescued by restoration of MeCP2, and found that 5 out of 6 were reverted by tamoxifen treatment. The same was also true when they used the TM- treatment in Stop/+, cre females with notable neurological symptomes, having a reversion close to the wild- type. Authors supposed that TM- treatment induced active X- chromosome recombination in neurons. Finally, they showed that the defect in long- term potential present in RTT conditions, was also reverted by restoration of the MeCP2. Their results fit with the proposal that MeCP2 is required during the mature neuronal state, and thus RTT is not a neurodevelopmental disorder. This also opens the possibility for a gene therapy in humans via the restoration of the wild- type copy of the allele.

The authors added to their conclusions that:

[...] The restoration of neuronal function by late expression of MeCP2suggests that the molecular preconditions for normal MeCP2 activityare preserved in its absence. To explain this, we propose thatessential MeCP2 target sites in neuronal genomes are encodedsolely by patterns of DNA methylation that are established andmaintained normally in cells lacking the protein. Accordingto this hypothesis, newly synthesized MeCP2 molecules home totheir correct chromosomal positions as dictated by methyl-CpGpatterns and, once in place, resume their canonical role asinterpreters of the DNA methylation signal [...].

Guy, J. et al. 2007. Science 315:1143-1147

Comment in the same issue of Science:

"[...This study] suggests that the damage to the nervous system may not be permanent. After the mice had received five weekly tamoxifen injections, the Rett-like symptoms all but disappeared. [...] It was a pleasant surprise, because researchers had feared that the developmental loss of Mecp2 led to missing or permanently disabled neural connections. [...] Still, the study doesn't point to an obvious strategy for treating Rett syndrome. Human gene mutations can't be repaired by the technique the Edinburgh team used, and simply boosting MECP2, either by gene therapy or administering the protein, is not likely to work, [Huda] Zoghbi [from Baylor College of Medicine, Texas] and others say. That's because girls with Rett syndrome already make MECP2 protein in about half their cells, thanks to a good copy of MECP2 on their second X chromosome. These cells would end up with a surplus of MECP2, which appears to be just as damaging as a deficit is. Finding alternative strategies won't be easy, but the mouse work suggests that such efforts are well worth pursuing." (Greg Miller, Science)

More comments can be found at the Faculty of 1000 site. Short reports about this paper can also be found at the IRSA, RSRF, EurekAlert, White Coat Notes, and MDVU websites.
epigenetics

Epigenetics and human disease: translating basic biology into clinical applications

2007-04-06T12:25:00.000-06:00

This review is an introduction to Epigenetics, for an audience of health- related professionals. It first presents an historic approach, then explains the basic concepts and mechanisms (DNA methylation, histone modification, enzymes involved), and the relationships between the acquired characters and evolution of human disorders. But the most interesting part of the paper comes when the authors make short explanations of the clinical consequences of epigenetic errors, such as those related to genomic imprinting disorders, ART, cancer, aging, immune diseases, neuropsychiatric conditions and pedriatic disorders.

Normal cellular functions regulated by epigenetic mechanisms and abnormalities caused by epigenetic errors (From: CMAJ)

D.Rodenhiser and M.Mann. 2006 CMAJ 174 (3):341-348.
epigenetics

Association of In Vitro Fertilization with Beckwith-Wiedemann Syndrome and Epigenetic Alterations of LIT1 and H19

2007-04-05T10:58:00.000-06:00

Beckwith- Wiedemann syndrome (BWS) is a congenital disease that is associated to a condition of loss of imprinting, involving hyper- or hypo- methylation depending on the affected gene; most patients with a BWS condition show aberrant methylation and imprinting of H19, IGF2 and LIT1 genes. To the date of this research, evidences were suggesting that assisted reproductive technologies (ART) could affect the methylation patterns during early embryogenesis, causing further birth defects, for example in the Angelman syndrome. Other common observations in humans were low birth weight associated also with ART, and an increased risk to birth defects was described after intracytoplasmic sperm injection (ICSI). More basic information about genomic imprinting could be found in a previous post. In this paper, DeBaun et al.(2003), report the first evidence that ART is associated with a human disorder, BWS. To this end, they used data obtained from medical histories of families in the BWS Registry, and then they evaluated the methylation status of the above mentioned imprinted loci by methylation- sensitive restriction enzyme digestion followed by Southern hybridization. They estimated that there is a sixfold increase in BWS in children born after ART, compared with the general population; and in basis of their own findings and the observations of the occurrence of the Angelman syndrome after ICSI, they suggested that ART increases the frequency of imprinting abnormalities leading to congenital disorders. Besides, they hypothesized that ART is also associated with embryonal cancers in which epigenetic impairments were observed. DeBaun et al.(2003), related the problem to the disturbance of the maternal genome in the oocyte because both in Angelman and BW syndromes it is the maternal allele that is epigenetically- modified, and they discussed that factors such as the culture conditions for the ovum, lenght of exposure to specific media or growth factors, stage of differentiation of sperm at the time of ICSI, or the ICSI itself, could be responsible for the association of ART with BWS.

DeBaun M.R., et al. 2003. Am J Hum Genet 72:156-160
epigenetics

Limited evolutionary conservation of imprinting in the human placenta

2007-04-04T18:15:00.000-06:00

Genomic imprinting is a feature of the genomes of angiosperm plants, marsupials and eutherian mammals, in which only an allele is expressed, depending on the parental origin. Although common to several different taxa, it has been suggested that imprinting has an important role in the development and function of placenta. An interesting characteristic of imprinted genes is that they are mostly clustered in what has been called as imprinted control regions (ICRs). Some of these clusters are notable because of their implication in diseases like the Beckwith- Wiedemann and Silver- Russell syndromes, malignancies, and fetal growth disorders. Here, Monk et al. (2006), have analyzed the imprinting status of four human chromosome regions, orthologues of all placental- specific imprinted genes from mice. They found that, instead of the exclusively maternal expression in the mouse (with placenta- specific expression regulated by histone modifications), human gene expression is biallelic, and they showed that this correlates with lack of chromatin modifications (no promoter DNA methylation or histone modifications found). This differences are consistent with the observation that Xist, a gene required for imprinted chromosome X inactivation, is imprinted in mouse placental expression but it is biallelic in humans, showing a lack of a common mechanism that could have diverged in the last 85 million years via either alternative species- specific imprinting mechanisms, or they share imprinting systems not yet known. This also suggest that, while in humans most paternally expressed imprinted genes enhance fetal growth whereas maternally expressed genes supress it (the "parental conflict hypothesis" of the origin of genomic imprinting), it is possible that mice have developed different control mechanisms, taking into account that they have shorter pregnancy periods and have a greater number of descendants per gestation. Authors also mentioned that imprinted control in humans would probably be more important in postnatal adaptation.

Monk et al. (2006) Proc Natl Acad Sci USA 103(17):6623-6628

Commment from the same issue of PNAS:

Genomic imprinting differentially marks maternal and paternal genes during the formation of the gametes and appears to play an important role during embryonic development, regulating whether the maternal or paternal allele of a given gene is expressed in the offspring. Although many imprinted genes are found in both the embryo and placenta in humans, the mouse has a number of genes specifically imprinted in the placenta only, suggesting that this imprinting helps to mediate the allocation of resources between the fetus and mother. David Monk et al. have found that a mechanism that regulates gene expression in the placenta is not conserved between mice and humans, a finding with important evolutionary implications. Monk et al. analyzed the human versions of these genes in human placentae and found that, in contrast to the mouse, these genes were expressed biallelically, i.e., not imprinted. Histone modifications thought to regulate imprinting in the mouse were absent in humans. These differences suggest that the imprinting of placental genes is not evolutionarily conserved, perhaps because humans typically carry only one fetus at a time, thus relaxing the need for enhanced allocation of resources from the mother.

epigenetics

More comments can be found at the Faculty of 1000 website.

RNA-mediated non-mendelian inheritance of an epigenetic change in the mouse

2007-04-03T20:37:00.000-06:00

This article provides the first observation of the phenomenon of paramutation in animals. Described before in plants (and discussed on a previous post), paramutation is often referred as a heritable epigenetic change in the phenotype by cross-talk between allelic loci (a paramutable allele), and initiated by interaction in heterozygotes with a paramutagenic form of the locus (Rassoulzadegan et al., 2006). In order to demonstrate the existance of paramutation in animals, the authors used the Kit tyrosine kinase gene as model, and whose tm1Alf mutation supresses the synthesis of the encoded protein, that has an important role in several developmental processes. Using transgenic mice, they observed an abnormal (non- mendelian) distribution of the phenotype in the progeny from heterozygous parents (Kit^tm1Alf/+). Most F1 mice that were genetically wild- type maintained the paramutated phenotype, and the inheritance of this trait had a variable phenotypic extent depending on the crosses.

Model of paramutation at Kit locus (Soloway/Nature, 2006)

They were not succesful in showing an epigenetic molecular basis of this paramutational phenotype variation, at the level of CpG islands methylation or histone modification. Instead, they found a reduced Kit RNA expression in trangenic paramutants, and later its stable inheritance was attributed to the higher levels of Kit RNA expression in heterozygotic sperm cells and they confirmed their assumptions by showing that Kit^tm1Alf/+RNA microinjection in early embryos produced the paramutated phenotype as well. Their explanation is that the paramutated state is induced by degradation of Kit RNA in microRNA pathways. Clearly, as they also recognize, the complete explanation of the phenomenon remains to be determined. But besides of demonstrating the phenomenon of paramutation in animals, this work also gave more clues to the epigenetic non- nuclear transmission of information coming from the sperm cells, which until recent times have been denied in favour of the egg cytoplasm.

Rassoulzadegan M. et al., 2006. Nature 441: 469-474.

An editorial and a comment can be found in the same issue of Nature:

[..] What might be occurring in this system, and possibly in other paramutation models, is that small RNAs produced from a paramutagenic allele are acting on the corresponding paramutable allele or on its transcribed mRNA, effectively silencing it. Because RNAi-mediated degradation of mRNAs produces more siRNAs, the silencing might be propagated if these small RNAs are packaged into germ cells and carried into the next generation. This could allow successive generations to display a certain characteristic, even if the paramutagenic allele that caused it was not transmitted [See figure]. Rassoulzadegan and colleagues' proposal that RNAs are involved in paramutation is strongly supported by work from Vicki Chandler's group showing that paramutation requires an RNA metabolizing enzyme that is involved in other epigenetic phenomena [...]. A particularly intriguing possibility is that such RNAs regulate other non-genetic modes of inheritance, such as metabolic or behavioural imprinting. (Paul D. Soloway, College of Agriculture and Life Sciences, Cornell University)

epigenetics

Analysis of Chromatin Structure by in vivo Formaldehyde Cross-Linking

2007-04-02T13:56:00.000-06:00

Looking for the original paper that first describes the ChIP method was not an easy task: Most labs often cite their own adaptations of the technique, others simply quote to Alexander Varshavsky, who was the pioneer in the studies of histones using chromatin fixation; and yet other people just make reference to the original development of formaldehyde fixation of DNA and proteins. Here is a paper by Orlando et al. (1997) of what it could be considered the very first original description of the modern ChIP as we know it now.

Chemical crosslinking of cytosine and lysine by formaldehyde. In reaction I, a Schiff base is formed between the nucleotide and HCHO, and then this intermediate can react with a second amino group (from lysine in this case) and a condensation occurs (From Orlando et al., 1997).

Taking the explanation of the authors and as it was mentioned in the above paragraph, this method is based on the formaldehyde fixation, that could cross-link proteins bound to nucleic acids or protein complexes in a reversible reaction (but does not react on dsDNA alone), with a very good resolution (range 2A). Cells are first fixed between 5 min - 1 hr, by addition of this reagent directly to the growth medium, then chromatin is solubilized by mechanical shearing (tipically sonication), because fixed cells are very resistant to nuclease treatment. Careful standardization of this sonication step should be carried out before further processing: variables such as time, immersion depth and others need to be precisely adjusted. In this original paper, CsCl- isopycnic centrifugation is employed to purify the chromatin; in the present times, after immuno- precipitation with monoclonal antibodies, DNA is extracted using silica- based commercial kits, or using first RNase - Proteinase K treatments followed by organic purification, and later a PCR amplification is performed to identify the sequence captured with the joined protein. Also, Orlando et al. (1997) use a Southern hybridization analysis, a step that is not carried out anymore, unless especially needed. As expected, the sensitivity of this method relies mostly on the quality and specificity of the monoclonal antibodies used; also the sonication step is determinant. We have already discussed here other ChIP adaptations such as ChIP-on-chip on a previous post ("High-throughput methods of regulatory element discovery") and there is also a good review of the ChIP method (Trends Biochem Sci 25:99 - 104) by the author himself.

Description of the steps in the original ChIP development; the Southern analysis has been intentionally ommited (Adapted from Orlando, 2000).

Orlando V., et al. 1997. Methods 11: 205-214
epigenetics

Plant epigenetics

2007-04-01T17:05:00.000-06:00

This short review is a primer in the field for plant scientists. The paper begins with the basics in epigenetics, and the persistance of changes in methylation patterns through development and generations is mentioned and compared to the -then- inexistant examples in mammals (We have already discussed here two more recent papers by Anway et al. and Cropley et al. showing epigenetic inheritance in this taxa). Then, the author present precise instances of epigenetic research in plants, using maize and Arabidopsis as models, but the importance of the stable maintenance of transgene expression that could be epigenetically altered is also mentioned.

In maize, the main concern to the date is changes due to the domestication. Here the phenomenon of paramutation (the "ability of one locus to alter the epigenetic patterning of a related locus elsewhere in the genome", after the author) using the B gene as model is discussed. She suggests that the effect of this transcription factor could be by (i) via direct interaction between the two involved chromosomes; or (ii) using an RNA mediator, as it has been observed that some RNAs can induce DNA methylation. In Arabidopsis, the model for studying paramutation is the PAI gene, and enzyme needed for the synthesis of triptophan that is also related to changes in DNA sequence promoted by different chromatin patterns; the same paramutational hypothesis presented for maize are discussed here for PAI. Another model is the ddm1 "epimutator" gene, an homolog of the yeast SWI2/SNF2 chromatin remodelling factor and the mammalian LSH1, that promotes global methylation changes, although gene- specific effects in methylation have been also observed.

Bender, J. 2002. Curr Biol 12(12):R412-R414
epigenetics

Germ-line epigenetic modification of the murine Avy allele by nutritional supplementation

2007-03-30T12:30:00.000-06:00

Changes in somatic cells almost never influence the phenotype displayed by descendants; here, Cropley et al. showed that this old genetic rule has some exceptions, because some environmental factors can cause variation in the epigenotype, and they demonstrated that this change could be stably maintained in the subsequent generations. To this end, they used agouti (A^vy) mice as model, whose epigenotype is controlled by methylation of the inserted IAP retrotransposon producing mosaic A^vy/a offspring (with phenotypes from yellow to fully agouti "pseudoagouti"), and the range of expressivity is correlated with levels of methylation (pseudoagoutiis heavily methylated, yellow unmethylated). After the observation that supplemental nutrition with methyl donors (folate, vit.B12, choline, betaine) in pregnant mice influenced the phenotypes of the offspring, they wondered if those changes could be seen on the next generation. They first found that the agouti allele is only nutritionally affected when it comes from the paternal line. Then they provided the methyl donor diet to pregnant mice only from E8.5 to E15.5 (period when somatic epigenetic marks have been set, and also germ cells differentiate and reset the same marks), and separated the F1 females to mate them with recessive wild- type males without the methyl- enriched diet. Now the F2 phenotypes were still influenced by the original parental diet exposure, suggesting an environmental intervention in the germ line.

The pattern of mosaicism in A^vy mice is consistent with an epigeneticstate that is set during early embryogenesis and is stable (insomatic cells) thereafter. This stability resembles that seenwith other epigenetic phenomena such as parental imprintingand X-chromosome inactivation. [...] Despite the restricted period of supplementation,the spectrum of phenotypes in F₁ mice was shifted significantly, indicating that the A^vy allele is susceptible toinduced epigenetic change even after the early embryonic periodwhen epigenetic resetting takes place. We selected (F₁) pseudoagouti A^vy/a females that had been exposedto methyl donors in utero from E8.5 to E15.5, and mated themto a/a males without any further methyl donor supplementation;this strategy takes advantage of the tendency for the A^vy epigenotypeto be partially stable in the female germ line. [...] Phenotypesof these F₂ mice were significantly shifted toward the pseudoagouti. Thus a pseudoagouti dam who was exposed to methyldonor supplementation only when she was in utero gives riseto phenotypically different offspring than does an otherwise(genetically and phenotypically) identical female who had noexposure to methyl donor supplementation; this grandparentaleffect is directly attributable to the epigenetic state of theA^vy allele. Because nutritional supplementationceased when the F₁ mice were still in utero, our evidence indicatesthat the effect on A^vy epigenotype in these primordial germcells is retained throughout gametogenesis as well as duringthe fertilization and development of the F₂ embryo.

They conclude that this is a definitive demonstration that an environmental factor could influence the epigenotype of germ cells and this could be stably transferred to the subsequent generations, surviving through the processes of gametogenesisand embryogenesis. The hypothesis previously formulated by Marcus Pembrey (a few months before) was thus validated.

[...T]he epigenotype of the paternal A^vy allele undergoes completeepigenetic resetting during embryogenesis, whereas the maternalA^vy allele partly retains its epigenotype. This relative stabilityof the A^vy epigenetic state in the female germ line may relateto its resistance to the influence of increased methyl donors. Thousands of retroelements have the potential, if active, tobehave as controlling elements, with unpredictable effects onphenotype. The susceptibility of these elements to perturbationby environmental agents provides another way in which epigeneticscan mediate environmental influence on phenotype. Moreover, in lightof the roughly 20-year generation time of humans, our resultssuggest that current dietary habits may have an influence ongrandchildren who will be born decades from now, independentof the diets that their parents consume.

Cropley JE, et al. 2006. Proc Natl Acad Sci USA 103(46): 17308-17312

Comment on the same issue of PNAS:

[...] We do not know what range of phenotypes to expect when epigeneticsystems that evolved over millions of years respond to new environmentalvariables such as refined foods, drugs, and xenobiotics. Glucoseand endocrine disrupters are examples offactors leading to apparent epigenetic transgenerational effectsin mammals; however, the genes responsible for the effects arenot known. Now Cropley et al. show that methyl donors havetransgenerational effects attributed to a known allele, A^vy. Cropley et al. used a diet supplementing betaine, choline,folic acid, vitamin B12, methionine, and zinc. Even withoutsupplements, human diets span a huge range for these nutrients [and t]hese nutrient levels can be much lower in diets relyingon refined foods. In humans, the possibility [...] that grandmaternaldiets contributed to the incidence of obesity and diabetes inthe current generation and that today's dietary habits willhave effects for generations to come make [this] work especially important (Craig A. Cooney, University of Arkansas for Medical Sciences).

Comment from one of the authors (press release):

[...] Our study highlights a layer of complexity about human development that needs to be thoroughly investigated. [...] We found that even when we stopped providing specific supplements during pregnancy, the past effect of supplements persisted. Therefore, it is possible that the maternal diet could have implications that stretch over decades, perhaps even centuries. (Kenneth Beckman, Children’s Hospital Oakland Research Institute)

The (Dual) Origin of Epigenetics

2007-03-29T06:03:00.000-06:00

A short paper worth reading, tracing the first appearences of the word epigenetics, from the definition related to developmental biology, until the more recent and well-known concept by Riggs et al. (1996).

Epigenetics was coined by Waddington (1942) to refer to the study of the “causal mechanisms” by which “the genes of the genotype bring about phenotypic effects.” Waddington had earlier used epigenotype to refer to “the set of organizers and organizing relations to which a certain piece of tissue will be subject during development.” He believed that genotype and phenotype referred to “differences between whole organisms... [and were] not adequate or appropriate for the consideration of differences within a single organism.” [...] Molecular biologists are probably most familiar with a definition of epigenetics as “the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence”

Waddington targeted what he saw as the naive view of many geneticists that there was a simple correspondence between genes and characters. [...] Neo-Darwinism, he believed, involved “a breach between organism and nature as complete as the Cartesian dualism of mind and matter; an epigenetic consideration of evolution would go some way toward healing it”. [...] Because Waddington claimed to provide a richer paradigm for studying the interaction between organism and environment than the impoverished view of genetics, an epigenetic approach has appealed to critics of evolutionary “orthodoxy,” whether these be biologists who feel that there is something lacking in the neo-Darwinian synthesis or philosophers who favor a less gene-centric, more holistic, view of biology

Another relevant point of view was that by Nanney, who was ahead of his time. His model of molecular inter- dependence ("a dynamic self-perpetuating organization of a variety of molecular species which owes its specific properties not to the characteristics of any one kind of molecule, but to the functional interrelationships of these molecular species") was distantly but significantly related to the Ganti's chemoton (briefly discussed here before)

Nanney (1958) [...] believed that “auxiliary mechanisms with different principles of operation are involved in determining which specificities are to be expressed in any particular cell.” These auxiliary mechanisms he called epigenetic control systems. He saw them as accounting for the observation that cells with the same genotype could have different phenotypes. [...] He adopted the terms genetic and epigenetic because they more clearly delimited “the notion of true genetic change [...] Unfortunately, I must remind them that, as a corollary, we must admit that not everything that is inherited is genetic.”

Finally, the recent interest on the field was credited to Robin Holliday.

Widespread use of epigenetics to refer to heritable changes that do not involve changes in DNA sequence did not occur until the 1990s [...] Holliday (1979) contains the earliest description of DNA methylation as an “epigenetic” process that I have found, but I suspect that “The inheritance of epigenetic defects” (Holliday 1987) was the critical paper that lit the fuse for the explosion in use of “epigenetic” in the 1990s.

Haig, D. 2004. Cold Spring Harbor Symp Quant Biol 69: 1-4.