Regulation of Replication Fork Progression Through Histone Supply and Demand

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

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

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

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

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