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 p16INK4a 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 p16INK4a 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 p16INK4a silencing was essential for growth. Two triple mutants (DKO and p16INK4a disrupted, called TKOmut/−, and other with DKO and the mutant gene disrupted, TKOwt/−) were generated. As expected TKOwt/− re- expressed p16INK4a but TKOwt/− grew slower than TKOmut/−, and the authors thought that altogether, these results meant that:
epigenetics
[...] 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 p16INK4a 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 p16INK4a 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
