Early studies have reported that TSG silenced by promoter DNA hypermethylation could be reactivated only after the removal of methylation marks. In these studies, treatment with TSA, an HDACi, could not produce gene reactivation of genes silenced by promoter DNA hypermethylation (6
). Based on these indirect observations, the function of promoter DNA methylation, as a signal for gene silencing, has been considered as a ‘lock’ for gene expression. However, other studies have reported that hypermethylated genes can be reactivated by TSA and other HDACi without any loss in promoter DNA methylation (9
). These reports put in jeopardy the lock hypothesis which has been the paradigm for more than a decade. Hence, we chose to investigate this issue by looking at the effects of more than 20 different HDACi on reactivation of genes silenced by promoter DNA methylation.
Using the well-characterized YB5 system and other cancer cell lines, we discovered that most of the HDACi-tested could reactivate hypermethylated genes in a dose-dependent pattern regarding their chemical class and HDAC affinity. DNA methylation analysis revealed that gene reactivation was generated without any loss of promoter DNA methylation. Methylation levels were carefully assessed before and after treatment by pyrosequencing and bisulfite cloning sequencing since it was reported that HDACi could potentially reduce DNA methylation levels by non-specific mechanisms (16
). However, our study, as well as others (19
) show that methylation levels did not change 24h after HDACi exposure or several days post-treatment. Therefore, these data confirm that HDACi can reactivate gene expression through hypermethylated promoters, which demonstrates that DNA methylation does not lock gene expression in that it does not prevent reactivation by chromatin remodeling. It is not clear why previous studies reported that HDACi do not reactivate the expression of hypermethylated genes, though it may relate to the use of low doses of HDACi for short periods of time, and the use of insensitive methods for gene expression analysis. Alternately, it is possible that some genes/cell lines are resistant to this effect, though we observed it for most genes and most cell lines tested.
The fact that DNA methylation does not lock gene expression raises the question of the relative contribution of DNA methylation and chromatin modifications to gene silencing. The YB5 system was particularly suitable to investigate this issue since after treatment with either Depsi or with 5-AZA-CdR, we were able to sort the GFP-expressing cells and monitor GFP fluorescence for several months. We discovered that a treatment with HDACi can transiently reactivate hypermethylated genes (GFP and other TSG) for up to 2 weeks without any changes in DNA methylation level in their promoter regions. On the other hand, treatment with 5-AZA-CdR leads to gene reactivation of GFP and other TSG for several months. Moreover, the decline in GFP-expressing cells after 5-AZA-CdR, thought largely to be due to remethylation, is in fact attributable in part to clonal replacement by YB5 cells that are methylated and do not express GFP. Indeed, cell sorting and single cell cloning 9 weeks after drug removal led to clones where the promoter region was completely demethylated, and expression permanently on. Thus, efficient demethylation leads to permanent gene reactivation, showing that DNA methylation provides a memory signal for the silent state.
Our data show that the respective roles of DNA methylation and chromatin remodeling can be completely separated using the YB5 selectable system. The chromatin state determines the immediate gene expression potential, while DNA methylation provides a long-term memory for gene silencing. Thus, DNA methylation does not provide a ‘lock’ function as previously thought, because gene expression can be restored by drug-induced chromatin modifications without any DNA demethylation (i.e. without breaking the lock). Rather, DNA methylation provides a ‘spring’ function, which does not suppress gene expression but brings back silencing, presumably through the previously defined order of events: methyl-binding protein recruitment, histone deacetylation, histone methylation, HP1 binding and so on (3
). This explains why physiologically, DNA methylation at promoter CGIs is only involved when very long-term silencing is required, and why it provides such a selective advantage to cancer cells when TSG are silenced by this mechanism (3
). Interestingly, after treatment with HDACi, gene expression is not sufficient to lead to permanent expression and DNA demethylation. It is possible that gene reactivation induced by HDACi may be caused by either i) bypassing transcription factors, whereby histone acetylation will directly trigger RNA pol II activation leading to reactivation or ii) transient binding of transcription factors to promoter regions, with gene silencing rapidly restored by repressive signals arising from DNA methylation. Restoring a silenced state is likely when histones are replaced during cell divisions in the face of persistent DNA methylation. Importantly after the treatment with hypomethylating drugs, it has been previously demonstrated that removal of DNA methylation marks will allow the binding of transcription factors leading to permanent epigenetic resetting promoting the emergence of stably reactivated clones. This was shown by 5-AZA-CdR-induced DNA demethylation in YB5 cells where the CREB transcription factor bound only the CMV promoter only when it was hypomethylated (13
These data have implications for therapeutic intervention. We show that genes silenced by DNA hypermethylation in cancer can be significantly but transiently reactivated through chromatin remodeling without any changes in DNA methylation, and this may be part of the clinical mechanisms of action of HDACi. Moreover, our results provide a molecular explanation for the synergy between decitabine and HDACi (6
) in which the combination induces more complete epigenetic reprogramming. Finally, while these findings validate chromatin as a key target for therapeutic intervention in cancer, they also suggest that stable reprogramming may require the removal of DNA methylation signals.