In this study, we describe a new model system YB5, which contains a hypermethylated and silenced CMV driven GFP gene with stably inherited epigenetic properties established over time. In this model, the promoter is DNA hypermethylated and has a closed chromatin structure characterized by histone H3K9 deacetylation and H3K27 hypermethylation. Expression is suppressed at the transcriptional level and can be restored by using the demethylating agent Decitabine or by inhibiting DNMT1 expression. Thus, this system mimics many of the features of typical gene silencing in mammalian cells, including cancer cells. CMV is a strong CpG island containing promoter, and it can be efficiently silenced as previously reported (27
). Because reactivated gene expression can be easily visualized and selected for, this model allowed us to ask important questions regarding the minimal requirements for gene reactivation, as well as to track re-silencing after epigenetic modulation. These questions have previously been investigated primarily from the perspective of entire (mixed) cell populations, though one study used sub-cloning and gene expression selection to ask questions about remethylation (9
We first asked about the association between hypomethylation induction and gene reactivation. Surprisingly, methylation studies showed similar demethylation levels between GFP positive cells and negative cells, which suggests that pharmacological uptake of DAC is uniform and not rate-limiting in these cells. In sorted cells that had high GFP expression, DNA methylation did not decrease to normal levels (nearly zero) suggesting that a previous hypothesis about heterogeneous reexpression being explained by a mixture of cells demethylated to 0% (and reexpressing) while others remain hypermethylated and silenced is incorrect. In fact, only 6 of 24 alleles sequenced had complete demethylation in these cells with high GFP expression. Thus, only a moderate degree of DNA hypomethylation is required for gene reactivation. In our experiments, we ruled out activation of an alternative host promoter as a possible explanation. However, further experiments may be needed to verify that methylated CpG islands can still have promoter activity if they retain an open chromatin. Bisulfite-sequencing analysis of H3K9ac histones showed about 30% DNA methylation, confirming that the gene can be activated despite residual DNA methylation, but also suggesting that unmethylated alleles are enriched in open chromatin. Moreover, in our data the GFP negative cells after DAC also had decreased DNA methylation levels, demonstrating that DNA hypomethylation, per-se, is not sufficient for gene reactivation. Rather, the main molecular difference between GFP-positive and GFP-negative cells post-DAC was the differential histone modifications and histone H3 densities revealed by ChIP assays. Thus, chromatin resetting is the prime factor determining gene reexpression state after DAC induced DNA demethylation. Interestingly, it has previously been reported that DAC induces nuclease sensitivity changes in the human HPRT gene locus prior to gene reexpression (29
), and decreased nucleosome occupancy of hMLH1 promoter after DAC (30
), which are consistent with our data.
It is interesting to ask why a relatively small degree of DNA demethylation would naturally, but not uniformly, bring histone changes and chromatin resetting. A possible explanation is that in the absence of DNMTs, the coupled assembly of newly synthesized histone octamers during cellular replication may be disrupted. The nascent histone octamers lose some original repressive histone tail marks, the promoter nucleosome assembly is disabled and the chromatin alters to a locally open structure, resulting in transcription of this strand of DNA (31
). Indeed, DNMT1 and DNMT3b have been reported to bind to HDACs, histone methyltransferases and chromatin scaffold proteins, and it is possible that this binding is important to replication of histone marks. It is interesting to note that after withdrawal of DAC, about 12% of sorted GFP negative cells transform to expressing cells after 24 hours in regular growth medium, which indicates the continuing chromatin resetting. This model also explains the observed synergistic effect between DNA demethylating agents and low-dose histone deacetylases inhibitors (12
The cell sorting strategy also allowed us to address the vexing problem of gene resilencing and remethylation after DAC withdrawal. Using a mixed cell population, it was previously reported that the repressive histone mark H3K27me3 persists or increases after DAC treatment (13
), and thus serves as a nidus for resilencing. Our data using purified cells are not consistent with these observations. Rather, we find that resilencing and remethylation is independent of gene expression levels or of local chromatin structure. The kinetics of DNA remethylation is quite flat, which can be cell division related (33
). However, loss of expression after DAC withdrawal is very rapid in the first several days (seen at day 2 and day 5). Notably, the speed of histone H3 gains up to day 5 is also prompt, which appears to be coincident with rapid GFP loss. Thus, it is highly likely the first re-silencing is due to the reassembly of nucleosomes, though the driving force of re-packaging DNA is unknown. It is possible that a closed chromatin configuration persists in-cis
away from the CMV promoter and underlies gene resilencing.
It remains to be seen how much the data we have generated with this in vitro
system are applicable to endogenous gene silencing. All indications are that they would be – the transgene is stably integrated, shows the typical methylation-associated closed chromatin configuration and is reactivated by Decitabine or DNMT1 knock-down with similar kinetics and patterns as endogenous genes. Nevertheless, it is likely that there will be gene or locus specific events that influence reactivation. Indeed, some genes are silenced without detectable H3K27me3 (33
), and those may behave differently. Similarly, promoters vary in their CpG density and degree of DNA methylation, and this could influence gene reactivation patterns.
Our data have clinical implications for the use of DNA methylation inhibitors. In treated patients, relatively small decreases in DNA methylation were observed (34
), but these were accompanied by significant gene reactivation and clinical responses. Moreover, as would be predicted from the current data, gene reactivation was a better predictor of response than hypomethylation induction (36
). Just like the in vitro
situation, gene remethylation was observed, and a higher extent of remethylation was associated with resistance to therapy (35
In summary, we found that DNA hypomethylation is necessary but not sufficient for gene reactivation after DAC. Rather, local chromatin structure resetting, which can happen at a low level of DNA demethylation, is a key determinant of actual gene re-expression. These data have implications for the use of hypomethylating drugs in the clinic. In addition, the YB5 system can be useful for evaluating potential demethylating compounds and epigenetic synergy studies to boost gene reactivation as well as prevent resilencing and remethylation.