Long-term stability of demethylation after transient exposure to 5-aza-2'-deoxycytidine correlates with sustained RNA polymerase II occupancy

Abstract

DNA methyltransferase inhibitors are currently the standard of care for myelodysplastic syndrome and are in clinical trials for leukemias and solid tumors. However, the molecular basis underlying their activity remains poorly understood. Here, we studied the induction and long-term stability of gene reactivation at three methylated tumor suppressor loci in response to the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-azaCdR) in human breast cancer cells. At the TMS1/ASC locus, treatment with 5-azaCdR resulted in partial DNA demethylation, the reengagement of RNA polymerase II (Pol II), and a shift from a repressive chromatin profile marked with H3K9me2 and H4K20me3 to an active profile enriched in H3ac and H3K4me2. Using a single-molecule approach coupling chromatin immunoprecipitation with bisulfite sequencing, we show that H3ac, H3K4me2, and Pol II selectively associated with the demethylated alleles, whereas H3K9me2 preferentially marked alleles resistant to demethylation. H4K20me3 was unaffected by DNA demethylation and associated with both unmethylated and methylated alleles. After drug removal, TMS1 underwent partial remethylation, yet a subset of alleles remained stably demethylated for over 3 months. These alleles remained selectively associated with H3K4me2, H3ac, and Pol II and correlated with a sustained low level of gene expression. TMS1 alleles reacquired H3K9me2 over time, and those alleles that became remethylated retained H3ac. In contrast, CDH1 and ESR1 were remethylated and completely silenced within approximately 1 week of drug removal, and failed to maintain stably unmethylated alleles. Our data suggest that the ability to maintain Pol II occupancy is a critical factor in the long-term stability of drug-induced CpG island demethylation.

Figure 1. TMS1 expression and DNA methylation following the removal of 5-azaCdR

A. Diagram of the TMS1 gene. Exons, open boxes; transcription start site, arrow. The CpG island is indicated by the bracket. Primers utilized for MSP, GBS, and COBRA analyses are indicated below the gene, as is the position of the Fnu4HI restriction site used in COBRA analysis. B. TMS1 mRNA abundance was measured immediately after treatment (5-azaCdR) or at the indicated time points after drug removal (pp, passages post 5-azaCdR) by reverse transcriptase qPCR and normalized to 18S rRNA. Shown is the fold change in expression (mean ± standard deviation) relative to untreated cells from three independent time-course experiments assayed in triplicate. C. COBRA analysis of DNA methylation following the removal of 5-azaCdR at the TMS1 locus. Data represents the mean percent methylation ( ± standard deviation) from three independent time course experiments D. DNA methylation was analyzed by bisulfite sequencing at the indicated time points. Each line represents a single colony isolate (8–13 isolated per sample). Open circles, unmethylated CpG; filled circles, methylated CpG.

Figure 2. Histone modifications and RNA Pol II occupancy at TMS1 after the removal of 5-azaCdR

MDA-MB231 cells were left untreated or treated with 0.5uM 5-azaCdR for six days. Chromatin was isolated immediately after treatment (5-azaCdR) or at the indicated time after drug removal (pp, passages post 5-azaCdR). Histone modifications and RNA Pol II occupancy were analyzed by ChIP followed by qPCR. Percent enrichment was determined by comparison of immunoprecipitated DNA relative to input DNA at each time point using primer set 3 of the TMS1 locus (11). Plotted is the mean (±standard deviation) of the fold change in enrichment relative to untreated MDA-MB231 cells from a single time course experiment assayed in triplicate. Similar results were obtained from a second independent time course (Supplemental Figure 1).

Figure 3. Single molecule association between DNA methylation and chromatin modifications at the TMS1 locus

MDA-MB231 cells were left untreated or treated with 0.5uM 5-azaCdR for six days (5-azaCdR) A. Chromatin was isolated immediately after (5-azaCdR) or at the indicated time after drug removal and immunoprecipitated with the indicated antibodies. Precipitated DNA was eluted, bisulfite modified, and amplified with the bisulfite sequencing primers indicated in Figure 1A. For each immunoprecipitation, 9–17 individual clones were sequenced. Open circles, unmethylated CpGs; filled circles, methylated CpGs. Vertical hash marks represents missing data. B. Overall methylation density was determined as the total number of methylated CpGs relative to total number of CpGs in all alleles analyzed.

Figure 4. Analysis of CDH1 during and following treatment with 5-azaCdR

A. Diagram of CDH1 CpG island. Open boxes, exons; arrow, transcription start site. The position of primers used for methylation analyses and the Fnu4H1 restriction enzyme site used for COBRA analysis are indicated. B. CDH1 mRNA expression was determined by reverse transcriptase qPCR and was normalized to 18S rRNA. Shown is the fold change in expression (mean ± standard deviation) relative to untreated cells from three independent experiments assayed in triplicate. C. COBRA analysis of DNA methylation following the removal of 5-azaCdR at the CDH1 locus. Data represents the mean percent methylation ( ± standard deviation) from three independent time course experiments D. DNA methylation was further analyzed by bisulfite sequencing at the indicated time points. Each line represents a single colony isolate (8–11 isolated per sample). Open circles, unmethylated CpG; filled circles, methylated CpG. pp, passages post 5-azaCdR treatment. E. Histone modifications and RNA Pol II occupancy were determined by ChIP as described in the legend to Figure 2. Plotted is the mean (±standard deviation) of the fold change in enrichment relative to untreated MDA-MB231 cells from a single time course experiment assayed in triplicate. Similar results were obtained from a second independent time course (Supplemental Figure 2).

Figure 5. Analysis of ESR1 during and following treatment with 5-azaCdR

A. Diagram of ESR1 CpG island. Open boxes, exons; arrow, transcription start site. The position of primers used for methylation analyses and the Mlu1restriction enzyme site used for COBRA analysis are indicated. B. mRNA expression was determined using primers specific to ESR1. Shown is the fold change in expression (mean ± standard deviation) relative to untreated cells after internal normalization to 18S rRNA from three independent experiments assayed in triplicate. C. COBRA analysis of DNA methylation at time points following the removal of 5-azaCdR at the ESR1 locus as described in the legend to Figure 1 D. DNA methylation was analyzed by bisulfite sequencing at the indicated time points. Each line represents a single colony isolate (9–12 isolated per sample). Open circles, unmethylated CpG; filled circles, methylated CpG. pp, passages post 5-azaCdR treatment. E. Histone modifications and RNA Pol II occupancy were determined by ChIP as described in the legend to Figure 2. Plotted is the mean (±standard deviation) of the fold change in enrichment relative to untreated MDA-MB231 cells from a single time course experiment assayed in triplicate. Similar results were obtained from a second independent time course (Supplemental Figure 3).