title: 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.
authors.affiliation: Graduate Program in Genetics and Molecular Biology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
authors.forename: Jacob D
authors.initials: JD
authors.lastname: Kagey
authors.forename: Priya
authors.initials: P
authors.lastname: Kapoor-Vazirani
authors.forename: Michael T
authors.initials: MT
authors.lastname: McCabe
authors.forename: Doris R
authors.initials: DR
authors.lastname: Powell
authors.forename: Paula M
authors.initials: PM
authors.lastname: Vertino
mesh_terms: D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation / Q000187:drug effects*; D000077209:Decitabine; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D016147:Genes, Tumor Suppressor; D006801:Humans; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*
chemical_list: D000077209:Decitabine; D012319:RNA Polymerase II; D001374:Azacitidine