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Author Notes:

To whom correspondence should be addressed at: 1365-C Clifton Rd, NE, Rm 4086, Atlanta, GA, 30322, pvertin@emory.edu

Current address: Cancer Research, Oncology R&D, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426

Acknowledgments The authors wish to thank Pritty Patel, Harold Saavedra, and Arsene Adon for technical assistance and helpful discussions.

Subject:

Research Funding:

This work was supported by NIH grants 2RO1 CA077337 (to PMV), a Department of Defense CDMRP Breast Cancer Program predoctoral fellowship W81XWH-08-1-0362 (to JDK) and an American Cancer Society postdoctoral fellowship PF-07-130-01-MGO (to MTM)

Keywords:

  • DNA methylation
  • epigenetics
  • TMS1/ASC
  • chromatin
  • Decitabine
  • breast cancer

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

Tools:

Journal Title:

Molecular Cancer Research

Volume:

Volume 8, Number 7

Publisher:

, Pages 1048-1059

Type of Work:

Article | Post-print: After Peer Review

Abstract:

DNA methyltransferase (DNMT) 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 DNMT inhibitor 5-aza-2′-deoxycytidine (5-azaCdR)in human breast cancer cells. At the TMS/ASC locus, treatment with 5-azaCdR resulted in partial DNA demethylation, the re-engagement 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 unmethylated and methylated alleles. After drug removal, TMS1 underwent partial remethylation yet a subset of alleles remained stably demethylated for over three months. These alleles remained selectively associated with H3K4me2, H3ac, and Pol II and correlated with a sustained low level of gene expression. TMS1 alleles reacquire H3K9me2over time and those alleles that became remethylated retained H3ac. In contrast, CDH1and ESR1 were remethylated and completely silenced within ~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.

Copyright information:

© 2010 American Association for Cancer Research

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