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Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer

Nature Genetics volume 21, pages 103–107 (1999)Cite this article

Abstract

Densely methylated DNA associates with transcriptionally repressive chromatin characterized by the presence of underacetylated histones1,2. Recently, these two epigenetic processes have been dynamically linked. The methyl-CpG-binding protein MeCP2 appears to reside in a complex with histone deacetylase activity3,4. MeCP2 can mediate formation of transcriptionally repressive chromatin on methylated promoter templates in vitro, and this process can be reversed by trichostatin A (TSA), a specific inhibitor of histone deacetylase3,4,5. Little is known, however, about the relative roles of methylation and histone deacetylase activity in the stable inhibition of transcription on densely methylated endogenous promoters, such as those for silenced alleles of imprinted genes6, genes on the female inactive X chromosome7 and tumour-suppressor genes inactivated in cancer cells8,9. We show here that the hypermethylated genes MLH1, TIMP3 (TIMP-3), CDKN2B (INK4B, p15) and CDKN2A (INK4, p16) cannot be transcriptionally reactivated with TSA alone in tumour cells in which we have shown that TSA alone can upregulate the expression of non-methylated genes. Following minimal demethylation and slight gene reactivation in the presence of low dose 5-aza-2´deoxycytidine (5Aza-dC), however, TSA treatment results in robust re-expression of each gene. TSA does not contribute to demethylation of the genes, and none of the treatments alter the chromatin structure associated with the hypermethylated promoters. Thus, although DNA methylation and histone deacetylation appear to act as synergistic layers for the silencing of genes in cancer, dense CpG island methylation is dominant for the stable maintenance of a silent state at these loci.

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Figure 1: Histone deacetylase inhibition by TSA is not sufficient to reactivate genes silenced in association with promoter region hypermethylation, but can upregulate expression of transcribed genes in the same cells.
Figure 2: Histone deacetylase inhibition by TSA facilitates transcriptional reactivation induced by the demethylating agent 5Aza-dC and is effective within 6 h of treatment.
Figure 3: TSA treatment does not further extend demethylation induced by 5Aza-dC.
Figure 4: Neither TSA, 5Aza-dC nor the combination of both agents results in gross chromatin change at the MLH1 CpG island region as monitored by accessibility to the restriction endonucleases MspI, XbaI, PvuII and SacI.

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Acknowledgements

The authors thank A. Wolffe, K. Kinzler and J. Bender for helpful advice and comments on this work; M. Rountree for scientific advice; K. Polyak for helpful advice and for providing the RT primer sequences for MLH1; B. Vogelstein for providing the RT primer sequences for CDKN1A; B. Vogelstein and K. Kinzler for initial supplying of RKO and SW480 cells; and F. Ruscetti for supplying HL60 cells.

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Authors and Affiliations

  1. The Oncology Center, The Johns Hopkins University School of Medicine, 424 N. Bond Street, Baltimore, 21231, Maryland, USA

    Elizabeth E. Cameron, Kurtis E. Bachman, Sanna Myöhänen, James G. Herman & Stephen B. Baylin

  2. Department of Medicine, The Johns Hopkins University School of Medicine, 424 N. Bond Street, Baltimore , 21231, Maryland, USA

    Stephen B. Baylin

  3. Predoctoral Training Program in Human Genetics, The Johns Hopkins University School of Medicine, 424 N. Bond Street , Baltimore, 21231, Maryland, USA

    Elizabeth E. Cameron & Stephen B. Baylin

  4. Graduate Program in Cellular and Molecular Medicine, The Johns Hopkins University School of Medicine, 424 N. Bond Street, Baltimore, 21231, Maryland, USA

    Kurtis E. Bachman & Stephen B. Baylin

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Correspondence to Stephen B. Baylin.

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Cameron, E., Bachman, K., Myöhänen, S. et al. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet 21, 103–107 (1999). https://doi.org/10.1038/5047

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