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5-Aza-2′-deoxycytidine-mediated reductions in G9A histone methyltransferase and histone H3 K9 di-methylation levels are linked to tumor suppressor gene reactivation

Oncogene volume 26, pages 77–90 (2007)Cite this article

Abstract

The epigenetic silencing of tumor suppressor genes is a common event during carcinogenesis, and often involves aberrant DNA methylation and histone modification of gene regulatory regions, resulting in the formation of a transcriptionally repressive chromatin state. Two examples include the antimetastatic, tumor suppressor genes, desmocollin 3 (DSC3) and MASPIN, which are frequently silenced in this manner in human breast cancer. Treatment of the breast tumor cell lines MDA-MB-231 and UACC 1179 with 5-aza-2′-deoxycytidine (5-aza-CdR) induced transcriptional reactivation of both genes in a dose-dependent manner. Importantly, DSC3 and MASPIN reactivation was closely and consistently linked with significant decreases in promoter H3 K9 di-methylation. Moreover, 5-aza-CdR treatment also resulted in global decreases in H3 K9 di-methylation, an effect that was linked to its ability to mediate dose-dependent, post-transcriptional decreases in the key enzyme responsible for this epigenetic modification, G9A. Finally, small interfering RNA (siRNA)-mediated knockdown of G9A and DNMT1 led to increased MASPIN expression in MDA-MB-231 cells, to levels that were supra-additive, verifying the importance of these enzymes in maintaining multiple layers of epigenetic repression in breast tumor cells. These results highlight an additional, complimentary mechanism of action for 5-aza-CdR in the reactivation of epigenetically silenced genes, in a manner that is independent of its effects on DNA methylation, further supporting an important role for H3 K9 methylation in the aberrant repression of tumor suppressor genes in human cancer.

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Acknowledgements

We thank Y Rodriguez, D Thompson and the Arizona Respiratory Sciences Center for their efforts in cloning and sequencing our sodium bisulfite-modified PCR products and for creating and maintaining a database to aid in the visualization and analysis our resultant sequence data. We thank T Monks and his laboratory for assistance of acid extraction of histones. We also thank G Tsaprailis and the Arizona Cancer Center/Southwest Environmental Health Sciences Center (AZCC/SWEHSC) Proteomics Core for technical advice in the development of a method to measure the global 5-methylcytosine content of our samples using LC-MS. NIH grant CA65662 to BWF supported this work. RJW was supported, in part, by Southwest Environmental Health Sciences Center training grant ES007091 and Cancer Biology training grant T32-CA09213. LC-MS data acquired in the AZCC/SWEHSC Proteomics Core supported by NIEHS grant P30 ES06694 and NIH/NCI grant P30 CA023074.

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Authors and Affiliations

  1. Department of Pharmacology and Toxicology, Arizona Cancer Center, University of Arizona, Tucson, AZ, USA

    R J Wozniak, S S Lau & B W Futscher

  2. Arizona Respiratory Sciences Center, University of Arizona, Tucson, AZ, USA

    W T Klimecki

  3. Arizona Cancer Center/Southwest Environmental Health Sciences Center Proteomics Core, University of Arizona,, Tucson, AZ, USA

    S S Lau & Y Feinstein

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  1. R J Wozniak
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Correspondence to B W Futscher.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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Wozniak, R., Klimecki, W., Lau, S. et al. 5-Aza-2′-deoxycytidine-mediated reductions in G9A histone methyltransferase and histone H3 K9 di-methylation levels are linked to tumor suppressor gene reactivation. Oncogene 26, 77–90 (2007). https://doi.org/10.1038/sj.onc.1209763

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