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

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 0 -deoxycytidine (5-azaCdR) 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, posttranscriptional 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.

Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts

“…This role has been demonstrated for histone H3 associated with the maspin gene. Treatment of P4 corneal stromal cells with 5-Aza-dC demethylated histone H3 at K9 in the same manner as was observed in mammary carcinoma cells (Wozniak et al, 2007).…”

“…As suggested by Wozniak et al (Wozniak et al, 2007), the lack of demethylation of the maspin promoter when maspin is re-expressed in response to 5-Aza-dC could be due to gene reactivation from a few alleles that become demethylated in the cell population. This might account for the really low maspin levels (mRNA and protein) observed in the presence of 5-Aza-dC.…”

“…Low levels of maspin reexpression were observed with 5-Aza-dC in mammary carcinoma cell lines without demethylation of the maspin promoter (Wozniak et al, 2007). Use of small interfering RNA to the DNA methyltransferase, DNMT1 increased the expression of maspin in MDA-MB-231 breast mammary carcinoma cells supporting DNA methylation in the regulation of maspin expression.…”

“…Even though the primary function of 5-Aza-dC at 10 μM or less is to inhibit the DNA methyltransferase, DNMT-1, it could have another role as a histone methyltransferase inhibitor (Kondo et al, 2003;Murakami et al, 2004;Wozniak et al, 2007;Zhu et al, 2001). This role has been demonstrated for histone H3 associated with the maspin gene.…”

Reprogramming epigenetic silencing: artificial transcription factors synergize with chromatin remodeling drugs to reactivate the tumor suppressor mammary serine protease inhibitor

“…18), valproic acid (19), and pivaloyloxymethyl butyrate (20,21)] are presently in phase I and II clinical trials. The small-molecule inhibitors 5-aza-2 ¶-dC and SAHA have been used to reactivate tumor suppressor genes aberrantly methylated in aggressive tumor cells, such as desmocollin 3 (22), gelsolin (23), and mammary serine protease inhibitor (maspin; refs. 13,22).…”