We found that E and GEN
We found that E2 and GEN each has mostly similar effects on recruitment of the epigenetic modulators to the MB-COMT distal promoter (summarized in Table 2). These effects are associated with increased promoter methylation and gene suppression of COMT. It is noted that the array of epigenetic modulators recruited by E2 is not exactly the same as that by GEN. GEN was shown to modulate E2 cellular bioavailability that may contribute to the regulations of E2/E1 metabolism (Hanet et al., 2008). It has been suggested that differential estrogenicity of GEN (either through ER-dependent or independent pathways) leads to distinct expression patterns of E2-responsive genes (Lin et al., 2008; Maggiolini et al., 2004). Thus, GEN may trigger alternative epigenetic or transcriptional machinery that contributes to COMT transcription although future study is required to support this claim. We showed that the epigenetic effects of E2 and GEN on COMT transcription are region-specific. Both E2 and GEN induced DNA methylation at specific CpG cluster (CpG sites 12–16). Increased DNA methylation at this CpG cluster was associated with the decrease in COMT transcription. This CpG cluster encompassed transcription factor C/EBPα (illustrated in Supplementary Fig. 4). Methylation at this cluster may either interfere the binding of C/EBPα or disrupt the tak tak australia remodeling which results in gene suppression (Singal and Ginder, 1999; Zhang et al., 2005). Our finding is consistent with the report suggesting the possible role of the transcription factor, C/EBP, on suppression of E2-induced COMT transcription (Xie et al., 1999). Our data showing that E2 or GEN increased binding of DNMT3B and MBD2 further supports the presence of DNA methylation in this CpG cluster. Moreover, we showed E2 and GEN induced increased HDAC1 binding and decreased recruitment of HAT/p300 at this cluster. Furthermore, we demonstrated E2 and GEN decreased levels of H3K4-, H3K9- and H3K27-acetylation at this CpG cluster. It suggests that histone deacetylation might also contribute to the suppression of COMT transcription. In addition to this CpG-rich region (R3), another CpG cluster (R1) at the MB-COMT distal promoter was found to be responsive to histone modifications (H3K4/H3K9/H3K27-acetylation). Strikingly, we did not find any recruitment of DNA methylation-related proteins at this region. We acknowledged that we could not examine the effect of every epigenetic modification enzyme individually on COMT transcription as there are a large number of epigenetic modification enzymes. Perhaps, there are alternative epigenetic mechanisms contributed to COMT transcription but it requires further mechanistic study to prove this hypothesis. In this study, we showed that AZA and TSA completely reversed the E2-induced COMT suppression (Supplementary Fig. 3). Hence, we speculate that in MCF-7 cells, E2 and GEN epigenetically regulate COMT transcription, at least through promoter methylation and histone de- and acetylation along the MB-COMT distal promoter. We demonstrated that phytoestrogen, GEN, inhibit COMT transcription epigenetically. GEN which is an isoflavone found in a soy-based diet is proposed to have the potential to prevent tumorgenesis and/or cancer progression through its selective estrogen receptor modulators (SERM) activity. Nevertheless, whether use of GEN is beneficial for chemoprevention of BCa is controversial. There are both in vitro and in vivo studies that reported adverse effects of GEN on mammary tissues/cells via epigenetic mechanisms (Dolinoy et al., 2006; Tang et al., 2008; Greathouse et al., 2012). In the present study, we further suggest that there are epigenetic modifications (promoter methylation and histone de- and acetylation) underlying regulation of COMT expression in response to GEN. The questions of whether soy isoflavones, such as GEN, could epigenetically regulate transcription of genes involved in estrogen metabolism and tumorigenesis, deserve further studies both in vivo and in vitro.