Epigenetic Mechanisms Of The Tissue-specific Expression Pattern Of CYP2C Subfamily

CYP2C subfamily, encoding CYP2C8, CYP2C9 and CYP2C19 proteins, constitutes an important part of cytochrome P450. The CYP2C subfamily is expressed in a tissue-specific manner, with highest expression in liver and weak expression in duodenum. It has been well documented that liver-enriched transcriptional factors are playing a pivotal role in CYP2C subfamily transcriptional regulation. Research also revealed a decrease of CYP2C8, CYP2C9 and CYP2C19 at both mRNA and protein level in tumorous tissues of hepatocellular carcinoma (HCC), indicating a potential relationship between CYP2C subfamily and HCC tumorigenesis, but the underlying mechanism remains unclear. Elucidating the epigenetic modulation of CYP2C subfamily might help improve our understanding of HCC. Epigenetics, including DNA methylation, histone modification, non-coding RNA regulation and other mechanisms, plays a key role in tumorigenesis, aging, cell proliferation and differentiation. It would help build a comprehensive regulation network of the CYP2C subfamily to elucidate whether and how epigenetics works in the tissue-specific expression of CYP2C subfamily.In our research, by analyzing DNA methylation sequencing (WGBS) and chromatin immunoprecipitation sequencing (ChIP-seq) datasets, CpG sites around transcriptional start site (TSS) of CYP2C8, CYP2C9 and CYP2C19, respectively, were identified differentially methylated between liver tissue and other non-liver tissues. Regarding histone modification, H3K9ac and H3K27ac were highly enriched on CYP2C8 and CYP2C9 promoters in liver, but not in any other tissues. CYP2C19 promoter was moderately acetylated and did not show difference between liver and non-liver tissues as strongly as CYP2C8 and CYP2C9. These differentially methylated or acetylated sites might be related with the tissue-specific expression of CYP2C subfamily.To investigate the regulation of DNA methylation and histone acetylation on CYP2C subfamily, either tricostatin A (TSA) or 5-Aza-2'-deoxycytidine (5-aza-dC) was applied to primary cultured hepatocytes. Consequently, CYP2C8, CYP2C9 and CYP2C19 were induced to different extents under both treatments. Moreover, constitutive androstane receptor (CAR) was the only central transcriptional factor that was induced by 5-aza-dC. Bisulfite sequencing PCR (BSP) revealed a decreased DNA methylation frequency on the promoters of both CYP2C subfamily members and CAR after 5-aza-dC treatment, which suggested that histone acetylation and DNA methylation are potential regulatory mechanisms of CYP2C subfamily in liver, in which CAR might also be involved.Based on results above, quantitative real-time PCR (qRT-PCR) and bisulfite sequencing PCR (BSP) was applied to determine whether the suppression of CYP2C subfamily in HCC is related with DNA methylation. No discrepancy of DNA methylation was detected on the promoters of CYP2C subfamily members between the tumorous and adjacent normal tissues. However, methylation frequency on the e-box site, which is closed to the TSS of CAR, was significantly elevated in tumorous tissues. The elevated methylation frequency was shown to be negatively correlated with the decreased expression of CAR and CYP2C subfamily. Reporter gene assay further demonstrated that the e-box hypermethylation is involved in the decreased C4AR-mediated CYP2C subfamily expression.Subsequently, ChIP-PCR was performed to investigate if histone acetylation also contributed to the suppressed CYP2C subfamily expression in HCC. Results revealed significant decrease of H3K9ac and H3K27ac on the promoters of CYP2C8 and CYP2C9 in HCC tumorous tissues. Compared with CYP2C8 and CYP2C9, CYP2C19 possessed a relatively lower H3K9ac and H3K27ac level in normal liver tissues, which was not significantly altered in HCC tumorous tissues. This was consistent with the differential acetylation status between liver and other non-liver tissues revealed by ChIP-seq analysis.In summary, we conducted preliminary research on epigenetic mechanisms of the tissue-specific expression of CYP2C subfamily. Moreover, these mechanisms also contribute to the altered gene expression in HCC, nevertheless, exact pathways might be different, which remains to be further elucidated. These findings will help us understanding how epigenetics contribute to hepatocytes formation and HCC tumorigenesis.