Analysis of DNA Methylation and Histone Modification Profiles of Liver-Specific Transporters

Abstract

Tissue-specific expression of transporters is tightly linked with their physiological functions through the regulation of the membrane transport of their substrates. We hypothesized that epigenetic regulation underlies the tissue-specific expression of mouse liver-specific transporters (Oatp1b2/ Slco1b2, Ntcp/Slc10a1, Bsep/Abcb11, and Abcg5/g8). We examined their DNA methylation and histone modification profiles near the transcriptional start site (TSS) in the liver, kidney, and cerebrum. Genome-wide DNA methylation profiling with tissue-dependent differentially methylated region profiling with restriction tag-mediated amplification and subsequent bisulfite genomic sequencing demonstrated that the CpG dinucleotides around the TSS of Oatp1b2 (from Ϫ515 to ϩ149 CpGs), Ntcp (from Ϫ481 to ϩ495 CpGs), Bsep (from Ϫ339 to ϩ282 CpGs), and Abcg5/g8 (from Ϫ161 to ϩ5 CpGs for Abcg5, i.e., from Ϫ213 to Ϫ48 CpGs for Abcg8) were hypomethylated in the liver and hypermethylated in the kidney and cerebrum. The opposite pattern was observed for Pept2/ Slc15a2 (from Ϫ638 to ϩ4 CpGs), which was expressed in the kidney and cerebrum but not in the liver. These DNA methylation profiles are consistent with the tissue distribution of these transporters. A chromatin immunoprecipitation assay demonstrated that the histone H3 associated with Oatp1b2, Ntcp, Bsep, and Abcg5/g8 promoters was hyperacetylated in the liver but was acetylated very little in the kidney and cerebrum, whereas the upstream region of Pept2 was hyperacetylated only in the kidney and cerebrum. These results suggest the involvement of epigenetic systems in the tissue-specific expression of mouse transporters Oatp1b2, Ntcp, Bsep, Abcg5/ g8, and Pept2.During the past two decades, extensive research has sought to uncover the molecular characteristics of transporters to explain their physiological and pathological functions. These transporters comprise more than 300 genes that are now classified into the solute carrier (SLC) and ATP-binding cassette (ABC) transporter superfamilies. Transporters show tissue-specific expression patterns that have critical implications for the physiological function of certain tissues (Borst and Elferink, 2002;Kusuhara and Sugiyama, 2002;Hediger et al., 2004; Stefkova et al., 2004;Shitara et al., 2006).Liver tissue is one of the most important tissues in terms of the physiological turnover of endogenous compounds, such as bile acids and sterols, and the clearance of exogenous substances, including clinically used drugs. Several transporters are closely related to the hepatobiliary transport of these compounds. SLC-type transporters mediate the hepatic uptake of bile acids and xenobiotic organic anions, and ABC transporters then contribute to the biliary efflux of bile acids, sterols, and xenobiotic compounds and their metabolites (Faber et al., 2003;Chandra and Brouwer, 2004). Numerous reports suggest the involvement of liver-enriched transcription factors, such as hepatocyte nuclear factors (HNFs) 1, 3, and 4, and proteins of the CCAA...

DNA Methylation and Histone Modification Profiles of Mouse Organic Anion Transporting Polypeptides

“…The degree of DNA methylation in these regions was calculated and compared among the tissues (Table 1). The corresponding data for Oatp1b2 were cited from our previous study (Imai et al, 2009) for comparison. In Oatp1c1, one CpG site located at +149 relative to the TSS was regarded as T-DMR, and the other 2 sites upstream of the TSS were similarly methylated among tissues (Fig.…”

“…In various types of genes, the widespread existence of tissue-dependent differentially methylated regions (T-DMRs) around their transcription start sites (TSS) has been described, which can potentially explain tissue-specific expression of respective genes (Shiota et al, 2002;Yagi et al, 2008). Recently, we and others have demonstrated an association of DNA methylation with the tissue-specific expression of human and mouse transporters (Aoki et al, 2008;Imai et al, 2009;Jin et al, 2012;Kikuchi et al, 2007;Kikuchi et al, 2006;Kikuchi et al, 2010). In particular, genome-wide DNA methylation analysis using a methylation-sensitive restriction enzyme HpyCH4IV and mouse promoter tiling array revealed that 53 of 297 SLC transporter genes in mouse, including Oatp1b2/Slco1b2, were associated with T-DMRtags in the liver (Imai et al, 2009).…”

“…The chromatin immunoprecipitation (ChIP) assay was performed using anti-acetylated histone H3 antibody (Upstate Biotechnology, Lake Placid, NY) according to a procedure described previously (Imai et al, 2009). Normal rabbit IgG was used as a negative control to verify the immunoprecipitation specificity.…”

“…Recently, we and others have demonstrated an association of DNA methylation with the tissue-specific expression of human and mouse transporters (Aoki et al, 2008;Imai et al, 2009;Jin et al, 2012;Kikuchi et al, 2007;Kikuchi et al, 2006;Kikuchi et al, 2010). In particular, genome-wide DNA methylation analysis using a methylation-sensitive restriction enzyme HpyCH4IV and mouse promoter tiling array revealed that 53 of 297 SLC transporter genes in mouse, including Oatp1b2/Slco1b2, were associated with T-DMRtags in the liver (Imai et al, 2009). TDMRtags in the liver are defined as the HpyCH4IV restriction sites that are hypomethylated in the liver, compared with other tissues.…”

Regulation of Tissue-Specific Expression of Renal Organic Anion Transporters by Hepatocyte Nuclear Factor 1 α/β and DNA Methylation

“…We have previously demonstrated that DNA methylation-dependent gene silencing underlies the kidney-specific expression of human OAT3 and mouse Urat1 (Kikuchi et al, 2006(Kikuchi et al, , 2007. Subsequently, a genomewide DNA methylation analysis together with bioinformatics approaches located tissue-dependent differentially methylated regions (T-DMRs) that were hypomethylated in the liver but hypermethylated in the extrahepatic tissues in a number of liver-specific genes, including transporters, and many of those genes contained HNF1-binding sequences in their promoters (Yagi et al, 2008;Imai et al, 2009). Most recently, we also identified T-DMRs in the kidney-specific amino acid transporters that are potentially transactivated by Hnf1␣/␤ (Kikuchi et al, 2010).…”