Differences in the Epigenetic Regulation of Cytochrome P450 Genes between Human Embryonic Stem Cell-Derived Hepatocytes and Primary Hepatocytes

<div><p>Human pluripotent stem cell-derived hepatocytes have the potential to provide <i>in vitro</i> model systems for drug discovery and hepatotoxicity testing. However, these cells are currently unsuitable for drug toxicity and efficacy testing because of their limited expression of genes encoding drug-metabolizing enzymes, especially cytochrome P450 (CYP) enzymes. Transcript levels of major <i>CYP</i> genes were much lower in human embryonic stem cell-derived hepatocytes (hESC-Hep) than in human primary hepatocytes (hPH). To verify the mechanism underlying this reduced expression of <i>CYP</i> genes, including <i>CYP1A1</i>, <i>CYP1A2</i>, <i>CYP1B1</i>, <i>CYP2D6</i>, and <i>CYP2E1</i>, we investigated their epigenetic regulation in terms of DNA methylation and histone modifications in hESC-Hep and hPH. CpG islands of <i>CYP</i> genes were hypermethylated in hESC-Hep, whereas they had an open chromatin structure, as represented by hypomethylation of CpG sites and permissive histone modifications, in hPH. Inhibition of DNA methyltransferases (DNMTs) during hepatic maturation induced demethylation of the CpG sites of <i>CYP1A1</i> and <i>CYP1A2</i>, leading to the up-regulation of their transcription. Combinatorial inhibition of DNMTs and histone deacetylases (HDACs) increased the transcript levels of <i>CYP1A1</i>, <i>CYP1A2</i>, <i>CYP1B1</i>, and <i>CYP2D6</i>. Our findings suggest that limited expression of <i>CYP</i> genes in hESC-Hep is modulated by epigenetic regulatory factors such as DNMTs and HDACs.</p></div

Transcriptional regulation of <i>CYP</i> genes by inhibition of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) during hepatic differentiation.

<p>(A) Methylation frequencies in the promoter and gene body regions of <i>CYP</i> genes were examined by bisulfite sequencing in hESC-Hep treated with DMSO or a DNMT inhibitor (DAC or RG108). Data represent methylation frequencies from two independent experiments. (B) Expression levels of <i>CYP</i> genes were examined by real-time RT-PCR in hESC-Hep treated with DMSO, DAC, or RG108. Data represent mean ± SD from three independent experiments. * p<0.05, ** p<0.01, significant values in comparison with DMSO (ANOVA followed by Dunn’s multiple comparison test). (C) Expression levels of <i>CYP</i> genes were examined by real-time RT-PCR in hESC-Hep treated with DMSO or a HDAC inhibitor (1 mM sodium butyrate (SB)) with or without a DNMT inhibitor (DAC or RG108). Data represent mean ± SD from three independent experiments. * p<0.05, ** p<0.01, *** p<0.001, significant values in comparison with DMSO (ANOVA followed by Bonferroni’s multiple comparison test).</p

Gene expression levels of drug-metabolizing enzymes.

<p>Expression of genes encoding nuclear receptors (A), phase I enzymes (B), phase II enzymes (C), and phase III transporters (D) was examined by real-time RT-PCR in human primary hepatocytes (hPH) and human embryonic stem cell-derived hepatocytes (hESC-Hep, day 20). Data represent mean ± SD from three independent experiments. * p<0.05, ** p<0.01, significant values in comparison with hPH (t-test followed by Wilcoxon matched pairs test).</p

DNA methylation and histone modifications in regulatory regions of <i>CYP</i> genes.

<p>Each diagram shows the locations of the sites of <i>CYP1A1</i> (A), <i>CYP1A2</i> (B), <i>CYP1B1</i> (C), <i>CYP2D6</i> (D), and <i>CYP2E1</i> (E) within gene promoters, which were examined by bisulfite sequencing and chromatin immunoprecipitation (ChIP). The methylation status of CpG dinucleotides in target regions was examined in hPH and hESC-Hep (day 20) by bisulfite sequencing (upper panel). Each row represents the methylation status of each CpG in one bacterial clone. A series of 9–10 clones is shown. Black circles represent metyhylated CpG sites while white circles represent unmethylated CpG sites. Numbers indicate nucleotide positions in relation to the transcription start site (TSS, +1). ChIP analysis of histone modifications including two active histone marks H3Ac and H3K4me3 and one repressive histone mark H3K27me3 in hPH and hESC-Hep is shown (lower graph). Data validated by real-time PCR are presented as fold enrichment of precipitated DNA associated with a given histone modification relative to a 100-fold dilution of input chromatin. Data represent mean ± SD from two independent experiments. * p<0.05, ** p<0.01, *** p<0.001, significant values in comparison with hPH (t-test followed by Wilcoxon matched pairs test).</p

Intermediate cells of in vitro cellular reprogramming and in vivo tissue regeneration require desmoplakin

Amphibians and fish show considerable regeneration potential via dedifferentiation of somatic cells into blastemal cells. In terms of dedifferentiation, in vitro cellular reprogramming has been proposed to share common processes with in vivo tissue regeneration, although the details are elusive. Here, we identified the cytoskeletal linker protein desmoplakin (Dsp) as a common factor mediating both reprogramming and regeneration. Our analysis revealed that Dsp expression is elevated in distinct intermediate cells during in vitro reprogramming. Knockdown of Dsp impedes in vitro reprogramming into induced pluripotent stem cells and induced neural stem/progenitor cells as well as in vivo regeneration of zebrafish fins. Notably, reduced Dsp expression impairs formation of the intermediate cells during cellular reprogramming and tissue regeneration. These findings suggest that there is a Dsp-mediated evolutionary link between cellular reprogramming in mammals and tissue regeneration in lower vertebrates and that the intermediate cells may provide alternative approaches for mammalian regenerative therapy.11Nsciescopu