Alterations of Epigenetic Signatures in Hepatocyte Nuclear Factor 4α Deficient Mouse Liver Determined by Improved ChIP-qPCR and (h)MeDIP-qPCR Assays

Qinghao Zhang,Xiaohong Lei,Hong Lu,Frances M Sladek

doi:10.1371/journal.pone.0084925

Abstract

Hepatocyte nuclear factor 4α (HNF4α) is a liver-enriched transcription factor essential for liver development and function. In hepatocytes, HNF4α regulates a large number of genes important for nutrient/xenobiotic metabolism and cell differentiation and proliferation. Currently, little is known about the epigenetic mechanism of gene regulation by HNF4α. In this study, the global and specific alterations at the selected gene loci of representative histone modifications and DNA methylations were investigated in Hnf4a-deficient female mouse livers using the improved MeDIP-, hMeDIP- and ChIP-qPCR assay. Hnf4a deficiency significantly increased hepatic total IPed DNA fragments for histone H3 lysine-4 dimethylation (H3K4me2), H3K4me3, H3K9me2, H3K27me3 and H3K4 acetylation, but not for H3K9me3, 5-methylcytosine,or 5-hydroxymethylcytosine. At specific gene loci, the relative enrichments of histone and DNA modifications were changed to different degree in Hnf4a-deficient mouse liver. Among the epigenetic signatures investigated, changes in H3K4me3 correlated the best with mRNA expression. Additionally, Hnf4a-deficient livers had increased mRNA expression of histone H1.2 and H3.3 as well as epigenetic modifiers Dnmt1, Tet3, Setd7, Kmt2c, Ehmt2, and Ezh2. In conclusion, the present study provides convenient improved (h)MeDIP- and ChIP-qPCR assays for epigenetic study. Hnf4a deficiency in young-adult mouse liver markedly alters histone methylation and acetylation, with fewer effects on DNA methylation and 5-hydroxymethylation. The underlying mechanism may be the induction of epigenetic enzymes responsible for the addition/removal of the epigenetic signatures, and/or the loss of HNF4α per se as a key coordinator for epigenetic modifiers.

Highlights

Hepatocyte nuclear factor 4a (HNF4a) is a conserved member of the nuclear receptor superfamily of ligand-dependent transcription factors [1]
We successfully developed the improved methylated DNA immunoprecipitation (MeDIP), hydroxymethylated DNA immunoprecipitation (hMeDIP), and ChIP-qPCR assays to elucidate the impact of Hnf4a deficiency on the histone modifications as well as DNA methylation and 5-hydroxymethylation in the female mouse livers
The marked changes in hepatic epigenetic signatures in Hnf4a-LivKO mice are associated with changes in hepatic mRNA expression of epigenetic modifiers

Summary

Introduction

HNF4a is a conserved member of the nuclear receptor superfamily of ligand-dependent transcription factors [1]. As a liver-enriched transcription factor, HNF4a is expressed in the kidney, small intestine, colon, stomach, and pancreas in which the mutation of HNF4A gene causes maturity-onset diabetes of the young in humans (MODY) [2,3]. HNF4a is essential in liver development and differentiation, lipid homeostasis, bile acid synthesis, as well as the expression of phase I, II, and III drug processing genes [4,5,6]. HNF4a may interact with other nuclear receptors such as chicken ovalbumin upstream promoter-transcription factor, retinoid X receptor, peroxisome proliferatoractivated receptor (PPAR), farnesoid X receptor, constitutive androstane receptor, glucocorticoid receptor, Vitamin D receptor, and small heterodimer partner to directly or indirectly regulate gene expression [1,2,12]

Objectives

Methods

Results

Conclusion