Mapping of HNF4α target genes in intestinal epithelial cells

Abstract

BackgroundThe role of HNF4α has been extensively studied in hepatocytes and pancreatic β-cells, and HNF4α is also regarded as a key regulator of intestinal epithelial cell differentiation. The aim of the present work is to identify novel HNF4α target genes in the human intestinal epithelial cells in order to elucidate the role of HNF4α in the intestinal differentiation progress.MethodsWe have performed a ChIP-chip analysis of the human intestinal cell line Caco-2 in order to make a genome-wide identification of HNF4α binding to promoter regions. The HNF4α ChIP-chip data was matched with gene expression and histone H3 acetylation status of the promoters in order to identify HNF4α binding to actively transcribed genes with an open chromatin structure.Results1,541 genes were identified as potential HNF4α targets, many of which have not previously been described as being regulated by HNF4α. The 1,541 genes contributed significantly to gene ontology (GO) pathways categorized by lipid and amino acid transport and metabolism. An analysis of the homeodomain transcription factor Cdx-2 (CDX2), the disaccharidase trehalase (TREH), and the tight junction protein cingulin (CGN) promoters verified that these genes are bound by HNF4α in Caco2 cells. For the Cdx-2 and trehalase promoters the HNF4α binding was verified in mouse small intestine epithelium.ConclusionThe HNF4α regulation of the Cdx-2 promoter unravels a transcription factor network also including HNF1α, all of which are transcription factors involved in intestinal development and gene expression.

Modification in Oxidative Stress, Inflammation, and Lipoprotein Assembly in Response to Hepatocyte Nuclear Factor 4α Knockdown in Intestinal Epithelial Cells

“…The literature examination revealed HNF4␣ capacity to bind to promoters of many genes involved in lipid metabolism, glutathione metabolism, oxidative stress, inflammatory processes, and transcription factor status (47)(48)(49). Because our study reveals potential HNF4␣ target genes that have not been previously identified in the literature (e.g.…”

Genome-wide Analysis of CDX2 Binding in Intestinal Epithelial Cells (Caco-2)

“…Cell Cultures and Transfections-The cells were grown, and reporter gene transfections were carried out as described in Boyd et al (12). The cells were stably transfected with SureSilencing TM shRNA plasmid for Human CDX2 (SABiosciences Corp.) according to the manufacturer's recommendations.…”

“…ChIP qPCR Analysis-Verification of enrichment due to immunoprecipitation was done as previously described (12). The primers listed in supplemental Data S1 were used to amplify human genomic sequences at CDX2-target loci.…”

“…Previous work by Lussier et al (44) has shown that CDX2 is essential in initiating differentiation and that this is coordinated with the activation of HNF4␣ and HNF1␣ expression, which in turn requires CDX2 action. Furthermore, we have previously shown that HFN4␣ regulates the expression of CDX2; thus, a loop that is necessary for the initiation and completion of differentiation of intestinal cells appears to exist (12). The promoter of HNF4␣ has previously been shown to contain a functional CDX2 binding site (30).…”

“…Some of the pathways identified to date include the Wnt pathway (2), the Notch pathway (3), the Hedgehog system (4), members of the transforming growth factor-␤ family including the bone morphogenetic proteins (5), and the phosphatidylinositol 3-kinase pathway (6). The intestine specific gene expression requires the tightly regulated activity of transcription factors, including the well characterized key factors HNF1␣/␤, 2 HNF4␣, GATA factors, ETS, CDX1, and CDX2 (7)(8)(9)(10)(11)(12)(13). These transcription factors often cooperate to control the expression of the intestinal specific genes; until now, in particular HNF1␣, GATA4 -6, and CDX2 have been found to interact in order to regulate the activity of several promoters in the intestine (7, 14 -20).…”