Abstract
The hepatic nuclear factor HNF4α is a versatile transcription factor and controls expression of many genes in development, metabolism and disease. To delineate its regulatory gene network in colon cancer and to define novel gene targets a comprehensive genome-wide scan was carried out at a resolution of 35 bp with chromatin IP DNA obtained from the human colon carcinoma cell line Caco-2 that is a particularly rich source of HNF4α. More than 90% of HNF4α binding sites were mapped as promoter distal sequences while enhancer elements could be defined to foster chromatin loops for interaction with other promoter-bound transcription factors. Sequence motif analysis by various genetic algorithms evidenced a unique enhanceosome that consisted of the nuclear proteins ERα, AP1, GATA and HNF1α as cooperating transcription factors. Overall >17,500 DNA binding sites were identified with a gene/binding site ratio that differed >6-fold between chromosomes and clustered in distinct chromosomal regions amongst >6600 genes targeted by HNF4α. Evidence is presented for nuclear receptor cross-talk of HNF4α and estrogen receptor α that is recapitulated at the sequence level. Remarkably, the Y-chromosome is devoid of HNF4α binding sites. The functional importance of enrichment sites was confirmed in genome-wide gene expression studies at varying HNF4α protein levels. Taken collectively, a genome-wide scan of HNF4α binding sites is reported to better understand basic mechanisms of transcriptional control of HNF4α targeted genes. Novel promoter distal binding sites are identified which form an enhanceosome thereby facilitating RNA processing events.
Highlights
Hepatic nuclear factor HNF4a is a member of the nuclear receptor superfamily and an extremely versatile transcription factor [1]
We were interested in exploring an HNF4a genomic footprint in the human colon adenocarcinmoa Caco-2 cell line that has been widely used to explore HNF4a activity [5] thereby identifying a network of regulated genes
Chromatin IP experiments were performed with Caco-2 cell cultures and an antibody highly specific for HNF4a
Summary
Hepatic nuclear factor HNF4a is a member of the nuclear receptor superfamily and an extremely versatile transcription factor [1]. This zinc finger protein is expressed in liver, intestine, pancreas and other tissues, and binds to cognate DNA sequences as a homodimer [2]. We were interested in exploring an HNF4a genomic footprint in the human colon adenocarcinmoa Caco-2 cell line that has been widely used to explore HNF4a activity [5] thereby identifying a network of regulated genes. We studied the consequences of HNF4a protein induction on transcriptional activity of de novo identified genes and demonstrate good agreement between novel gene targets and their expression in Caco-2 cells. We analyzed HNF4a binding sites for enriched binding motifs and identified cooperating transcription factors that appeared to act in concert with HNF4a in an enhanceosome of transcriptional regulation
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