Project description:Background and Aims: Hepatocyte nuclear factor 1 (HNF1) transcription factors direct tissue specific gene regulation in liver, pancreas and kidney and are associated with diabetes. Here we investigate the transcriptional network governed by HNF1 in an intestinal epithelial cell line. Methods: Chromatin immunoprecipitation followed by direct sequencing (ChIP-seq) was used to identify HNF1 binding sites genome-wide. Direct targets of HNF1 were validated using conventional ChIP assays. siRNA-mediated depletion of HNF1 followed by RT-qPCR further confirmed target genes. The effect of HNF1 reduction on glucose uptake was measured by fluorescence activated cell sorting (FACS) following treatment of intestinal epithelial cells with 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG, a fluorescent glucose mimic). Results: HNF1 controls multiple pathways that are critical for intestinal epithelial cell function, including properties of the cell membrane, cellular response to hormones, and regulation of biosynthetic processes. Approximately 50% of HNF1 binding sites are also occupied by hepatocyte nuclear factor 4A (HNF4A), caudal type homeobox 2 (CDX2), and forkhead box A2 (FOXA2). Depletion of HNF1 increases FOXA2 abundance and decreases levels of CDX2. Moreover, loss of HNF1 inhibits glucose uptake by the intestinal epithelial cell line. Conclusions: These data show that HNF1 plays a critical role in regulating intestinal epithelial cell functions including glucose absorption. HNF1 interacts with other tissue-specific transcription factors to regulate differentiated properties of these cells.
Project description:Chromatin from mouse hepatocytes was immunoprecipitated with antibodies against Hepatocyte Nuclear Factor 4 alpha (Hnf4a) and IgG. Amplified and labelled ChIP DNA and input DNA were hybridyzed to Mouse PromoterChip 5A.1 arrays.
Project description:Although hepatocyte-nuclear-factor-1α (Hnf1α) is crucial for pancreas and liver functions, it is believed to play a limited functional role for intestinal epithelial functions. The aim of this study was to assess the consequences of abrogating Hnf1α on the maintenance of adult small intestinal epithelial functions. Methodology/Principal Findings An Hnf1α knockout mouse model was used. Assessment of histological abnormalities, crypt epithelial cell proliferation, epithelial barrier, glucose transport and signalling pathways were measured in these animals. Changes in global gene expression were also analyzed. Mice lacking Hnf1α displayed increased crypt proliferation and intestinalomegaly as well as a disturbance of intestinal epithelial cell lineages production during adult life. This phenotype was associated with a decrease of the mucosal barrier function and lumen-to-blood glucose delivery. The mammalian target of rapamycin (mTOR) signalling pathway was found to be overly activated in the small intestine of adult Hnf1α mutant mice. The intestinal epithelium of Hnf1α null mice displayed a reduction of the enteroendocrine cell population. An impact was also observed on proper Paneth cell differentiation with abnormalities in the granule exocytosis pathway. Conclusions/Significance Together, these results unravel a functional role for Hnf1α in regulating adult intestinal growth and sustaining the functions of intestinal epithelial cell lineages. HNF1alpha was knocked out. A total of 3 control and 3 mutant littermate individuals were sacrificed at 4 months of age. The jejunum was harvested and total RNA was isolated from each individual. Each RNA sample was independently used to generate probes to screen Affymetrix chips.
Project description:Pancreatic islets were isolated from Hnf1a+/- (Hepatocyte nuclear factor 1 alpha) and wild-type mice and cultured ex vivo for two days in RPMI medium with 10% FBS before RNA extraction. The transcription profiles were obtained using Affymetrix GeneChip Mouse Genome 430 2.0 arrays [Mouse430_2].
