Hepatocyte Nuclear Factor 1 coordinates multiple functions of intestinal epithelial cells. Homo sapiens

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. Overall design: To determine the role of HNF1 in regulating gene expression in intestinal epithelial cells, ChIP-seq was performed for HNF1 in Caco2 (colorectal adenocarcinoma) cells.

背景与目的：肝细胞核因子1（Hepatocyte nuclear factor 1, HNF1）转录因子可介导肝脏、胰腺与肾脏的组织特异性基因调控，并与糖尿病发生密切相关。本研究旨在探究肠上皮细胞系中HNF1所调控的转录网络。方法：采用染色质免疫共沉淀测序（ChIP-seq）在全基因组范围内鉴定HNF1的结合位点；通过常规ChIP实验验证HNF1的直接靶基因；利用siRNA介导HNF1敲低后联合实时定量PCR（RT-qPCR）进一步确认靶基因；将肠上皮细胞用2-[N-(7-硝基苯并-2-氧杂-1,3-二唑-4-基)氨基]-2-脱氧-D-葡萄糖（2-NBDG，一种荧光葡萄糖类似物）处理后，采用荧光激活细胞分选（FACS）检测HNF1表达降低对葡萄糖摄取的影响。结果：HNF1调控多条对肠上皮细胞功能至关重要的通路，包括细胞膜特性、细胞对激素的应答以及生物合成过程的调控。约50%的HNF1结合位点同时被肝细胞核因子4A（Hepatocyte nuclear factor 4A, HNF4A）、尾型同源盒转录因子2（Caudal type homeobox 2, CDX2）以及叉头框蛋白A2（Forkhead box A2, FOXA2）所占据。HNF1敲低可升高FOXA2的表达丰度，并降低CDX2的蛋白水平。此外，HNF1缺失会抑制肠上皮细胞系的葡萄糖摄取能力。结论：本研究数据表明，HNF1在调控肠上皮细胞功能（包括葡萄糖吸收）中发挥关键作用；HNF1可与其他组织特异性转录因子相互作用，以调控该类细胞的分化特性。整体实验设计：为明确HNF1在肠上皮细胞基因表达调控中的作用，本研究在Caco2（结直肠腺癌细胞）中针对HNF1开展了ChIP-seq实验。