| Summary: | Hepatocyte nuclear factor 1A (HNF1A) is the master regulator of liver homeostasis and organogenesis and regulates many aspects of hepatocyte functions. It acts as a tumor suppressor in the liver, evidenced by the increased proliferation in HNF1A knockout (KO) hepatocytes. Hence, we postulated that any loss-of-function variation in the gene structure or composition (mutation) could trigger dysfunction, including disrupted transcriptional networks in liver cells. From the International Cancer Genome Consortium (ICGC) database of cancer genomes, we identified several <i>HNF1A</i> mutations located in the functional Pit-Oct-Unc (POU) domain. In our biochemical analysis, we found that the <i>HNF1A</i> POU-domain mutations Y122C, R229Q and V259F suppressed <i>HNF4A</i> promoter activity and disrupted the binding of HNF1A to its target <i>HNF4A</i> promoter without any effect on the nuclear localization. Our results suggest that the decreased transcriptional activity of HNF1A mutants is due to impaired DNA binding. Through structural simulation analysis, we found that a V259F mutation was likely to affect DNA interaction by inducing large conformational changes in the N-terminal region of HNF1A. The results suggest that POU-domain mutations of HNF1A downregulate <i>HNF4A</i> gene expression. Therefore, to mimic the HNF1A mutation phenotype in transcription networks, we performed siRNA-mediated knockdown (KD) of HNF4A. Through RNA-Seq data analysis for the HNF4A KD, we found 748 differentially expressed genes (DEGs), of which 311 genes were downregulated (e.g., <i>HNF1A</i>, <i>ApoB</i> and <i>SOAT2</i>) and 437 genes were upregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping revealed that the DEGs were involved in several signaling pathways (e.g., lipid and cholesterol metabolic pathways). Protein–protein network analysis suggested that the downregulated genes were related to lipid and cholesterol metabolism pathways, which are implicated in hepatocellular carcinoma (HCC) development. Our study demonstrates that mutations of HNF1A in the POU domain result in the downregulation of HNF1A target genes, including <i>HNF4A,</i> and this may trigger HCC development through the disruption of HNF4A–HNF1A transcriptional networks.
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