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Reversal of Xenopus Oct25 function by disruption of the POU domain structure.

ABSTRACT: Xenopus Oct25 is a POU family subclass V (POU-V) transcription factor with a distinct domain structure. To investigate the contribution of different domains to the function of Oct25, we have performed gain of function analyses. Deletions of the N- or C-terminal regions and of the Hox domain (except its nuclear localization signal) result in mutants being indistinguishable from the wild type protein in the suppression of genes promoting germ layer formation. Deletion of the complete POU domain generates a mutant that has no effect on embryogenesis. However, disruption of the alpha-helical structures in the POU domain, even by a single amino acid mutation, causes reversal of protein function. Overexpression of such mutants leads to dorsalization of embryos and formation of secondary axial structures. The underlying mechanism is an enhanced transcription of genes coding for antagonists of the ligands for ventralizing bone morphogenetic protein and Wnt pathways. Corresponding deletion mutants of Xenopus Oct60, Oct91, or mouse Oct4 also exhibit such a dominant-negative effect. Therefore, our results reveal that the integrity of the POU domain is crucial for the function of POU-V transcription factors in the regulation of genes that promote germ layer formation.

SUBMITTER: Cao Y

PROVIDER: S-EPMC2832990 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 HNF1A mutations located in the functional Pit-Oct-Unc (POU) domain. In our biochemical analysis, we found that the HNF1A POU-domain mutations Y122C, R229Q and V259F suppressed HNF4A promoter activity and disrupted the binding of HNF1A to its target HNF4A 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 HNF4A 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., HNF1A, ApoB and SOAT2) 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 HNF4A, and this may trigger HCC development through the disruption of HNF4A-HNF1A transcriptional networks.

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Reversal of Xenopus Oct25 function by disruption of the POU domain structure.

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