Browsing by Subject "Hepatocyte Nuclear Factor 1-beta"
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Item Sumo and Ubiquitin Ligases Regulate Hepatocyte Nuclear Factor - 1 Beta Transcriptional Activity(2007-05-27) McNally, Brian Timothy; Igarashi, PeterHepatocyte nuclear factor-1beta (HNF-1β ) is a homeodomain-containing transcription factor that regulates tissue-specific gene expression in the kidney, liver, and other organs. Humans with heterozygous mutations of HNF-1β develop maturity-onset diabetes of the young type 5 (MODY5) associated with cystic abnormalities of the kidney. A yeast two-hybrid screen was performed to identify proteins that interact with HNF-1β The SUMO E2 ligase, Ubc9, SUMO E3 ligases, PIAS1 and PIASgamma , and the ubiquitin E3 ligase, Arkadia, were isolated as putative interacting proteins. SUMOylation of transcription factors affects their subcellular localization, intracellular transport, stability, and transcriptional activity. Ubiquitination of transcription factors has been shown to alter their stability and transcriptional activity. Immunostaining of the adult mouse kidney showed that HNF-1β co-localized with Ubc9, PIAS1, and PIASgamma in the nuclei of renal tubular epithelial cells. Immunostaining also showed that Arkadia localized to the nuclei of renal tubules. Overexpression of Ubc9, PIAS1, or PIASgamma inhibited the activity of the Pkhd1 promoter, a known HNF-1β target. Expression of catalytically inactive Ubc9 or PIASgamma mutants also inhibited Pkhd1 promoter activity, suggesting that repression of HNF-1β transcriptional activity is SUMOylation-independent. In contrast to the repressive effect of SUMO ligases, overexpression of Arkadia stimulated Pkhd1 promoter activity in an HNF-1β;-dependent fashion. Immunoprecipitation of epitope-tagged SUMO or ubiquitin resulted in co-precipitation of HNF-1β , indicating that HNF-1β is both SUMOylated and ubiquitinated in vivo. In summary, the E2 and E3 SUMO ligases Ubc9, PIAS1, and PIASgamma repressed HNF-1β transcriptional activity via SUMOylation-dependent and -independent mechanisms. The E3 ubiquitin ligase Arkadia stimulated HNF-1β transcriptional activity.Together, these results demonstrated that HNF-1β is covalently modified by SUMO and ubiquitin, and these modifications had opposing effects on HNF-1β transcriptional activity. In addition, insights into the regulation of HNF-1β and ubsequent effects on target gene expression may lead to a greater understanding of the mechanisms that lead to renal cystogenesis.Item Understanding HNF-1β through Identification of Interacting Proteins and Target Genes in the Kidney(2010-11-02) Choi, Yun-Hee; Igarashi, PeterHepatocyte nuclear factor-1Beta (HNF-1Beta) is a POU/homeodomain-containing transcription factor that regulates tissue-specific gene expression in the liver, kidney, pancreas, and other epithelial organs. During kidney development, HNF-1Beta is expressed in renal collecting ducts and all segments of the nephron. Mutation of HNF-1Beta causes maturity-onset diabetes of the young type 5 (MODY5) and kidney developmental anomalies including renal agenesis, hypoplasia, and cysts. Here, I studied interacting proteins and target genes to understand the function of HNF-1Beta in the kidney. Yeast two-hybrid screening was performed to identify binding partners of HNF-1Beta in the kidney. Zyxin and LPP were isolated as putative interacting proteins. The LIM-containing proteins, Zyxin and LPP, are focal adhesion proteins that shuttle between the cytoplasm and nucleus and play a role in the architectural organization of cells. Both Zyxin and LPP interact with HNF-1Beta and stimulate the transcriptional activity of HNF-1Beta in mIMCD3 renal epithelial cells. Epidermal growth factor (EGF), which plays a role in the progression of polycystic kidney disease, induces translocation of zyxin into the nucleus. These studies identify a novel pathway by which signals may be transmitted from the cell surface to regulate the activity of a nuclear transcription factor that is essential for epithelial differentiation in the kidney. Chromatin immunoprecipitaion and DNA chip analysis (ChIP-chip) were performed to identify direct target genes of HNF-1Beta in the kidney. Phosphodiesterase 4C (PDE4C) was identified as an HNF-1Beta target gene. PDE4C belongs to the phosphodiesterase superfamily of enzymes that control the intracellular concentration of cyclic adenosine monophosphate (cAMP) by catalyzing its hydrolysis. cAMP may play a role in cystogenesis by stimulating fluid secretion and cell proliferation. PDE4C is transcriptionally activated by HNF-1Beta and regulates cAMP levels in mIMCD3 renal epithelial cells. Antibody staining showed that PDE4C is localized in the primary cilium and there interacts with a protein complex containing AKAP150, adenylyl cyclase 5/6 (AC5/6), protein kinase A (PKA), and polycystin-2 (PKD2). These results identify a cAMP-regulating protein complex that is localized in the primary cilium and is disrupted in PKD.