Sumo and Ubiquitin Ligases Regulate Hepatocyte Nuclear Factor - 1 Beta Transcriptional Activity




McNally, Brian Timothy

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Hepatocyte 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.

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