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Mutation of hepatocyte nuclear factor–1β inhibits Pkhd1 gene expression and produces renal cysts in mice
Thomas Hiesberger, … , Stefan Somlo, Peter Igarashi
Thomas Hiesberger, … , Stefan Somlo, Peter Igarashi
Published March 15, 2004
Citation Information: J Clin Invest. 2004;113(6):814-825. https://doi.org/10.1172/JCI20083.
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Article Nephrology

Mutation of hepatocyte nuclear factor–1β inhibits Pkhd1 gene expression and produces renal cysts in mice

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Abstract

Hepatocyte nuclear factor–1β (HNF-1β) is a Pit-1, Oct-1/2, UNC-86 (POU)/homeodomain-containing transcription factor that regulates tissue-specific gene expression in the liver, kidney, and other organs. Humans with autosomal dominant mutations of HNF-1β develop maturity-onset diabetes of the young type 5 (MODY5) and congenital cystic abnormalities of the kidney. Autosomal recessive polycystic kidney disease (ARPKD) is an inherited cystic disorder that produces renal failure in infants and children and is caused by mutations of PKHD1. The proximal promoter of the mouse Pkhd1 gene contains an evolutionarily conserved HNF-1–binding site that is located near a region of deoxyribonuclease hypersensitivity. HNF-1β and the structurally related HNF-1α bind specifically to the Pkhd1 promoter and stimulate gene transcription. Mutations of the HNF-1 site or expression of a dominant-negative HNF-1β mutant inhibit Pkhd1 promoter activity in transfected cells. Transgenic mice expressing a dominant-negative HNF-1β mutant under the control of a kidney-specific promoter develop renal cysts, similarly to humans with MODY5. Pkhd1 transcripts are absent in the cells lining the cysts but are present in morphologically normal surrounding tubules. These studies identify a link between two cystic disease genes, HNF1β (MODY5) and PKHD1 (ARPKD). HNF-1β directly regulates the transcription of Pkhd1, and inhibition of PKHD1 gene expression may contribute to the formation of renal cysts in humans with MODY5.

Authors

Thomas Hiesberger, Yun Bai, Xinli Shao, Brian T. McNally, Angus M. Sinclair, Xin Tian, Stefan Somlo, Peter Igarashi

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Figure 4

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Effects of DN-HNF1β on DNA binding and promoter activity. (A) EMSA perfo...
Effects of DN-HNF1β on DNA binding and promoter activity. (A) EMSA performed using a 44-bp DNA fragment containing the consensus HNF-1 site and nuclear extracts from HeLa cells transfected with expression plasmids encoding V5-tagged HNF-1β (lanes 1 and 2), E12 (lanes 3 and 4), or DN-HNF1β (lanes 5 and 6). Binding reactions were performed in the presence of anti-V5 Ab (lanes 2, 4, and 6) or no Ab (lanes 1, 3, and 5). Arrow indicates retarded band, and arrowhead indicates supershifted band. (B) HEK293 cells were transfected with plasmids encoding FLAG-tagged HNF-1β (lanes 1–3), V5-tagged HNF-1β (lanes 1, 3, 4, and 6), or FLAG-tagged DN-HNF1β (lanes 4–6). Cell lysates were immunoprecipitated (IP) with anti-V5 Ab (lanes 1, 2, 4, and 5) or anti-Myc Ab (lanes 3 and 6), and the immune complexes were subjected to immunoblot analysis with HRP-conjugated anti-FLAG Ab. (C) EMSA performed with the 44-bp DNA fragment and reticulocyte lysates programmed with the indicated amounts (nanograms) of plasmids encoding V5-tagged HNF-1β or FLAG-tagged DN-HNF1β. Binding reactions were performed in the presence of anti-V5 Ab (lanes 6–9), anti-FLAG Ab (lanes 10–13), or no Ab (lanes 1–5). Arrow indicates retarded band, and arrowhead indicates supershifted band. (D) Luciferase activity in mIMCD-3 cells cotransfected with a reporter plasmid containing the 444-bp Pkhd1 promoter and the indicated amounts (nanograms) of an expression plasmid encoding DN-HNF1β. Data presented are mean ± SE of nine independent transfections. *P < 0.05 compared with cells transfected with empty expression plasmid.
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