Mutation of hepatocyte nuclear factor–1β inhibits Pkhd1 gene expression and produces renal cysts in mice
Thomas Hiesberger, Yun Bai, Xinli Shao, Brian T. McNally, Angus M. Sinclair, Xin Tian, Stefan Somlo, Peter Igarashi
Thomas Hiesberger, Yun Bai, Xinli Shao, Brian T. McNally, Angus M. Sinclair, Xin Tian, Stefan Somlo, Peter Igarashi
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Article Nephrology

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

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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Mutational analysis of the Pkhd1 promoter and binding of HNF-1. (A) Luci...
Mutational analysis of the Pkhd1 promoter and binding of HNF-1. (A) Luciferase activity in mIMCD-3 cells transfected with reporter plasmids containing the WT 444-bp Pkhd1 promoter (WT), mutated promoter (M1–M3), or empty pGL3-Basic (Vector). Data are presented as mean ± SE of nine independent transfections. *P < 0.01 compared with WT promoter. (B) EMSA performed with a 44-bp DNA fragment containing the consensus HNF-1 site and reticulocyte lysates programmed with HNF-1α (lanes 2–7), HNF-1β (lanes 9–14), or unprogrammed lysates (lanes 1 and 8). Binding reactions were performed in the presence of anti–HNF-1α Ab (lane 3), anti–HNF-1β Ab (lane 10), or 100-fold excess unlabeled competitor (Comp.) DNA fragment (lanes 4–7, 11–14). (C) EMSA performed using the 44-bp DNA fragment and nuclear (Nuc) extracts from mIMCD-3 cells (lanes 2–8) or no protein (lane 1). Binding reactions were performed in the presence of anti–HNF-1β Ab (lane 3), irrelevant Ab (lanes 4), or 100-fold excess unlabeled DNA fragment (lanes 5–8). In B and C, arrows indicate retarded band, and arrowheads indicate supershifted band. †Complex that does not contain HNF-1β. (D) Luciferase activity in HeLa cells cotransfected with reporter plasmids containing the WT 444-bp Pkhd1 promoter (WT), mutated promoter (M1–M3), or empty pGL3-Basic (Vector) and expression plasmids encoding HNF-1α, HNF-1β, or empty pcDNA3. Data are presented as mean ±SE of six independent transfections. *P < 0.01 compared with cells cotransfected with empty expression plasmid.