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

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 2

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DNase hypersensitive site mapping and deletion analysis of the mouse Pkh...
DNase hypersensitive site mapping and deletion analysis of the mouse Pkhd1 promoter. (A) Structure of the 5′ end of the Pkhd1 gene. Boxes indicate exons. Bent arrow indicates the transcription initiation site at +1. Bar indicates the 3′ probe used for indirect end labeling. Vertical arrows indicate hypersensitive sites. (B) Southern blot of genomic DNA from mIMCD-3 cells (right) and 10T1/2 cells (left) after digestion with graded concentrations of DNase I. Open arrow indicates the parental 8.1-kb EcoRI fragment. Closed arrows indicate sub-bands corresponding to hypersensitive sites located at the positions indicated on the right. (C) Northern blot showing endogenous expression of Pkhd1 (upper panel) and HNF-1β (middle panel) in mIMCD-3 cells (lane 2) and absence of expression in 10T1/2 cells (lane 1). Lower panel shows expression of GAPDH as a loading control. (D) Deletion analysis of the Pkhd1 promoter. Left panel shows plasmids containing fragments of the Pkhd1 promoter linked to a promoterless luciferase (Luc) reporter gene. Bent arrow indicates the transcription initiation site at +1, gray boxes indicate exons, and black boxes indicate the consensus HNF-1 site. Right panel shows luciferase activity in transfected mIMCD-3 cells (white bars) and 10T1/2 cells (gray bars). Data are presented as mean ± SE of six to nine independent transfections. *P < 0.05 compared with empty pGL3-Basic.