Hepatocyte nuclear factor 1β suppresses canonical Wnt signaling through transcriptional repression of lymphoid enhancer–binding factor 1
2020; Elsevier BV; Volume: 295; Issue: 51 Linguagem: Inglês
10.1074/jbc.ra120.015592
ISSN1083-351X
AutoresSiu Chiu Chan, Sachin Hajarnis, Sophia M. Vrba, Vishal Patel, Peter Igarashi,
Tópico(s)Epigenetics and DNA Methylation
ResumoHepatocyte nuclear factor-1β (HNF-1β) is a tissue-specific transcription factor that is required for normal kidney development and renal epithelial differentiation. Mutations of HNF-1β produce congenital kidney abnormalities and inherited renal tubulopathies. Here, we show that ablation of HNF-1β in mIMCD3 renal epithelial cells results in activation of β-catenin and increased expression of lymphoid enhancer–binding factor 1 (LEF1), a downstream effector in the canonical Wnt signaling pathway. Increased expression and nuclear localization of LEF1 are also observed in cystic kidneys from Hnf1b mutant mice. Expression of dominant-negative mutant HNF-1β in mIMCD3 cells produces hyperresponsiveness to exogenous Wnt ligands, which is inhibited by siRNA-mediated knockdown of Lef1. WT HNF-1β binds to two evolutionarily conserved sites located 94 and 30 kb from the mouse Lef1 promoter. Ablation of HNF-1β decreases H3K27 trimethylation repressive marks and increases β-catenin occupancy at a site 4 kb upstream to Lef1. Mechanistically, WT HNF-1β recruits the polycomb-repressive complex 2 that catalyzes H3K27 trimethylation. Deletion of the β-catenin–binding domain of LEF1 in HNF-1β–deficient cells abolishes the increase in Lef1 transcription and decreases the expression of downstream Wnt target genes. The canonical Wnt target gene, Axin2, is also a direct transcriptional target of HNF-1β through binding to negative regulatory elements in the gene promoter. These findings demonstrate that HNF-1β regulates canonical Wnt target genes through long-range effects on histone methylation at Wnt enhancers and reveal a new mode of active transcriptional repression by HNF-1β. Hepatocyte nuclear factor-1β (HNF-1β) is a tissue-specific transcription factor that is required for normal kidney development and renal epithelial differentiation. Mutations of HNF-1β produce congenital kidney abnormalities and inherited renal tubulopathies. Here, we show that ablation of HNF-1β in mIMCD3 renal epithelial cells results in activation of β-catenin and increased expression of lymphoid enhancer–binding factor 1 (LEF1), a downstream effector in the canonical Wnt signaling pathway. Increased expression and nuclear localization of LEF1 are also observed in cystic kidneys from Hnf1b mutant mice. Expression of dominant-negative mutant HNF-1β in mIMCD3 cells produces hyperresponsiveness to exogenous Wnt ligands, which is inhibited by siRNA-mediated knockdown of Lef1. WT HNF-1β binds to two evolutionarily conserved sites located 94 and 30 kb from the mouse Lef1 promoter. Ablation of HNF-1β decreases H3K27 trimethylation repressive marks and increases β-catenin occupancy at a site 4 kb upstream to Lef1. Mechanistically, WT HNF-1β recruits the polycomb-repressive complex 2 that catalyzes H3K27 trimethylation. Deletion of the β-catenin–binding domain of LEF1 in HNF-1β–deficient cells abolishes the increase in Lef1 transcription and decreases the expression of downstream Wnt target genes. The canonical Wnt target gene, Axin2, is also a direct transcriptional target of HNF-1β through binding to negative regulatory elements in the gene promoter. These findings demonstrate that HNF-1β regulates canonical Wnt target genes through long-range effects on histone methylation at Wnt enhancers and reveal a new mode of active transcriptional repression by HNF-1β. Hepatocyte nuclear factor-1β (HNF-1β) is a tissue-specific, homeodomain-containing transcription factor that is expressed in epithelial organs, such as the kidney, liver, and pancreas (1Igarashi P. Shao X. McNally B.T. Hiesberger T. Roles of HNF-1β in kidney development and congenital cystic diseases.Kidney Int. 2005; 68 (16221171): 1944-194710.1111/j.1523-1755.2005.00625.xAbstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Human full-length HNF-1β consists of 557 amino acids comprising an N-terminal dimerization domain, DNA-binding POU/homeodomain, and C-terminal transactivation domain (2Chi Y.I. Frantz J.D. Oh B.C. Hansen L. Dhe-Paganon S. Shoelson S.E. Diabetes mutations delineate an atypical POU domain in HNF-1α.Mol. Cell. 2002; 10 (12453420): 1129-113710.1016/S1097-2765(02)00704-9Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). HNF-1β binds to genomic DNA containing the consensus sequence (5′-GTTAATNATTAAC-3′) as a homodimer or heterodimer with the paralogous protein HNF-1α and regulates gene transcription (3Mendel D.B. Hansen L.P. Graves M.K. Conley P.B. Crabtree G.R. HNF-1α and HNF-1β (vHNF-1) share dimerization and homeo domains, but not activation domains, and form heterodimers in vitro.Genes Dev. 1991; 5 (2044952): 1042-105610.1101/gad.5.6.1042Crossref PubMed Scopus (240) Google Scholar). HNF-1β is required for the embryonic development of the kidney where it plays important roles in branching morphogenesis, nephron patterning, and tubular epithelial differentiation (4Shao A. Chan S.C. Igarashi P. Role of transcription factor hepatocyte nuclear factor-1β in polycystic kidney disease.Cell Signal. 2020; 71 (32068086)10956810.1016/j.cellsig.2020.109568Crossref PubMed Scopus (4) Google Scholar). In the adult kidney, HNF-1β is highly expressed in all renal tubules, including proximal tubules, loops of Henle, distal tubules, and collecting ducts (5Ferrè S. Igarashi P. New insights into the role of HNF-1β in kidney (patho)physiology.Pediatr. Nephrol. 2018; 34 (29961928): 1325-133510.1007/s00467-018-3990-7Crossref PubMed Scopus (29) Google Scholar). HNF-1β activates kidney-specific gene transcription by binding to its cognate consensus sequence in the proximal promoters of genes such as Cdh16 and Pkhd1 and recruiting coactivators including Zyxin, CBP, and P/CAF (6Bai Y. Pontoglio M. Hiesberger T. Sinclair A.M. Igarashi P. Regulation of kidney-specific Ksp-cadherin gene promoter by hepatocyte nuclear factor-1β.Am. J. Physiol. Renal Physiol. 2002; 283 (12217876): F839-F85110.1152/ajprenal.00128.2002Crossref PubMed Scopus (43) Google Scholar, 7Hiesberger T. Bai Y. Shao X. McNally B.T. Sinclair A.M. Tian X. Somlo S. Igarashi P. Mutation of hepatocyte nuclear factor-1β inhibits Pkhd1 gene expression and produces renal cysts in mice.J. Clin. Invest. 2004; 113 (15067314): 814-82510.1172/JCI200420083Crossref PubMed Scopus (136) Google Scholar, 8Aboudehen K. Kim M.S. Mitsche M. Garland K. Anderson N. Noureddine L. Pontoglio M. Patel V. Xie Y. DeBose-Boyd R. Igarashi P. Transcription factor hepatocyte nuclear factor-1β regulates renal cholesterol metabolism.J. Am. Soc. Nephrol. 2016; 27 (26712526): 2408-242110.1681/ASN.2015060607Crossref PubMed Scopus (11) Google Scholar, 9Choi Y.H. McNally B.T. Igarashi P. Zyxin regulates migration of renal epithelial cells through activation of hepatocyte nuclear factor-1β.Am. J. Physiol. Renal Physiol. 2013; 305 (23657850): F100-F11010.1152/ajprenal.00582.2012Crossref PubMed Scopus (13) Google Scholar). Although HNF-1β has also been shown to inhibit transcription of genes such as SOCS3, the mechanism of transcriptional repression is poorly understood (10Ma Z. Gong Y. Patel V. Karner C.M. Fischer E. Hiesberger T. Carroll T.J. Pontoglio M. Igarashi P. Mutations of HNF-1β inhibit epithelial morphogenesis through dysregulation of SOCS-3.Proc. Natl. Acad. Sci. U.S.A. 2007; 104 (18077349): 20386-2039110.1073/pnas.0705957104Crossref PubMed Scopus (50) Google Scholar). Mutations of HNF1B encoding human HNF-1β were first identified as a cause of maturity-onset diabetes of the young type 5 (MODY5), an autosomal dominant disorder characterized by early-onset diabetes associated with congenital kidney abnormalities (11Horikawa Y. Iwasaki N. Hara M. Furuta H. Hinokio Y. Cockburn B.N. Lindner T. Yamagata K. Ogata M. Tomonaga O. Kuroki H. Kasahara T. Iwamoto Y. Bell G.I. Mutation in hepatocyte nuclear factor-1β gene (TCF2) associated with MODY.Nat. Genet. 1997; 17 (9398836): 384-38510.1038/ng1297-384Crossref PubMed Scopus (705) Google Scholar). Subsequently, germline mutations of HNF1B have been shown to cause congenital anomalies of the kidney and urinary tract, autosomal dominant tubulointerstitial kidney disease, and hypoplastic glomerulocystic disease (4Shao A. Chan S.C. Igarashi P. Role of transcription factor hepatocyte nuclear factor-1β in polycystic kidney disease.Cell Signal. 2020; 71 (32068086)10956810.1016/j.cellsig.2020.109568Crossref PubMed Scopus (4) Google Scholar). A common phenotype of HNF1B mutations is the formation of cystic kidneys and progressive impairment in kidney function. Disturbances in renal epithelial transport produce hypokalemia, hyperuricemia, and hypomagnesemia. Extrarenal manifestations of HNF1B mutations include pancreatic abnormalities, abnormal liver function tests, cognitive impairment, and hyperparathyroidism (12Verhave J.C. Bech A.P. Wetzels J.F. Nijenhuis T. Hepatocyte nuclear factor 1β-associated kidney disease: more than renal cysts and diabetes.J. Am. Soc. Nephrol. 2016; 27 (26319241): 345-35310.1681/ASN.2015050544Crossref PubMed Scopus (64) Google Scholar). Genomewide association studies have linked HNF1B to prostate cancer, chromophobe renal cell carcinoma, and clear cell ovarian cancer (13Yu D.D. Guo S.W. Jing Y.Y. Dong Y.L. Wei L.X. A review on hepatocyte nuclear factor-1β and tumor.Cell Biosci. 2015; 5 (26464794): 5810.1186/s13578-015-0049-3Crossref PubMed Scopus (29) Google Scholar). To understand the pathophysiology of HNF-1β–associated kidney diseases, we have generated Hnf1b mutant mice by kidney-specific deletion of Hnf1b or transgenic expression of dominant-negative HNF-1β mutants (7Hiesberger T. Bai Y. Shao X. McNally B.T. Sinclair A.M. Tian X. Somlo S. Igarashi P. Mutation of hepatocyte nuclear factor-1β inhibits Pkhd1 gene expression and produces renal cysts in mice.J. Clin. Invest. 2004; 113 (15067314): 814-82510.1172/JCI200420083Crossref PubMed Scopus (136) Google Scholar, 14Gresh L. Fischer E. Reimann A. Tanguy M. Garbay S. Shao X. Hiesberger T. Fiette L. Igarashi P. Yaniv M. Pontoglio M. A transcriptional network in polycystic kidney disease.EMBO J. 2004; 23 (15029248): 1657-166810.1038/sj.emboj.7600160Crossref PubMed Scopus (251) Google Scholar). Similar to humans with HNF1B mutations, mutant mice develop cystic kidneys and kidney failure. Molecular analysis of mutant kidneys has revealed that HNF-1β regulates a network of cystic disease genes, including PKD2, PKHD1, KIF12, and UMOD (7Hiesberger T. Bai Y. Shao X. McNally B.T. Sinclair A.M. Tian X. Somlo S. Igarashi P. Mutation of hepatocyte nuclear factor-1β inhibits Pkhd1 gene expression and produces renal cysts in mice.J. Clin. 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U.S.A. 2011; 108 (21670265): 10679-1068410.1073/pnas.1016214108Crossref PubMed Scopus (83) Google Scholar). Collecting duct-specific deletion of Hnf1b produces a more slowly progressive disease and has revealed roles of HNF-1β in urinary concentration and renal fibrosis (17Aboudehen K. Noureddine L. Cobo-Stark P. Avdulov S. Farahani S. Gearhart M.D. Bichet D.G. Pontoglio M. Patel V. Igarashi P. Hepatocyte nuclear factor-1β regulates urinary concentration and response to hypertonicity.J. Am. Soc. Nephrol. 2017; 28 (28507058): 2887-290010.1681/ASN.2016101095Crossref PubMed Scopus (16) Google Scholar). Transcriptomic analysis of Hnf1b mutant cells suggests that HNF-1β regulates a broad range of genetic networks involving cellular metabolism, cancer, and fibrosis (8Aboudehen K. Kim M.S. Mitsche M. Garland K. Anderson N. Noureddine L. Pontoglio M. Patel V. Xie Y. DeBose-Boyd R. Igarashi P. Transcription factor hepatocyte nuclear factor-1β regulates renal cholesterol metabolism.J. Am. Soc. Nephrol. 2016; 27 (26712526): 2408-242110.1681/ASN.2015060607Crossref PubMed Scopus (11) Google Scholar, 18Chan S.C. Zhang Y. Shao A. Avdulov S. Herrera J. Aboudehen K. Pontoglio M. Igarashi P. Mechanism of fibrosis in HNF1B-related autosomal dominant tubulointerstitial kidney disease.J. Am. Soc. Nephrol. 2018; 29 (30097458): 2493-250910.1681/ASN.2018040437Crossref PubMed Scopus (16) Google Scholar). Recently, new functions of HNF-1β in the regulation of noncoding RNAs, cholesterol metabolism, and epithelial-mesenchymal transition have been identified (17Aboudehen K. Noureddine L. Cobo-Stark P. Avdulov S. Farahani S. Gearhart M.D. Bichet D.G. Pontoglio M. Patel V. Igarashi P. Hepatocyte nuclear factor-1β regulates urinary concentration and response to hypertonicity.J. Am. Soc. Nephrol. 2017; 28 (28507058): 2887-290010.1681/ASN.2016101095Crossref PubMed Scopus (16) Google Scholar, 18Chan S.C. Zhang Y. Shao A. Avdulov S. Herrera J. Aboudehen K. Pontoglio M. Igarashi P. Mechanism of fibrosis in HNF1B-related autosomal dominant tubulointerstitial kidney disease.J. Am. Soc. Nephrol. 2018; 29 (30097458): 2493-250910.1681/ASN.2018040437Crossref PubMed Scopus (16) Google Scholar, 19Hajarnis S.S. Patel V. Aboudehen K. Attanasio M. Cobo-Stark P. Pontoglio M. Igarashi P. Transcription factor hepatocyte nuclear factor-1β (HNF-1β) regulates microRNA-200 expression through a long noncoding RNA.J. Biol. Chem. 2015; 290 (26292219): 24793-2480510.1074/jbc.M115.670646Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Wnt signaling is an essential signal transduction pathway that is required for normal embryonic development and tissue homeostasis (20Nusse R. Clevers H. Wnt/β-catenin signaling, disease, and emerging therapeutic modalities.Cell. 2017; 169 (28575679): 985-99910.1016/j.cell.2017.05.016Abstract Full Text Full Text PDF PubMed Scopus (1396) Google Scholar). Wnts are secreted glycolipoproteins that bind to cell surface receptors and signal through at least three different pathways (21MacDonald B.T. Tamai K. He X. Wnt/β-catenin signaling: components, mechanisms, and diseases.Dev. Cell. 2009; 17 (19619488): 9-2610.1016/j.devcel.2009.06.016Abstract Full Text Full Text PDF PubMed Scopus (3597) Google Scholar). The canonical (β-catenin–dependent) pathway consists of three main steps: 1) Wnt ligands activate the Frizzled (Fzd) and low-density lipoprotein–related protein complex, 2) sequestration of the Axin–APC–GSK3β destruction complex leads to accumulation of cytosolic β-catenin, and 3) β-catenin translocates to the nucleus where it interacts with TCF/LEF1 transcription factors bound to Wnt-responsive elements (WREs) and promotes Wnt enhanceosome formation to activate or repress Wnt target genes (22Gammons M. Bienz M. Multiprotein complexes governing Wnt signal transduction.Curr. Opin. Cell Biol. 2018; 51 (29153704): 42-4910.1016/j.ceb.2017.10.008Crossref PubMed Scopus (81) Google Scholar). Canonical Wnt signaling plays important roles in normal kidney development and is dysregulated in kidney diseases (23Wang Y. Zhou C.J. Liu Y. Wnt signaling in kidney development and disease.Prog. Mol. Biol. Transl. Sci. 2018; 153 (29389516): 181-207Crossref PubMed Scopus (44) Google Scholar). We have recently shown that ablation of HNF-1β in renal epithelial cells produces hyperresponsiveness to Wnt ligands and activation of canonical Wnt/β-catenin signaling (24Chan S.C. Zhang Y. Pontoglio M. Igarashi P. Hepatocyte nuclear factor-1β regulates Wnt signaling through genome-wide competition with β-catenin/lymphoid enhancer binding factor.Proc. Natl. Acad. Sci. U.S.A. 2019; 116 (31712448): 24133-2414210.1073/pnas.1909452116Crossref PubMed Scopus (4) Google Scholar). We identified one mechanism for Wnt pathway activation involving competition between HNF-1β and β-catenin/TCF/LEF1 for binding to a novel composite DNA element that is present in a subset of Wnt target genes. Loss of HNF-1β promotes β-catenin/TCF/LEF1 binding and leads to overexpression of Wnt target genes. Here, we identify a second mechanism for activation of the Wnt pathway. We show that HNF-1β functions as a transcriptional repressor of the Wnt effector LEF1 through long-range chromosomal effects on histone methylation and β-catenin binding. Ablation of HNF-1β in kidney epithelial cells leads to increased accumulation of nuclear LEF1, which contributes to Wnt hyperresponsiveness through a feed-forward loop mechanism. Previous studies from our laboratory have shown that ablation of Hnf1b in kidney epithelial cells and kidney-specific knockout of Hnf1b in transgenic mice lead to activation of the canonical Wnt pathway (24Chan S.C. Zhang Y. Pontoglio M. Igarashi P. Hepatocyte nuclear factor-1β regulates Wnt signaling through genome-wide competition with β-catenin/lymphoid enhancer binding factor.Proc. Natl. Acad. Sci. U.S.A. 2019; 116 (31712448): 24133-2414210.1073/pnas.1909452116Crossref PubMed Scopus (4) Google Scholar). To identify Wnt pathway genes that are directly regulated by HNF-1β, we compared our previous RNA-Seq and ChIP-Seq data sets (18Chan S.C. Zhang Y. Shao A. Avdulov S. Herrera J. Aboudehen K. Pontoglio M. Igarashi P. Mechanism of fibrosis in HNF1B-related autosomal dominant tubulointerstitial kidney disease.J. Am. Soc. Nephrol. 2018; 29 (30097458): 2493-250910.1681/ASN.2018040437Crossref PubMed Scopus (16) Google Scholar) and identified Lef1, a downstream effector in the canonical Wnt signaling pathway, as a potential HNF-1β target gene. To determine whether expression of Lef1 depends on HNF-1β, we performed quantitative RT-PCR analysis on mIMCD3 cells in which Hnf1b was deleted by CRISPR/Cas9 gene editing (18Chan S.C. Zhang Y. Shao A. Avdulov S. Herrera J. Aboudehen K. Pontoglio M. Igarashi P. Mechanism of fibrosis in HNF1B-related autosomal dominant tubulointerstitial kidney disease.J. Am. Soc. Nephrol. 2018; 29 (30097458): 2493-250910.1681/ASN.2018040437Crossref PubMed Scopus (16) Google Scholar). Under basal conditions, the expression of Lef1 was 4.7-fold higher in HNF-1β–deficient cells compared with WT mIMCD3 cells. Treatment with exogenous Wnt3a ligand for 240 min to stimulate canonical Wnt signaling produced a 3.9-fold increase in Lef1 transcripts in WT cells and a significantly greater 37-fold increase in HNF-1β–deficient cells (Fig. 1A). Analysis of our previous RNA-Seq data (GSE130164) showed that Lef1 was the most up-regulated member of the TCF/LEF1 family following treatment with Wnt3a (Fig. S1A). Stimulation of the canonical Wnt pathway leads to cytosolic accumulation of N-terminal dephosphorylated β-catenin, which translocates to the nucleus where it binds to TCF/LEF1 transcription factors and activates Wnt target genes (25Cadigan K.M. Waterman M.L. TCF/LEFs and Wnt signaling in the nucleus.Cold Spring Harb. Perspect. Biol. 2012; 4 (23024173)a00790610.1101/cshperspect.a007906Crossref PubMed Scopus (379) Google Scholar). We performed subcellular fractionation and immunoblot analysis to measure the abundance of LEF1 protein and activated (dephosphorylated) β-catenin in HNF-1β–deficient cells. Treatment with Wnt3a produced a greater accumulation of LEF1 protein in the nuclear fraction in HNF-1β–deficient cells compared with WT cells (Fig. 1B). Treatment with Wnt3a also resulted in greater accumulation of activated β-catenin in the cytosol and nucleus of HNF-1β–deficient cells. To validate these findings in vivo, we analyzed kidneys from Hnf1b mutant mice in which the expression of HNF-1β in renal tubular epithelial cells was ablated by Cre/loxP recombination. Previous studies have shown that kidney tubule–specific deletion of Hnf1b results in the postnatal formation of kidney cysts and leads to kidney failure (14Gresh L. Fischer E. Reimann A. Tanguy M. Garbay S. Shao X. Hiesberger T. Fiette L. Igarashi P. Yaniv M. Pontoglio M. A transcriptional network in polycystic kidney disease.EMBO J. 2004; 23 (15029248): 1657-166810.1038/sj.emboj.7600160Crossref PubMed Scopus (251) Google Scholar). To measure Lef1 expression, we performed qRT-PCR analysis on kidneys at postnatal day 28 (P28), an age at which cysts are present. The levels of Lef1 transcripts were increased 1.6-fold in Hnf1b mutant kidneys compared with WT kidneys (Fig. 1C). Immunohistochemical staining showed increased LEF1 protein in the nuclei of cyst-lining epithelial cells (Fig. 1D and Fig. S1B). Collectively, these results showed that ablation of HNF-1β in renal epithelial cells led to activation of canonical Wnt signaling associated with increased nuclear expression of LEF1. To confirm the above findings using a different experimental model, we measured Wnt pathway activity in cells expressing a dominant-negative mutant form of HNF-1β (DN–HNF-1β) that lacks the C-terminal activation domain but retains the dimerization and DNA-binding domain (10Ma Z. Gong Y. Patel V. Karner C.M. Fischer E. Hiesberger T. Carroll T.J. Pontoglio M. Igarashi P. Mutations of HNF-1β inhibit epithelial morphogenesis through dysregulation of SOCS-3.Proc. Natl. Acad. Sci. U.S.A. 2007; 104 (18077349): 20386-2039110.1073/pnas.0705957104Crossref PubMed Scopus (50) Google Scholar). This mutant is similar to a disease-causing HNF-1β mutant in humans (26Hiesberger T. Shao X. Gourley E. Reimann A. Pontoglio M. Igarashi P. Role of the hepatocyte nuclear factor-1β (HNF-1β) C-terminal domain in Pkhd1 (ARPKD) gene transcription and renal cystogenesis.J. Biol. Chem. 2005; 280 (15647252): 10578-1058610.1074/jbc.M414121200Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). We used a stable cell line derived from mIMCD3 cells (53A) in which expression of the DN–HNF-1β mutant can be induced by treatment with mifepristone. RNA profiling showed that the expression of Lef1 was 2.1-fold higher in 53A cells treated with mifepristone compared with uninduced cells (data not shown). To measure Wnt pathway activity, 53A cells were transfected with an 8× TOPFlash reporter plasmid containing eight copies of a TCF/LEF1-binding site linked to a luciferase reporter gene. Induction of the DN–HNF-1β mutant increased both basal and Wnt3a-stimulated luciferase activity compared with uninduced cells (Fig. 2A). Treatment with LiCl, which inhibits the phosphorylation of β-catenin by GSK3β, further increased luciferase activity. Mutation of the TCF/LEF1–binding sites in the 8× FOPFlash reporter plasmid largely abolished the stimulation of luciferase activity (Fig. 2B). The significantly higher luciferase activity in DN-HNF-1β–expressing cells treated with LiCl compared with uninduced cells suggested that the Wnt pathway was activated downstream to GSK3β, possibly at the level of LEF1. To test this possibility, we inhibited LEF1 expression in 53A cells using siRNA. Knockdown of LEF1 in cells expressing DN–HNF-1β reduced the magnitude of the increased luciferase activity following treatment with Wnt3a or LiCl (Fig. 2C). Collectively, these results demonstrated that the activation of the canonical Wnt pathway observed in HNF-1β mutant cells was mediated in part by increased expression of Lef1. We previously performed genomewide ChIP-Seq analysis to identify HNF-1β–binding sites in native chromatin from mIMCD3 renal epithelial cells (8Aboudehen K. Kim M.S. Mitsche M. Garland K. Anderson N. Noureddine L. Pontoglio M. Patel V. Xie Y. DeBose-Boyd R. Igarashi P. Transcription factor hepatocyte nuclear factor-1β regulates renal cholesterol metabolism.J. Am. Soc. Nephrol. 2016; 27 (26712526): 2408-242110.1681/ASN.2015060607Crossref PubMed Scopus (11) Google Scholar). Inspection of the HNF-1β ChIP-Seq data using the IGV program revealed two novel HNF-1β–binding sites at the Lef1 locus (see Fig. 4A). One site was located 94 kb upstream to the transcription start site (TSS), and a second site was located 30 kb downstream within the third intron. DNA sequence alignment showed that the HNF-1β–binding sites were evolutionarily conserved in vertebrates (Fig. S2, A and B). Binding peaks at these locations were not detected when ChIP-Seq was performed using control IgG. To validate these findings, we performed quantitative ChIP using an antibody against HNF-1β (Fig. 3, B–D). These studies confirmed that HNF-1β binds to the 94-kb upstream site and the 30-kb downstream site in chromatin from mIMCD3 cells. In contrast, no significant binding was detected to an irrelevant Lef1 site or when ChIP was performed using isotype control IgG. As an additional test of specificity, no significant binding was detected when ChIP was performed using an HNF-1β antibody and chromatin from HNF-1β–deficient mIMCD3 cells.Figure 3Lef1 is a novel HNF-1β–regulated Wnt target gene. A, ChIP-Seq analysis of WT mIMCD3 cells showing HNF-1β–binding sites located 94 kb upstream and 30 kb downstream from the transcription initiation site of Lef1 (red). ChIP-Seq with isotype IgG was used as a negative control (black). The data were visualized using IGV software. B–D, quantitative ChIP demonstrated binding of HNF-1β to the upstream enhancer and intron 3 enhancer in the Lef1 locus (open bars). No binding was detected to an irrelevant control DNA sequence (C). ChIP was performed on HNF-1β mutant cells as an additional negative control (filled bars). The data shown represent the means ± S.D. E–G, luciferase reporters containing the DNA sequences of the upstream enhancer, control region, and intron 3 enhancer were cotransfected into HeLa cells with expression plasmids encoding full-length HNF-1β (WT), DN–HNF-1β mutant (DN), or empty plasmid (Ctr). The data shown represent means ± S.D. chr, chromosome.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To test whether the HNF-1β genomic binding sites were functional, we cloned the corresponding DNA sequences into a plasmid containing a luciferase reporter gene driven by a minimal promoter. The reporter plasmids were cotransfected with expression plasmids encoding WT or mutated HNF-1β into HeLa cells, which do not express endogenous HNF-1β. Luciferase measurements showed that reporter plasmids containing the upstream 94-kb enhancer or downstream 30-kb enhancer were transactivated by full-length HNF-1β (Fig. 3, E–G). In contrast, no transactivation was observed using a reporter plasmid containing an irrelevant Lef1 sequence. Cotransfection with an expression plasmid encoding the DN–HNF-1β mutant also failed to stimulate luciferase activity. These results indicated that both distal HNF-1β–occupied sites were transcriptionally active. In addition to its function as a downstream Wnt effector, Lef1 itself is also a well-characterized canonical Wnt target gene (27Eastman Q. Grosschedl R. Regulation of LEF-1/TCF transcription factors by Wnt and other signals.Curr. Opin. Cell Biol. 1999; 11 (10209158): 233-24010.1016/S0955-0674(99)80031-3Crossref PubMed Scopus (450) Google Scholar). Previous studies have suggested that the 5′ UTR of the human LEF1 gene contains several WRE that are occupied by β-catenin/TCF/LEF1 and mediate transcriptional activation in response to Wnt signaling (28Filali M. Cheng N. Abbott D. Leontiev V. Engelhardt J.F. Wnt-3A/β-catenin signaling induces transcription from the LEF-1 promoter.J. Biol. Chem. 2002; 277 (12052822): 33398-3341010.1074/jbc.M107977200Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). We have recently performed ChIP-Seq to identify β-catenin–occupied sites in WT and HNF-1β–deficient renal epithelial cells treated with Wnt3a (24Chan S.C. Zhang Y. Pontoglio M. Igarashi P. Hepatocyte nuclear factor-1β regulates Wnt signaling through genome-wide competition with β-catenin/lymphoid enhancer binding factor.Proc. Natl. Acad. Sci. U.S.A. 2019; 116 (31712448): 24133-2414210.1073/pnas.1909452116Crossref PubMed Scopus (4) Google Scholar). Surprisingly, inspection of the ChIP-Seq data showed only a minor increase in β-catenin occupancy in the 5′ UTR of mouse Lef1 (Fig. 4A). Instead, we identified several novel β-catenin–binding peaks located ∼4-kb upstream to the Lef1 TSS. Three evolutionarily conserved consensus TCF/LEF1-binding motifs were identified within this region (Fig. S3). Occupancy of the 4-kb upstream sites by β-catenin was strongly induced by Wnt3a in HNF-1β–deficient cells. In contrast, minimal β-catenin occupancy was observed in WT cells. Together with the results shown in Fig. 3, these findings indicate that HNF-1β normally binds to the 94-kb upstream site and the 30-kb downstream site, where it inhibits β-catenin occupancy and represses Lef1 transcription. Ablation of HNF-1β induces β-catenin occupancy at the novel 4-kb upstream site and derepresses Lef1 transcription. To further elucidate the mechanism of transcriptional repression by HNF-1β, we compared histone methylation in WT and HNF-1β–deficient cells. Trimethylation of lysine 27 of histone H3 (H3K27me3) is a characteristic epigenetic mark of gene repression (29Cao R. Zhang Y. The functions of E(Z)/EZH2-mediated methylation of lysine 27 in histone H3.Curr. Opin. Genet. Dev. 2004; 14 (15196462): 155-16410.1016/j.gde.2004.02.001Crossref PubMed Scopus (681) Google Scholar). HNF-1β–deficient cells and WT cells were treated with Wnt3a or vehicle, and quantitative ChIP was performed using an antibody that recognizes H3K27me3. Treatment of WT cells with Wnt3a produced a modest 22.3% reduction in H3
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