Transcription Factor Hepatocyte Nuclear Factor–1Β Regulates

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Transcription Factor Hepatocyte Nuclear Factor–1b Regulates Renal Cholesterol Metabolism

† ‡ Karam Aboudehen,* Min Soo Kim, Matthew Mitsche,§ Kristina Garland,§ | ‡ Norma Anderson,§ Lama Noureddine,* Marco Pontoglio, Vishal Patel,* Yang Xie, † Russell DeBose-Boyd,§¶ and Peter Igarashi* **

Departments of *Internal Medicine, ‡Clinical Sciences, §Molecular Genetics, and **Pediatrics and ¶Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas; †Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota; and |Department of Development, Reproduction and Cancer, National Institute of Health and Medical Research (INSERM) U1016, The National Center for Scientiﬁc Research (CNRS) Joint Research Unit (UMR) 8104, University of Paris Descartes, Institut Cochin, Paris, France

ABSTRACT HNF-1b is a tissue–specific transcription factor that is expressed in the kidney and other epithelial organs. Humans with mutations in HNF-1b develop kidney cysts, and HNF-1b regulates the transcription of several cystic disease genes. However, the complete spectrum of HNF-1b–regulated genes and pathways is not known. Here, using chromatin immunoprecipitation/next generation sequencing and gene expression profiling, we identified 1545 protein-coding genes that are directly regulated by HNF-1b in murine kidney epithelial cells. Pathway analysis predicted that HNF-1b regulates cholesterol metabolism. Expression of dominant negative mutant HNF-1b or kidney-specificinactivationofHNF-1b decreased the expression of genes that are essential for cholesterol synthesis, including sterol regulatory element binding factor 2 (Srebf2) and 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr). HNF-1b mutant cells also expressed lower levels of cholesterol biosynthetic intermediates and had a lower rate of cholesterol synthesis than control cells. Addi- tionally, depletion of cholesterol in the culture medium mitigated the inhibitory effects of mutant HNF-1b on the proteins encoded by Srebf2 and Hmgcr, and HNF-1b directly controlled the renal epithelial expression of proprotein convertase subtilisin–like kexin type 9, a key regulator of cholesterol uptake. These findings reveal a novel role of HNF-1b in a transcriptional network that regulates intrarenal cholesterol metabolism.

J Am Soc Nephrol 27: 2408–2421, 2016. doi: 10.1681/ASN.2015060607

Hepatocyte nuclear factor-1b (HNF-1b) is a tissue– transcriptional activation involves the recruitment of speciﬁc transcription factor that is expressed in epithe- coactivators that include P/CAF, CBP, p300, lial cells in the liver, kidney, genital tract, pancreas, and zyxin.2,5 b lung, and intestine.1 In the mammalian kidney, HNF-1 is essential for the proper embryonic 6–8 HNF-1b is expressed in tubular epithelial cells in all development of the kidney. In the developing segments of the nephrons and renal collecting ducts. Received June 1, 2015. Accepted November 11, 2015. HNF-1b contains an N–terminal dimerization Published online ahead of print. Publication date available at domain, a Pit-1/Oct-1/Unc-86 homeodomain that www.jasn.org. mediates binding to the consensus sequence Present address: Dr. Lama Noureddine, Department of Internal (59-RGTTAATNATTAACM-39), and a C–terminal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa. transactivation domain.2 HNF-1b has been shown Correspondence: Dr. Peter Igarashi, Department of Medicine, to function as either a transcriptional activator or a 420 Delaware Street SE, MMC 194, Minneapolis, MN 55455. transcriptional repressor depending on the target Email: [email protected] – gene and cellular context.2 4 One mechanism for Copyright © 2016 by the American Society of Nephrology

2408 ISSN : 1046-6673/2708-2408 JAmSocNephrol27: 2408–2421, 2016 www.jasn.org BASIC RESEARCH mouse kidney, HNF-1b is expressed in nephron precursors IgG.16 Quality control of the ChIP-seq experiments is shown and the branching ureteric bud that gives rise to the renal in Supplemental Table 1. We found a total of 10,250 peaks collecting system. Loss-of-function mutations in Hnf-1b representing significantly enriched HNF-1b binding sites cause renal agenesis, in part, because of reduced expression (FDR,0.01). We then determined the spatial distribution of of Wnt9b, a ureteric bud–derived factor that is required for the HNF-1b binding peaks relative to annotated genes in the the induction of new nephrons.7 Expression of dominant mouse genome. The majority of HNF-1b binding sites were negative mutant HNF-1b disrupts renal tubulogenesis be- located close to or within genes (48% gene bodies, 6% gene cause of deregulated expression of the target gene Socs3.4 promoters, 7% upstream regions, and 4% downstream re- HNF-1b also plays a role in nephron patterning through gions) (Figure 1A). The remainder of the peaks (35%) mapped regulation of Notch signaling.9 to intergenic domains. Humans with heterozygous mutations in HNF-1b de- Next, we mapped the HNF-1b binding sites to known velop congenital kidney anomalies, including renal agenesis, mRNA and miRNA transcripts. Binding sites were linked hypoplasia/dysplasia, multicystic renal dysplasia, and glomer- to a gene if they were located within 50 kb upstream of the ulocystic kidney disease.10,11 A common feature is the forma- transcription start site or within the body of the gene. Binding tion of kidney cysts derived from the renal tubules. This cystic sites were linked to an miRNA if there was no intervening phenotype is recapitulated in the mouse by transgenic expres- gene between the binding site and the miRNA. On the basis sion of dominant negative mutant HNF-1b or kidney-specific of these criteria, in total, 4725 mRNAs and 85 miRNAs were inactivation of Hnf-1b.12,13 HNF-1b regulates the expression mapped to the HNF-1b binding peaks (Figure 1B). We per- of genes encoding ciliary proteins that have been implicated in formed quantitative ChIP to validate the ChIP-seq results cyst formation, including PKD2 and PKHD1.12,13 Moreover, for a representative sample of the target genes (Supplemental we have recently reported that HNF-1b regulates the activity Figure 1A). of the Pkhd1 promoter in the kidney in vivo.14 Active enhancers can be distinguished by epigenetic In the adult kidney, HNF-1b is expressed in renal tubular marks, such as histone H3 lysine 4 monomethylation and epithelial cells composing the nephron and collecting ducts, lysine 27 acetylation,17,18 as well as binding of RNA poly- where it regulates the expression of tissue-specific genes, in- merase 2.19 We determined whether the HNF-1b binding cluding Ksp-cadherin, collectrin, and solute transporters.1,15 sites that were identified by ChIP-seq overlapped with active Several physiologically relevant gene targets have been identi- enhancer marks in the kidney identified from the mouse fied in the kidney, primarily through identification of the con- ENCODE Project.20 Using this approach, in total, 6501 en- sensus recognition sequence in candidate gene promoters hancers were identified in the mouse kidney, of which 680 (e.g., NKCC2, FXYD2, OAT3/4,andURAT1). However, the were occupied by HNF-1b (Figure 1C). Supplemental Figure complete spectrum of genes and networks that are directly 1C depicts an example of an active enhancer located between regulated by HNF-1b is still not known. Psat1 and Cep78 genes showing colocalization with HNF-1b Here, we used chromatin immunoprecipitation (ChIP) binding. followed by next generation sequencing (ChIP sequencing To determine the consensus HNF-1b binding sequence in [ChIP-seq]) combined with gene expression profiling to our ChIP-seq dataset, we extracted the sequence elements and identify genes that are directly regulated by HNF-1b in renal performed motif analysis. When we examined binding sites in epithelial cells. These studies unexpectedly revealed that HNF- gene bodies and intergenic domains, only the half–site con- 1b directly regulates the expression of multiple genes that sensus motif for HNF-1b was over-represented. However, are required for cholesterol synthesis. We also found evidence when we examined peaks extracted from promoter regions, for a role of HNF-1b in the regulation of cholesterol uptake by the full HNF-1b consensus motif was significantly enriched transcriptional activation of proprotein convertase subtilisin–like (Supplemental Figure 1D). kexin type 9 (Pcsk9). Identification of Genes That Are Directly Regulated by HNF-1b RESULTS To identify genes that are regulated by HNF-1b,weper- formed microarray analysis on HNF-1b mutant cells. RNA Identification of HNF-1b Binding Sites in Kidney Cells was extracted from renal epithelial cells (53A cells) To identify HNF-1b binding sites at the whole-genome level, expressing a dominant negative HNF-1b mutant lacking we performed ChIP-seq analysis on mIMCD3 renal epithelial the C–terminal transcriptional activation domain (HNF- cells. Chromatin was isolated from mIMCD3 cells, cross- 1bDC),2,4 and gene expression profiles were compared linked, and immunoprecipitated with an anti–HNF-1b anti- with uninduced cells. Expression of the HNF-1bDCmu- body. After reversing the crosslinks, the immunoprecipitated tant altered the expression of 4878 mRNAs, of which 2610 DNA was subjected to next generation sequencing. Binding were downregulated and 2268 were upregulated (Figure sites were identified by enrichment of genomic sequences 1D). We validated the microarray results by performing compared with input and immunoprecipitation with control real–time quantitative RT-PCR (qRT-PCR) analysis on a

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Figure 1. Genome-wide identiﬁcation of genes that are directly regulated by HNF-1b in kidney cells. (A) Genome-wide identiﬁcation of HNF-1b binding sites in chromatin from mIMCD3 renal epithelial cells. The pie chart shows the distribution of HNF-1b binding sites in the indicated genomic regions: promoter regions extending from the transcription start site (TSS) to 21 kb, upstream regions (21to 210 kb), gene bodies extending from the TSS to the transcription termination site (TTS), downstream regions (TTS to +5 kb), and

2410 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 2408–2421, 2016 www.jasn.org BASIC RESEARCH representative subset of mRNAs targets. We found a high Table 1. Deregulated pathways in HNF-1b mutant cells fi correlation (Pearson coef cient =0.89) between the two Pathways Count P Value FDR datasets (Supplemental Figure 2A). Metabolic pathways 125 ,0.001 7.59E-37 fi Next, we combined the gene expression pro les with the Pathways in cancer 57 ,0.001 1.38E-27 ChIP-seq data to identify direct HNF-1b targets. Genes that Wnt signaling 31 ,0.001 9.79E-17 were differentially expressed in HNF-1b mutant cells and Endocytosis 35 ,0.001 1.20E-15 contained a nearby HNF-1b binding site were considered to Exon guidance 27 ,0.001 4.98E-15 be direct targets. Genes that were differentially expressed but Focal adhesion 30 ,0.001 7.40E-13 did not contain a nearby HNF-1b binding site were consid- Basal cell carcinoma 16 ,0.001 1.76E-11 ered to be indirect targets. Figure 1E shows that 1545 protein- Adherens junction 18 ,0.001 2.19E-11 , coding genes were identified as direct targets of HNF-1b.Of Melanogenesis 20 0.001 4.22E-11 Phosphatidylinositol signaling 16 ,0.001 3.65E-09 these genes, 591 were upregulated and 954 were downregu- , b Glycerophospholipid metabolism 16 0.001 4.98E-09 lated in HNF-1 mutant cells. This unbiased method identi- , fi fi Hedgehog signaling 13 0.001 1.60E-08 ed multiple genes that have previously been identi ed as Leukocyte endothelial migration 18 ,0.001 4.01E-08 b direct HNF-1 targets, including Pkhd1, Glis2, Pde4c,and MAPK signaling 27 ,0.001 4.90E-08 Socs3, further establishing the validity of the approach (Sup- The analysis was performed on direct HNF-1b targets with P,0.05. Columns plemental Figure 2B).12 indicate KEGG pathway, gene count, P value, and FDR for the enrichment. We also analyzed HNF-1b binding to active enhancers and FDR, false discovery rate; MAPK, mitogen-activated protein kinase, KEGG, Kyoto Encyclopedia of Genes and Genomes. correlated binding with changes in gene expression (Figure 1F). Active enhancers bound by HNF-1b were distributed be- to be indirect targets. Supplemental Figure 3 shows the loca- tween intragenic (n=360) and intergenic (n=320) domains. In tions of HNF-1b binding peaks within or near target genes. addition, we found 341 unique genes that mapped to intra- The enrichment of HNF-1b binding at the respective site for genic enhancers, of which 138 (40%) were differentially ex- each target gene was confirmed by ChIP-quantitative PCR pressed in HNF-1b mutant cells. In total, 558 unique genes (qPCR) both in mIMCD3 cells and in vivo in the kidney mapped 59 or 39 to the intergenic enhancers, of which 157 (Figure 2, A and B). We also performed ChIP-qPCR analysis (28%) were differentially expressed. These data indicate that of the identified target genes in uninduced 53A cells and b HNF-1 binds to active enhancers and regulates a subset of found similar enrichment of HNF-1b binding (Supplemen- nearby genes. tal Figure 1B). The changes in the expression of direct and indirect target genes were confirmed by qRT-PCR in cells HNF-1b Directly and Indirectly Regulates Genes expressing the HNF-1bDCmutant(Figure2C).qRT-PCR Involved in Cholesterol Synthesis expression analysis was also performed on kidneys from fl fl To gain insight into the biologic functions of the genes that are Ksp-Cre;Hnf-1b ox/ ox mice, in which HNF-1b is specifically b directly regulated by HNF-1 , we performed KEGG pathway inactivated in renal tubules.13 Kidney-specific inactivation 21 analysis using WebGestalt software. Table 1 lists the highest of HNF-1b inhibited the expression of key genes involved in scoring pathways. Genes that function in metabolic pathways cholesterol synthesis, including Hmgcr and Srebf2 (Figure were over-represented, and within this category, cholesterol 2D). To determine if the reduction in mRNA levels resulted biosynthesis was the top scoring pathway. Seven genes (Cyp51, in lower protein abundance, we performed Western blot as- Dhcr24, 3-hydroxy-3-methylglutaryl-CoA reductase [Hmgcr], bD fi Lbr, Msmo1, Sqle,andsterol regulatory element binding factor says. Expression of the HNF-1 Cmutantsigni cantly 2 [Srebf2]) representing 26% of the genes in the cholesterol reduced the amount of active sterol regulatory element bind- biosynthesis pathway were identified as direct HNF-1b tar- ing protein-2 (SREBP-2) in the nucleus and HMGCR in gets. Seven additional genes in the cholesterol biosynthesis membranes (Figure 2E, FBS). These findings indicate that pathway (Fdft1, Hmgcs1, Idi1, Lss, Mvd, Mvk,andNsdhl) HNF-1b directly and indirectly regulates the renal expres- were downregulated in HNF-1b mutant cells but did not sion of genes involved in cholesterol synthesis both in vitro contain a nearby binding site and therefore, were considered and in vivo. intergenic regions (peaks outside of the classified regions). (B) Number of protein-coding genes and miRNAs that are mapped to HNF-1b binding sites. (C) Total number of active enhancers in the kidney identified in the mouse ENCODE Project (upper bar) and number of active enhancers corresponding to HNF-1b binding sites (lower bar). (D) Microarray analysis of mRNA expression in kidney cells expressing HNF- 1bDC. Heat map depicting genes that are differentially expressed in response to induction of the HNF-1bDC mutant (FDR,0.05). Data shown are from three independent experiments. The numbers of upregulated and downregulated genes are indicated in the histogram. (E) Venn diagrams combining the ChIP-seq data and microarray data to identify direct mRNA targets that are differentially expressed in response to induction of the HNF-1bDC mutant. (F) Mapping of enhancers to intergenic and intragenic domains. Venn diagrams show the number of genes located in close proximity to enhancers occupied by HNF-1b and differentially expressed in response to induction of the HNF-1bDCmutant.

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Figure 2. HNF-1b regulates the expression of genes involved in cholesterol synthesis. ChIP showing occupancy of the indicated genes by endogenous HNF-1b in chromatin from (A) mIMCD3 cells and (B) 28-day-old mouse kidney. Enrichment of HNF-1b binding was calculated using the percentage input method and compared with control IgG. Error bars represent SEM (n=3). *P,0.05. (C) qRT-PCR analysis showing altered expression of genes involved in cholesterol synthesis in cells expressing the HNF-1bDC mutant (gray bars) compared with wild-type cells (black bars). Direct targets indicate genes that are located near HNF-1b binding sites. Data shown are means6SEMs of three independent experiments. All pairwise comparisons were signiﬁcantly different (P,0.05). (D) qRT-PCR analysis showing altered expression of genes involved in cholesterol synthesis in kidneys from 28-day-old HNF-1b mutant mice (Ksp/Cre;Hnf-1bF/F; gray bars) compared with

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HNF-1b Regulates Cholesterol Synthesis To determine if the observed changes in gene expression and protein levels affected sterol metabolism, we measured the level of sterols andoxysterolsby HPLC-tandem mass spectrometry (MS).22 The total levels of cholesterol were not detectably different between HNF-1b mutant cells and controls. In contrast, the levels of major intermediates in sterol synthesis, including zymosterol, 7-dehydrocholesterol, and follicular fluid meiosis–activating sterol, were significantly decreased in HNF-1b mutant cells (P,0.05) (Figure3A).Conversely,threeoxysterols(7a- hydroxycholesterol, 5a-hydroxycholesterol, and 25-hydroxycholesterol) were increased (P,0.05) (Figure 3A). The decreased levels of cholesterol synthetic intermediates are consistent with the decreased expression of cholesterol biosynthetic genes in HNF-1b mutant cells (Supplemental Figure 4). Next, we directly measured the rate of cholesterol synthesis using deuterium 23 (D2O) labeling. D2O (5%) was added to the culture media, and incorporation into cholesterol was measured at different time points by HPLC/MS. Mutant cells exhibited a 55% reduction in the cholesterol synthesis rate 36 hours after induction Figure 3. Altered sterol levels and cholesterol synthesis rate in HNF-1b mutant cells. of HNF-1bDC (Figure 3B). Taken together, (A) Levels of cholesterol intermediates and oxysterols were measured by HPLC/MS in these findings show that HNF-1b plays a wild-type cells (black bars) and cells expressing the HNF-1bDC mutant (gray bars). , significant role in regulating cholesterol Cells were cultured in 10% FBS. Error bars indicate SEM. *P 0.05. (B) Cholesterol synthesis rate in wild-type cells (▪) and cells expressing the HNF-1bDCmutant(○). synthesis in mouse kidney cells. Mutation b Cells were labeled with D2O, and the incorporation of the isotope into cholesterol was of HNF-1 inhibits cholesterol synthesis, measured at the indicated time points. Data shown are the means6SDs of three which alters the levels of cholesterol inter- separate experiments. *P,0.05. (C) Serum cholesterol in 35-day-old HNF-1b mutant mediates and metabolites. mice (Pkhd1/Cre;Hnf-1bF/F; gray bars) compared with control littermates (black bars). To determine if the changes in renal Data shown are means6SEMs of six independent experiments. FFMAS, follicular fluid cholesterol synthesis affected circulating meiosis-activating sterol. cholesterol levels, we measured serum cholesterol in kidney–specificHNF-1b mutant mice. For these confirming that the kidney is not a major source of circulating fl fl experiments, we used Pkhd1/Cre;Hnf-1b ox/ ox mice with cholesterol. more slowly progressive cystic kidney disease, in which serum cholesterol could be measured before the onset of renal failure HNF-1b Regulates Pcsk9, a Regulator of Cholesterol and its confounding effects on lipid metabolism. As shown in Uptake Figure 3C, serum cholesterol levels were not significantly dif- Because the presence of lipoproteins and cholesterol in the cell ferent between HNF-1b mutant mice and control littermates, media can affect the expression of cholesterol biosynthetic control littermates (black bars). Data shown are means6SEMs of three independent experiments. All pairwise comparisons were significantly different (P,0.05). (E) Western blot analysis showing the expression of SREBP-2, SREBP-1, and HMGCR in wild-type cells (WT) and cells expressing the HNF-1bDC mutant (Mut). Cells were cultured in FBS or LPDS for 48 hours before analysis. Right panel shows densitometric analysis of the HMGCR and nuclear fractions of SREBP1 and SREBP2 normalized to the levels of actin. (F) Expression of Hmgcr, Srebf1,andSrebf2 in wild-type cells (black bars) and cells expressing the HNF-1bDC mutant (gray bars). Cells were cultured in either 10% FBS or 10% LPDS, induced for 48 hours, and then, subjected to qRT-PCR analysis. Error bars represent SEM. *P,0.05.

J Am Soc Nephrol 27: 2408–2421, 2016 HNF-1b Controls Renal Cholesterol Synthesis 2413 BASIC RESEARCH www.jasn.org genes,24 we performed qRT-PCR analysis on cells cultured in contained the HNF-1b half-site. HNF-1b binding and regu- lipoprotein-depleted serum (LPDS). Incubation of HNF-1b latory activity may be strongest at gene promoters. We also mutant cells in LPDS prevented the downregulation of found that approximately 10% of the total active enhancers in SREBP-2 and partially restored the expression of HMGCR at the kidney are occupied by HNF-1b.Moreoccupieden- both the mRNA and protein levels (Figure 2, E and F). SREBP- hancers are located in intragenic regions than intergenic re- 1 protein was slightly downregulated under both conditions. gions. Only a minority of genes (,50%) in close proximity to These findings suggested that HNF-1b regulates cholesterol the mapped enhancers showed changes in expression in HNF- synthesis and that this regulation may be influenced by cho- 1b mutant cells. It is possible that some of the identified en- lesterol uptake from the growth medium. hancers may regulate genes through long–range chromatin To further explore the mechanism, we re-examined the interactions and may not necessarily regulate the genes that ChIP-seq dataset and identified a prominent HNF-1b binding are in closest proximity to HNF-1b–bound enhancers. Alter- site in the first exon of Pcsk9 in chromatin from renal epithelial natively, the epigenetic marks of active chromatin in whole cells (Figure 4A). This site was located 419 bp upstream from kidney may differ from the histone modifications in the start codon in close proximity to the sterol regulatory mIMCD3 cells. element. PCSK9 inhibits the expression of the LDL receptor By combining the mRNA microarray results with the ChIP- on the cell surface and thereby, regulates cellular uptake of seq results, we identified 1545 protein-coding genes as direct LDL cholesterol.25 We verified that Pcsk9 is a direct target targets of HNF-1b. Some of the identified HNF-1b targets gene of HNF-1b by quantitative ChIP-qPCR of chromatin have been previously shown to play a role in cyst formation from mIMCD3 cells, uninduced 53A cells, and kidney tissue and tubulogenesis (Pkhd1, Glis2, Cys1,andSocs3).4,27–29 To (Figure 4B). To determine whether HNF-1b binding results in understand the biologic functions of the genes that are directly altered expression of Pcsk9, we measured Pcsk9 mRNA tran- regulated by HNF-1b, we performed KEGG pathway analysis. scripts. qRT-PCR analysis showed that induction of mutant Not surprisingly given the increased cell proliferation in kid- HNF-1b reduced the expression of Pcsk9 by approximately ney cysts, many HNF-1b target genes have known functions in 50% (P,0.05). The decrease in Pcsk9 mRNA levels persisted cancer. Specifically, genes in the Wnt and hedgehog signaling in mutant cells cultured in LPDS (Figure 4C). PCSK9 is syn- pathways were identified. Consistent with this result, previous thesized as an inactive proenzyme that undergoes proteolytic studies have shown that HNF-1b regulates the expression of cleavage, and the catalytically active fragment is secreted from Wnt9b, Glis2,andGlis3.7,30 An unexpected finding was that the cell.25,26 HNF-1b mutant cells exhibited a 50% reduction the largest number of protein-coding genes that are directly in the levels of active PCSK9 secreted in the media, which was regulated by HNF-1b (n=125) functions in metabolic path- similar under both FBS and LPDS conditions (Figure 4D). ways. In particular, genes that are involved in cholesterol syn- These results indicate that the regulation of Pcsk9 by HNF- thesis were over-represented in the dataset. 1b is independent of the presence of lipoproteins in the cul- Cholesterol synthesis has been extensively studied in the ture media. liver, the source of circulating LDL. Less is known about the To test whether HNF-1b functions as a transcriptional ac- regulation of synthesis in peripheral tissues, such as the kidney. tivator of Pcsk9, we performed reporter gene assays. A genomic Here, we show that multiple genes in the cholesterol bio- fragment containing the promoter and HNF-1b binding site synthetic pathway, including Lbr, Cyp51, Dhcr24, Hmgcr, was cloned into a luciferase reporter plasmid and transfected Msmo1,andSqle, contain binding sites for HNF-1b and are into mIMCD3 cells. Luciferase activity was increased 3.5-fold downregulated in HNF-1b mutant cells, which suggests that compared with the empty reporter plasmid (Figure 4E). Mu- HNF-1b directly regulates their expression. The HNF-1b gene tation of the HNF-1b binding sites reduced luciferase activity targets include Hmgcr, which encodes HMG-CoA reductase, by 50%. These results indicate that HNF-1b directly stimu- the enzyme that catalyzes the rate-limiting step in cholesterol lates Pcsk9 transcription in renal epithelial cells. synthesis. Seven additional genes involved in cholesterol synthesis (Fdft1, Hmgcs1, Idi1, Lss, Mvd, Mvk,andNsdhl) were indirectly regulated by HNF-1b. The expression of these genes DISCUSSION is regulated by SREBP-2. The gene encoding SREBP-2 (Srebf2) contains a prominent HNF-1b binding site in the first intron In this study, we used an unbiased approach combining ChIP- and a smaller peak in the promoter, and its expression is de- seq and gene expression profiling to identify genes and creased in HNF-1b mutant cells and kidneys. Collectively, networks that are directly regulated by the transcription factor these findings show that HNF-1b regulates cholesterol syn- HNF-1b in renal epithelial cells. ChIP-seq revealed a wide thesis directly through transactivation of genes, such as Hmgcr, range of HNF-1b binding sites throughout the mouse ge- as well as indirectly through transactivation of Srebf2 nome. Most HNF-1b binding sites are located within or (Figure 5). near protein-coding genes. In addition, the majority of sites Consistent with the alterations in gene transcription, the within gene promoters contained the full HNF-1b consensus levels of HMGCR protein were reduced in HNF-1b mutant motif, whereas sites outside promoters predominantly cells. This reduction resulted in a lower rate of cholesterol

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Figure 4. HNF-1b regulates Pcsk9 expression and promoter activity. (A, upper panel) Schematic diagram of the mouse Pcsk9 promoter region showing the HNF-1b binding site and sterol regulatory element (SRE) relative to the ATG start codon. The gray bar indicates the first exon, the dashed line indicates the 59 flanking sequence, and the line indicates the first intron. (A, lower panel) Genomic coordinates and size bar are shown at the top. HNF-1b binding peaks from ChIP-seq are shown in black. Blue boxes indicate Pcsk9 exons, and arrowheads indicate the direction of transcription. The bottom line indicates evolutionary sequence conservation. Data were vi- sualized using the UCSC Genome Browser.54 (B) Quantitative ChIP-qPCR showing binding of HNF-1b to the indicated region of Pcsk9 in mIMCD3 cells, uninduced 53A cells, and adult kidney. Data shown are means6SEMs of three independent experiments. *P,0.05. (C) Expression of Pcsk9 mRNA in wild-type cells (black bars) and cells expressing the HNF-1bDC mutant (gray bars) cultured in either

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metabolism in the liver.32 Inactivation of HNF-1a increases hepatic cholesterol synthesis accompanied by upregulation of Hmgcr and Fdft1, two key cholesterol synthetic genes. In contrast, we found that inhibition of HNF-1b in kidney cells leads to downregulation of Hmgcr and Fdft1 and reduces cholesterol synthesis. These findings indicate that HNF-1a and HNF-1b have opposing effects on cholesterol synthesis in the liver and kidney, respectively. The expression of Pcsk9 also seems to be Figure 5. Multilevel regulation of renal cholesterol metabolism by HNF-1b. Loss of a b differentially regulated by HNF-1 and HNF-1 inhibits the transcription of genes involved in cholesterol synthesis either b directly (middle line) or indirectly through inhibition of Srebf2 (top line). In addition, HNF-1 . In the liver, expression of Pcsk9 a b loss of HNF-1b inhibits the transcription of Pcsk9, which may increase cholesterol is regulated by HNF-1 , whereas HNF-1 uptake (bottom line). Not shown is that loss of HNF-1b also leads to increased levels of has no effect.33,34 In contrast, we found 25-hydroxycholesterol, which may further inhibit SREBP-2 activity. that HNF-1b is a strong activator of Pcsk9 in kidney cells. In the adult rat kidney, Pcsk9 is primarily expressed in the inner synthesis and reduced levels of major cholesterol biosynthetic medulla,35 which overlaps with the expression of HNF-1b. intermediates. These findings show that HNF-1b plays a phys- In contrast, HNF-1a is restricted to proximal tubules and iologically significant role in cholesterol synthesis in the kid- therefore, unlikely to be involved in expression of Pcsk9 in ney. In addition, the observed increase in oxysterols, such as the inner medulla. Although the function of PCSK9 in the 25-hydroxycholesterol, may contribute to the reduction in kidney is unclear, PCSK9 may regulate cholesterol uptake in cholesterol synthesis in HNF-1b mutant cells. By antagonizing renal epithelial cells similar to its function in hepatocytes. In SREBP-2 transcriptional activity and promoting its retention in the ER, 25-hydroxycholesterol exerts a negative feedback on addition, PCSK9 produced by transgenic overexpression in cholesterol synthesis.31 the kidney has also been shown to enter the bloodstream 36 ChIP-seq also identified Pcsk9 as a direct transcriptional and inhibit the uptake of LDL cholesterol in the liver. target of HNF-1b. PCSK9 is a serine protease that plays a cru- Because the liver is the major source of LDL cholesterol, it is b– cial role in regulating cholesterol influx by degradation of the unlikely that HNF-1 dependent renal cholesterol synthesis fl LDL receptor.25 Wild–type HNF-1b transactivates the Pcsk9 will in uence circulating cholesterol levels. Indeed, we found promoter, whereas mutation of HNF-1b inhibits the expres- that serum cholesterol levels were unaffected by deletion of b b sion of Pcsk9 and reduces the amount of secreted active HNF-1 in the kidney. Instead, HNF-1 seems to regulate PCSK9. Secreted PCSK9 binds to the EGF repeats of the intrarenal cholesterol metabolism. Previous studies have iden- fi LDL receptor and promotes its degradation, which reduces ti ed two other transcription factors, SREBP-2 and liver X cellular uptake of LDL cholesterol. Therefore, reduced pro- receptor, that are expressed in the kidney and have known duction of active PCSK9 in HNF-1b mutant cells would be roles in cholesterol metabolism.37 In response to low choles- predicted to promote cholesterol uptake (Figure 5), which terol levels, proteolytic processing of SREBP-2 releases an would mitigate the effects of inhibition of cholesterol synthesis N-terminal domain that binds sterol response elements and and may explain why we did not detect changes in total cho- activates cholesterol synthetic genes. Liver X receptor is an lesterol content. Consistent with a dependence on exogenous orphan hormone receptor that mediates cholesterol efflux cholesterol, incubation of HNF-1b mutant cells in lipoprotein- through transcriptional regulation of ABC transporters. Our deficient serum prevented the decrease in nuclear SREBP-2 and studies show that intrarenal cholesterol metabolism is also reduced the magnitude of the inhibition of HMGCR mRNA regulated by the transcription factor HNF-1b and that this and protein. regulation is physiologically relevant, because mutations of HNF-1b is structurally related to HNF-1a, a transcription HNF-1b alter sterol levels and the rate of cholesterol synthesis. factor that has previously been shown to play a role in lipid Moreover, we found that the gene encoding SREBP-2 is itself

FBS or LPDS. (D) ELISA of cleaved PCSK9 protein in wild-type cells (black bars) and cells expressing the HNF-1bDC mutant (gray bars) cultured in FBS or LPDS. (E) Luciferase assays of Pcsk9 promoter activity. A DNA fragment extending 1019 bp upstream to the ATG start codon and containing 419 bp of the first exon and 600 bp of the 59 flanking sequence was cloned into a luciferase reporter plasmid. mIMCD3 cells were transfected with equimolar amounts of wild-type (wt-pcsk9) or mutant (m-pcsk9) reporter plasmid and an HNF-1b or control (pcDNA5) expression plasmid. Luciferase activity was measured 48 hours after transfection. Luciferase activity was significantly reduced in m-pcsk9 transfected cells. Data shown are means6SEMs of three independent experiments. *P,0.05.

2416 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 2408–2421, 2016 www.jasn.org BASIC RESEARCH activated by HNF-1b, which indicates that HNF-1b acts up- the HNF-1b binding site was generated by site-directed mutagenesis stream in a transcriptional network that regulates cholesterol using the Quick-Change XL II Site-Directed Mutagenesis Kit (Stratagene) synthesis and uptake in kidney cells. using the following primers: 59-ccccatcggaagatcctctctgagttac- Cholesterol is an essential constituent of the plasma catgcaagggccccggtactaaaggatca-39 and 59-ctgatcctttagtaccggggcccttg- membrane, and its metabolic precursors play important roles catggtaactcagagaggatcttccgatgggg-39. The sequences of wild-type and in signaling pathways, such as hedgehog and Wnt signaling.38 mutant plasmids were verified by DNA sequencing. Reporter gene These pathways are deregulated in HNF-1b mutant cells assays were performed by plating cells in six–well plastic dishes at a (Table 1), although whether the perturbations are directly density of 1.53105 cells per dish. After 24 hours, when cells had related to the loss of HNF-1b activity or consequences of the reached 50% confluence, cells were transfected with equimolar abnormal sterol profile remains to be determined. Expression amounts of plasmid DNA using the Lipofectamine Plus Reagent (In- of HNF-1b is increased after ischemic kidney injury,39 a con- vitrogen). Cells were lysed 48 hours after transfection and assayed for dition that is also associated with stimulation of renal choles- luciferase activity as previously described.3 terol synthesis.40,41 One possibility is that the increase in renal cholesterol synthesis, which seems to have a cytoprotective Real-Time PCR function, is mediated by HNF-1b through activation of cho- The 53A cells were cultured for 24 hours followed by induction of lesterol biosynthetic genes. mutant HNF-1bDC protein for 48 hours. Total RNA from cells or In summary, we have identified a novel role of HNF-1b in postnatal day 28 adult wild–type or HNF-1b mutant mouse kidneys the regulation of multiple steps in renal cholesterol metabo- was extracted using the RNeasy Mini Kit (Qiagen, Germantown, MD) lism (Figure 5). The apparently paradoxical effect of wild–type according to the manufacturer’s protocol. cDNA was synthesized us- HNF-1b to both stimulate cholesterol synthesis and inhibit ing the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA), and real- cholesterol uptake is reminiscent of the role of SREBP-2, time qPCR was performed with the iTAG Universal SYBER Green which also activates transcription of cholesterol biosynthetic Supermix (Bio-Rad) using the CFX Connect Real-Time System (Bio- genes and Pcsk9. In the case of SREBP-2, the inhibition of Rad). Gene expression levels were normalized to 18S rRNA. Primers cholesterol uptake is thought to militate against cholesterol used for qRT-PCR are listed in Supplemental Table 2. overload in the liver, and it is possible that HNF-1b plays a similar role in the kidney. Additional studies will be needed to Gene Expression Profiling define the contribution of abnormal cholesterol metabolism Microarray experiments on total RNA were performed at the to the pathogenesis of cystic kidney disease and other HNF-1b Genomics and Microarray Core Facility at the University of Texas mutant phenotypes. Southwestern Medical Center. Briefly, total RNA from three different samples of induced and uninduced 53A cells was extracted as described above. RNA was reversed transcribed, and the cDNA was CONCISE METHODS fluorescently labeled and hybridized with Mouse Gene 1.0 ST Array (Affymetrix, Santa Clara, CA). Normalization of probe–level gene Cell Lines and Animals expression data across experiments was done using the RMA method Wild–typemIMCD3cellsanditsderivedcellline(53A)thatex- available within the Bioconductor R package.42 An FDR value cutoff presses HNF-1bDC3 were grown to confluence in growth medium of 0.05 was applied to identify genes significantly expressed between consisting of low-glucose DMEM (Invitrogen, Carlsbad, CA) supple- conditions. Annotation and summary of genes were done using the mented with 10% FBS (US Biotechnologies); 53A cells were treated Bioconductor R package.43 Microarray data were deposited in the with mifepristone to induce expression of the HNF-1bDC mutant as Gene Expression Omnibus (accession no. GSE72033). described previously,4 and vehicle-treated cells were used as a negative control. Kidney-specific inactivation of HNF-1b was achieved Quantitative ChIP-qPCR using Cre/LoxP recombination by crossing Ksp-Cre mice or Pkhd1- ChIP assays were performed using the ChIP-IT High Sensitivity Kit fl fl Cre mice with Hnf-1b ox/ ox mice as previously described.13,14 All (Active Motif) or the EZ ChIP Kit (EMD Millipore, Billerica, MA) animal procedures were performed in accordance with the guidelines according to the manufacturer’s protocol. Briefly, mIMCD3 cells, unin- of the Institutional Animal Care and Use Committees of the Univer- duced 53A cells, or mouse kidney tissue were crosslinked with 1% sity of Texas Southwestern Medical Center and the University of formaldehyde for 15 minutes at room temperature. Crosslinked tissues Minnesota Medical School. were homogenized into a single-cell suspension, and chromatin samples were extracted from the nuclei and sonicated. Immunoprecipitationwas Plasmids and Reporter Gene Assay performed with 5 mgrabbitanti–HNF-1b (sc-22840; Santa Cruz Bio- The HNF-1b binding region in the promoter of Pcsk9 was amplified technology, Santa Cruz, CA) and rabbit IgG (sc-2027; Santa Cruz Bio- from genomic DNA using the following primers: 59-cggggtaccctgcc- technology) antibody as a negative control. Immunoprecipitated DNA gaacagtgccagactggg-39 and 59-cccaagcttcggggcgaggagaggtgcgc-39.The or 1% of the input was diluted 1:20 in dH2O, and real-time PCR was 1019-bp amplified region was digested with KpnIandHindIII and performed in triplicate using gene-specific primers and SOSadvanced cloned into pGL3-Basic to produce the wt-Pcsk9-Luc reporter plas- Cyber Green Supermix (Bio-Rad). ChIP-qPCR data were normalized mid. A mutant Pcsk9-Luc reporter plasmid containing a mutation of using the percentage input method according to the following formula:

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2 2 b 10032((Ct(input) 6.644) Ct(HNF-1 )). Mock immunoprecipitation (IgG) and subsequently processed for HPLC/MS analysis as previously was used as a negative control; t test was used for statistical analysis. described.22 Primers used for ChIP-qPCR are listed in Supplemental Table 3. Serum Cholesterol Measurements ChIP Sequencing Mice were anesthetized according to approved protocols, and Immunoprecipitation of chromatin bound to HNF-1b in mIMCD3 blood from 35-day-old kidney–specificHNF-1b mutant mice fl fl cells was carried out as described above. Two control (anti-IgG) and (Pkhd1/Cre;Hnf-1b ox/ ox) and control littermates was collected by two experimental (anti–HNF-1b) samples from the immunopreci- cardiac puncture. Blood was separated by centrifugation, and 40 ml pitated DNA were sent for next generation sequencing. Sequencing serum was sent to the University of Texas Southwestern Mouse Met- was performed by the Genomics and Microarray Core Facility at the abolic Phenotype Core for determination of cholesterol concentra- University of Texas Southwestern Medical Center using an Illumina tion. Total cholesterol was measured using VITROS CHOL Slides platform. The quality of the raw sequencing data was assessed using and the VITROS Chemistry Products Calibrator Kit 2 (Vitros 250; FASTQC. Reads with .70% bases with quality lower than phred reference no. 166 9829; Vitros Chemistry Systems) as previously score of 20 were removed. Quality score–filtered reads were then described.52 aligned to the mouse reference genome NCBI37 (mmu9) using Burrows–Wheeler aligner.44 Identification of transcription factor Cell Fractionation and Immunoblot Analyses binding sites was performed using the peak calling algorithm Pooled cell pellets from triplicate 10-cm plates were washed in PBS; QuEST.45 A fold change cutoff of three was applied to identify sig- resuspended in 0.5 ml buffer containing 10 mM HEPES-KOH fi ni cant peaks between experimental and background samples. The (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 5 mM EDTA, 5 mM EGTA, identified peaks were annotated using HOMER.46 The ChIP-seq 5 mM dithiothreitol, 0.1 mM leupeptin, and 250 mM sucrose; and data have been deposited in the Gene Expression Omnibus supplemented with a protease inhibitor cocktail consisting of 1 mM (accession no. GSE71250). dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM Pefa- bloc, 10 mg/ml leupeptin, 5 mg/ml pepstatin A, 25 mg/ml ALLN, and Motif Analyses 10 mg/ml aprotinin. The cell suspension was homogenized by passing Sequence elements from the ChIP-Seq data were extracted, and through a 22-gauge needle 30 times and centrifuged at 2200 rpm for prediction of the consensus binding motifs was performed using 7 minutes at 4°C. The resulting postnuclear supernatants were fur- MEME-Chip.47 MEME software was used to predict de novo motifs ther subjected to centrifugation at 100,0003g for 30 minutes at 4°C. that were statistically over-represented within a 200-bp region cen- The pellet fraction obtained from this spin (designated membranes) tered on the genomic coordinates where HNF-1b was bound. Anal- was resuspended in 100 ml buffer containing 10 mM Tris-HCl ysis was carried out on peaks derived from three separate HNF-1b (pH 6.8), 100 mM NaCl, 1% (wt/vol) SDS, 1 mM EDTA, and binding regions (promoter, intergenic, and intragenic) for which 1 mM EGTA. The pellet obtained from the original 2200-rpm spin common and novel binding motifs were discovered. was resuspended in 0.3 ml buffer containing 20 mM HEPES-KOH

(pH 7.6), 2.5% (vol/vol) glycerol, 0.42 M NaCl, 1.5 mM MgCl2,1mM HPLC-MS and Cholesterol Synthesis Rate EDTA, 1 mM EGTA, and the above mentioned protease inhibitor Cholesterol synthesis rate was analyzed in wild-type and HNF-1bDC cocktail. The resuspended pellet was then rotated at 4°C for 1 hour –expressing cells. Briefly, cells were seeded in 6-mm dishes, cultured and centrifuged at 100,0003g for 30 minutes at 4°C. The supernatant in 10% FBS for 24 hours, and induced for 16 hours. D2O(5%)was (designated nuclear extract) was precipitated overnight in 1.5 ml then added to the media, and cells were harvested at different time acetone at 220°C. The precipitated material was pelleted by centri- points after D2O labeling (time =0, 1, 2, 4, 6, 8, 12, 24, and 32 hours). fugation at 17,0003g for 15 minutes at 4°C and resuspended in 80 ml After harvesting, the cells were saponified in 1% KOH at 60°C for 2 buffer containing 10 mM Tris-HCl (pH 6.8), 100 mM NaCl, 1% hours. After saponification, the lipids were isolated using a Bligh– (wt/vol) SDS, 1 mM EDTA, and 1 mM EGTA. Protein concentrations Dyer extraction48 and reconstituted in 300 ml methanol. To deter- of the membrane and nuclear extract fractions were measured using mine the synthesis rate, the isotopomer pattern of cholesterol was the BCA Protein Assay Kit (Pierce, Rockford, IL). Before SDS-PAGE, measured using HPLC/MS. A Shimadzu LC20A HPLC System (Shi- the membranes were mixed with an equal volume of buffer contain- madzu, Tokyo, Japan) was used with an Agilent C18 Poroshell Col- ing 62.5 mM Tris-HCl (pH 6.8), 15% (wt/vol) SDS, 8 M urea, 10% umn (Agilent Technologies, Santa Clara, CA) with a linear gradient (vol/vol) glycerol, and 100 mM dithiothreitol as well as 66.7 ml43 transitioning from 93% methanol:7% H2O to 100% methanol over SDS loading buffer. The nuclear extracts were mixed with 26.7 ml43 10 minutes.49 The cholesterol isotopic pattern was measured using an SDS loading buffer. All samples were incubated at 37°C for 30 minutes AB Sciex Qtrap 4000 (AB Sciex, Framingham, MA).49 The fraction of and subjected to 10% SDS-PAGE, after which the proteins were trans- the cholesterol newly synthesized at each point was determined using ferred to nitrocellulose membranes (GE Healthcare, Waukesha, WI). isotopic spectral analysis by evaluating the M=0 to M=3 isotopom- Immunoblot analysis was carried out with the following primary anti- ers.50,51 Cholesterol synthesis rate was estimated by fitting the rela- bodies: IgG-211, a rabbit polyclonal antibody against SREBP-1 (amino tionship between fraction of newly synthesized and time to a first– acids 32–250); IgG-22D5, a rabbit mAb against SREBP-2 (amino acids order kinetic models.23 For sterols and oxysterols measurements, 32–250); IgG-A9, a mouse mAb against the catalytic domain of hamster cells were seeded in six–well cell culture plates, induced for 48 hours, HMGCR (amino acids 450–887)53; and rabbit polyclonal antiactin

2418 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 2408–2421, 2016 www.jasn.org BASIC RESEARCH antibody (Sigma-Aldrich, St. Louis, MO). Primary antibodies were de- DISCLOSURES tected with horseradish peroxidase–conjugated donkey anti–mouse or None. anti–rabbit (Jackson ImmunoResearch Laboratories, West Grove, PA) using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Vernon Hills, IL) according to the manufacturer’s REFERENCES instructions. 1. Igarashi P, Shao X, McNally BT, Hiesberger T: Roles of HNF-1beta in kidney development and congenital cystic diseases. Kidney Int 68: ELISA 1944–1947, 2005 A LumiNunc Maxisorp White Assay Plate (NalgeNunc) was coated 2. Hiesberger T, Shao X, Gourley E, Reimann A, Pontoglio M, Igarashi P: – with rabbit anti mouse PCSK9 polyclonal antibody (IgG-551C) di- Role of the hepatocyte nuclear factor-1beta (HNF-1beta) C-terminal luted to 5 mg/ml in 100 ml 20 mM sodium phosphate (pH 7.5) and domain in Pkhd1 (ARPKD) gene transcription and renal cystogenesis. J 100 mM sodium chloride (buffer A) and incubated overnight at 4°C. Biol Chem 280: 10578–10586, 2005 The plate was then washed three times with 350 mlPBSwithTween20 3. Gong Y, Ma Z, Patel V, Fischer E, Hiesberger T, Pontoglio M, Igarashi P: fi (pH 7.4) and blocked with 150 ml 0.5% BSA in buffer A for 1 hour at HNF-1beta regulates transcription of the PKD modi er gene Kif12. J Am Soc Nephrol 20: 41–47, 2009 room temperature with shaking. All subsequent plate washes were 4. Ma Z, Gong Y, Patel V, Karner CM, Fischer E, Hiesberger T, Carroll TJ, conducted three times in PBS with Tween 20 on a BioTek ELv405 Pontoglio M, Igarashi P: Mutations of HNF-1beta inhibit epithelial Plate Washer (BioTek Instruments, Winooski, VT). All samples, stan- morphogenesis through dysregulation of SOCS-3. Proc Natl Acad Sci U dards, and antibodies were diluted in buffer A and 0.5% BSA. Media SA104: 20386–20391, 2007 were diluted 1:10, and 100 ml were added in duplicate to the assay 5. Choi YH, McNally BT, Igarashi P: Zyxin regulates migration of renal epithelial cells through activation of hepatocyte nuclear factor-1b. Am J plate. Purified mouse PCSK9 was serially diluted in the range of 0.39– Physiol Renal Physiol 305: F100–F110, 2013 50 ng/ml and added in duplicate as standards. The plate was incu- 6. Coffinier C, Gresh L, Fiette L, Tronche F, Schütz G, Babinet C, Pontoglio bated 2.5 hours at 37°C with shaking. The plate was again washed, M, Yaniv M, Barra J: Bile system morphogenesis defects and liver and 100 mlrabbitanti–mouse PCSK9 polyclonal antibody (IgG- dysfunction upon targeted deletion of HNF1beta. Development 129: – 552C; biotinylated with the EZ-Link Sulfo-NHS-Biotin Kit; Pierce) 1829 1838, 2002 7. Lokmane L, Heliot C, Garcia-Villalba P, Fabre M, Cereghini S: vHNF1 was added and incubated for 2 hours at room temperature with shak- functions in distinct regulatory circuits to control ureteric bud branching m ing. After an additional wash, 100 l Avidin-HRP (1:40,000; Pierce) and early nephrogenesis. Development 137: 347–357, 2010 was added and incubated for 1 hour at room temperature. After a 8. Haumaitre C, Barbacci E, Jenny M, Ott MO, Gradwohl G, Cereghini S: final wash, 100 ml SuperSignal ELISA Pico Substrate (Pierce) was Lack of TCF2/vHNF1 in mice leads to pancreas agenesis. 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PJ, Clark PM, Lindner T, Bell GI, Ryffel GU, Nicholls AJ, Hattersley AT: Abnormal nephron development associated with a frameshift mutation in the transcription factor hepatocyte nuclear factor-1 beta. Kidney Int 57: 898–907, 2000 ACKNOWLEDGMENTS 12. Hiesberger T, Bai Y, Shao X, McNally BT, Sinclair AM, Tian X, Somlo S, Igarashi P: Mutation of hepatocyte nuclear factor-1beta inhibits Pkhd1 gene expression and produces renal cysts in mice. JClinInvest113: Wethank Patricia Cobo-Stark and SachinHajarnisforexpert technical 814–825, 2004 assistance, Zhendong Ma for assistance with chromatin immuno- 13. Gresh L, Fischer E, Reimann A, Tanguy M, Garbay S, Shao X, Hiesberger precipitation sequencing, and Jeffery McDonald for assistance with T, Fiette L, Igarashi P, Yaniv M, Pontoglio M: A transcriptional network in liquid chromatography-tandem mass spectrometry. Wealso thank Jay polycystic kidney disease. EMBO J 23: 1657–1668, 2004 Horton and David Russell for helpful discussions. 14. Williams SS, Cobo-Stark P, Hajarnis S, Aboudehen K, Shao X, Richardson JA, Patel V, Igarashi P: Tissue-specific regulation of the This work was supported by National Institutes of Health (NIH) mouse Pkhd1 (ARPKD) gene promoter. Am J Physiol Renal Physiol 307: Grant R37DK042921 (to P.I.) and University of Texas Southwestern F356–F368, 2014 O’Brien Kidney Research Core Center NIH Grant P30DK079328. K.A. 15. Bai Y, Pontoglio M, Hiesberger T, Sinclair AM, Igarashi P: Regulation of and L.N. were supported by NIH Training Grant T32DK007257. M.P. kidney-specific Ksp-cadherin gene promoter by hepatocyte nuclear was supported by Fondation pour la recherche médicale (FRM), factor-1beta. Am J Physiol Renal Physiol 283: F839–F851, 2002 European Community’s Seventh Framework Programmme FP7/2009 16. 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