Genome-Wide Mapping of DNA Accessibility and Binding Sites for CREB and C/Ebpb in Vasopressin- Sensitive Collecting Duct Cells

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Genome-Wide Mapping of DNA Accessibility and Binding Sites for CREB and C/EBPb in Vasopressin- Sensitive Collecting Duct Cells

Hyun Jun Jung,1 Viswanathan Raghuram,1 Jae Wook Lee ,2 and Mark A. Knepper1

1Epithelial Systems Biology Laboratory, Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; and 2National Cancer Center, Goyang, Gyeonggi-do, Republic of Korea

ABSTRACT Background Renal water excretion is controlled by vasopressin, in part through regulation of the transcription of the aquaporin-2 gene (Aqp2).

Methods To identify enhancer regions likely to be involved in the regulation of Aqp2 and other principal cell–specific genes, we used several next generation DNA-sequencing techniques in a well characterized cultured cell model of collecting duct principal cells (mpkCCD). To locate enhancers, we performed the assay for transposase-accessible chromatin using sequencing (ATAC-Seq) to identify accessible regions of DNA and integrated the data with data generated by chromatin immunoprecipitation followed by next generation DNA-sequencing (ChIP-Seq) for CCCTC binding factor (CTCF) binding, histone H3 lysine-27 acetylation, and RNA polymerase II. Results We identified two high-probability enhancers centered 81 kb upstream and 5.8 kb downstream from the Aqp2 transcriptional start site. Motif analysis of these regions and the Aqp2 promoter identified several potential transcription factor binding sites, including sites for two b-ZIP transcription factors: CCAAT/enhancer binding protein-b (C/EBPb) and cAMP-responsive element binding protein (CREB). To identify genomic binding sites for both, we conducted ChIP-Seq using well characterized antibodies. In the presence of vasopressin, C/EBPb,a pioneer transcription factor critical to cell-specific gene expression, bound strongly at the identified enhancer downstream from Aqp2. However, over multiple replicates, we found no detectable CREB binding sites within 390 kb of Aqp2.Thus,anyroleforCREBintheregulationofAqp2 gene transcription is likely to be indirect.

Conclusions The analysis identiﬁed two enhancer regions pertinent to transcriptional regulation of the Aqp2 gene and showed C/EBPb (but not CREB) binding.

J Am Soc Nephrol 29: 1490–1500, 2018. doi: https://doi.org/10.1681/ASN.2017050545

Vasopressin regulates water excretion largely found to be responsible for many acquired water through control of water permeability in the renal balance disorders,11 including polyuric disorders collecting duct. At a molecular level, water trans- (e.g., lithium-induced nephrogenic diabetes port is controlled through regulation of the water channel protein, aquaporin-2 (AQP2). AQP2 is Received May 19, 2017. Accepted February 18, 2018. regulated in at least two ways: (1)controloftraf- ﬁ Published online ahead of print. Publication date available at cking of AQP2 to the apical plasma membrane of www.jasn.org. collecting duct principal cells1,2 and (2) control of the absolute abundance of the AQP2 protein.3 The Correspondence: Dr. Mark A. Knepper, Epithelial Systems Biology Laboratory, Systems Biology Center, National Heart, latter process results largely from the regulation of Lung, and Blood Institute, National Institutes of Health, Aqp2 gene transcription,4–7 although regulation of 10 Center Drive, MSC-1603, Bethesda, MD 20892-1603. AQP2 protein half life also plays a role.8–10 Defec- Email: [email protected] tive regulation of Aqp2 gene transcription has been Copyright © 2018 by the American Society of Nephrology

1490 ISSN : 1046-6673/2905-1490 JAmSocNephrol29: 1490–1500, 2018 www.jasn.org BASIC RESEARCH insipidus, hypercalcemic polyuria, hypokalemic polyuria) Significance Statement and dilutional hyponatremia as seen with the syndrome of inappropriate antidiuresis and congestive heart failure. Renal water excretion is controlled by vasopressin, in part through To understand the pathophysiologic basis of these disor- transcriptional regulation of the Aqp2 gene in collecting duct cells. ders, it is important to discover the normal physiologic Defects in this mechanism are responsible for several water balance disorders. Using mouse collecting duct cells, the authors used mechanisms by which vasopressin, working by binding several next generation sequencing (NGS) techniques to in- and activation of the vasopressin V2 receptor, stimulates vestigate Aqp2 regulation. These approaches identified two en- Aqp2 gene transcription. hancer regions vicinal to the Aqp2 gene centered 81 kb upstream TheV2receptorcouplestotheheterotrimericG-protein and 5.8 kb downstream from the Aqp2 transcriptional start site. b fi a-subunit Gas, which in collecting duct principal cells, ac- ChIP-Seq analysis for the transcription factor C/EBP identi ed a strong binding site within the second region. ChIP-Seq analysis for tivates adenylyl cyclase 6, thereby increasing intracellular another transcription factor CREB, which widely held to regulate cAMP levels. Actions of cAMP are mediated chieflythrough Aqp2 gene transcription, identified many canonical CREB binding activation of one or both protein kinase A catalytic proteins sites throughout the genome but none within 390 kb of Aqp2. (coded by Prkaca and Prkacb genes). When these two genes were deleted from mpkCCD cells using CRISPR-Cas9, AQP2 METHODS mRNA and protein were nearly undetectable even in the presence of vasopressin, suggesting that Aqp2 gene transcrip- Cell Culture tion depends on protein kinase A.12 AQP2-expressing mpkCCD clone 11 (mpkCCD) cells were The mouse mpkCCD cell line has been used extensively in grown on permeable membrane supports as described previ- the investigation of regulation of collecting duct function.6,13–15 ously.15 Cells were exposed to 0.1 nM [deamino-Cys1, It manifests both major elements of vasopressin-mediated D-Arg8]-Vasopressin (dDAVP) or vehicle for 30 minutes. regulation of AQP2, namely regulated AQP2 trafficking and regulated Aqp2 gene transcription.15 On the basis of Preparation of Nuclear and Cytosolic Fractions RNA-Seq and RNA polymerase II chromatin immunopre- Nuclear and cytosolic fractions were prepared from mpkCCD cipitation followed by DNA sequencing (ChIP-Seq) in cells using the NE-PER Nuclear and Cytoplasmic Extraction mpkCCD cells, it seems that vasopressin mediates highly Reagent Kit (Pierce) as described previously.28 selective regulation of Aqp2 gene transcription, requiring the joint action of at least four DNA binding transcription factors.4 An intermediate goal on the way to an understand- Immunoblotting 29 ing of the mechanism of vasopressin-mediated Aqp2 gene Immunoblotting was performed as described. Primary antibodies were CREB (17–600; Millipore), C/EBPb (sc-150; transcription is to identify these transcription factors. A – number of hypothesis-driven studies have proposed roles Santa Cruz), p-CREB (S133, 17 10131; Millipore), ATF1 (sc-243; Santa Cruz), b-catenin (9562; Cell Signaling), and for a variety of transcription factors, including cAMP-responsive b element binding (CREB) protein,16,17 AP-1,16 NF-kB,18 GATA p- -catenin (S552, 9566; Cell Signaling). family,19–21 ETS family,15,22 NFAT5, and NFATc proteins.23–25 However, a general solution to the problem is challenging, be- ATAC-Seq cause the mammalian genome codes for approximately 1500 ATAC-Seq was performed following a method published by transcription factors and a trial and error approach are unlikely Buenrostro et al.27 Nuclear pellets (from 50,000 cells) were incu- “ ” to be definitive. In this paper, we present a systems-level anal- bated in transposition reaction mixture using Tagment DNA – – ysis using both prior data (histone H3 acetylation at lysine 27 Buffer (FC-121 1030; Illumina) and Tn5 transposase (FC-121 [H3K27Ac] ChIP-Seq and RNA polymerase II ChIP-Seq) and 1030) for 30 minutes at 37°C. The transposed DNA fragments fi fi fi new data from assay for transposase-accessible chromatin us- were puri ed and ampli ed using PCR. Puri ed DNA libraries ing sequencing (ATAC-Seq) experiments to identify enhancers weresequencedonanIlluminaHiSeq3000platform. near the Aqp2 gene. Enhancers are segments of DNA that are typically a few hundred base pairs in length, and they are oc- ChIP-Seq cupied by multiple transcription factors that can recruit RNA Chromatin immunoprecipitation (ChIP) was performed using the polymerase II and transcriptional coactivators to target truChIP Chromatin Shearing Reagent Kit (Covaris) and the Sim- genes.26 ATAC-Seq is a new methodology designed to precisely pleChIP Enzymatic Chromatin IP Kit (Cell Signaling) following the identify regions of DNA that are open, allowing access to manufacturer’s protocols. Protein/DNA was crosslinked using transcription factors and other DNA binding proteins.27 After 1.1% formaldehyde. Nuclei were isolated and sheared using an enhancer identification, transcription factor ChIP-Seq exper- ultrasonicator. The sheared chromatin was incubated with 4 mg iments focused on two b-ZIP transcription factors, CCAAT/ of antibody (CREB, 17–600 or C/EBPb, sc-150) and additionally enhancer binding protein-b (C/EBPb)andCREB,thatare incubated with Protein G magnetic beads. After elution of ChIP predicted to bind to either the identified enhancers or the products, DNA was purified and subjected to library preparation Aqp2 promoter. using the Ovation SP Ultralow Library System (NuGEN) following

J Am Soc Nephrol 29: 1490–1500, 2018 Genomic Binding Sites for CREB and C/EBPb 1491 BASIC RESEARCH www.jasn.org the manufacturer’sprotocol.TheDNAlibrariesweregelpuriﬁed for histone H3K27 acetylation ChIP-Seq12 and GSE79584 for and sequenced on an Illumina HiSeq 2000 platform. RNA polymerase II ChIP-Seq.4 Data from ATAC-Seq, H3K27Ac ChIP-Seq, and RNA polymerase II ChIP-Seq were transformed to Data Processing obtain minimum Bayes factors (range, 0–1) as described35 on the The sequence reads were mapped on the mouse reference genome basis of signal-to-noise ratios as a function of genomic position. (mm10) using Burrows–Wheeler Aligner30 and Samtools.31 All The noise level for a given insulated region was estimated as twice uniquely mapped reads were used in downstream peak calling the median signal across the region. The data from ATAC-Seq, (MACS2; Bedtools), motif analysis (MEME-ChIP),32 and bioin- H3K27Ac ChIP-Seq, and RNA polymerase II ChIP-Seq were in- formatic analysis (R package ChIPpeakAnno).33 tegrated by multiplying the complements of their minimum Bayes For ATAC-Seq data processing, adapter sequences were factors. trimmed for both forward and reverse reads using cutadapt ATAC-Seq and ChIP-Seq data have been deposited at Gene and aligned on mouse reference genome mm10 using Bowtie2. Expression Omnibus (GEO; GSE98076 and GSE108786). Duplicates were removed from mapped reads using Picard. Peaks were identiﬁed using MACS2. Read distribution was visualized on the UCSC genome browser using UCSC bedClip RESULTS and bedGraphToBigWig. Transcription factor proteins that regulate transcription of Data Mining and Bioinformatic Analyses Aqp2 and other genes in the renal collecting duct may bind A dataset of CCCTC binding factor (CTCF) ChIP-Seq was down- directly to gene promoters or enhancers that are not immedi- loaded from the mouse ENCODE database (GSE36027).34 ately adjacent to the regulated gene. Recent studies summa- mpkCCD ChIP-Seq datasets were downloaded from GSE95009 rized by Hnisz et al.36 provide information that narrows the

Figure 1. CCCTC binding factor (CTCF) insulated loops. (A) Hypo- Figure 2. The mpkCCD cells are responsive to vasopressin. (A) thetical insulated loop. Two adjacent DNA-bound CTCF molecules Example immunoblots for b-catenin phosphorylated at S552, dimerize to create a loop. Genes within an insulated loop are regu- total b-catenin, cAMP-responsive element binding protein lated by their own promoters plus enhancers (and repressors) within (CREB) phosphorylated at S133, ATF1 phosphorylated at S63, the same insulated loop. (B) CTCF binding sites flanking the total CREB, and total ATF1 as well as a Coomassie blue–stained aquaporin-2 (Aqp2) gene from mouse ENCODE data. Two other gel showing equality of loading. (B) Quantification of immuno- genes, Faim2 (neuron specific) and Aqp5 (specific to secretory ep- blots (n=3 for each). *Statistically significant difference was found ithelia), are within the defined insulated loop. ChIP-Seq, chromatin between cells treated with vehicle or [deamino-Cys1, D-Arg8]- immunoprecipitation followed by DNA sequencing. Vasopressin (dDAVP) for 30 minutes (P,0.05 by t test).

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possible locations of transcription factor binding sites that regulate a particular gene, such as Aqp2.Thisconstraintis on the basis of the concept of CTCF-bounded loops that define insulated neighborhoods. CTCF is a DNA binding protein that binds to DNA as a monomer during interphase but homodi- merizes with upstream or downstream DNA-bound CTCF molecules to create CTCF loops (Figure 1A). The locations of these loops are highly conserved among mammalian species and relatively invariant among cell types.36 There are approximately 13,000 such insulated loops across the genome, with an average of two to three genes per insulated loop. The enhancers that regulate a given gene are found within the same CTCF loop. Prior studies done as part of the mouse ENCODE project have used ChIP-Seq to identify CTCF binding sites.34 These data provide the information needed to identify the insulated loop that contains Aqp2 (Figure 1B). As expected, CTCF binding sites were the same among kidney, liver, lung, and small intestine. The loop that contains Aqp2 is 99 kb in length and also contains two other genes: Faim2 and Aqp5. Thus, enhancers that control Aqp2 gene transcription are highly likely to reside within this 99-kb insulated loop. To assure that the mpkCCD cells used in this study respond appropriately to vasopressin, we carried out Western blotting for known vasopressin-regulated phosphorylation sites. We tested the ability of dDAVP (0.1 nM for 30 minutes) to increase phosphorylation of the transcriptional coregulator b-catenin at Ser552, a vasopressin-responsive site12,28,37 (Figure 2). Band density corresponding to this target was more than doubled. In the same samples, we probed for protein kinase A target sites on CREB and ATF1, which are recognized by the same antibody but can be discriminated on the basis of molecular weight. Both sites showed increases in phosphorylation in response to vasopressin (Figure 2), confirming the viability and vasopressin responsiveness of the cells. We previously found evidence that 714 distinct transcription factors are expressed in mpkCCD cells12 (Supplemental Dataset 1). All could be considered candidates to play roles in regulation of Aqp2 gene transcription. However, 714 transcription factors are too many to allow efficient one by one evaluation by ChIP-Seq. To narrow the possible candidates, it

are indicated by the track MACS2 Peaks. (General feature of ATAC- Seq analysis are in Supplemental Figure 1.) Read distribution normalized by signal per million reads was visualized on the UCSC genome browser using two-reference genome information: mouse mm9 for CTCF ChIP-Seq in kidney downloaded from mouse EN- CODE database and mouse mm10 for ATAc-Seq, H3K27Ac ChIP- Seq, and RNA polymerase II ChIP-Seq in mpkCCD cells. Gray shading indicates the region corresponding to gene body. dDAVP, [deamino-Cys1, D-Arg8]-Vasopressin. CTCF, CCCTC binding factor; ATAC-Seq, assay for transposase-accessible chromatin us- Figure 3. Mapping ChIP-Seq data to three collecting duct genes: (A) ing sequencing; H3K27Ac, histone H3 acetylation at lysine 27; adenylyl cyclase 6 (Adcy6), (B) V2 vasopressin receptor (Avpr2), and (C) ChIP-Seq, chromatin immunoprecipitation followed by DNA se- aquaporin-2 (Aqp2). The ATAC-Seq sites indicating accessible DNA quencing; RNA PolII, RNA polymerase II.

J Am Soc Nephrol 29: 1490–1500, 2018 Genomic Binding Sites for CREB and C/EBPb 1493 BASIC RESEARCH www.jasn.org is desirable to identify subregions of the Faim2-Aqp2-Aqp5 surrounding Adcy6 encloses a major ATAC-Seq peak centered insulated loop that are likely to contain enhancers. Doing so 1200 bases upstream from Adcy6 plus several less prominent will allow us to identify potential transcription factor binding peaks that are indicated by the “MACS2 Peaks” track. The sites in these regions. We present three types of data that are 97.5-kb putative CTCF insulated region surrounding Avpr2 informative in this regard, viz. ATAC-Seq, histone H3K27Ac encloses three discernable ATAC-Seq peaks centered at ChIP-Seq, and RNA polymerase II ChIP-Seq (explained be- 11,300, 43,800, and 67,200 bases upstream from Avpr2.The low). Relevant proﬁles for these measures in mpkCCD cells are Faim2-Aqp2-Aqp5 insulated loop contains a major peak over- shown in Figure 3. lapping the unexpressed Faim2 gene 81 kb upstream from the ATAC-Seq is a recently devised method27 for assessing DNA Aqp2 transcriptional start site. It also contains several less accessibility, providing better spatial resolution than DNase I prominent peaks between Faim2 and Aqp2 as well as between hypersensitivity proﬁling. Figure 3 shows ATAC-Seq data for Aqp2 and Aqp5. Full ATAC-Seq data can be downloaded from mpkCCD cells mapped to the regions around three collecting GEO (GSE108786). duct genes: Adcy6, Avpr2,andAqp2. Adcy6 and Avpr2 are Figure 3 also shows data from H3K27Ac ChIP-Seq exper- strongly expressed in collecting duct principal cells, but their iments for the same three regions. (A small subset of these data transcription is not regulated by vasopressin.4 Mouse EN- was previously reported in the work by Isobe et al.12) CODE data for CTCF ChIP-Seq are included to provide a ref- H3K27Ac mapping is widely used to identify open regions erence. The 37-kb putative CTCF insulated region of DNA overlapping both promoters and enhancers.38 To a

Figure 4. Identiﬁcation of enhancer elements for aquaporin-2 (Aqp2) gene. High open probability regions in the Faim2-Aqp2-Aqp5 insulated loop were identiﬁed using minimum Bayes factors to integrate ATAC-Seq, histone H3K27Ac ChIP-Seq, and RNA polymerase

II ChIP-Seq data into single “open probability” (Po) values as described in Supplemental Methods. The calculation is on the basis of the signal-to-noise ratio for each measure at each point in the loop. Noise values for this calculation were estimated as two times the median read density value across the loop. (Supplemental Figures 2 and 3 show sensitivity analysis.) The Po calculation identiﬁed two regions of high probability as indicated. These regions were analyzed using MEME-ChIP to identify JASPAR transcription factor motifs present. CTCF, CCCTC binding factor; dDAVP, [deamino-Cys1, D-Arg8]-Vasopressin. ATAC-Seq, assay for transposase-accessible chromatin using sequencing; H3K27Ac, histone H3 acetylation at lysine 27; ChIP-Seq, chromatin immunoprecipitation followed by DNA sequencing; RNA PolII, RNA polymerase II.

1494 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1490–1500, 2018 www.jasn.org BASIC RESEARCH large extent, the H3K27Ac peaks overlap the ATAC-Seq peaks, providing redundancy for identification of potential enhancers. In response to dDAVP, there was a demonstrable increase in H3K27 acetylation in the Aqp2 promoter, gene body, and downstream from the gene body. Figure 3 also shows binding sites for RNA polymerase II (Polr2a subunit) mined from Sandoval et al.4 RNA polymerase II has been shown to localize not only to gene bodies of actively transcribed genes but also, to active enhancers. dDAVP increases RNA polymerase II occupancy over the Aqp2 gene body as previously shown4 and also in two other regions of the Faim2-Aqp2-Aqp5 insulated loop, namely a region just downstream from Aqp2 and a region overlapping the last exon of Faim2. (Of note, the Faim2 mRNA is not detectable in mpkCCD cells.4) In Figure 4, we integrate the three measures (ATAC-Seq, histone H3K27Ac ChIP-Seq, and RNA polymerase II ChIP- Seq) to obtain an aggregate “open probability” (Po) indicating the regions in the Faim2-Aqp2-Aqp5 insulated loop that are most likely to be enhancers. Among the open regions, two stand out as being the densest and longest as highlighted in Figure 4. We analyzed these two enhancers using the JASPAR database of transcription factor binding motifs to identify DNA sequence motifs associated with specific transcription factors. In the upstream enhancer, potential sites for several transcription factors were identified, including some known to be expressed in mpkCCD cells viz. NFYA, RREB1, E2F3, YY1, CTCF, and SMAD4 (Supplemental Dataset 1). In the identified enhancer between the Aqp2 and Aqp5 genes, potential sites for the transcription factors TFAP2A, PBX3, CEBPB, CEBPG, NRF1, ELF1, ELF4, KLF9, GLIS2, NFIX, NFIA, and HIF1A were identified. Some of these have previously been implicated in regulation of Aqp2 gene transcription: AP2 (TFAP2A),15 ETS family (ELF1 and ELF4),15 and KLF (KLF9).39 Among the sites found, C/EBPb is a so-called “pioneer” transcription factor that can initiate regulated changes in chromatin structure before binding of other transcription Figure 5. C/EBPb ChIP-Seq: general findings. (A) Quantification of – factors.40,41 peak read densities in dDAVP treated samples and vehicle controls To identify whether C/EBPb binds anywhere in the (n=2). (These observations were made with 30 minutes of exposure to dDAVP or vehicle; additional observations at 24 hours are re- Faim2-Aqp2-Aqp5 insulated loop or elsewhere in the genome ported in Supplemental Figure 5.) (B) Locations of peaks relative to (Supplemental Figure 4), we carried out ChIP-Seq experi- the nearest gene. The majority of C/EBPb binding sites were found b 42 ments (Figure 5) using a well tested C/EBP antibody. The within the promoters and introns of genes and intergenic regions. full datasets are provided permanently at https://hpcwebapps. UTR, untranslated region. (C) C/EBPb binding motif derived from cit.nih.gov/ESBL/Database/ChIP-Seq_CREB_CEBPB/ and ChIP-Seq data. Motifs matched previously reported motifs for the GEO (GSE98076). As shown in Figure 5, 2401 C/EBPb C/EBPa and C/EBPb found in the JASPAR motif database; e values binding sites were identified in dDAVP-treated cells (0.1 nM, in parentheses are the motif P values (computed by a Fisher exact 30 minutes), whereas 209 reproducible sites were found in test for enrichment of the motif in the sequences) times the number of vehicle-treated cells. Read densities for many of the 209 sites candidate motifs tested. dDAVP, [deamino-Cys1, D-Arg8]-Vasopres- b b common to the dDAVP and vehicle samples were increased in sin; C/EBP , CCAAT/enhancer binding protein- ;ChIP-Seq,chromatin immunoprecipitation followed by DNA sequencing. the dDAVP-treated versus vehicle-treated cells: mean log2(d- DAVP/vehicle) =1.14 (Figure 5A) as expected on the basis of the observation that dDAVP causes translocation of C/EBPb limited to gene promoters, but they are also frequently found into the nucleus of mpkCCD cells (Supplemental Figure 4) in intergenic regions and introns (Figure 5B), contrasting with (https://hpcwebapps.cit.nih.gov/ESBL/Database/QuantNuc- CREB (see below). The consensus motif identified from Proteomics/). In mpkCCD cells, C/EBPb binding sites are not C/EBPb binding sites matched canonical binding motifs for

J Am Soc Nephrol 29: 1490–1500, 2018 Genomic Binding Sites for CREB and C/EBPb 1495 BASIC RESEARCH www.jasn.org the C/EBP family. These data support the fidelity of the ChIP- their promoters as well as protein kinase and transporter genes Seq methodology applied to C/EBPb binding site localization. with C/EBPb binding sites (Supplemental Figure 6). Vasopressin-sensitive C/EBPb binding sites were identified in The fraction of C/EBPb binding sites associated with col- the vicinity of several known C/EBPb-regulated genes, namely lecting duct–specific genes was greater than in the whole tran- Hif1a,43 Itpr2,44 Smg7,42 and Idh145 (Figure 6, A–E), further scriptome, suggesting a role in tissue-specific gene expression supporting the fidelity of the method. (Supplemental Figure 7). Tissue-specific genes tend to have AC/EBPb binding site was identified just 400 bp down- TATA boxes rather than CpG islands in their promoter prox- stream from Aqp2, and the read density appeared to be imal regions.46,47 Consistent with this, genes with TATA boxes increased by dDAVP (Figure 6F). This corresponds to a pre- are more frequent among genes with C/EBPb binding sites dicted C/EBPb binding site in one of the high open probability than would be expected from a random selection of expressed DNA regions identified in Figure 4. The same site was identi- genes, whereas genes with promoter proximal CpG islands are fied in two additional samples treated for 24 hours with less frequent (Supplemental Figure 7). These observations dDAVP (Supplemental Figure 5). Supplemental Tables 1 and lend support to the possible role of C/EBPb in cell-specific 2 list all transcription factors with C/EBPb binding sites at gene expression in collecting duct principal cells.

Figure 6. C/EBPb ChIP-Seq: examples of binding sites found. Read distribution normalized by signal per million reads was visualized on the UCSC genome browser using two-reference genomes: mouse mm9 for CTCF ChIP-Seq in kidney downloaded from the mouse ENCODE database and mouse mm10 for C/EBPb ChIP-Seq in mpkCCD cells. Gray shading indicates the region corresponding to gene body. (A–E) Results confirmed previous findings for Hif1a, Itpr2, Smg7, Idh1,andCebpb.(F)AC/EBPb binding site was identified just 400 bp downstream from the aquaporin-2 (Aqp2) gene in the dDAVP–treated sample. C/EBPb, CCAAT/enhancer binding protein-b; CTCF, CCCTC binding factor; dDAVP, [deamino-Cys1, D-Arg8]-Vasopressin; ChIP-Seq, chromatin immunoprecipitation followed by DNA sequencing.

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Because vasopressin signals chiefly through increases in cAMP in collecting duct principal cells, it has been widely assumed that the transcription factor CREB is responsible for vasopressin-mediated regulation of Aqp2 gene transcription.11,48–51 AcAMPresponse element has been reported to be present within the promoter of Aqp2,16,17 although CREB binding to this site has not been directly shown. To identify CREB binding sites throughout the genome, we have carried out ChIP-Seq experiments in mpkCCD cells (four control-dDAVP pairs done on different days). For ChIP-Seq, we used a CREB antibody previously used for ChIP-Seq in other tissues52 (Supplemental Figure 4). Binding sites were identified as genomic positions in which mapped reads for ChIP samples significantly exceeded the input control using a strin- gent cutoff (q,0.01 using MACS2). The full datasets are provided at https://hpcwebapps.cit.nih.gov/ESBL/Database/ChIP- Seq_CREB_CEBPB/ and at GEO (GSE98076). A total of 793 CREB binding sites were identified in mpkCCD, and most of them were seen under both conditions. In general, normalized read densities were virtually unchanged or slightly reduced in the dDAVP-treated cells (0.1 nM for 30 minutes) versus vehicle-treated cells (Figure 7A). This finding is consistent with prior studies showing that CREB is regulated chiefly by changes in phosphorylation in its “pKID domain” rather than by differential binding or nuclear translocation.53,54 Over 90% of total CREB binding sites were lo- cated at promoter regions (1 kb upstream to +100 bp downstream from the transcription start site) (Figure 7B). Sequences extracted from binding site peak centers were used to identify binding motifs (Figure 7C). A consensus half-cAMP response element sequence was identified that matches prior data for b-ZIP transcription factors CREB3, ATF1, and ATF4, confirming the fidelity of the method. The motif is the same for vehicle- and dDAVP-treated cells, although shifted by three bases. CREB binding sites identified in our ChIP-Seq analysis matched known target genes (Figure 8). For example, a canonical CREB binding site in the promoter of Fos55 matches the binding site found in this study (Figure 8A). fi CREB binding was also found at previously identi ed sites in the Figure 7. CREB ChIP-Seq: general findings. (A) Quantification of 56 57 57 – promoters of Mcm5, Cbwd1, and Jund (Figure 8, B D). In- peak read densities in dDAVP–treatedsamplesandvehiclecontrols terestingly, CREB also bound the promoter of the Creb1 gene itself (n=4). There was no significant difference in peak read densities (Figure 8E), suggesting a feedback relationship. between dDAVP- and vehicle-treated samples (30 minutes of ex- Interestingly, no CREB binding sites were found within 390 kb posure). (B) Locations of peaks relative to nearest gene. The majority of the Aqp2 gene, including the 99-kb Faim2-Aqp2-Aqp5 insulated of CREB binding sites were found within the promoters of genes. loop (Figure 8F), indicating that any role of CREB in transcrip- UTR, untranslated region. (C) CREB binding motif derived from tional regulation of Aqp2 is likely to be indirect. For example, its ChIP-Seq data. Motifs were the same for dDAVP- and vehicle- regulatory role may be through regulation of expression of other treated samples, although shifted by three bases. Motifs matched transcription factors. Supplemental Tables 3 and 4 list transcrip- previously reported motifs for CREB3, ATF1, and ATF4 found in the JASPAR motif database; e values in parentheses are the motif P tion factors with CREB binding sites at their promoters as well as values (computed by a Fisher exact test for enrichment of the motif protein kinase and transporter genes with CREB binding sites in the sequences) times the number of candidate motifs tested. (Supplemental Figure 8). CREB, cAMP-responsive element binding protein; dDAVP, [deamino-Cys1, D-Arg8]-Vasopressin; ChIP-Seq, chromatin immunoprecipitation followed by DNA sequencing. DISCUSSION Sandoval et al.4 have estimated that a minimum of four tran- Studies in model organisms have shown a high level of scription factors are required to explain selective regulation of complexity in transcriptional networks with multiple cross- Aqp2 gene transcription in collecting duct cells. mpkCCD cells regulated transcription factors affecting transcription.58 have been found to express .700 transcription factors,

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Figure 8. CREB ChIP-Seq: examples of binding sites found. Read distribution normalized by signal per million reads was visualized on the UCSC genome browser using two-reference genomes: mouse mm9 for CTCF ChIP-Seq in kidney downloaded from the mouse ENCODE database and mouse mm10 for CREB ChIP-Seq in mpkCCD cells. Gray shading indicates the region corresponding to gene body. (A–E) Results confirmed previous findings for Fos, Mcm5, Cbwd1, Jund,andCreb1. (F) No CREB binding sites were found within the Faim2-aquaporin-2 (Aqp2)-Aqp5 insulated loop. (The closest CREB binding site from the Aqp2 gene was 390 kb upstream.) CREB, cAMP-responsive element binding protein; CTCF, CCCTC binding factor; dDAVP, [deamino-Cys1, D-Arg8]-Vasopressin; ChIP-Seq, chromatin immunoprecipitation followed by DNA sequencing. making a protein by protein identification of relevant tran- investigators interested in regulation of genes other than scription factors using ChIP-Seq and CRISPR-Cas9 deletions Aqp2. Future studies can use next generation sequencing tech- impractical. Here, we have used an unbiased, systems-level niques, advanced reporter systems, and CRISPR-Cas9 to ex- approach using several next generation sequencing modalities amine roles of all transcription factor candidates in the Aqp2 to identify likely enhancers within the same insulated neigh- gene regulatory network. borhood as Aqp2.TheseAqp2-associated enhancers are likely to bind the transcription factors involved in cell type–specific expression and regulation by vasopressin. Motif analysis of the ACKNOWLEDGMENTS enhancers identified relatively few transcription factors, thus reducing the number of candidates that will require investiga- Next generation sequencing was done in the National Heart, Lung and tion in future studies (Figure 4). We have carried out tran- Blood Institute DNA Sequencing Core Facility (Yuesheng Li, Director). scription factor ChIP-Seq for two candidates, viz.CREBand The work was primarily funded by Division of Intramural Re- C/EBPb, identifying a strong binding site for the latter in one search, National Heart, Lung, and Blood Institute projects ZIA- of the identified enhancer regions. We provide comprehensive HL001285 (to M.A.K.) and ZIA-HL006129 (to M.A.K.). genome-wide binding data as an online resource for All authors approved the final version of the manuscript.

1498 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1490–1500, 2018 www.jasn.org BASIC RESEARCH

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