A Grainyhead-Like 2/Ovo-Like 2 Pathway Regulates Renal Epithelial Barrier Function and Lumen Expansion

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† ‡ | Annekatrin Aue,* Christian Hinze,* Katharina Walentin,* Janett Ruffert,* Yesim Yurtdas,*§ | Max Werth,* Wei Chen,* Anja Rabien,§ Ergin Kilic,¶ Jörg-Dieter Schulzke,** †‡ Michael Schumann,** and Kai M. Schmidt-Ott*

*Max Delbrueck Center for Molecular Medicine, Berlin, Germany; †Experimental and Clinical Research Center, and Departments of ‡Nephrology, §Urology, ¶Pathology, and **Gastroenterology, Charité Medical University, Berlin, Germany; and |Berlin Institute of Urologic Research, Berlin, Germany

ABSTRACT Grainyhead transcription factors control epithelial barriers, tissue morphogenesis, and differentiation, but their role in the kidney is poorly understood. Here, we report that nephric duct, ureteric bud, and collecting duct epithelia express high levels of grainyhead-like homolog 2 (Grhl2) and that nephric duct lumen expansion is defective in Grhl2-deficient mice. In collecting duct epithelial cells, Grhl2 inactivation impaired epithelial barrier formation and inhibited lumen expansion. Molecular analyses showed that GRHL2 acts as a transcriptional activator and strongly associates with histone H3 lysine 4 trimethylation. Integrating genome-wide GRHL2 binding as well as H3 lysine 4 trimethylation chromatin immunoprecipitation sequencing and gene expression data allowed us to derive a high-confidence GRHL2 target set. GRHL2 transactivated a group of genes including Ovol2, encoding the ovo-like 2 zinc finger transcription factor, as well as E-cadherin, claudin 4 (Cldn4), and the small GTPase Rab25. Ovol2 induction alone was sufficient to bypass the requirement of Grhl2 for E-cadherin, Cldn4,andRab25 expression. Re-expression of either Ovol2 or a combination of Cldn4 and Rab25 was sufficient to rescue lumen expansion and barrier formation in Grhl2-deficient collecting duct cells. Hence, we identified a Grhl2/Ovol2 network controlling Cldn4 and Rab25 expression that facilitates lumen expansion and barrier formation in subtypes of renal epithelia.

J Am Soc Nephrol 26: 2704–2715, 2015. doi: 10.1681/ASN.2014080759

The renal collecting duct’s vital electrolyte and water- is clear that these epithelia share several cell biologic regulatory functions are carried out by highly special- properties such as a uniform tubular appearance ized cell populations.1 The collecting duct itself is characterized by a cuboidal epithelium surround- composed of a tight epithelium, which separates the ing a fluid-filled lumen and a molecular composi- urinary compartment from a hypertonic interstitium tion of the apical junctional complex that includes and maintains a barrier to concentration gradients. The collecting duct derives from the ureteric bud, Received August 8, 2014. Accepted December 30, 2014. which emanates from the nephric duct and then un- dergoes branching morphogenesis in response to sig- A.A. and C.H. contributed equally to this work. 2 nals from the adjacent metanephric mesenchyme. Published online ahead of print. Publication date available at Many of the genes that are critical for aspects of neph- www.jasn.org. ric duct development continue to be expressed in the Present address: Max Werth, Department of Medicine, Columbia ureteric bud and collecting duct, serving roles in de- University, New York, New York. velopment, differentiation, and maintenance of these Correspondence: Dr. Kai M. Schmidt-Ott, Max Delbrueck Cen- cells (e.g., Pax2/8, Gata3, Emx2,andHnf1b).3 Little is ter for Molecular Medicine, Robert-Rössle-Strasse 10, 13125 known about the molecular pathways governing the Berlin, Germany. Email: [email protected] specific epithelial properties of these cells, although it Copyright © 2015 by the American Society of Nephrology

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E-cadherin–based adherence junctions and CLDN4-containing tight junctions, both of which are characteristic of a tight epithelial barrier. The grainyhead transcription factor family regulates various aspects of epithelial function, including differentiation and morphogenesis.4–6 Grainyhead-like 2 (Grhl2)isone of three mammalian homologs of Drosophila grainyhead. It is expressed in the surface ectoderm, the gut tube, and the otic vesicle, which are among the earliest barrier-forming epithelia of the embryo.7,8 Grhl2 expression remains uniformly high in E-cadherin– positive epithelia throughout development and adulthood.8 Grhl2 is required for important morphogenetic events during embryonic development, with gene deletion in mice leading to defective neural tube closure, split face malformations, exencephaly, and death at embryonic day (E) 11.5.8–10 Grhl2 was reported to participate in epithelial morphogenesis and differentiation in cultured airway,11 in biliary and hepatic epithelia,12,13 and in tumorigenesis of skin and mammary epithelial cells.14,15 However, the underlying genetic networks are incompletely understood. Here, we show that epithelia of the nephric duct, ureteric bud, and collecting duct express high levels of GRHL2. Grhl2 inactivation results in defects of epithelial barrier formation and lumen expansion. We combine gene expression and genome-wide chromatin immunoprecipitation data to show that GRHL2 strongly associates with H3K4 trimethylation, a chromatin mark important for transcriptional activity. Next, we derive a high-confidence target gene set and show that GRHL2 activates genes encoding the apical junctional complex proteins CDH1, CLDN4, Figure 1. GRHL2 is localized in epithelial nuclei in the nephric duct, ureteric bud, col- and the small GTPase RAB25, as well as the lecting duct, and distal nephron. (A) Immunofluorescence staining of a cross-section of an zinc finger transcription factor ovo-like 2 E10.0 mouse embryo at the level of the urogenital ridges revealing GRHL2-positive nuclei (OVOL2). In functional studies, we identify a in the nephric ducts. (B) Immunofluorescence staining of murine embryonic kidney at novel Grhl2/Ovol2 pathway that reinforces E15.5 shows GRHL2 expression in the branching ureteric bud. (C and D) Immunofluo- CDH1, CLDN4, and RAB25, and regulates rescence staining of murine embryonic (E18) (C) and adult (P30) (D) kidney tissue using lumen expansion and barrier formation in antibodies against GRHL2 (colors as indicated), the ureteric bud marker keratin 8 (KRT8), collecting duct epithelia. and the collecting duct principal cell marker aquaporin 2 (AQP2). (E and F) Immunohis- tochemical staining of human adult kidney sections using a GRHL2 antibody (purple staining). The section in E is slightly counterstained with hematoxylin and eosin, and the section in F is without counterstaining. ND, nephritic duct; UB, ureteric bud; G, glo- RESULTS merulus; PT, proximal tubule; DT, distal tubule; CD, collecting duct. Bar, 100 mminA,E, and F; 50 mminB;300mminC;20mminD. GRHL2 Localization and DNA Binding Sites in the Kidney In mice, strong GRHL2 immunoreactivity was detected in nuclei of nephric ducts in

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immunoprecipitation with a GRHL2 antibody and sequencing (ChIP-seq) on kidneys from E18 and adult mice, as well as on murine inner medullary collecting duct (IMCD-3) cells. We detected genomic regions bound by GRHL2 (peaks) with model-based analysis for ChIP-seq (MACS) using default parameters17 and found 7366, 7890, and 10,621 peaks in IMCD-3 cells, adult mouse kidney, and E18 mouse kidney, respectively. The three data sets revealed a marked overlap, which became even more pronounced when only the top 25% scoring peaks were considered (Figure 2A, Supplemental Figure 1A). The distance distribution of IMCD-3 cell GRHL2 peaks from adult kidney peaks revealed two distinct large groups with ChIP peaks that either overlapped or occurred at large distances from each other (Supple- mental Figure 1B). The overlap of in vivo GRHL2 ChIP peaks with IMCD-3 GRHL2 peaks was highly nonrandom (P,0.001). Peak annotation of all three GRHL2 ChIP-seq experiments showed a statistically significant (P,0.001) clustering near transcriptional start sites, promoters, and in- tronic regions (Figure 2B). De novo motif Figure 2. GRHL2 binding in IMCD-3 cells, adult kidney, and embryonic kidney, and gene discovery with MEME revealed a sequence expression data. (A) Venn diagram for the top 25% GRHL2 ChIP peaks shows a significant overlap of peaks between IMCD-3 cells, adult, and E18 kidney with a substantial number (Figure 2C) that is identical to previously 18 of peaks present in all three scenarios. (B) Promoters/TSSs (blue), exons (yellow), and published GRHL2 binding motifs. introns (orange) show a statistically significant enrichment for GRHL2 ChIP peaks compared with random peaks, whereas intergenic regions (green) display a significant de- GRHL2 Acts as a Transcriptional enrichment. (C) De novo motif discovery from the top 10% of IMCD-3 GRHL2 peaks Activator and Strongly Associates yields a consensus sequence identical to previously published grainyhead binding motifs with Regions of H3K4 Trimethylation (P value provided by MEME). (D) Volcano plot of the expression data of all genes dis- To gain further insight into the transcriptional playing log-transformed P values and fold changes. The solid lines represent the fold network downstream of GRHL2, we gener- change thresholds of 1.4 (orthogonal) and the P value threshold of 0.05 (horizontal). Ac- ated IMCD-3 cells with a Grhl2 knockdown cording to the axes labels, genes colored in red are downregulated in Grhl2-KD (n=214) (Grhl2-KD) using lentiviral gene transfer of a and genes in green are upregulated in Grhl2-KD (n=207) compared with Grhl2-Con. Gray short hairpin RNA (shRNA) expression cas- data points refer to nondifferential genes or differentially expressed genes not rescued in sette (Supplemental Figure 2, A and B). To Grhl2-KD; Grhl2-Res. The dotted line marks the border to the top 250 differentially ex- fi pressed genes depicted in E. (E) Heatmap of the top 250 differentially regulated genes control the speci city of the shRNA, we also from microarray data for control (n=3), Grhl2 knockdown (n=3), and Grhl2 rescue (n=3) generated IMCD-3 cells expressing a control in IMCD-3 cells. Expression values are maximum normalized for each gene. *P,0.05; shRNA (Grhl2-Con), and IMCD-3 cells ex- **P,0.01; ***P,0.001. TSS, transcriptional start site; UTR, untranslated region. pressing the Grhl2 shRNA and a rescue construct consisting of a full-length Grhl2 cDNA with silent mutations at the shRNA target E10.0 mouse embryos, of ureteric bud epithelia in E15.5 mouse site rendering it insensitive to shRNA-mediated degradation embryos, and of distal nephron epithelia andcollecting ductsin (Grhl2-KD; Grhl2-Res). GRHL2 protein was reduced to E18 mouse embryonic kidney, adult mouse kidney, and adult 26.3%64.9% in Grhl2-KD compared with Grhl2-Con and rein- human kidney (Figure 1). GRHL2 was not detectable in meta- duced up to 348.4%649.5% in Grhl2-KD; Grhl2-Res (Supple- nephric mesenchymal cells, in renal stromal cells, in glomer- mental Figure 2, D and E). We performed gene expression analysis uli, and in proximal segments of the nephron. GRHL2 binds on Grhl2-Con, Grhl2-KD, and Grhl2-KD; Grhl2-Res IMCD-3 to a DNA consensus sequence highly similar to the DNA ma- cells using microarrays. We found 421 genes that were differentially trix for Drosophila grainyhead.11,16 We used chromatin expressed (P,0.05, $1.4-fold change, Grhl2-Con versus Grhl2-KD).

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Figure 3. GRHL2 acts as a transactivator and strongly associates with regions of H3K4 trimethylation. (A) Differential H3K4me3 peaks (significantly more H3K4me3 in Grhl2-Con compared with Grhl2-KD) strongly cluster at genes, TSSs, and GRHL2 ChIP peaks (P,0.001 for 0 bp distance compared with random peaks). (B) Integrated analysis of ChIP-seq and gene expression data. The first two columns display regions of H3K4me3 enrichment in Grhl2-Con and Grhl2-KD cells (26,500 regions corresponding to H3K4me3 peaks detected). Row-wise, a window of 2 kb around the peak summit is shown. Peaks are sorted by read count ratio (Grhl2-Con/Grhl2-KD). The horizontal black lines mark read count fold change thresholds. Note that in response to Grhl2 deficiency, H3K4 trimethylation is more frequently lost than gained. The third column shows a density plot indicating the frequency of GRHL2 ChIP peaks within the respective H3K4me3 regions. The last two columns show density plots representing the frequency of differentially expressed genes downregulated (column 4) or upregulated (column 5) in Grhl2-KD cells adjacent to the respective H3K4me3 regions. A H3K4me3 region is considered associated with a gene if it is #500 bp away from the gene’s transcriptional start site or/and intersected with the gene body. In total, 175 downregulated and 170 upregulated genes are associated with some H3K4me3 region shown. Note that a gene can associate with multiple H3K4me3 regions. The density plots are generated by calculating a sliding average over 500 H3K4me3 regions. (C) Detailed view of ChIP-seq peak locations in proximity to the genes encoding Ovol2, Cdh1, Rab25,andCldn4. The red bar indicates the position of a significant GRHL2 binding motif (P,0.0001, sequence depicted). TSS, transcriptional start site. ***P,0.001.

Of these genes, 207 were upregulated and 214 were downregu- development, and cell organization (Supplemental Table 2). lated upon Grhl2 deﬁciency (Figure 2, D and E, see Supple- These genes included previously reported GRHL2 targets mental Table 1 for a list of these genes). Expression of 372 such as Rab25 (encoding the GTPase Rab 25), Cdh1 (encoding (88.4%) of these genes was rescued toward control levels in E-cadherin), and Cldn4 (encoding claudin 4; Supplemental Grhl2-KD; Grhl2-Res (Supplemental Table 1). Gene ontology Figure 2, C and D).8,12 analysis of differentially expressed genes in Grhl2-KD, using Histone H3 lysine 4 trimethylation (H3K4me3) is a chromatin the HOMER software package,19 revealed a high enrichment in mark associated with actively transcribed genes.20,21 We previ- key epithelial cell programs such as differentiation, ously reported that H3K4 trimethylation at the Cdh1 promoter

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2 2 Figure 4. GRHL2 regulates lumen expansion and barrier formation. (A) Hematoxylin and eosin staining of E10.0 Grhl2-deficient (Grhl2 / ) 2 2 and control embryo cross-sections at the level of the caudal mesonephros. Grhl2 / nephric ducts show a reduced (middle) or collapsed (right) lumen compared with control embryos. (B) Statistical analysis of E10.0 nephric duct lumen area with a significantly re- 2 2 duced lumen area in Grhl2 / embryos is shown (mean6SD). (C) Immunofluorescence staining of E10.0 mouse embryo sections at the

2708 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 2704–2715, 2015 www.jasn.org BASIC RESEARCH is dependent on GRHL2.8 To better understand the association expression levels in Grhl2-KD; Grhl2-Res cells, (3) differential of GRHL2 and H3K4 trimethylation in a genome-wide context, GRHL2-dependent H3K4 trimethylation in the gene or #500 bp we utilized H3K4me3 ChIP-seq from Grhl2-Con and Grhl2-KD from the transcriptional start site, and (4) GRHL2-binding peaks cells. Using MACS, we found 19,904 absolute H3K4me3 peaks in IMCD-3 and adult kidney chromatin overlapping with the site corresponding to regions of strong H3K4 trimethylation in of differential H3K4 trimethylation. The rationale for these rig- Grhl2-Con cells. Of these regions, 1440 displayed significantly orous criteria was derived from our observation that GRHL2- reduced H3K4 trimethylation in Grhl2-KD (MACS on Grhl2- dependent H3K4me3 peaks either intersect with or appear at a Con using Grhl2-KD as background model) and 417 displayed large distance from GRHL2 peaks and GRHL2-dependent genes significantly enhanced H3K4 trimethylation in Grhl2-KD cells (Figure 3A). This approach yielded a set of 17 genes. Twelve of (MACS on Grhl2-KD using Grhl2-Con as a background model). these genes exhibited a peak center related (650 bp from peak This indicated widespread modulation of H3K4 trimethylation summit) significant GRHL2 binding motif (P,0.001) (Supple- by GRHL2, particularly toward a loss of H3K4 trimethylation in mental Figure 1, C and D, Supplemental Table 3). Among these the absence of GRHL2. Regions of significantly reduced H3K4 core target genes, we found previously reported targets, Cdh1 trimethylation in Grhl2-KD were strongly overlapping with and Rab25,8,12 and additional novel targets, including the gene GRHL2 peaks, genes, and their transcriptional start sites (Figure encoding the zinc finger transcription factor Ovol2.Claudin4is 3A). In fact, GRHL2 binding sites were observed in more than a known GRHL2 target gene,8,22 butwasnotrepresentedwithin one third of the regions that displayed significantly reduced the core target gene set due to a lack of a differential H3K4me3 H3K4 trimethylation in Grhl2-KD compared with Grhl2-Con, peak, although all other criteria were fulfilled. However, visual and among more than half of the top differentially regulated inspection indicated a reduced H3K4 trimethylation level at the regions (Figure 3B). Conversely, they were rarely found in other Cldn4 locus in Grhl2-KD cells compared with Grhl2-Con, regions of H3K4 trimethylation, particularly in regions that dis- which did not fulfill our strict statistical criteria (Figure 3C). played increased H3K4 trimethylation in Grhl2-KD cells (Figure 3B). We observed a tight association of GRHL2 binding with GRHL2 is Required for Lumen Expansion and Epithelial genes downregulated in Grhl2-KD compared with Grhl2-Con, Barrier Function but not with genes that were upregulated (Figure 3B). Hence, Tostudy therole ofGRHL2 inrenal epithelial morphogenesis, we our data are consistent with a model in which GRHL2 associates analyzed Grhl2-deficient mice8 and Grhl2-deficient IMCD-3 with H3K4 trimethylation and acts mainly as a transcriptional cells. Because Grhl2 mutant mice exhibit embryonic lethality activator. Conversely, although several genes were negatively reg- at E11.5, we focused our analysis on the nephric duct epithelium ulated by GRHL2, including epithelial-to-mesenchymal tran- at E10.0, a stage at which Grhl2-deficient embryos and their sition (EMT)–associated genes encoding vimentin (Vim), zinc control littermates are still comparable in size and developmen- finger E-box binding homeobox 1 (Zeb1), and twist basic helix- tal stage. We analyzed nephric duct morphology in the posterior loop-helix transcription factor 2 (Twist2), we did not find ev- part of the mesonephros where mesonephric tubules are visible idence for direct physical interaction of GRHL2 with these but not connected to the nephric duct. There, the nephric duct genes (Figure 3B and Supplemental Figure 3 showing GRHL2 epithelium forms a uniform straight tubule with a relatively ChIP-seq data at the Vim, Zeb1, and Twist2 loci), suggesting constant lumen area, which can be quantitated in cross-sections that GRHL2 may regulate their expression by indirect effects. of the embryo. In control embryos, the epithelium was cuboidal and formed a fluid-filled lumen at this stage, expressing the The Core Target Gene Set of GRHL2 Includes the Gene nephric duct marker PAX2 (Figure 4C). In Grhl2 mutants, the Encoding Ovol2 nephric duct still revealed a cuboidal epithelium, but the lumen To search for relevant GRHL2 targets, we combined the data of was markedly reduced and occasionally displayed a complete our experiments and defined strict criteria for possible target collapse (Figure 4, A and C). Average lumen area was reduced to genes, including the following: (1) differential expression be- 27% in these ducts compared with control littermate nephric ducts tween Grhl2-Con and Grhl2-KD cells, (2) successful rescue of (Figure 4B). Epithelial polarization of Grhl2 mutant nephric ducts

2 2 same level as in A using antibodies against PAX2, b-catenin (CTNNB1), and PAR3 in control and Grhl2 / nephric ducts. (D–I) IMCD-3 collecting duct cell cysts formed in Matrigel after 5 days of culture are immunoﬂuorescence stained and analyzed by confocal microscopy. (D) To evaluate lumen expansion cysts are stained with antibodies against GRHL2 and podocalyxin (PODXL). Nuclei are counterstained using SYTOXGreen. (E) Percentages of cysts that did not develop a single open lumen are shown (mean6SEM). (F) The area bounded by the apical, podocalyxin-marked membrane of the cells is determined at the level of the largest lumen diameter of each cyst (median and IQR). Cysts that did not develop an open lumen are assigned an area of 0. (G) Cysts are analyzed for the number of cells surrounding the lumen at the largest diameter (mean6SEM). (H) Volumes of individual cells are determined using Imaris software after three-dimensional reconstruction (median and IQR). (I) Representative cysts are shown after immunoﬂuorescence staining using antibodies against PAR3, b-catenin (CTNNB1), and PODXL. Grhl2-Con, Grhl2-KD, and Grhl2-KD; Grhl2-Res cells are grown on polycarbonate membranes. Measurements of TERs are shown for the indicated days (mean6SEM). *P,0.05; **P,0.01; ***P,0.001. Con, control; ND, nephric duct; MT, mesonephric tubules; IQR, interquartile range. Bar, 25 mminAandC;38mminD;35mminI.

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Figure 5. Re-expression of Ovol2 after Grhl2 knockdown rescues lumen formation and reinduces GRHL2-dependent genes. (A) Val- idation of differential H3K4 trimethylation at the Ovol2 TSS between Grhl2-KD and Grhl2-Con cells using ChIP-quantitative PCR. Shown are mean values of percent recovery 6SD. Primers amplified a region in the H3K4me3 peak area, 0.5 kb downstream of Ovol2 TSS. Chromatin is amplified for a negative control locus. IgG is used as negative antibody control. (B) ChIP-quantitative PCR using an antibody against GRHL2 reveals an enrichment at the Ovol2 promoter (0.1 kb upstream of Ovol2 TSS) compared with a negative control locus in IMCD-3 chromatin. (C) mRNA expression of Ovol2 is analyzed using quantitative RT-PCR in Grhl2-KD, Grhl2-Con, and Grhl2-KD; Grhl2-Res cells (mean6SEM). (D) After 5 days of culture in Matrigel, cysts from Grhl2-Con, Grhl2-KD, and Grhl2-KD; Ovol2- Res cells are immunofluorescence stained and analyzed by confocal microscopy. Representative cysts stained with antibodies against GRHL2 and PODXL are shown. Nuclei are counterstained using SYTOXGreen. (E) Grhl2-Con, Grhl2-KD, and Grhl2-KD; Ovol2-Res cells are grown on polycarbonate membranes. Measurements of TER are shown for the indicated days (mean6SEM). (F and G) Cysts grown in Matrigel for 5 days are analyzed for lumen formation. (F) Percentages of cysts that did not develop a single open lumen are shown (mean6SEM). (G) Areas generated from sections with the largest lumen diameter are calculated for every cyst (median and IQR). (H)

2710 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 2704–2715, 2015 www.jasn.org BASIC RESEARCH appeared intact based on the localization of b-catenin and PAR3 and the tight junction protein zonula occludens 1 (Figure 4I and (Figure 4C). Supplemental Figure 4E), revealing no overt abnormalities in We next examined IMCD-3 epithelial morphogenesis in Grhl2-KD cysts compared with Grhl2-Con and Grhl2-KD; three-dimensional cultures and barrier formation through Grhl2-Res cysts. Together, these analyses indicate that Grhl2- measurements of transepithelial resistance (TER) of IMCD-3 deficient collecting duct cells display a selective defect of lumen monolayers in transwells. Grhl2-Con, Grhl2-KD, and Grhl2-KD; expansion without evidence of mitotic axis deviation, multiple Grhl2-Res IMCD-3 cells were seeded in Matrigel. During a lumen formation, or disruption of cell polarity. culture period of 5 days, Grhl2-Con cells formed epithelial cysts, Next, we analyzed the effect of Grhl2 inactivation on barrier consisting of polarized epithelial cells surrounding a central lu- formation in IMCD-3 monolayers by measuring TER over a 3- men outlined by podocalyxin, a marker of the apical membrane. day culture period. Grhl2-KD cells displayed a significantly Conversely,Grhl2-KD cells displayed a collapse of the cyst lumen. lower TER than Grhl2-Con cells, which was appropriately res- Although the apical plasma membranes of the Grhl2-KD cells cued in Grhl2-KD; Grhl2-Res cells (Figure 4J). Extending the were formed and podocalyxin was localized apically, the lumen culture period for up to 7 days did not alter this finding (Sup- frequently failed to expand with an undetectable or markedly plemental Figure 4F). Together, these data suggest that GRHL2 diminished luminal space (Figure 4, D and E). Expression of regulates collecting duct cell lumen expansion independent Grhl2-Res in Grhl2-KD cells restored the number of open lumen- of cell number or alteration of apicobasal cell polarization, but forming cysts, indicating that GRHL2 specifically mediates this by a mechanism that may involve enforcement of the epithelial effect (Figure 4, D and E). Extending the culture period to 9 days barrier. did not substantially alter these observations, indicating a qual- itative defect of lumen expansion rather than a temporal delay Ovol2 Reinduces Central Components of the GRHL2 (Supplemental Figure 4, A and B). Of note, we observed mul- Network and Rescues TER and Lumen Expansion in tiple lumen formation only in rare instances, both in Grhl2- Grhl2-Deficient Cells Con cysts and in Grhl2-KD cysts. To quantitate the defect in The gene encoding OVOL2, a mammalian homolog of Drosophila lumen expansion, the lumen area bounded by the apical, ovo,26 was among the core targets of GRHL2. Deletion of Ovol2 podocalyxin-marked membrane at the center and at maximal in mice results in an embryonic phenotype and death by E10.5,27 lumen diameter of each cyst was analyzed. This revealed profound similar to the phenotype observed in Grhl2 mutant mice. Ovol2 differences in lumen size between Grhl2-Con and Grhl2-KD cells expression correlates with hallmark genes of epithelial differen- and their rescue in Grhl2-KD; Grhl2-Res cells (Figure 4F). tiation in cancer cell lines.28 Therefore, we decided to further To determine whether a reduced number of cells or differences investigate the potential functional role of Ovol2 downstream of in the cell volume of Grhl2-KD cells relative to controls GRHL2. Downregulation of Ovol2 and H3K4 trimethylation at contributed to the defective lumen expansion in Grhl2-KD the Ovol2 promoter in Grhl2-KD cells and GRHL2-binding to cysts, we analyzed the number of cells surrounding the lumen at the Ovol2 promoter in IMCD-3 cells were validated by quanti- the largest cyst diameter and the single cell volume within each tative RT-PCR and ChIP-quantitative PCR, respectively (Figure 5, cyst. Analysis exhibited no significant differences in the number A–C). We next analyzed the effect of ectopically expressed full- of cyst-lining cells between Grhl2-Con, Grhl2-KD, and Grhl2- length Ovol2 in Grhl2-KD cells on lumen formation and barrier KD; Grhl2-Res cells (Figure 4G). Grhl2-KD cells showed no function by generating Grhl2-KD; Ovol2-Res cells (Supplemen- significant reduction in single cell volume compared with con- tal Figure 5A). Remarkably, Ovol2 re-expression significantly trol cells, whereas Grhl2-KD; Grhl2-Res cells displayed a signif- increased the percentage of cysts establishing a single open lu- icantly increased single cell volume compared with Grhl2-KD men and resulted in a higher TER compared with Grhl2-KD and Grhl2-Con cells (Figure 4H). Recent investigations showed cells (Figure 5, D–G), indicating that reinduction of Ovol2 ex- that a defective single lumen formation might be explained by a pression in Grhl2-deficient cells restored the hallmarks of disrupted orientation of the mitotic spindle during cell divi- GRHL2 inactivation. To analyze the molecular basis of these sion.23–25 We found that in Grhl2-KD cells, the mitotic spindle effects, we performed gene expression profiling using microar- axis was mainly perpendicular to the podocalyxin-marked cen- rays in Grhl2-Con, Grhl2-KD, and Grhl2-KD; Ovol2-Res cells. ter of the cysts, comparable to Grhl2-Con and Grhl2-KD; Grhl2- Unexpectedly, this revealed that 75.7% of the genes downregu- Res cells (Supplemental Figure 4, C and D). We next analyzed lated in Grhl2 deficiency were reinduced by Ovol2 overexpres- markers of apicobasal polarity by staining for the basolateral sion (Figure 5H). Among these genes were Cldn4, Cdh1,and marker b-catenin, the cell polarity complex component PAR3, Rab25, which were rescued to baseline levels in Grhl2-KD;

Microarray gene expression analysis is performed on Grhl2-Con (n=3), Grhl2-KD (n=3), and Grhl2-KD; Ovol2-Res (n=3) cells. The top 250 differentially expressed genes are hierarchically clustered (Pearson correlation, average linkage clustering). Note substantial reinduction of Grhl2-dependent genes in Grhl2-KD cells after Ovol2 overexpression. (I) Western blots for CDH1, GRHL2, CLDN4, and RAB25 are performed on Grhl2-Con, Grhl2-KD, and Grhl2-KD; Ovol2-Res cells. a-Tubulin is used as the loading control. *P,0.05; **P,0.01; ***P,0.001. TSS, transcriptional start site; IQR, interquartile range; DDCt, difference in cycle threshold. Bar, 38 mminD.

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Figure 6. CLDN4 and RAB25 are critical mediators of GRHL2 function. Cdh1, Cldn4,andRab25 are re-expressed in Grhl2-KD cells. Protein expression is evaluated using Western blots (left panels). Effects on lumen formation (center panels) and TER (right panels) are quantiﬁed as described above. Grhl2-Con, Grhl2-KD, and Grhl2-KD cells overexpressing Cdh1 (Grhl2-KD; Cdh1-Res) (A), Cldn4 (Grhl2-KD; Cldn4-Res) (B), Rab25 (Grhl2-KD; Rab25-Res) (C), or a combination of Cldn4 and Rab25 (Grhl2-KD; Cldn4-Res; Rab25-Res) (D) were analyzed. *P,0.05; **P,0.01; ***P,0.001.

2712 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 2704–2715, 2015 www.jasn.org BASIC RESEARCH

Ovol2-Res cells based on mRNA and protein levels (Figure 5I, Supplemental Figure 5B). Interestingly, re-expression of Ovol2 also resuppressed the expression of 85.5% of genes that were upregulated in Grhl2-KD cells, including the EMT-associated genes Vim, Zeb1,andTwist2. Recent studies implicated CDH1, RAB25, and CLDN4 in epithelial lumen formation.12,29,30 Although CDH1 and CLDN4 are components of the apical junctional complex, RAB25 appears to be important for cargo shuttling to the apical domain. Therefore, we decided to re-express each of Figure 7. A putative transcriptional network downstream of Grhl2 these genes in Grhl2-KD cells to assay for their sufficiency to regulating barrier formation and lumen expansion in collecting replace GRHL2 function. Re-expression of Cldn4 in Grhl2-KD duct cells. Asterisks indicate direct regulations, whereas inter- cells was sufficient to completely restore TER in transwell actions indicated by open arrowheads may be direct or indirect. monolayers, whereas Cdh1 and Rab25 were insufficient to rescue TER (Figure 6, A–C). However, re-expression of neither of these genes alone was sufficient to rescue lumen expansion in Grhl2- previously been associated with defective MDCK cyst lumen for- KD cells (Figure 6, A–C). This suggested that GRHL2 drove mation.29 Inactivation of Rab25 or the related Rab11a resulted barrier formation via Cldn4 activation but that this alone was in a multiple lumen phenotype in epithelial cysts, which is char- insufficient to explain the effect of GRHL2 on lumen expansion. acterized by failure of lumen expansion and retention of podo- Because Rab25 had previously been implicated in epithelial lu- calyxin in subapical vesicles.29,33 Multiple lumen formation has men formation,29 we re-expressed a combination of Cldn4 and been linked with defective CDC42-mediated orientation of the Rab25 in Grhl2-KD cells. Notably, this rescued both lumen for- mitotic spindle.23,24 Interestingly, in response to Grhl2 inactiva- mation and TER (Figure 6D). Together these data identify a tion in IMCD-3 cells, we found no evidence of multiple lumen novel Grhl2/Ovol2 pathway that reinforces Cldn4 and Rab25 formation or misorientation of the mitotic spindle. In fact, even expression and thereby regulates lumen expansion and barrier extended culture of Grhl2-deficient IMCD-3 cysts for 9 days formation. produced regularly aligned polarized cells surrounding a single large patch of podocalyxin without any visible lumen (Supple- mental Figure 4A). This may be an intrinsic property of collect- DISCUSSION ing duct cells and distinct from the MDCK or Caco-2 cell systems. However, it suggests that Grhl2-deficient collecting We present the first functional and molecular analysis of GRHL2 duct cells exhibit a defect of lumen expansion that occurs sub- and its downstream signaling events in renal epithelia. Through sequent to adequate cell alignment and polarization. Because an integration of gene expression data, genome-wide GRHL2 previous studies indicated that lumen expansion is driven by binding in vivo and in vitro, and global analysis of H3K4 trime- fluid accumulation34–37 and requires the formation of tight thylation, we were able to derive GRHL2 downstream targets paracellular barriers,36 we propose that GRHL2 regulates lu- and discovered a Grhl2/Ovol2 network controlling lumen expan- men expansion by stabilizing the epithelial barrier via CLDN4 sion and barrier formation. We show that GRHL2 expression is and facilitating apical fluid excretion via RAB25. However, this high in nephric ducts, in ureteric bud epithelia, and in distal hypothesis will require additional experimental support. nephron epithelia and collecting ducts. A loss of Grhl2 leads Our study also provides novel insights into the molecular to a reduced or even collapsed lumen in E10.0 nephric ducts. regulation of target genes by GRHL2. We show that GRHL2 acts Functional analyses using IMCD-3 cells showed that GRHL2 as a predominant activator of target genes, supporting and controls epithelial barrier formation (determined by TER) and expanding our previous finding of GRHL2 transactivation of lumen expansion. Molecular analyses indicate a network involv- Cdh1 and Cldn4.8 This conclusion is based on genome-wide ing GRHL2-mediated activation of Ovol2,bothofwhichrein- evidence of strong association of GRHL2 with genes that are force expression of important epithelial effectors, including downregulated when GRHL2 function is lost and with regions CDH1, CLDN4, and RAB25. In addition, our data suggest that of decreased H3K4 trimethylation in Grhl2-KD. Conversely, we GRHL2-induced CLDN4 expression mediates increased barrier found little evidence of a direct repressor function of GRHL2, formation,butthisisinsufficient to completely explain the effect because we saw no evidence of a direct association of GRHL2 on lumen formation. Only a joint re-expression of CLDN4 and with genes that are upregulated in Grhl2-deficient cells or with RAB25 was able to rescue lumen expansion in Grhl2-deficient corresponding regions of upregulated H3K4 trimethylation in IMCD-3 cells, suggesting that RAB25 mediates important as- Grhl2-KD. This finding is in contrast with previous reports of a pects of lumen formation downstream of GRHL2 that go be- direct suppressor effect of GRHL2 at for instance EMT-associated yond increasing the epithelial barrier. genes, including the mesenchymal transcription factor ZEB1.14,38 Rab25,aRab11 GTPase family member, has been reported to Although we observed an activation of EMT-associated genes control apical trafficking and transcytosis.31,32 and has Zeb1, Vim,andTwist2 in Grhl2-deficient cells, we did not find

J Am Soc Nephrol 26: 2704–2715, 2015 Grhl2/Ovol2 in Lumen and Barrier Formation 2713 BASIC RESEARCH www.jasn.org direct association of GRHL2 with their promoters or other adja- recombinant lentiviruses, lentiviral gene transfer, PCR, microarray cent gene-regulatory regions. These discrepancies may be related analysis, ChIP-seq technology, immunofluorescence and immuno- to differences in tissues or cell types between different studies. histochemical staining, Western blot, and measurements of TERs as Our identified core target Ovol2 is a member of an evolution- outlined in detail in the Supplemental Materials and Methods. ary conserved family of zinc finger transcription factors. Ovol2 is involved in diverse processes such as keratinocyte differentiation and cell fate decisions, as well as embryonic development.27,39,40 Our current data reveal that most differentially regulated genes ACKNOWLEDGMENTS in Grhl2-deficient cells were rescued to their original expression levels, when Ovol2 was re-expressed in these cells. Furthermore, We thank Antje Sommer, Tatjana Luganskaja, Gabriel Kirchgraber, we show that Ovol2 is sufficient to functionally replace Grhl2 in Kirstin Rautenberg, and Siegrun Blauhut for their excellent technical lumen expansion and barrier function through induction of support. We also thank Dr. Nora Mecklenburg for help with paraffin Cldn4 and Rab25. Potentially, OVOL2 may mediate the previ- embedding of murine embryos, Dr. Friedrich C. Luft for critical ously identified function of GRHL2 as a suppressor of EMT. In a comments concerning the manuscript, Dr. Hans-Peter Rahn (Max recent publication, it was shown that OVOL2 is necessary to Delbrück Center Preparative Flow Cytometry Facility), and Dr. Anje suppress EMT in mammary cells with OVOL2 ChIP peaks Sporbert (Max Delbrück Center Advanced Light Microscopy Facility). near the Vim, Twist1,andZeb1 promoters.41 Consistently, our This work was funded by the German Research Foundation data show that Ovol2 overexpression can abolish the upregula- (Emmy-Noether grant (to K.S.), Research Unit 667 grant (to K.S.), tion of EMT genes Vim, Zeb1,andTw ist2 in Grhl2-deficient cells. and Research Unit 1368 grant (to K.S.)) and the Urological Research The fact that CLDN4 and RAB25 together were sufficient to Foundation. functionally replace GRHL2 in lumen formation and barrier function suggests that suppression of EMT genes is not a man- datory prerequisite for these processes. DISCLOSURES The novel gene network we derived has two input transcrip- None. tionfactors,oneofwhich(GRHL2)regulatestheother(OVOL2), both jointly regulating effector target genes (e.g., Cdh1, Cldn4, and Rab25; see Figure 7 for a putative transcriptional network). REFERENCES This system bears the characteristics of a feed-forward signaling 42 system. Feed-forward loops of this type are widespread regu- 1. Madsen KM, Clapp WL, Verlander JW: Structure and function of the latory elements and are implicated in sign-sensitive delay and inner medullary collecting duct. Kidney Int 34: 441–454, 1988 preventing inactivation of the target gene program when input 2. Little M, Georgas K, Pennisi D, Wilkinson L: Kidney development: Two – signals are lost (i.e., they serve as stabilizers of target gene ex- tales of tubulogenesis. Curr Top Dev Biol 90: 193 229, 2010 3. 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J Am Soc Nephrol 26: 2704–2715, 2015 Grhl2/Ovol2 in Lumen and Barrier Formation 2715 A Grainyhead-Like 2/Ovo-Like 2 Pathway Regulates Renal Epithelial Barrier Function and Lumen Expansion

Annekatrin Aue,*† Christian Hinze,*‡ Katharina Walentin,* Janett Ruffert,* Yesim Yurtdas,*§| Max Werth,* Wei Chen,* Anja Rabien,§| Ergin Kilic,** Jörg-Dieter Schulzke,†† Michael Schumann,†† and Kai M. Schmidt-Ott*†‡ *Max Delbrueck Center for Molecular Medicine, Berlin, Germany; † Experimental and Clinical Research Center, and Departments of ‡ Nephrology, § Urology, **Pathology, and †† Gastroenterology, Charité Medical University, Berlin, Germany; | Berlin Institute of Urologic Research, Berlin, Germany

A.A. and C.H. contributed equally to this work

#Corresponding author:

Prof. Dr. Kai M. Schmidt-Ott

Max Delbrueck Center for Molecular Medicine

Robert-Rössle-Str 10

13125 Berlin, Germany

Phone:+49 30 9406 2512

Fax: +49 30 9406 2536

Email: [email protected] Supplements

Materials and Methods...... 3 Supplemental Figure 1 Grhl2 binding through ChIP-seq and derived target genes by combining ChIP-seq and gene expression data...... 10 Supplemental Figure 2 Validation of IMCD-3 Grhl2-KD cells...... 11 Supplemental Figure 3 Grhl2 ChIP tracks at selected core targets and EMT genes ...... 13 Supplemental Figure 4 Characterization of IMCD-3 Grhl2-KD cysts at several time ponits...... 14 Supplemental Figure 5 Validation of IMCD-3 Grhl2-KD;Ovol2-Res cells...... 16 Supplemental Table 1 Differentially expressed genes between Grhl2-Con and Grhl2-KD...... 17 Supplemental Table 2 Gene ontology analysis on differentially expressed genes...... 27 Supplemental Table 3 Grhl2 core target module...... 35 Supplemental Table 4 Primers used for quantitative RT-PCR...... 37 Supplemental Table 5 Primers used for quantitative ChIP-PCR...... 37 Supplemental References...... 38

2 Materials and Methods

Human tissue samples

Paraffin material was chosen from the archive of the Institute of Pathology of the Charité Berlin and used when adequate tissue was available. The study was undertaken according to approved institutional review board protocols. It was conducted within the framework of the declaration of

Helsinki.

Mouse nephric duct samples

Paraffin-embedded E10.0 embryos were cut into 5μm sections. For lumen area quantification, HE stained sections were analyzed at the level of the posterior part of the mesonephros where mesonephric tubules were visible but not connected to the nephric duct. The measurements were performed using ImageJ.

Antibodies

The following antibodies were used: Anti-Grhl2 (HPA004820, Sigma-Aldrich, Munich, Germany), anti-Aquaporin 2 (SC-9882, Santa Cruz Biotechnology, Heidelberg, Germany), Anti-Podocalyxin

(AF1556, R&D Systems, Abingdon, United Kingdom), Anti-a-Tubulin (T9026, SIGMA-Aldrich), Anti-

Claudin4 (36-4800, Life technologies, Darmstadt, Germany), Anti-E-Cadherin (610182, BD

Bioscience, Heidelberg, Germany), Anti-Par-3 (07-330, Merck Millipore, Darmstadt, Germany),

Anti-Rab25 (R8407, SIGMA-Aldrich), Ant-b-Catenin (610154, BD Bioscience) and Anti-Cytokeratin

8 (TROMA-I, Developmental Studies Hybridoma Bank, DSHB), that was developed by Philippe

Brulet and Rolf Kemler under the auspices of the NICHD and maintained by the University of Iowa,

Department of Biology, Iowa City.

Cloning procedure/ lentiviral particles

All plasmids for the production of lentiviral particles were purchased from Addgene (Cambridge,

USA) Lentiviral particles were generated using the 2nd generation packaging system, comprising the envelope plasmid psPAX2 (Addgene plasmid 12260) and packaging plasmid pMD2.G

(Addgene plasmid 12259) established in the laboratories of Didier Trono (EPFL, Lausanne,

Switzerland). 1 The Wiznerowicz laboratories (lentiweb.com) provided protocols for the production

3 and titration of lentiviral particles. To establish a lentiviral mediated KD of Grhl2, H1 promoter- shRNA cassettes were cloned from pSUPER directly into the vector plasmid pLVTHM (Addgene plasmid 12247) using EcoRI and ClaI restriction sites. shRNAs directed against Grhl2 target the sequence 5´-GACTTCCCCTGATGATTCA-3´. A shRNA targeting 5′-

CATCACGTACGCGGAATACT-3′ of firefly luciferase was used as a negative control.

Full-length cDNA of Cldn4 and Rab25 were amplified from IMCD3 cDNA using Phusion High-

Fidelity DNA Polymerase (New England BioLabs, Frankfurt am Main, Germany). Full-length cDNA of Ovol2A was generated from cDNA of wild-type kidney. The coding sequence of Cdh1 was kindly provided from the Daniel Besser laboratories.2 A Grhl2 rescue construct, which is resistant to the

Grhl2-shRNA was generated in our lab.3 We used full-length cDNA of click beetle luciferase as control cDNA. Vector plasmid pLVUT-tTR-KRAB (Addgene plasmid 11651) was utilized to produce lentiviral particles to integrate expression cassettes of cDNAs in IMCD3 Grhl2-KD cells. The GFP-

EMCV IRES-tetR-KRAB domain was replaced by cDNA-IRES-mCherry.

Cell culture/ three-dimensional (3D) cultivation of IMCD-3 cells

IMCD-3 cells were purchased from ATCC (CRL-2123, Manassas, USA) and maintained in

DMEM+GlutaMAX (Life technologies) supplemented with 10% fetal bovine serum (FBS) and penicillin/ streptomycin (100 Units/ ml/ 100 µg/ ml). Cells were sub-cultured two to three times per week in a ratio of 1:4 to 1:8 using 0.25% trypsin, 0.5 mM EDTA in 1xPBS.

IMCD3 cells were transduced with lentiviral particles generated from pLVTHMshRNA at a multiplicity of infection (MOI) of 5. At the same time cells were infected with a second type of lentiviral particle, generated from pLVUT-cDNA-mCherry (MOI of 0.5). Cells were expanded for at least six days and EGFP- and mCherry-expressing cells were isolated using Fluorescence- activated cell sorting (FACS, BD FACSAriaII, BD Biosciences).

For 3D cultivation, IMCD-3 Grhl2-Con, Grhl2-KD, Grhl2-KD;Grhl2-Res were trypsinized and centrifuged to remove the complete growth medium. A suspension of 1.5x104 cells in 150µl serum- free medium were given to 150 µl Matrigel (Basement Membrane Matrix, BD Biosciences) and plated in Nunc® LabTek® 8-well chamber slides (Thermo Fisher Scientific, Bonn, Germany). After incubation for 30 min at 37°C complete growth medium was added to cover the Matrigel. Medium

4 was changed every second day.

RNA extraction and cDNA synthesis

RNA was extracted from IMCD-3 cells using RNeasy Mini-kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions including treatment with RNase-free DNase I (Qiagen). 500 ng total

RNA of each sample was utilized for cDNA synthesis using RevertAid First Strand cDNA Synthesis

Kit (Fermentas, St. Leon-Rot, Germany) according to manufacturer’s instructions.

Real-time PCR

Quantitative Real-time PCR was performed using FastStart Universal SYBR Green Master (Rox)

(Roche Diagnostics, Mannheim, Germany) with cDNA as template and the primers listed in Table

S4. Relative mRNA expression was normalized to -actin and calculated according to Ct method.

Microarray

Illumina TotalPrep RNA Amplification kit (Life technologies) was used to synthesis cRNA out of

RNA isolated from IMCD3 cells according to manufacturer’s instructions. Quality of RNA and cRNA was evaluated using Agilent 2100 Bioanalyzer and the Agilent RNA 6000 Nano kit (Agilent

Technologies, Böblingen, Germany). We used MouseWG-6 v2.0 Expression BeadChips (Illumina,

Essex, UK) to analyze genome-wide gene expression profiles.

Chromatin Immunoprecipitation Sequencing (ChIP-Seq)

We performed ChIP analysis using an antibody directed against H3K4me3 (pAB-003-010, diagenode, Seraing (Ougrée), Belgium) on IMCD-3 Grhl2-KD and Grhl2-Con using the iDeal ChIP- seq kit (diagenode) according to manufacturer’s instructions. The protocol was modified as follows.

Cells were grown to confluence and cowered with DMEM. Cross-linking was done with 1% formaldehyde on a shaking platform for 10 min at room temperature (RT). The reaction was quenched with 125 mM glycine. DMEM was removed from the plates and cells were washed with ice-cold 1xPBS than following the kit’s manual. ChIP experiments were performed on IMCD-3 cells, embryonic (embryonic day E15.5) and adult kidney of mice using an antibody against Grhl2.

IMCD-3 cells were grown to 100% confluence, 5×106 cells were cross-linked with 1% formaldehyde for 20 min at room temperature (RT). Kidneys were cut into small pieces and

5 homogenized in Lysis Buffer (15mM HEPES (pH=7.8), 1.5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM DTT, 10% Glycerol) with 0.5% IGEPAL and proteinase inhibitors. Nuclei were fixed in Lysis

Buffer with 1% formaldehyde for 20 min (RT). Chromatin was fragmented to an average size of

300-500 bp. Chromatin (100 μg) and 5 μg of antibody was used per assay using Chromatin

Immunoprecipitation (ChIP) Assay Kit (Merck Millipore, Darmstadt, Germany). IP’d DNA concentration was determined using the Qubit system (Qubit ds DNA HS Assay kit, Molecular probes/ Life Technologies) and quality was controlled using Agilent 2100 Bioanalyzer and the

Agilent High Sensitivity DNA kit (Agilent Technologies). For library preparation the TruSeq ChIP-

Seq Sample Prep Kit (illumina) was used according to manufacturer’s instructions. Flow cell was sequenced using illumina HiSeq 2000 (50 bp reads). Primers used for quantitative ChIP-PCR are listed in Table S5.

Immunofluorescence/ immunohistochemical staining

Immunohistochemical staining on human tissue was performed on 5 µm paraffin-embedded, dewaxed and rehydrated sections. Samples were blocked in 5% bovine serum albumin (BSA) in

1xTBS, 0.025% Tween20 and stained with primary antibodies and secondary antibodies labelled to alkaline phosphatase. Enzyme activity was detected using SIGMA FASTTM Fast Red TR/Naphthol

AS-MX Alkaline Phosphatase Substrate Tablets Set (SIGMA-Aldrich) according to manufacturer’s instructions.

Embryonic (E18) or adult (P30) kidneys of C57BL/6 mice were cut in slices and fixed in 4% paraformaldehyde (PFA). 16 µm cryosections were incubated with 1% bovine serum albumin

(BSA), 0.1% Triton X-100, stained with primary antibodies and secondary antibodies labeled with

Cy2, Cy3, Cy5, Alexa488 or Alexa 647 (JacksonImmunoResearch, Newmarket, UK). Staining for

Grhl2 required preliminary treatment with PBS/citric acid (10 mM).

IMCD-3 cysts grown in 3D culture were fixed with 4% PFA for 30 min on ice while rotating gently.

After incubation with 50mM NH4Cl, cells were permeabilized with 0.5% TritonX-100 in 1xPBS.

Cysts were blocked with 1%BSA, 0.25% TritonX-100 in 1xPBS and incubated with primary antibodies and secondary antibodies in combination with nuclei counterstaining substance

SYTOX® Green (Life Technologies). Cells were mounted using Dako Mounting Medium (Agilent

6 Technologies).

Confocal images were obtained with an inverted Zeiss LSM700 (Carl Zeiss Microscopy, Jena,

Germany) or TCS SP5 Tandem Microscope (Leica microsystems, Wetzlar, Germany).

Western Blot

Whole-cell lysates of IMCD-3 cells were prepared using RIPA buffer (50 mM Tris-HCl, 150 mM

NaCl, 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS, 1X Protease inhibitor from Roche

Diagnostics, Mannheim, Germany). Total protein was quantitated using Pierce BCA Protein Assay kit (Thermo Fisher Scientific) according to manufacturer’s instructions.

Protein samples were incubated with NuPAGE loading buffer (10% -mercaptoethanol added) at

95°C for 10 min. For sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), samples were loaded on Novex Bis-Tris 4-12% precast gels and electrophoresis was performed according to the manufacturer's instructions using MOPS SDS running buffer (50 mM MOPS, 50 mM TrisBase, 3.5 mM SDS, 1.0 mM EDTA) and the XCell SureLock® Mini-Cell (Life technologies).

Proteins were transferred onto PVDF membrane using 1.25 mM Bicine, 1.25 mM BisTris, 0.05 mM

EDTA, 10% methanol. Blocking was performed in 1xTris-buffered saline (TBS, 50 mM Tris-Cl, pH

7.6, 150 mM NaCl) and 0.05% Tween20. Membranes were incubated with primary antibodies and secondary antibodies conjugated to horseradish peroxidase. Chemilumenscence was detected using the SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific).

Relative signal intensity was analyzed using Adope Photoshop.

Transepithelial Resistance (TER) Measurements

IMCD-3 cell were grown on 0.4 µm PCF membranes. TER measurements were done using Ussing chambers and chopsick electrodes as described previously.4 Statistical analysis was done using

Student’s t-test.

Statistical analysis

Data represent analysis of at least three independent experiments. Statistical analysis was done using Student’s t-test or Mann-Whitney-U Test.

Microarray data analysis

After quantile normalization of the data, we considered genes as differentially expressed if a fold

7 change larger than or equal to 1.4 (control vs. KD) and a p-value less than 0.05 using Student's t test were present. Heatmap visualization of the microarray data was done using MultiExperiment

Viewer (http://www.tm4.org/). Heatmap visualized data has been maximum normalized on a per- gene basis. Gene names are according to the used Illumina microarray chip (MouseWG-6 v2). A gene was considered “successfully rescued” in one of the rescue constructs, if:

1.) the p-value after t-test control vs. rescue expression values was <= 0.05 [no significant difference between control and rescue] or

2.) p-value t-test rescue vs. Grhl2-KD <0.05 and average(rescue)/average(control)>=1/1.4 for differential genes downregulated in KD (that is, average(rescue)/average(control)>=1.4) [significant difference between rescue and KD + right fold change tendency for DE genes downregulated in

KD] or

3.) p-value t-test rescue vs. Grhl2-KD <0.05 and average(rescue)/average(control)<=1.4 for differential genes upregulated in KD (that is, average(rescue)/average(control)<=1/1.4) [significant difference between rescue and KD + right fold change tendency for DE genes upregulated in KD].

ChIP data analysis

The alignment was done using Bowtie2. The reads were then filtered by applying a mapping quality threshold (MAPQ>20), removing PCR duplicates, and considering only uniquely mapping reads with less than 1bp mismatch. We used MACS for peak calling with default parameters

(P<1e-05). Foreground and background models used for peak finding are mentioned in the main paper as well as total peak numbers. The peaks were ranked according to the intrinsic scores provided by MACS (negative log10 transform of the peak's p-value). Motif finding was then performed using MEME on the top 10% of all peaks in a 50 bp window around the peak's summit and optimized for de novo detection of 8-9 base pair motifs.5 Motif enrichment in a given sequence set was done using MAST and FIMO.6,7 Visualization of the ChIP data was carried out using the

Integrative Genome Viewer provided by the Broad Institute.8,9

Miscellaneous Bioinformatics

Most of the plots of the bioinformatics related figures were generated with custom-made python scripts using matplotlib.10 Operations on genomic intervals (e.g. generation of random peaks,

8 distance calculations) were based on bedtools and its python interface pybedtools.11,12

Analagously, operations on sam and bam files were performed with samtools and pysam,13 respectively. Gene ontology and ChIP peak annotation was done using HOMER.14

9 Supplemental Figure 1 Grhl2 binding through ChIP-seq and derived target genes by combining ChIP-seq and gene expression data. A Venn diagram for all Grhl2 ChIP peaks shows a significant overlap between IMCD-3 cells, adult, and E18 kidney. B The Distance distribution of IMCD-3 Grhl2 ChIP peaks from adult kidney Grhl2 ChIP peaks reveal two distinct groups with peaks that either intersect or occur at larger distances (>10 kb) from each other. The bottom orange subplot shows the distance distribution from a random set of control peaks based on adult kidney Grhl2 ChIP peaks. C Overview of the Grhl2 target gene set with scores and key values from ChIP-seq experiments. The Grhl2 peak score is referring to MACS generic peak score. In case of multiple peak occurrences, the scores refer to the most significant peaks fulfilling the criteria mentioned in the main text. D Grhl2 target genes with ChIP tracks at the Grhl2 and differential H3K4me3 peak areas which have been used for Grhl2 target identification. Target genes were obtained based on differential expression in microarray data, differential H3K4 trimethylation, and Grhl2 ChIP peak occurrence (with Grhl2 binding motif) in IMCD-3 cells and adult kidney (differential H3K4me3 peak occuring ±500bp from TSS and/or within genomic region, Grhl2 ChIP peaks intersecting each other and H3K4me3 peak area). Note that most of the targets also exhibit a ChIP peak in E18 kidney.

10 Supplemental Figure 2 Validation of IMCD-3 Grhl2-KD cells. IMCD-3 cells were infected with lentiviral particles to stably integrate expression cassettes for a Grhl2 shRNA (or control shRNA) and the marker gene GFP A. Lentiviral particles were used in a multiplicity of infection (MOI) of 5. At the same time, cells were transduced to integrate a cassette for synthesis of a Grhl2 rescue (Grhl2-Res) construct (or control cDNA), followed by mCherry (MOI: 0.5). B At least six days after infection, GFP- and mCherry-expressing cells were isolated using Fluorescence-activated cell sorting (FACS; IMCD-3 negative: non-infected cells; Control (Grhl2-Con): control shRNA, GFP plus control cDNA and mCherry; Grhl2-KD: Grhl2 shRNA, GFP plus cDNA and mCherry; Grhl2-KD/Grhl2-Res: Grhl2 shRNA, GFP plus Grhl2-Res and mCherry).

11 Isolated cells were expanded and used for further investigations. Expression of Grhl2 and known targets Cdh1, Cldn4, and Rab25 was confirmed in IMCD-3 Grhl2-KD, control cells (Grhl2-Con), and Grhl2-KD;Grhl2-Res cells using quantitative RT-PCR C and Western blots D. E Shown is a quantification of GRHL2 expression from Western blots (mean SD, ***p<0.001, **p<0.01, *p<0.05).

12 Supplemental Figure 3 Grhl2 ChIP tracks at selected core targets and EMT genes. IGV snapshots of raw read count tracks from Grhl2 ChIP-seq experiments for murine E18, adult kidney, and IMCD-3 cells. The plots on the left side show 3 genes of the Grhl2 core target module (Ovol2, Cdh1, Rab25). On the right, raw read count tracks for Vim, Twist2, and Zeb1 are displayed with read count signal at noise level even at long distance around the respective gene. All tracks are scaled to the peak height of the Ovol2 tracks.

13 Supplemental Figure 4 Characterization of IMCD-3 Grhl2-KD cysts at several time points. A Culture period of Grhl2-Con, Grhl2-KD, and Grhl2-KD;Grhl2-Res was prolonged to nine days in Matrigel. Cysts were immunofluorescence-stained with antibodies against GRHL2 and podocalyxin (PODXL). Nuclei were counterstained using SYTOXGreen; scale bar: 25 µm. B Still most of Grhl2- KD cysts did not form open lumen cysts compared to Grhl2-Con and Grhl2-KD;Grhl2-Res (mean ± SEM). C, D Grhl2-Con, Grhl2-KD, and Grhl2-KD; Grhl2-Res cells were grown in Matrigel for four days. Cells were immunofluorescence-stained using antibodies against -tubulin and PODXL. Nuclei were counterstained using DAPI. C Orientation of the mitotic spindle towards the lumen, for Grhl2-Con and Grhl2-KD-Res cysts, or the podocalyxin-marked center of the whole cyst, for Grhl2- KD cysts, were analyzed (median and IQR). D Representative mitotic spindles in Grhl2-Con, Grhl2-KD and Grhl2-KD, Grhl2-Res cysts are shown; scale bar: 15 µm. E Grhl2-Con, Grhl2-KD,

14 and Grhl2-KD;Grhl2-Res cells were grown in Matrigel for five days and immunofluorescence- stained using antibodies against PODXL and tight junction protein zonula occludens-1 (ZO-1). Staining revealed no overt abnormalities in ZO-1 localization in Grhl2-KD cysts, compared to Grhl2-Con and Grhl2-KD;Grhl2-Res cysts; scale bar: 38 µm. F Grhl2-Con and Grhl2-KD cells were grown on polycarbonate membranes. Measurements of transepithelial resistances (TER) are shown for seven days (mean SEM).

15 Supplemental Figure 5 Validation of IMCD-3 Grhl2-KD;Ovol2-Res cells. A, B IMCD-3 cells were infected with lentiviral particles to integrate expression cassettes for a Grhl2 shRNA, or control shRNA, and for the marker gene GFP. At the same time, cells were transduced to integrate a cassette for synthesis of full-length cDNA of Ovol2A (Grhl2-KD; Ovol2- Res), or Luciferase control gene, followed by mCherry. GFP- and mCherry-expressing cells were isolated using FACS. A Expression of Grhl2 and Ovol2 were confirmed in Grhl2-Con, Grhl2-KD, and Grhl2-KD;Ovol2 using quantitative RT-PCR. B Shown are expression data for Cdh1, Rab25, and Cldn4 in Grhl2-Con, Grhl2-KD, and Grhl2-KD;Ovol2-Res using quantitative RT-PCR (mean SD, ***p<0.001, **p<0.01, *p<0.05).

16 Supplemental Table 1 Differentially expressed genes between Grhl2-Con and Grhl2-KD. List of differentially expressed genes with expression levels in Grhl2-Con, Grhl2-KD, and the rescue constructs for Grhl2 and Ovol2. Fold Averages changes Rescue success in Ovol2- Grhl2- ttest Res/ Res/ Con vs. Grhl2- Ovol2- Gene name (Illumina) Grhl2-Con Grhl2-KD Grhl2-Res Ovol2-Res Con/KD Con Con KD Res Res Atp8b1 596.64 244.42 586.35 225.71 2.44 0.38 0.98 0.0001 1 0 Calm3 9050.18 17566.95 17994.21 16783.07 0.52 1.85 1.99 0.0002 0 0 2310040B03Rik 562.49 898.48 540.07 712.44 0.63 1.27 0.96 0.0002 1 1 Ovol2 258.80 179.12 302.73 385.12 1.44 1.49 1.17 0.0002 1 1 Il28ra 845.52 588.41 1002.23 1101.72 1.44 1.30 1.19 0.0003 1 1 Atg9b 4526.00 1231.47 4313.38 6713.05 3.68 1.48 0.95 0.0003 1 1 LOC100046254 285.01 178.00 233.31 200.42 1.60 0.70 0.82 0.0004 1 0 Ap1m2 1015.24 312.64 967.75 1595.49 3.25 1.57 0.95 0.0004 1 1 Tmem54 3272.00 476.29 4298.71 487.02 6.87 0.15 1.31 0.0005 1 0 EG432448 6816.56 13124.74 11778.28 11879.01 0.52 1.74 1.73 0.0005 0 0 Prss8 1277.87 740.15 1045.18 1049.30 1.73 0.82 0.82 0.0005 1 1 Meis2 2043.83 2910.19 1827.38 1967.63 0.70 0.96 0.89 0.0006 1 1 Sprr1a 967.41 465.55 321.26 2090.60 2.08 2.16 0.33 0.0006 0 1 Bhlhb2 4033.83 2365.93 4860.47 5796.33 1.70 1.44 1.20 0.0006 1 1 Wisp2 159.86 241.61 238.43 155.26 0.66 0.97 1.49 0.0007 1 1 LOC100046802 282.11 598.72 187.63 633.44 0.47 2.25 0.67 0.0008 1 0 Eno3 500.11 881.53 352.73 1201.21 0.57 2.40 0.71 0.0008 1 0 Galnt3 232.26 158.01 224.47 290.53 1.47 1.25 0.97 0.0008 1 1 LOC100046211 5101.68 8774.14 6738.59 8947.54 0.58 1.75 1.32 0.0010 1 0 LOC214575 235.98 161.40 277.71 199.46 1.46 0.85 1.18 0.0010 1 1 Slc44a2 3517.47 2498.94 2938.12 2293.49 1.41 0.65 0.84 0.0010 1 0 Npal2 2019.51 769.11 1500.12 1417.14 2.63 0.70 0.74 0.0011 1 1 LOC100047167 1249.91 2194.93 1482.30 2136.44 0.57 1.71 1.19 0.0012 1 0 Dkk3 822.68 1510.65 706.57 665.69 0.54 0.81 0.86 0.0012 1 1 Ocrl 1017.34 687.22 937.51 748.83 1.48 0.74 0.92 0.0012 1 0 Marveld1 427.63 754.20 359.14 273.95 0.57 0.64 0.84 0.0012 1 1 Sc4mol 11040.51 7306.50 7110.08 6910.16 1.51 0.63 0.64 0.0013 1 0 Pi4k2b 1904.97 2769.45 1798.75 2185.47 0.69 1.15 0.94 0.0013 1 1 Ccdc68 700.59 281.21 338.72 508.67 2.49 0.73 0.48 0.0013 0 1 Syt8 426.54 184.71 214.19 242.04 2.31 0.57 0.50 0.0013 0 0 Gpbar1 190.71 559.89 168.89 322.36 0.34 1.69 0.89 0.0014 1 0 9930039L23Rik 4195.62 2144.35 3092.12 2745.79 1.96 0.65 0.74 0.0014 1 0 Mal 4346.98 1448.58 3173.67 3903.48 3.00 0.90 0.73 0.0015 1 1 Otud7b 570.81 398.92 504.76 510.43 1.43 0.89 0.88 0.0016 1 1 Epha1 548.09 261.79 701.32 488.67 2.09 0.89 1.28 0.0016 1 1 Stard10 2780.30 1382.27 3934.59 1372.92 2.01 0.49 1.42 0.0016 1 0 Inadl 404.91 279.45 453.05 410.20 1.45 1.01 1.12 0.0017 1 1 Ptpla 394.19 653.10 322.44 318.36 0.60 0.81 0.82 0.0017 1 1 Il17re 573.30 182.59 1346.34 151.87 3.14 0.26 2.35 0.0018 1 0 B3gnt3 567.92 331.33 444.73 635.51 1.71 1.12 0.78 0.0018 1 1 Phldb1 555.14 833.17 443.43 547.29 0.67 0.99 0.80 0.0019 1 1 Pcyt1b 1023.66 651.25 725.16 891.54 1.57 0.87 0.71 0.0021 0 1 Eps8l1 289.24 190.36 361.10 399.94 1.52 1.38 1.25 0.0022 1 1

17 Fold Averages changes Rescue success in Ovol2- Grhl2- ttest Res/ Res/ Con vs. Grhl2- Ovol2- Gene name (Illumina) Grhl2-Con Grhl2-KD Grhl2-Res Ovol2-Res Con/KD Con Con KD Res Res Rasef 230.72 157.31 292.30 158.48 1.47 0.69 1.27 0.0023 1 0 Usp43 404.42 271.88 443.56 440.57 1.49 1.09 1.10 0.0023 1 1 Cpe 9908.38 16885.12 6390.62 5632.92 0.59 0.57 0.64 0.0023 1 1 Zscan21 1264.11 1781.93 417.25 841.92 0.71 0.67 0.33 0.0023 1 1 2010004A03Rik 788.32 541.24 466.08 876.73 1.46 1.11 0.59 0.0024 1 1 Pkia 3440.06 5614.56 3303.00 3032.40 0.61 0.88 0.96 0.0025 1 1 Thbs3 286.07 427.83 435.12 377.53 0.67 1.32 1.52 0.0025 1 1 Myo9a 419.37 632.50 432.58 312.26 0.66 0.74 1.03 0.0025 1 1 scl0003547.1_6 339.60 561.07 298.56 283.26 0.61 0.83 0.88 0.0026 1 1 Rrad 653.75 974.58 460.03 985.36 0.67 1.51 0.70 0.0026 1 1 Cdh6 3737.95 6336.54 4940.14 3722.85 0.59 1.00 1.32 0.0027 1 1 Cldn4 27144.28 9168.79 27597.50 30441.99 2.96 1.12 1.02 0.0028 1 1 AI427122 1160.55 571.63 780.03 1134.80 2.03 0.98 0.67 0.0029 0 1 Batf3 182.82 268.33 205.33 206.49 0.68 1.13 1.12 0.0029 1 1 Cxcl16 2034.03 726.45 2609.12 2240.99 2.80 1.10 1.28 0.0029 1 1 Anapc5 9000.87 13215.64 10385.50 13060.71 0.68 1.45 1.15 0.0031 1 0 Marveld3 1183.92 622.90 1119.24 883.91 1.90 0.75 0.95 0.0031 1 1 Mobk1b 1656.47 1092.48 1308.97 1024.31 1.52 0.62 0.79 0.0032 0 0 Zeb1 343.33 1340.83 214.35 294.80 0.26 0.86 0.62 0.0032 1 1 Tnk1 1930.21 1052.81 2571.52 2947.82 1.83 1.53 1.33 0.0033 1 1 Tspan18 230.51 409.81 150.32 429.74 0.56 1.86 0.65 0.0034 1 0 Itgb4 4544.50 2472.02 5434.25 7267.41 1.84 1.60 1.20 0.0034 1 1 3110043J09Rik 548.60 373.42 521.41 486.33 1.47 0.89 0.95 0.0035 1 1 Hnt 496.51 710.05 332.28 280.48 0.70 0.56 0.67 0.0036 1 1 Cdh1 1354.63 515.19 1190.31 1637.61 2.63 1.21 0.88 0.0037 1 1 Stx2 395.34 627.37 319.75 379.97 0.63 0.96 0.81 0.0037 1 1 Sult2b1 537.83 374.16 1112.66 987.47 1.44 1.84 2.07 0.0038 1 1 Mvd 2754.42 1802.94 2016.96 1817.13 1.53 0.66 0.73 0.0038 0 0 Mapk13 2036.04 1033.38 4640.18 2696.44 1.97 1.32 2.28 0.0041 1 1 1700012H17Rik 1361.16 843.95 1287.04 558.86 1.61 0.41 0.95 0.0041 1 0 St8sia2 196.98 286.13 167.21 135.57 0.69 0.69 0.85 0.0042 1 1 Mtap7 1064.54 566.15 1133.79 1423.52 1.88 1.34 1.07 0.0042 1 1 A430088H15Rik 2610.05 1541.27 1939.54 1679.72 1.69 0.64 0.74 0.0043 1 1 Flrt3 552.09 293.56 316.37 297.44 1.88 0.54 0.57 0.0044 0 0 Cldn23 551.40 267.41 1387.03 636.26 2.06 1.15 2.52 0.0044 1 1 Bahcc1 1408.25 2068.36 1792.27 2184.47 0.68 1.55 1.27 0.0045 1 0 Kif21a 459.52 319.74 443.89 305.92 1.44 0.67 0.97 0.0045 1 0 Ehd2 193.09 274.07 149.48 238.41 0.70 1.23 0.77 0.0045 1 1 LOC100048721 398.12 229.50 232.32 227.36 1.73 0.57 0.58 0.0046 0 0 Slco4a1 2139.00 1461.05 1856.76 1973.61 1.46 0.92 0.87 0.0046 1 1 Gmfg 304.87 206.58 317.38 372.32 1.48 1.22 1.04 0.0049 1 1 Smox 1897.36 1073.07 2302.64 2528.74 1.77 1.33 1.21 0.0050 1 1 Gper 320.34 978.00 259.84 535.30 0.33 1.67 0.81 0.0050 1 1 Nfix 666.31 1482.63 836.19 1023.91 0.45 1.54 1.25 0.0051 1 1 Fut10 738.94 1101.89 687.15 398.69 0.67 0.54 0.93 0.0052 1 1 Foxc1 3368.75 5012.78 3653.60 3048.94 0.67 0.91 1.08 0.0053 1 1 St14 1679.98 712.99 1789.77 1754.53 2.36 1.04 1.07 0.0054 1 1

18 Fold Averages changes Rescue success in Ovol2- Grhl2- ttest Res/ Res/ Con vs. Grhl2- Ovol2- Gene name (Illumina) Grhl2-Con Grhl2-KD Grhl2-Res Ovol2-Res Con/KD Con Con KD Res Res LOC100040736 146.62 208.25 153.93 175.37 0.70 1.20 1.05 0.0054 1 1 Gsto1 23254.76 9391.20 15580.07 23633.54 2.48 1.02 0.67 0.0054 0 1 6430573F11Rik 513.11 823.49 533.84 328.08 0.62 0.64 1.04 0.0055 1 1 Nr2c1 503.45 345.85 365.95 359.45 1.46 0.71 0.73 0.0055 0 0 Pkd2 2167.22 3271.71 1470.09 1973.43 0.66 0.91 0.68 0.0057 1 1 Aacs 4020.60 5630.80 5926.12 4751.25 0.71 1.18 1.47 0.0058 0 1 Fmnl2 1452.34 2105.23 601.31 851.44 0.69 0.59 0.41 0.0058 1 1 H2-K1 556.98 395.41 706.21 706.44 1.41 1.27 1.27 0.0061 1 1 Car12 711.76 1329.96 614.44 478.84 0.54 0.67 0.86 0.0061 1 1 Ank 1219.68 2825.28 1186.97 1671.80 0.43 1.37 0.97 0.0062 1 1 Slc4a11 385.33 220.53 334.08 511.03 1.75 1.33 0.87 0.0063 1 1 Fam110b 1138.50 696.90 1043.03 483.96 1.63 0.43 0.92 0.0064 1 0 LOC100044622 369.56 521.14 400.15 251.52 0.71 0.68 1.08 0.0065 1 1 Trp53i11 330.84 575.72 349.01 647.42 0.57 1.96 1.05 0.0065 1 0 9530095P18Rik 808.27 425.90 632.19 607.53 1.90 0.75 0.78 0.0066 1 1 Ctla2b 191.41 131.61 128.54 167.32 1.45 0.87 0.67 0.0067 0 1 BC046418 477.99 322.42 436.77 497.65 1.48 1.04 0.91 0.0067 1 1 Ankrd56 2807.70 1906.27 3351.17 3022.00 1.47 1.08 1.19 0.0072 1 1 Slc29a1 648.06 1007.04 752.45 955.72 0.64 1.47 1.16 0.0076 1 1 Ppp1r14d 221.13 156.74 211.63 208.43 1.41 0.94 0.96 0.0078 1 1 A730040I05Rik 365.09 235.57 258.29 395.57 1.55 1.08 0.71 0.0079 0 1 Dclk2 387.38 684.60 305.44 268.99 0.57 0.69 0.79 0.0079 1 1 4732474O15Rik 1166.75 418.94 1025.16 714.23 2.78 0.61 0.88 0.0084 1 0 Rpl18a 13054.97 8693.33 14155.53 12170.41 1.50 0.93 1.08 0.0084 1 1 1700047I17Rik1 1711.35 1207.20 2179.28 1599.96 1.42 0.93 1.27 0.0084 1 1 9530027K23Rik 1776.84 904.25 1916.29 1161.14 1.96 0.65 1.08 0.0085 1 0 Anxa1 1946.66 882.37 1418.48 1185.35 2.21 0.61 0.73 0.0085 1 0 Rgl1 449.09 668.07 405.17 538.99 0.67 1.20 0.90 0.0085 1 1 Cldn7 1280.72 650.90 1346.79 1819.74 1.97 1.42 1.05 0.0086 1 1 Krt7 14541.59 5269.03 8977.73 16283.77 2.76 1.12 0.62 0.0086 0 1 Kntc1 2772.97 3923.74 3305.99 3712.30 0.71 1.34 1.19 0.0087 1 0 OTTMUSG00000015762 338.80 206.41 248.83 380.40 1.64 1.12 0.73 0.0087 1 1 Stat1 663.54 456.30 747.46 438.98 1.45 0.66 1.13 0.0088 1 0 Bspry 810.78 347.77 965.53 748.49 2.33 0.92 1.19 0.0088 1 1 Mpp2 194.65 288.46 143.48 159.89 0.67 0.82 0.74 0.0090 1 1 Rcan1 422.62 911.91 656.28 268.80 0.46 0.64 1.55 0.0091 1 1 Ildr1 285.38 183.43 276.33 292.12 1.56 1.02 0.97 0.0091 1 1 Rbm35a 7197.20 3190.41 7469.76 7954.26 2.26 1.11 1.04 0.0092 1 1 Nt5dc2 3876.46 7943.51 4585.02 4998.34 0.49 1.29 1.18 0.0095 1 1 Mpzl3 240.11 132.83 227.65 162.42 1.81 0.68 0.95 0.0097 1 0 Hr 512.99 758.04 2686.92 1915.88 0.68 3.73 5.24 0.0098 0 0 Sgpp2 498.43 312.60 203.98 739.11 1.59 1.48 0.41 0.0098 0 1 3110050N22Rik 2042.55 2869.51 1541.58 1838.36 0.71 0.90 0.75 0.0101 1 1 Cgn 860.91 451.12 633.11 852.97 1.91 0.99 0.74 0.0101 1 1 Sox12 954.35 1431.35 902.58 641.14 0.67 0.67 0.95 0.0103 1 1 Fam110c 1154.08 644.89 1020.14 1059.51 1.79 0.92 0.88 0.0105 1 1 Casp7 1181.16 1725.75 1154.15 1152.15 0.68 0.98 0.98 0.0106 1 1

19 Fold Averages changes Rescue success in Ovol2- Grhl2- ttest Res/ Res/ Con vs. Grhl2- Ovol2- Gene name (Illumina) Grhl2-Con Grhl2-KD Grhl2-Res Ovol2-Res Con/KD Con Con KD Res Res Krt19 6846.32 3086.42 5865.22 10099.60 2.22 1.48 0.86 0.0108 1 1 Lpin3 448.39 309.85 437.76 392.53 1.45 0.88 0.98 0.0108 1 1 Cda 511.55 301.82 408.36 449.99 1.69 0.88 0.80 0.0109 1 1 EG635702 234.96 162.65 224.52 138.87 1.44 0.59 0.96 0.0110 1 0 Rab25 7624.17 1739.95 14198.88 6976.77 4.38 0.92 1.86 0.0110 1 1 Mcam 708.62 1157.16 388.85 558.35 0.61 0.79 0.55 0.0111 1 1 D17H6S56E-5 804.21 515.90 825.44 669.46 1.56 0.83 1.03 0.0112 1 1 Ppbp 7756.87 3373.04 7125.99 5290.39 2.30 0.68 0.92 0.0113 1 1 Wnt2 169.82 389.45 151.43 165.07 0.44 0.97 0.89 0.0113 1 1 9830001H06Rik 540.76 971.76 238.45 423.77 0.56 0.78 0.44 0.0113 1 1 Chrnb1 5625.06 3532.76 5389.02 3639.63 1.59 0.65 0.96 0.0114 1 0 Atp2a1 268.07 182.51 234.33 527.22 1.47 1.97 0.87 0.0114 1 1 Arg1 235.66 162.07 767.98 171.08 1.45 0.73 3.26 0.0114 1 0 Gfm1 1728.48 1182.99 1379.82 1500.81 1.46 0.87 0.80 0.0115 0 1 Fbxo30 336.85 527.56 359.06 445.24 0.64 1.32 1.07 0.0115 1 0 Efemp2 3484.96 5485.39 3243.45 4073.13 0.64 1.17 0.93 0.0115 1 1 Stxbp1 727.98 1148.20 1693.08 428.65 0.63 0.59 2.33 0.0116 0 1 Wipf1 380.24 753.46 203.73 465.26 0.50 1.22 0.54 0.0119 1 1 Sfn 813.02 393.14 1137.71 1019.45 2.07 1.25 1.40 0.0120 1 1 Ttyh3 1754.95 3178.24 1127.70 1872.42 0.55 1.07 0.64 0.0120 1 1 AW548124 265.15 547.77 174.41 253.84 0.48 0.96 0.66 0.0120 1 1 8430415N23Rik 580.44 1332.95 1519.38 1083.15 0.44 1.87 2.62 0.0120 0 0 Maml1 330.28 499.07 376.26 404.97 0.66 1.23 1.14 0.0120 1 1 Tmem38b 2175.49 1541.80 1882.36 1559.23 1.41 0.72 0.87 0.0122 1 0 Msln 1046.06 423.53 742.98 1024.77 2.47 0.98 0.71 0.0125 1 1 6330578E17Rik 1597.45 2507.57 2687.42 2851.25 0.64 1.78 1.68 0.0125 0 0 scl0002694.1_23 350.25 204.18 323.37 357.34 1.72 1.02 0.92 0.0129 1 1 6330549D23Rik 289.87 500.96 197.67 209.36 0.58 0.72 0.68 0.0131 1 1 LOC383576 921.37 537.53 1002.49 649.04 1.71 0.70 1.09 0.0131 1 0 9930023K05Rik 923.04 231.48 541.46 1925.81 3.99 2.09 0.59 0.0131 0 1 Ccng1 2870.86 2042.54 3480.84 2699.94 1.41 0.94 1.21 0.0133 1 1 Ly6a 5516.55 1838.57 1687.62 8257.38 3.00 1.50 0.31 0.0133 0 1 4632417N05Rik 597.03 345.33 1171.98 263.19 1.73 0.44 1.96 0.0133 1 0 Klk8 1087.94 322.95 1047.39 2647.99 3.37 2.43 0.96 0.0134 1 1 Nacc2 1498.18 3546.54 855.73 2078.40 0.42 1.39 0.57 0.0135 1 1 Krt18 15048.22 7231.22 17235.49 16752.19 2.08 1.11 1.15 0.0138 1 1 Twist2 1714.36 4515.89 820.99 1473.57 0.38 0.86 0.48 0.0139 1 1 Tmem184a 1355.37 702.33 1841.21 1586.71 1.93 1.17 1.36 0.0140 1 1 Krt20 906.16 327.08 476.00 2518.75 2.77 2.78 0.53 0.0140 1 1 Fzd2 1084.02 1611.16 822.57 1030.47 0.67 0.95 0.76 0.0140 1 1 C920027I18Rik 1168.08 719.22 619.90 909.99 1.62 0.78 0.53 0.0141 0 0 Ube2e3 1320.44 1902.96 1114.20 1320.47 0.69 1.00 0.84 0.0141 1 1 Agl 596.58 888.50 715.98 544.11 0.67 0.91 1.20 0.0142 1 1 D11Bwg0414e 9131.16 13346.19 10854.49 9371.49 0.68 1.03 1.19 0.0144 1 1 Ctsh 1675.07 701.31 1487.47 1663.13 2.39 0.99 0.89 0.0146 1 1 Prkch 283.95 166.85 359.85 242.22 1.70 0.85 1.27 0.0147 1 1 Cep170 1586.57 2301.57 834.72 932.67 0.69 0.59 0.53 0.0148 1 1

20 Fold Averages changes Rescue success in Ovol2- Grhl2- ttest Res/ Res/ Con vs. Grhl2- Ovol2- Gene name (Illumina) Grhl2-Con Grhl2-KD Grhl2-Res Ovol2-Res Con/KD Con Con KD Res Res 2310007B03Rik 1966.73 307.45 1724.94 2361.37 6.40 1.20 0.88 0.0148 1 1 Tgfbr1 811.67 1482.95 1172.55 844.37 0.55 1.04 1.44 0.0151 1 1 Tmem40 826.55 228.12 1022.14 353.44 3.62 0.43 1.24 0.0152 1 0 LOC100045981 2143.92 3407.51 3123.91 4094.27 0.63 1.91 1.46 0.0155 1 0 Centd1 1181.36 610.49 1389.89 772.64 1.94 0.65 1.18 0.0155 1 0 Pou6f1 1367.29 2314.23 510.72 1043.18 0.59 0.76 0.37 0.0161 1 1 Kctd15 1023.42 1787.73 1085.50 1019.52 0.57 1.00 1.06 0.0162 1 1 Vgll3 466.29 204.08 368.32 334.51 2.28 0.72 0.79 0.0164 1 1 D130071O13Rik 396.65 584.48 223.09 312.97 0.68 0.79 0.56 0.0166 1 1 Bnc2 189.65 270.54 236.13 162.38 0.70 0.86 1.25 0.0167 1 1 Smarcd3 285.71 810.69 218.19 421.78 0.35 1.48 0.76 0.0169 1 0 Sept5 707.15 1108.52 561.63 989.85 0.64 1.40 0.79 0.0169 1 0 Ccbl1 1393.60 912.26 2440.33 1000.53 1.53 0.72 1.75 0.0172 1 0 Fosl2 1251.35 1975.83 1187.17 1549.13 0.63 1.24 0.95 0.0172 1 1 Htr5b 229.76 145.63 500.23 119.43 1.58 0.52 2.18 0.0173 1 0 Samd14 1832.30 2720.44 1726.71 1766.89 0.67 0.96 0.94 0.0174 1 1 Wnt7a 2319.23 668.78 1829.66 4527.93 3.47 1.95 0.79 0.0175 1 1 EG629383 989.51 1680.71 1906.11 1894.07 0.59 1.91 1.93 0.0176 0 0 Sparc 2321.54 4608.30 602.03 2437.60 0.50 1.05 0.26 0.0177 1 1 Ppp1r9b 748.08 1219.26 986.87 795.13 0.61 1.06 1.32 0.0177 1 1 Ctsa 344.64 504.22 331.06 314.90 0.68 0.91 0.96 0.0177 1 1 Zkscan1 521.96 731.94 553.62 573.73 0.71 1.10 1.06 0.0180 1 1 Gm22 366.21 603.67 626.28 791.15 0.61 2.16 1.71 0.0181 1 0 Tmie 439.81 282.76 532.07 482.08 1.56 1.10 1.21 0.0182 1 1 Dlx1 191.07 342.82 156.33 178.13 0.56 0.93 0.82 0.0184 1 1 Met 591.39 956.12 685.27 704.25 0.62 1.19 1.16 0.0185 1 1 Ripk4 6244.91 3818.70 5820.57 6463.02 1.64 1.03 0.93 0.0186 1 1 Tgfb1i1 225.83 341.22 219.18 396.90 0.66 1.76 0.97 0.0187 1 0 Tnc 261.11 494.71 1046.94 242.67 0.53 0.93 4.01 0.0189 0 1 Casp14 1859.67 840.16 318.09 2095.38 2.21 1.13 0.17 0.0192 0 1 4933428A15Rik 2742.81 1235.04 2454.46 928.70 2.22 0.34 0.89 0.0194 1 0 Dennd2a 2154.00 4814.95 1146.30 2816.45 0.45 1.31 0.53 0.0194 1 1 Abi2 1643.30 2572.67 2299.50 1450.12 0.64 0.88 1.40 0.0196 0 1 2300008H03Rik 319.65 220.45 252.22 234.33 1.45 0.73 0.79 0.0197 1 0 Fgf13 462.99 227.94 455.50 258.61 2.03 0.56 0.98 0.0197 1 0 Leprel1 397.12 691.22 253.02 286.71 0.57 0.72 0.64 0.0199 1 1 Rasl11b 12934.72 19759.72 9767.98 12571.04 0.65 0.97 0.76 0.0203 1 1 H2-Bl 182.64 124.52 284.22 199.25 1.47 1.09 1.56 0.0203 1 1 Mcf2l 209.13 313.32 216.09 232.69 0.67 1.11 1.03 0.0206 1 1 Serpinb6b 190.11 134.71 162.17 154.48 1.41 0.81 0.85 0.0212 1 1 Cttnbp2nl 3189.12 2209.78 3362.29 5675.13 1.44 1.78 1.05 0.0214 1 1 4933428D01Rik 197.26 139.35 159.96 143.59 1.42 0.73 0.81 0.0214 1 0 Serpina3h 397.46 1797.36 400.87 420.16 0.22 1.06 1.01 0.0215 1 1 Mslnl 254.09 156.41 414.18 421.85 1.62 1.66 1.63 0.0216 1 1 6330408J11Rik 511.67 326.79 545.99 584.53 1.57 1.14 1.07 0.0217 1 1 Pemt 539.55 379.19 580.20 397.21 1.42 0.74 1.08 0.0218 1 0 1600002K03Rik 2433.90 1669.39 2419.73 2311.54 1.46 0.95 0.99 0.0218 1 1

21 Fold Averages changes Rescue success in Ovol2- Grhl2- ttest Res/ Res/ Con vs. Grhl2- Ovol2- Gene name (Illumina) Grhl2-Con Grhl2-KD Grhl2-Res Ovol2-Res Con/KD Con Con KD Res Res Upk3b 500.98 314.05 2750.03 401.62 1.60 0.80 5.49 0.0219 1 1 Tox 256.95 410.79 207.39 208.00 0.63 0.81 0.81 0.0221 1 1 Scly 716.11 1283.78 806.80 756.22 0.56 1.06 1.13 0.0223 1 1 Depdc1b 726.82 1083.18 957.83 887.57 0.67 1.22 1.32 0.0225 1 1 Grhl2 2023.61 672.93 387.48 1006.21 3.01 0.50 0.19 0.0227 0 0 Mxd3 343.90 674.19 317.66 406.70 0.51 1.18 0.92 0.0227 1 1 LOC100044221 1639.90 1041.22 1865.41 1951.48 1.57 1.19 1.14 0.0227 1 1 Akr1c12 511.36 917.29 379.69 689.48 0.56 1.35 0.74 0.0228 1 1 9130211I03Rik 235.20 427.15 285.14 256.61 0.55 1.09 1.21 0.0229 1 1 1700016K19Rik 267.93 175.21 285.54 296.39 1.53 1.11 1.07 0.0231 1 1 1810019J16Rik 544.23 298.48 588.60 660.23 1.82 1.21 1.08 0.0232 1 1 Btn1a1 289.47 168.32 169.21 374.72 1.72 1.29 0.58 0.0235 0 1 Tmprss2 4776.12 1428.19 3370.01 2362.57 3.34 0.49 0.71 0.0238 1 0 Tmem102 855.06 566.65 1259.43 831.80 1.51 0.97 1.47 0.0238 1 1 Sav1 7734.73 12696.80 8378.33 8067.03 0.61 1.04 1.08 0.0239 1 1 Stxbp5 841.74 538.20 867.86 631.68 1.56 0.75 1.03 0.0239 1 1 Sgms2 640.73 413.67 534.56 849.05 1.55 1.33 0.83 0.0239 1 1 Apbb2 818.43 1160.26 703.69 922.70 0.71 1.13 0.86 0.0240 1 1 Tbc1d2 804.01 342.96 1062.19 867.51 2.34 1.08 1.32 0.0240 1 1 Pmvk 1825.48 1293.48 1572.16 1229.82 1.41 0.67 0.86 0.0241 1 0 A430106B04Rik 374.97 539.60 321.91 539.15 0.69 1.44 0.86 0.0242 1 0 D930046M13Rik 400.30 709.10 287.26 486.67 0.56 1.22 0.72 0.0243 1 1 Apcdd1 150.39 307.40 178.02 156.72 0.49 1.04 1.18 0.0243 1 1 Cdkn1a 4892.51 2405.32 4595.29 5408.69 2.03 1.11 0.94 0.0245 1 1 1700105P06Rik 236.64 352.19 287.70 242.69 0.67 1.03 1.22 0.0245 0 1 Prom2 476.68 169.81 567.14 176.57 2.81 0.37 1.19 0.0249 1 0 Moxd1 301.50 193.77 706.94 491.94 1.56 1.63 2.34 0.0251 1 1 Cic 391.61 631.37 446.68 405.04 0.62 1.03 1.14 0.0254 1 1 Dysf 481.50 739.55 481.89 323.23 0.65 0.67 1.00 0.0256 1 1 2310028O11Rik 250.85 354.97 315.84 298.98 0.71 1.19 1.26 0.0258 1 1 Ramp2 373.61 772.45 274.98 423.97 0.48 1.13 0.74 0.0259 1 1 4632428D17Rik 236.01 388.95 141.46 242.94 0.61 1.03 0.60 0.0259 1 1 EG666756 8374.86 5691.37 6766.79 7390.58 1.47 0.88 0.81 0.0261 1 1 Fam102a 2420.97 1476.93 2848.61 1980.33 1.64 0.82 1.18 0.0261 1 1 Fermt2 2004.72 2980.48 2112.09 2096.07 0.67 1.05 1.05 0.0267 1 1 1500002O10Rik 272.34 435.05 191.12 233.20 0.63 0.86 0.70 0.0269 1 1 Nnmt 845.14 1647.67 234.63 1156.05 0.51 1.37 0.28 0.0270 1 1 Sephs1 224.82 370.43 286.82 321.25 0.61 1.43 1.28 0.0270 0 0 Vim 919.22 3957.28 408.97 1463.84 0.23 1.59 0.44 0.0270 1 1 Rhpn2 685.37 341.07 259.24 857.26 2.01 1.25 0.38 0.0272 0 1 1110012J17Rik 1101.81 2026.12 451.71 692.50 0.54 0.63 0.41 0.0272 1 1 1110018J23Rik 1699.53 3157.95 1301.49 1387.02 0.54 0.82 0.77 0.0273 1 1 Dcbld1 1561.79 898.61 1740.85 1344.43 1.74 0.86 1.11 0.0274 1 1 Pus7l 1946.70 1277.82 2242.30 1869.28 1.52 0.96 1.15 0.0278 1 1 BC004044 377.28 764.12 334.65 381.01 0.49 1.01 0.89 0.0280 1 1 4631426J05Rik 246.41 516.22 212.79 201.89 0.48 0.82 0.86 0.0280 1 1 Npy 181.50 356.70 196.24 155.49 0.51 0.86 1.08 0.0280 1 1

22 Fold Averages changes Rescue success in Ovol2- Grhl2- ttest Res/ Res/ Con vs. Grhl2- Ovol2- Gene name (Illumina) Grhl2-Con Grhl2-KD Grhl2-Res Ovol2-Res Con/KD Con Con KD Res Res Zdhhc13 3346.01 2340.81 2472.83 2686.93 1.43 0.80 0.74 0.0281 0 1 Ctxn1 1389.75 2479.68 1550.83 2133.74 0.56 1.54 1.12 0.0281 1 1 6430514L14Rik 242.16 363.67 194.84 285.21 0.67 1.18 0.80 0.0282 1 1 Tnni2 527.57 204.56 255.29 301.13 2.58 0.57 0.48 0.0283 0 1 Slc45a3 671.08 477.24 761.03 809.35 1.41 1.21 1.13 0.0284 1 1 Plek2 1258.45 857.27 1694.90 2179.23 1.47 1.73 1.35 0.0285 1 1 Axud1 2286.68 1604.15 2641.86 2619.53 1.43 1.15 1.16 0.0285 1 1 Sgk2 280.15 186.42 219.17 202.94 1.50 0.72 0.78 0.0285 1 0 Tmem62 1427.00 981.45 1073.67 1085.40 1.45 0.76 0.75 0.0286 1 0 Klf5 3920.03 2047.78 4198.52 5677.42 1.91 1.45 1.07 0.0287 1 1 Adi1 3227.26 2289.06 3099.96 2046.32 1.41 0.63 0.96 0.0288 1 0 Rnf144a 599.36 1180.74 325.02 827.59 0.51 1.38 0.54 0.0289 1 1 Bex4 8089.15 4746.78 10412.28 6612.33 1.70 0.82 1.29 0.0290 1 1 G430005B15Rik 809.38 1167.71 437.46 718.11 0.69 0.89 0.54 0.0292 1 1 LOC545481 523.98 236.42 399.35 748.78 2.22 1.43 0.76 0.0293 1 1 3110018K01Rik 205.85 333.42 133.03 195.54 0.62 0.95 0.65 0.0294 1 1 Hebp2 817.86 567.47 1370.58 486.41 1.44 0.59 1.68 0.0294 1 0 LOC670044 995.10 1452.86 1117.95 1205.43 0.68 1.21 1.12 0.0294 1 1 Sim2 6282.45 12179.20 6061.98 5243.90 0.52 0.83 0.96 0.0299 1 1 Zbtb32 214.95 141.08 155.87 298.51 1.52 1.39 0.73 0.0299 0 1 2210011C24Rik 830.91 541.96 427.85 1155.73 1.53 1.39 0.51 0.0301 0 1 Ppif 6250.40 4046.84 7818.45 7113.82 1.54 1.14 1.25 0.0302 1 1 Plau 5915.56 1783.24 2892.10 4596.35 3.32 0.78 0.49 0.0306 1 1 Enc1 3678.34 5346.09 4435.33 3775.26 0.69 1.03 1.21 0.0307 1 1 Pdgfa 6602.75 9350.71 7950.47 6072.48 0.71 0.92 1.20 0.0311 1 1 Tmem79 3309.14 1869.32 6212.54 3096.39 1.77 0.94 1.88 0.0313 1 1 Lama3 1421.67 400.34 1226.41 2987.41 3.55 2.10 0.86 0.0313 1 1 Fkbp5 758.37 1071.89 894.58 641.93 0.71 0.85 1.18 0.0316 1 1 Itpk1 1491.08 2136.05 2161.23 1618.69 0.70 1.09 1.45 0.0319 1 1 Rhob 2290.39 3947.34 1895.14 3470.66 0.58 1.52 0.83 0.0319 1 1 Ivns1abp 3852.91 5721.81 4010.62 3877.99 0.67 1.01 1.04 0.0320 1 1 Grb10 611.85 1124.39 321.92 326.37 0.54 0.53 0.53 0.0321 1 1 Spsb2 555.36 851.43 627.47 581.57 0.65 1.05 1.13 0.0321 1 1 Spag1 462.66 323.31 965.25 436.06 1.43 0.94 2.09 0.0323 1 1 2610528J11Rik 334.17 155.78 325.76 505.09 2.15 1.51 0.97 0.0326 1 1 scl0002540.1_6 416.74 817.88 191.00 377.34 0.51 0.91 0.46 0.0326 1 1 Lhfpl2 2359.54 3787.57 1426.15 2159.25 0.62 0.92 0.60 0.0326 1 1 D10Ertd516e 799.26 1157.94 893.72 854.82 0.69 1.07 1.12 0.0330 1 1 Slc2a1 12256.50 7680.47 12396.61 17975.46 1.60 1.47 1.01 0.0337 1 1 Gipc2 1620.04 1025.61 1518.52 467.98 1.58 0.29 0.94 0.0340 1 0 LOC638301 454.15 264.34 355.61 404.03 1.72 0.89 0.78 0.0341 1 1 2610009E16Rik 3146.91 5630.57 3089.30 3093.67 0.56 0.98 0.98 0.0342 1 1 9130204C11Rik 322.68 480.02 341.17 464.58 0.67 1.44 1.06 0.0343 1 0 LOC100046395 3378.39 2341.54 2619.57 2359.80 1.44 0.70 0.78 0.0344 0 0 5930429A15Rik 276.98 450.29 208.67 369.58 0.62 1.33 0.75 0.0350 1 1 Dscr1 1526.76 2403.64 2086.57 1434.78 0.64 0.94 1.37 0.0350 1 1 Oas1g 475.39 299.44 751.39 456.27 1.59 0.96 1.58 0.0353 1 1

23 Fold Averages changes Rescue success in Ovol2- Grhl2- ttest Res/ Res/ Con vs. Grhl2- Ovol2- Gene name (Illumina) Grhl2-Con Grhl2-KD Grhl2-Res Ovol2-Res Con/KD Con Con KD Res Res Tgm1 1074.94 364.15 737.07 2315.24 2.95 2.15 0.69 0.0353 1 1 4930445K14Rik 441.63 658.24 498.27 565.67 0.67 1.28 1.13 0.0354 1 1 Ifit3 5213.28 9967.33 4937.22 4405.54 0.52 0.85 0.95 0.0354 1 1 Ddr1 2063.41 2939.59 1744.97 1951.65 0.70 0.95 0.85 0.0357 1 1 Dpysl2 2214.80 3334.80 1510.31 1804.15 0.66 0.81 0.68 0.0358 1 1 Fgd6 454.12 282.54 450.36 550.65 1.61 1.21 0.99 0.0359 1 1 Pias3 1657.76 2593.21 1468.32 1664.86 0.64 1.00 0.89 0.0361 1 1 Nedd4b 151.18 219.60 168.48 157.05 0.69 1.04 1.11 0.0361 1 1 4833409N03Rik 1800.93 2719.13 2065.25 1595.25 0.66 0.89 1.15 0.0361 1 1 Elfn1 169.82 310.07 287.23 185.24 0.55 1.09 1.69 0.0361 0 1 Ggt7 161.81 267.34 197.86 199.24 0.61 1.23 1.22 0.0363 1 1 2610528B01Rik 336.63 480.99 340.25 353.29 0.70 1.05 1.01 0.0364 1 1 Col16a1 481.10 826.13 564.94 626.59 0.58 1.30 1.17 0.0364 1 1 2310043J07Rik 1388.71 482.71 1609.16 1325.01 2.88 0.95 1.16 0.0366 1 1 Plekho2 371.52 577.34 321.06 409.28 0.64 1.10 0.86 0.0369 1 1 Itga2 199.87 139.75 242.32 239.23 1.43 1.20 1.21 0.0369 1 1 4933436C20Rik 314.51 540.34 267.13 169.57 0.58 0.54 0.85 0.0370 1 1 Smad6 624.42 1026.61 688.87 709.96 0.61 1.14 1.10 0.0371 1 1 Tgfb3 358.83 553.83 428.95 298.97 0.65 0.83 1.20 0.0375 1 1 Ankrd13d 374.06 573.42 886.61 377.83 0.65 1.01 2.37 0.0377 0 1 Tmem159 677.14 432.24 691.94 427.14 1.57 0.63 1.02 0.0378 1 0 Serpina3g 169.49 445.09 145.38 161.06 0.38 0.95 0.86 0.0381 1 1 AI480653 978.19 1423.48 1252.58 1209.51 0.69 1.24 1.28 0.0381 1 1 Sdcbp2 391.18 161.49 382.84 1217.35 2.42 3.11 0.98 0.0381 1 1 C130057N11Rik 583.49 824.52 670.41 670.89 0.71 1.15 1.15 0.0381 1 1 Mb 204.31 144.81 333.20 172.23 1.41 0.84 1.63 0.0382 1 1 Lbh 1397.14 2887.46 868.49 2227.01 0.48 1.59 0.62 0.0383 1 1 Cox7a2l 3208.95 5682.85 6005.51 5362.96 0.56 1.67 1.87 0.0386 0 0 C130073O12Rik 383.46 245.55 411.92 257.60 1.56 0.67 1.07 0.0386 1 1 Col4a3 1555.53 1009.28 1830.66 1042.78 1.54 0.67 1.18 0.0387 1 0 Cysltr1 205.14 129.08 222.59 149.40 1.59 0.73 1.09 0.0388 1 1 Lepre1 208.73 299.71 193.85 178.97 0.70 0.86 0.93 0.0388 1 1 Gata3 211.53 384.27 390.28 241.07 0.55 1.14 1.85 0.0389 1 1 Cdcp1 447.83 291.59 380.24 376.46 1.54 0.84 0.85 0.0389 1 1 Chst7 371.89 257.29 851.67 359.37 1.45 0.97 2.29 0.0390 1 1 1500005K14Rik 1152.89 719.88 1499.50 1099.76 1.60 0.95 1.30 0.0390 1 1 St3gal2 334.03 491.03 278.83 287.40 0.68 0.86 0.83 0.0391 1 1 Ppap2c 14917.20 10191.82 12215.81 12591.07 1.46 0.84 0.82 0.0392 1 1 Pmepa1 514.94 355.81 446.65 427.09 1.45 0.83 0.87 0.0393 1 1 Ddit4 4412.13 1921.04 4665.58 4184.31 2.30 0.95 1.06 0.0394 1 1 Sprr2g 611.67 308.18 381.77 937.99 1.98 1.53 0.62 0.0394 1 1 BC025546 4778.02 3311.96 3609.35 4158.56 1.44 0.87 0.76 0.0395 0 1 Gjb3 4662.03 2083.38 3929.04 4831.35 2.24 1.04 0.84 0.0397 1 1 Ckmt1 237.95 156.58 259.20 359.92 1.52 1.51 1.09 0.0397 1 1 Mcc 363.91 578.94 448.07 292.60 0.63 0.80 1.23 0.0398 1 1 4432405B04Rik 499.37 916.23 530.37 531.11 0.55 1.06 1.06 0.0399 1 1 Ntn4 217.85 318.28 248.77 191.35 0.68 0.88 1.14 0.0400 1 1

24 Fold Averages changes Rescue success in Ovol2- Grhl2- ttest Res/ Res/ Con vs. Grhl2- Ovol2- Gene name (Illumina) Grhl2-Con Grhl2-KD Grhl2-Res Ovol2-Res Con/KD Con Con KD Res Res 4732458O05Rik 437.47 625.27 441.19 1058.37 0.70 2.42 1.01 0.0400 1 0 Sh3rf2 251.85 156.86 238.94 192.16 1.61 0.76 0.95 0.0402 1 1 Fbxo33 1000.06 713.12 972.64 771.58 1.40 0.77 0.97 0.0404 1 1 Usp12 326.14 480.16 368.00 351.82 0.68 1.08 1.13 0.0405 1 1 Ramp3 1462.19 781.85 1272.85 1756.43 1.87 1.20 0.87 0.0406 1 1 Ankrd47 1510.70 748.16 1277.63 1511.36 2.02 1.00 0.85 0.0409 1 1 Sema5a 961.14 1930.65 699.01 563.87 0.50 0.59 0.73 0.0410 1 1 Trim47 824.30 1278.63 1347.47 1124.21 0.64 1.36 1.63 0.0412 0 1 Cxcl10 774.86 527.15 696.72 1045.00 1.47 1.35 0.90 0.0414 1 1 6430598A04Rik 247.90 349.30 300.35 355.03 0.71 1.43 1.21 0.0414 0 0 2700084L06Rik 1941.54 2970.74 1926.84 2106.73 0.65 1.09 0.99 0.0414 1 1 Palm 871.77 1413.69 747.77 952.47 0.62 1.09 0.86 0.0417 1 1 Gcnt3 304.84 179.64 215.19 192.99 1.70 0.63 0.71 0.0420 1 1 Cdon 456.57 722.60 447.60 386.90 0.63 0.85 0.98 0.0423 1 1 LOC100038882 2020.76 1029.22 3186.11 1704.64 1.96 0.84 1.58 0.0430 1 1 Cacnb3 624.56 911.62 657.71 746.73 0.69 1.20 1.05 0.0432 1 1 Pcolce 1717.19 4416.04 771.30 1990.61 0.39 1.16 0.45 0.0432 1 1 Marcks 3781.91 6033.78 6367.38 3994.79 0.63 1.06 1.68 0.0432 1 1 Col6a1 230.99 828.54 162.82 314.13 0.28 1.36 0.70 0.0436 1 1 Atoh8 184.58 506.81 210.69 150.45 0.36 0.82 1.14 0.0439 1 1 Id3 1495.49 2125.23 1375.94 1525.31 0.70 1.02 0.92 0.0440 1 1 Mpnd 2163.74 3153.35 2248.71 2100.41 0.69 0.97 1.04 0.0440 1 1 Prss22 1787.42 206.48 2601.04 631.79 8.66 0.35 1.46 0.0442 1 1 Krt17 333.32 216.55 636.61 2740.65 1.54 8.22 1.91 0.0442 1 1 2610202E01Rik 517.51 866.94 548.06 555.55 0.60 1.07 1.06 0.0444 1 1 Figf 165.88 365.97 142.91 192.16 0.45 1.16 0.86 0.0445 1 1 Pdlim4 4340.38 6490.67 5648.01 5893.55 0.67 1.36 1.30 0.0447 1 1 Ocln 411.90 263.49 440.68 401.92 1.56 0.98 1.07 0.0450 1 1 Cd40 519.04 344.84 1078.62 741.84 1.51 1.43 2.08 0.0451 1 1 Ece1 963.16 1507.64 1148.27 900.12 0.64 0.93 1.19 0.0455 1 1 9330132O05Rik 586.64 918.98 520.85 708.97 0.64 1.21 0.89 0.0459 1 1 LOC381770 210.73 300.65 201.13 175.69 0.70 0.83 0.95 0.0463 1 1 Tacstd2 17451.86 8508.10 16845.38 14238.21 2.05 0.82 0.97 0.0464 1 1 Trim8 6494.15 11981.84 7499.35 7097.34 0.54 1.09 1.15 0.0467 1 1 S100a14 677.09 295.37 873.37 1477.00 2.29 2.18 1.29 0.0471 1 1 Rab11fip5 5169.74 3314.83 5075.62 8175.23 1.56 1.58 0.98 0.0474 1 1 Apc2 158.24 226.35 167.41 179.36 0.70 1.13 1.06 0.0476 1 1 AI661453 460.60 304.20 485.69 631.81 1.51 1.37 1.05 0.0481 1 1 Ptms 1099.12 1649.92 1156.90 1169.85 0.67 1.06 1.05 0.0483 1 1 LOC236311 315.95 174.45 182.62 333.12 1.81 1.05 0.58 0.0483 1 1 Hmgcr 608.93 386.42 388.79 389.90 1.58 0.64 0.64 0.0486 0 1 Ppl 2239.14 1473.39 1744.24 2002.74 1.52 0.89 0.78 0.0486 1 1 5830411G16Rik 1441.91 2022.66 2189.38 2332.49 0.71 1.62 1.52 0.0488 0 0 Btbd6 2128.73 3222.50 2462.71 2553.08 0.66 1.20 1.16 0.0489 1 1 Nupr1 13745.86 8663.44 8245.42 12653.05 1.59 0.92 0.60 0.0490 0 1 Sox9 3251.60 1562.51 3588.43 5224.90 2.08 1.61 1.10 0.0491 1 1 Myl9 175.05 248.93 233.72 257.03 0.70 1.47 1.34 0.0493 1 0

25 Fold Averages changes Rescue success in Ovol2- Grhl2- ttest Res/ Res/ Con vs. Grhl2- Ovol2- Gene name (Illumina) Grhl2-Con Grhl2-KD Grhl2-Res Ovol2-Res Con/KD Con Con KD Res Res Sdc2 1073.55 1974.42 1182.44 742.40 0.54 0.69 1.10 0.0496 1 1 Gm1673 2286.69 3853.44 1388.55 1434.51 0.59 0.63 0.61 0.0496 1 1

26 Supplemental Table 2 Gene ontology analysis on differentially expressed genes. Gene ontology analysis for differentially expressed genes between Grhl2-Con and Grhl2-KD using the HOMER software package. GO terms with more than 500 genes or less than 2 genes in term have been excluded from the analysis as well as categories with less than 2 target genes in term.

Target Genes in Genes in TermID Term p-value logP Term Term Gene Symbols

Sfn, Tmem79, Cdh1, Sprr2g, Sprr1a, Tgfb1i1, Ppl, Vim, Gata3, Fzd2, Stx2, Fam20c, Tgm1, Sav1, Id3, Klf5, Krt17, GO:0030855 epithelial cell differentiation <0.0001 -14.5 391 21 Map7, Sox9, Wnt7a, Anxa1

Pkia, Sfn, Pkdcc, Cdh1, Tmem184a, Krt20, Tgfb3, Rab11fip5, Gata3, Tgfbr1, Ramp3, Btn1a1, Sema5a, Cd40, Apc2, Gper1, Wipf1, Cdkn1a, Pkd2, Nedd4l, GO:0032880 regulation of protein localization <0.0001 -12.4 448 21 Anxa1

Rbm20, Casp7, Ovol2, Mb, Cpe, Foxc1, Maml1, Tgfbr1, Gata3, Fzd2, Smarcd3, Ece1, Grhl2, Sav1, Id3, Smad6, Ramp2, Wnt2, Pkd2, GO:0007507 heart development <0.0001 -12.3 413 20 Sox9

Sfn, Tmem79, Tgm1, Apcdd1, Pdgfa, Sav1, Itga2, Sprr1a, Sprr2g, Prss8, Krt17, GO:0043588 skin development <0.0001 -12.1 249 15 Sox9, Wnt7a, Anxa1, Ppl Sprr1a, Sprr2g, Sfn, Krt17, GO:0031424 keratinization <0.0001 -11.5 34 6 Tgm1, Ppl Sfn, Tgm1, Sav1, Sprr2g, GO:0030216 keratinocyte differentiation <0.0001 -11.5 71 8 Sprr1a, Krt17, Ppl, Anxa1 Fzd2, Plau, Met, Cdh1, Pdgfa, Wnt2, Tacstd2, Sox9, GO:2000027 regulation of organ morphogenesis <0.0001 -11.2 147 11 Foxc1, Ntn4, Gata3 regulation of branching involved in salivary GO:0060693 gland morphogenesis <0.0001 -11.1 11 4 Met, Cdh1, Pdgfa, Ntn4

Pkia, Sfn, Pkdcc, Cdh1, Krt20, Tgfb3, Gata3, Tgfbr1, Ramp3, Btn1a1, Sema5a, regulation of establishment of protein Cd40, Apc2, Gper1, Wipf1, GO:0070201 localization <0.0001 -11.1 375 18 Cdkn1a, Pkd2, Anxa1 Arg1, Plau, Cdh1, Sav1, Ovol2, Vash1, Wnt2, Stat1, Sox9, Twist2, Mcc, Wnt7a, GO:0050678 regulation of epithelial cell proliferation <0.0001 -11.1 239 14 Gata3, Tgfbr1 regulation of morphogenesis of a branching Wnt2, Met, Cdh1, Tacstd2, GO:0060688 structure <0.0001 -10.9 56 7 Sox9, Pdgfa, Ntn4

Cdh1, Ehd2, Fgf13, Palm, GO:0072659 protein localization to plasma membrane <0.0001 -10.9 77 8 Krt18, Ramp2, Map7, Ramp3

Pkdcc, Met, Cdh1, Pdgfa, Ovol2, Tgfb3, Ddr1, Stat1, Ctsh, Foxc1, Gata3, Sparc, Sema5a, Grhl2, Sav1, Zeb1, GO:0035295 tube development <0.0001 -10.8 459 20 Sim2, Pkd2, Sox9, Wnt7a

Cxcl10, Plau, Cxcl16, Pdgfa, Fam110c, Figf, Gper1, S100a14, Itga2, Epha1, positive regulation of cellular component Sox9, Ctsh, Gata3, Tgfbr1, GO:0051272 movement <0.0001 -10.6 284 15 Mcam

27 Target Genes in Genes in TermID Term p-value logP Term Term Gene Symbols

Pkia, Tmem102, Sfn, Pkdcc, Gsto1, Cdh1, Ehd2, Tgfb3, Tgfbr1, Sema5a, Cxcl10, Apc2, Gper1, Wipf1, Cdkn1a, GO:0032386 regulation of intracellular transport <0.0001 -10.5 320 16 Pkd2

Cdh1, Cxcl16, Pdgfa, Fam110c, Figf, Itga2, Epha1, Tacstd2, Vim, Ctsh, Gata3, Mcam, Lama3, Cxcl10, Plau, Gper1, S100a14, Vash1, GO:0030334 regulation of cell migration <0.0001 -10.4 471 20 Sox9, Mcc regulation of endothelial cell apoptotic Ramp2, Sema5a, Cd40, GO:2000351 process <0.0001 -10.0 27 5 Gper1, Gata3 Cdh1, Ehd2, Dysf, Fgf13, Palm, Krt18, Ramp2, Map7, GO:0007009 plasma membrane organization <0.0001 -10.0 112 9 Ramp3

Cdh1, Cxcl16, Pdgfa, Fam110c, Figf, Itga2, Epha1, Tacstd2, Vim, Ctsh, Gata3, Mcam, Lama3, Cxcl10, Plau, Gper1, S100a14, Vash1, GO:2000145 regulation of cell motility 0.0001 -9.9 490 20 Sox9, Mcc

Pkia, Sfn, Pkdcc, Cdh1, Krt20, Tgfb3, Gata3, Tgfbr1, Btn1a1, Sema5a, Cd40, Apc2, Wipf1, Cdkn1a, Pkd2, GO:0051223 regulation of protein transport 0.0001 -9.8 339 16 Anxa1 Fzd2, Smarcd3, Grhl2, Sav1, Ovol2, Wnt2, Cpe, Foxc1, GO:0003206 cardiac chamber morphogenesis 0.0001 -9.8 115 9 Gata3

Cxcl10, Plau, Cxcl16, Pdgfa, Fam110c, Figf, Gper1, S100a14, Itga2, Epha1, GO:0030335 positive regulation of cell migration 0.0001 -9.7 271 14 Sox9, Ctsh, Gata3, Mcam Met, Enc1, Klk8, Fam110c, Itga2, Tgfb3, Tacstd2, Vim, Sdc2, Gata3, Tgfbr1, Ntm, GO:0031344 regulation of cell projection organization 0.0001 -9.6 346 16 Fgf13, Palm, Klf5, Wnt7a

Cxcl10, Plau, Cxcl16, Pdgfa, Fam110c, Figf, Gper1, S100a14, Itga2, Epha1, GO:2000147 positive regulation of cell motility 0.0001 -9.5 276 14 Sox9, Ctsh, Gata3, Mcam

Sfn, Tgm1, Apcdd1, Pdgfa, Sav1, Sprr2g, Sprr1a, Prss8, GO:0008544 epidermis development 0.0001 -9.4 211 12 Krt17, Sox9, Anxa1, Ppl

Cxcl10, Plau, Cxcl16, Pdgfa, Fam110c, Figf, Gper1, S100a14, Itga2, Epha1, GO:0040017 positive regulation of locomotion 0.0001 -9.1 288 14 Sox9, Ctsh, Gata3, Mcam

Pkia, Sfn, Pkdcc, Sema5a, Cdh1, Apc2, Wipf1, Cdkn1a, GO:0033157 regulation of intracellular protein transport 0.0001 -8.9 189 11 Tgfb3, Pkd2, Tgfbr1 negative regulation of epithelial cell Cdh1, Sav1, Vash1, Stat1, GO:0050680 proliferation 0.0001 -8.8 102 8 Sox9, Mcc, Tgfbr1, Gata3 Fzd2, Smarcd3, Grhl2, Sav1, Ovol2, Wnt2, Cpe, Foxc1, GO:0003205 cardiac chamber development 0.0002 -8.7 132 9 Gata3 Lama3, Marveld3, Cdh1, Fermt2, Ramp2, Tgfb3, Ocln, GO:0034330 cell junction organization 0.0002 -8.6 105 8 Itgb4 Pkia, Sfn, Sema5a, Cdh1, Apc2, Wipf1, Cdkn1a, Tgfb3, GO:0046822 regulation of nucleocytoplasmic transport 0.0002 -8.5 166 10 Pkd2, Tgfbr1

Tgfb3, Fzd2, Pkdcc, Csrnp1, GO:0060021 palate development 0.0002 -8.5 81 7 Bnc2, Wnt7a, Tgfbr1 28 Target Genes in Genes in TermID Term p-value logP Term Term Gene Symbols Cdh1, Ehd2, Fermt2, Fgf13, Palm, Krt18, Ramp2, Map7, GO:0072657 protein localization to membrane 0.0002 -8.4 168 10 Cpe, Ramp3 Cxcl10, Sema5a, Rhob, Col4a3, Ramp2, Vash1, GO:0045765 regulation of angiogenesis 0.0003 -8.2 172 10 Epha1, Stat1, Cysltr1, Ctsh Fzd2, Smarcd3, Grhl2, Sav1, Ovol2, Pkd2, Wnt2, Sox9, GO:0003007 heart morphogenesis 0.0003 -8.2 206 11 Foxc1, Cpe, Gata3 Sfn, Tgm1, Sav1, Sprr2g, GO:0009913 epidermal cell differentiation 0.0003 -8.1 114 8 Sprr1a, Krt17, Ppl, Anxa1 Wisp2, Sfn, Cda, Cxcl16, Fgf13, Ppp1r9b, Cdkn1a, Rrad, Ddr1, Krt17, Tnk1, GO:0001558 regulation of cell growth 0.0005 -7.6 294 13 Apbb2, Tgfbr1

Ddit4, Met, Csrnp1, Fermt2, Figf, Pdgfa, Tgfb3, Tgfb1i1, enzyme linked receptor protein signaling Epha1, Ddr1, Foxc1, Tgfbr1, GO:0007167 pathway 0.0005 -7.6 413 16 Smad6, Sox9, Grb10, Stub1 Cxcl10, Sema5a, Rhob, Col4a3, Ramp2, Vash1, GO:1901342 regulation of vasculature development 0.0005 -7.5 188 10 Epha1, Stat1, Cysltr1, Ctsh

Figf, Pdgfa, Ovol2, Epha1, Foxc1, Tgfbr1, Sema5a, Plau, Rhob, Smad6, Klf5, Ramp2, Pkd2, Wnt2, Vash1, GO:0001568 blood vessel development 0.0005 -7.5 416 16 Wnt7a

Figf, Pdgfa, Ovol2, Epha1, Foxc1, Tgfbr1, Fzd2, Sema5a, Plau, Rhob, Klf5, Smad6, Ramp2, Vash1, GO:0001944 vasculature development 0.0006 -7.5 458 17 Wnt2, Pkd2, Wnt7a Cdh1, Ehd2, Fermt2, Dysf, Fgf13, Palm, Gper1, Krt18, Ramp2, Ppif, Chrnb1, Map7, GO:0044802 single-organism membrane organization 0.0006 -7.4 338 14 Cpe, Ramp3

Dlx1, Sparc, Pkdcc, Thbs3, Csrnp1, Grhl2, Fam20c, Bnc2, Zeb1, Tgfb3, Sox9, GO:0001501 skeletal system development 0.0006 -7.4 379 15 Twist2, Wnt7a, Foxc1, Tgfbr1 Tgfb3, Tacstd2, Fam110c, GO:0060491 regulation of cell projection assembly 0.0006 -7.4 70 6 Palm, Klf5, Tgfbr1 GO:0032410 negative regulation of transporter activity 0.0006 -7.3 27 4 Ppif, Pkd2, Nedd4l, Gsto1

Cdon, Smarcd3, Krt19, Met, Nupr1, Id3, Klf5, Smad6, Ramp2, Wnt2, Bcl9, Chrnb1, GO:0061061 muscle structure development 0.0007 -7.3 383 15 Rcan1, Foxc1, Maml1

Sema5a, Plau, Rhob, Pdgfa, Figf, Klf5, Smad6, Ovol2, Ramp2, Vash1, Epha1, GO:0048514 blood vessel morphogenesis 0.0007 -7.2 346 14 Foxc1, Wnt7a, Tgfbr1 Sfn, Cxcl10, Sema5a, Cdh1, Ehd2, Gper1, Wipf1, Tgfb3, GO:0032388 positive regulation of intracellular transport 0.0008 -7.2 163 9 Tgfbr1 Ovol2, Pkdcc, Cdh1, Stx2, GO:0048565 digestive tract development 0.0008 -7.1 102 7 Pdgfa, Sav1, Klf5 Itga2, Vash1, Tacstd2, Sox9, GO:0010632 regulation of epithelial cell migration 0.0010 -6.9 105 7 Mcc, Ctsh, Gata3

Cda, Ppif, Arap2, Epha1, Tbc1d2, Fzd2, Cxcl10, Ocrl, Cd40, Ece1, Palm, Gper1, Ramp2, Pkd2, Mcf2l, GO:0006140 regulation of nucleotide metabolic process 0.0010 -6.9 483 17 Dennd2a, Arhgap8

Pkia, Sema5a, Cdh1, Apc2, GO:0042306 regulation of protein import into nucleus 0.0010 -6.9 136 8 Cdkn1a, Pkd2, Tgfb3, Tgfbr1 GO:0014812 muscle cell migration 0.0011 -6.9 14 3 Ddr1, Plau, Met

29 Target Genes in Genes in TermID Term p-value logP Term Term Gene Symbols GO:0060037 pharyngeal system development 0.0011 -6.9 14 3 Ece1, Tgfbr1, Gata3 Mvd, Hmgcr, Sc4mol, GO:0046165 alcohol biosynthetic process 0.0011 -6.8 78 6 Sgms2, Pmvk, Pemt

regulation of cellular response to growth Fam20c, Figf, Zeb1, Smad6, GO:0090287 factor stimulus 0.0011 -6.8 138 8 Tgfb3, Tgfb1i1, Grb10, Gata3 Thbs3, Pkdcc, Nupr1, Plau, Grhl2, Lepre1, Bnc2, Tgfb3, Bcl9, Map7, Sox9, Wnt7a, GO:0040007 growth 0.0011 -6.8 361 14 Foxc1, Gata3 Thbs3, Csrnp1, Grhl2, Bnc2, Zeb1, Tgfb3, Sox9, Twist2, GO:0048705 skeletal system morphogenesis 0.0011 -6.8 207 10 Wnt7a, Tgfbr1 Fzd2, Smarcd3, Grhl2, Cpe, GO:0003231 cardiac ventricle development 0.0012 -6.8 108 7 Sav1, Foxc1, Gata3

Prkch, Stxbp1, Cand1, Fam110c, Palm, Klf5, Kank3, Smad6, Tgfb3, Epha1, Nedd4l, Tacstd2, Wnt7a, GO:0044087 regulation of cellular component biogenesis 0.0012 -6.7 406 15 Stub1, Tgfbr1

GO:0016126 sterol biosynthetic process 0.0012 -6.7 32 4 Mvd, Hmgcr, Sc4mol, Pmvk regulation of protein localization to cell GO:2000008 surface 0.0013 -6.6 15 3 Nedd4l, Cdh1, Rab11fip5 positive regulation of nucleocytoplasmic Tgfb3, Sfn, Sema5a, Cdh1, GO:0046824 transport 0.0013 -6.6 81 6 Tgfbr1, Wipf1 Cdon, Smarcd3, Krt19, Met, Smad6, Ramp2, Bcl9, GO:0042692 muscle cell differentiation 0.0014 -6.6 213 10 Chrnb1, Rcan1, Maml1 Ovol2, Pkdcc, Cdh1, Stx2, GO:0055123 digestive system development 0.0014 -6.5 112 7 Pdgfa, Sav1, Klf5 Cdon, Ehd2, Sox9, Id3, GO:0010830 regulation of myotube differentiation 0.0014 -6.5 56 5 Maml1

Sema5a, Plau, Rhob, Pdgfa, Figf, Klf5, Ovol2, Ramp2, GO:0001525 angiogenesis 0.0015 -6.5 252 11 Vash1, Epha1, Tgfbr1

Pkia, Sema5a, Cdh1, Apc2, GO:1900180 regulation of protein localization to nucleus 0.0015 -6.5 145 8 Cdkn1a, Pkd2, Tgfb3, Tgfbr1 GO:0090102 cochlea development 0.0016 -6.5 34 4 Fzd2, Cdh1, Sox9, Zeb1 GO:0043403 skeletal muscle tissue regeneration 0.0016 -6.5 16 3 Bcl9, Plau, Wnt7a GO:0060575 intestinal epithelial cell differentiation 0.0016 -6.5 16 3 Cdh1, Sav1, Klf5 negative regulation of endothelial cell GO:2000352 apoptotic process 0.0016 -6.5 16 3 Ramp2, Sema5a, Gata3 Fzd2, Pkdcc, Krt19, Tmie, Grhl2, Zeb1, Ovol2, Wnt2, Tgfb3, Pkd2, Sox9, Twist2, GO:0048568 embryonic organ development 0.0016 -6.4 418 15 Foxc1, Gata3, Tgfbr1 Itga2, Mapk13, Pkd2, GO:0006970 response to osmotic stress 0.0017 -6.4 58 5 Nedd4l, Map7 Ddit4, Nupr1, Cd40, Rhob, Sav1, Gper1, Id3, Casp7, Cdkn1a, Ppif, Tgfb3, Hmgcr, GO:0010942 positive regulation of cell death 0.0017 -6.4 379 14 Anxa1, Apbb2 Cdh1, Ehd2, Fermt2, Fgf13, Palm, Krt18, Ramp2, Map7, GO:1902580 single-organism cellular localization 0.0018 -6.3 220 10 Cpe, Ramp3 Cdh1, Ehd2, Fermt2, Fgf13, Palm, Krt18, Ramp2, Map7, GO:1902578 single-organism localization 0.0018 -6.3 220 10 Cpe, Ramp3

organic hydroxy compound biosynthetic Mvd, Hmgcr, Sc4mol, GO:1901617 process 0.0018 -6.3 116 7 Sgms2, Pmvk, Pemt, Gata3 regulation of cyclic nucleotide metabolic Ramp2, Pkd2, Fzd2, Cxcl10, GO:0030799 process 0.0019 -6.3 117 7 Ece1, Palm, Gper1 Cdh1, Sox9, Apcdd1, Apc2, GO:0030178 negative regulation of Wnt signaling pathway 0.0019 -6.2 118 7 Grb10, Mcc, Dkk3 Prss8, Sox9, Pdgfa, Mpzl3, GO:0042303 molting cycle 0.0021 -6.1 89 6 Apcdd1, Sav1

30 Target Genes in Genes in TermID Term p-value logP Term Term Gene Symbols Prss8, Sox9, Pdgfa, Mpzl3, GO:0042633 hair cycle 0.0021 -6.1 89 6 Apcdd1, Sav1 Sema5a, Met, Grhl2, Ovol2, Pkd2, Ripk4, Tgfb1i1, Ddr1, Tnc, Krt17, Sox9, Wnt7a, GO:0002009 morphogenesis of an epithelium 0.0022 -6.1 388 14 Ctsh, Gata3

Ppif, Arap2, Epha1, Tbc1d2, Fzd2, Cxcl10, Cd40, Ocrl, Ece1, Palm, Gper1, Ramp2, regulation of purine nucleotide metabolic Pkd2, Mcf2l, Dennd2a, GO:1900542 process 0.0023 -6.1 478 16 Arhgap8

GO:0042312 regulation of vasodilation 0.0024 -6.0 38 4 Hmgcr, Ece1, Gper1, Smad6 Cdh1, Ehd2, Fermt2, Dysf, Fgf13, Palm, Gper1, Krt18, Ramp2, Ppif, Chrnb1, Map7, GO:0061024 membrane organization 0.0025 -6.0 395 14 Cpe, Ramp3 Cdh1, Apcdd1, Apc2, Wnt2, Sox9, Grb10, Mcc, Dkk3, GO:0030111 regulation of Wnt signaling pathway 0.0026 -6.0 194 9 Wnt7a negative regulation of ion transmembrane GO:0032413 transporter activity 0.0026 -5.9 19 3 Pkd2, Nedd4l, Gsto1

GO:2001259 positive regulation of cation channel activity 0.0026 -5.9 19 3 Pkd2, Nedd4l, Gsto1 Fzd2, Serpinb6b, Nupr1, Pcyt1b, Tnc, Sox9, Map7, GO:0048608 reproductive structure development 0.0027 -5.9 233 10 Wnt7a, Foxc1, Gata3 Lepre1, Col4a3, Ramp2, Sox9, Mpzl3, Foxc1, Tgfbr1, GO:0030198 extracellular matrix organization 0.0028 -5.9 160 8 Apbb2 Ovol2, Fam101b, Wnt2, GO:0014031 mesenchymal cell development 0.0028 -5.9 94 6 Sox9, Foxc1, Tgfbr1 Ramp2, Pkd2, Cxcl10, Ece1, GO:0030814 regulation of cAMP metabolic process 0.0028 -5.9 94 6 Palm, Gper1 Ramp2, Marveld3, Ocln, GO:0045216 cell-cell junction organization 0.0028 -5.9 94 6 Tgfb3, Cdh1, Fermt2 GO:0014902 myotube differentiation 0.0029 -5.9 40 4 Cdon, Bcl9, Met, Rcan1 Lepre1, Col4a3, Ramp2, Sox9, Mpzl3, Foxc1, Tgfbr1, GO:0043062 extracellular structure organization 0.0029 -5.8 161 8 Apbb2 Prkch, Ovol2, Sox9, Zeb1, GO:0030856 regulation of epithelial cell differentiation 0.0030 -5.8 95 6 Gata3, Tgfbr1 Fzd2, Nupr1, Serpinb6b, Pcyt1b, Tnc, Map7, Sox9, GO:0061458 reproductive system development 0.0031 -5.8 238 10 Foxc1, Wnt7a, Gata3

Pkia, Ddit4, Cda, Met, Pdgfa, negative regulation of phosphorus metabolic Palm, Smad6, Cdkn1a, Ppif, GO:0010563 process 0.0033 -5.7 321 12 Hmgcr, Epha1, Grb10

Pkia, Ddit4, Cda, Met, Pdgfa, negative regulation of phosphate metabolic Palm, Smad6, Cdkn1a, Ppif, GO:0045936 process 0.0033 -5.7 321 12 Hmgcr, Epha1, Grb10 Fzd2, Serpinb6b, Nupr1, Pcyt1b, Map7, Sox9, Foxc1, GO:0008406 gonad development 0.0034 -5.7 165 8 Gata3

GO:0060324 face development 0.0034 -5.7 42 4 Tgfb3, Csrnp1, Grhl2, Twist2 Ovol2, Fam101b, Wnt2, GO:0060485 mesenchyme development 0.0035 -5.7 131 7 Sox9, Bnc2, Foxc1, Tgfbr1

GO:0045684 positive regulation of epidermis development 0.0036 -5.6 21 3 Prkch, Tmem79, Krt17 GO:0045109 intermediate filament organization 0.0036 -5.6 21 3 Krt20, Krt17, Vim Fzd2, Tmie, Grhl2, Zeb1, Ovol2, Pkd2, Tgfb3, Sox9, GO:0048562 embryonic organ morphogenesis 0.0036 -5.6 283 11 Twist2, Gata3, Tgfbr1 Tgfb3, Sprr2g, Wnt7a, GO:0043627 response to estrogen 0.0036 -5.6 99 6 Gper1, Tgfbr1, Ramp3 Smarcd3, Grhl2, Foxc1, Cpe, GO:0003208 cardiac ventricle morphogenesis 0.0036 -5.6 69 5 Gata3

31 Target Genes in Genes in TermID Term p-value logP Term Term Gene Symbols Cdkn1a, Wnt2, Nupr1, Fosl2, GO:0048145 regulation of fibroblast proliferation 0.0036 -5.6 69 5 Pdgfa Lama3, Ramp2, Itgb4, Cdh1, GO:0034329 cell junction assembly 0.0036 -5.6 69 5 Fermt2 Plau, Stxbp1, Klk8, Atp2a1, Pdgfa, Sav1, Smad6, Hmgcr, negative regulation of multicellular Tgfb3, Tacstd2, Sox9, Twist2, GO:0051241 organismal process 0.0037 -5.6 368 13 Gata3 Fam101b, Wnt2, Sox9, GO:0001837 epithelial to mesenchymal transition 0.0038 -5.6 43 4 Tgfbr1

GO:0010464 regulation of mesenchymal cell proliferation 0.0038 -5.6 43 4 Wnt2, Sox9, Pdgfa, Zeb1 Gper1, Agl, Sprr2g, Tgfb3, Wnt7a, Anxa1, Ramp3, GO:0048545 response to steroid hormone 0.0040 -5.5 207 9 Nr2c1, Tgfbr1 Cdon, Ehd2, Sox9, Zeb1, GO:0051147 regulation of muscle cell differentiation 0.0040 -5.5 101 6 Id3, Maml1 GO:0042246 tissue regeneration 0.0041 -5.5 22 3 Bcl9, Plau, Wnt7a GO:0051491 positive regulation of filopodium assembly 0.0041 -5.5 22 3 Tgfb3, Palm, Tgfbr1 GO:0090103 cochlea morphogenesis 0.0041 -5.5 22 3 Fzd2, Sox9, Zeb1 GO:0048701 embryonic cranial skeleton morphogenesis 0.0041 -5.5 44 4 Tgfb3, Grhl2, Twist2, Tgfbr1 Ramp2, Epha1, Sema5a, GO:0045766 positive regulation of angiogenesis 0.0042 -5.5 102 6 Rhob, Cysltr1, Ctsh regulation of G-protein coupled receptor Ramp2, Ece1, Palm, Gper1, GO:0008277 protein signaling pathway 0.0042 -5.5 102 6 Ppp1r9b, Ramp3 Tnc, Klk8, Cdh1, Palm, Elfn1, GO:0050808 synapse organization 0.0044 -5.4 103 6 Wnt7a

GO:0048146 positive regulation of fibroblast proliferation 0.0044 -5.4 45 4 Cdkn1a, Wnt2, Fosl2, Pdgfa Prkch, Ovol2, Tmem79, GO:0045682 regulation of epidermis development 0.0044 -5.4 45 4 Krt17 Bspry, Zdhhc13, Atp2a1, Ramp2, Cacnb3, Pkd2, GO:0070838 divalent metal ion transport 0.0045 -5.4 211 9 Nipal2, Cysltr1, Ramp3

GO:0010171 body morphogenesis 0.0048 -5.3 46 4 Tgfb3, Cdon, Csrnp1, Twist2 Ramp2, Krt19, Rcan1, GO:0055001 muscle cell development 0.0049 -5.3 105 6 Chrnb1, Maml1, Smad6

Fam101b, Fzd2, Sema5a, Cdh1, Stxbp1, Fermt2, Flrt3, Wnt2, Epha1, Sox9, Tgfbr1, GO:0000904 cell morphogenesis involved in differentiation 0.0050 -5.3 426 14 Stxbp5, Apbb2, Gata3 Cdon, Klk8, Met, Enc1, Fgf13, Palm, Dpysl2, Zeb1, Sox9, Wnt7a, Vim, Ntm, GO:0045664 regulation of neuron differentiation 0.0050 -5.3 426 14 Sdc2, Gata3 Ovol2, Fam101b, Wnt2, GO:0048762 mesenchymal cell differentiation 0.0051 -5.3 106 6 Sox9, Foxc1, Tgfbr1 Nupr1, Rhob, Cdkn1a, Vash1, Pkd2, Foxc1, Apbb2, GO:0045786 negative regulation of cell cycle 0.0053 -5.2 178 8 Gata3 Ddit4, Met, Csrnp1, Figf, transmembrane receptor protein tyrosine Pdgfa, Epha1, Ddr1, Sox9, GO:0007169 kinase signaling pathway 0.0053 -5.2 257 10 Grb10, Foxc1 Tmem79, Cdh1, Sox9, Map7, GO:0002064 epithelial cell development 0.0054 -5.2 142 7 Wnt7a, Klf5, Gata3 regulation of canonical Wnt signaling Wnt2, Cdh1, Sox9, Apc2, GO:0060828 pathway 0.0054 -5.2 142 7 Mcc, Wnt7a, Dkk3 Tgfb3, Tgfb1i1, Tacstd2, GO:2000736 regulation of stem cell differentiation 0.0055 -5.2 76 5 Sox9, Sav1 positive regulation of intracellular protein Tgfb3, Sfn, Sema5a, Cdh1, GO:0090316 transport 0.0058 -5.1 109 6 Tgfbr1, Wipf1 Btn1a1, Pkd2, Pkia, Pkdcc, GO:0051224 negative regulation of protein transport 0.0058 -5.1 109 6 Apc2, Anxa1 Cdkn1a, Rrad, Cda, Fgf13, GO:0030308 negative regulation of cell growth 0.0059 -5.1 144 7 Tnk1, Apbb2, Ppp1r9b GO:0008299 isoprenoid biosynthetic process 0.0059 -5.1 25 3 Mvd, Hmgcr, Pmvk

32 Target Genes in Genes in TermID Term p-value logP Term Term Gene Symbols Dlx1, Cdon, Fgf13, Wnt2, Tgfb1i1, Sox9, Wnt7a, GO:0045165 cell fate commitment 0.0060 -5.1 221 9 Gata3, Tgfbr1 Itga2, Tnni2, Nedd4l, Ece1, GO:0090257 regulation of muscle system process 0.0061 -5.1 145 7 Atp2a1, Cald1, Gper1 Tgfb3, Fermt2, Sox9, Smad6, GO:0071559 response to transforming growth factor beta 0.0061 -5.1 78 5 Tgfbr1 positive regulation of cyclic nucleotide Ramp2, Fzd2, Cxcl10, Ece1, GO:0030801 metabolic process 0.0061 -5.1 78 5 Gper1 cellular response to transforming growth Tgfb3, Sox9, Fermt2, Smad6, GO:0071560 factor beta stimulus 0.0061 -5.1 78 5 Tgfbr1

Dlx1, Fgf13, Zeb1, Gper1, Id3, Ovol2, Meis2, Bcl9, Tgfb1i1, Tacstd2, Sox9, GO:0045596 negative regulation of cell differentiation 0.0064 -5.1 485 15 Twist2, Wnt7a, Gata3, Tgfbr1 Arg1, Wnt2, Vash1, Stat1, GO:0001936 regulation of endothelial cell proliferation 0.0065 -5.0 79 5 Tgfbr1 Sparc, Ddit4, Fermt2, Smad6, Ramp2, Tgfb3, GO:0071363 cellular response to growth factor stimulus 0.0066 -5.0 224 9 Sox9, Tgfbr1, Gata3 Bspry, Zdhhc13, Atp2a1, Ramp2, Cacnb3, Pkd2, GO:0072511 divalent inorganic cation transport 0.0066 -5.0 224 9 Nipal2, Cysltr1, Ramp3 GO:0048708 astrocyte differentiation 0.0066 -5.0 26 3 Nfix, Sox9, Vim GO:0061326 renal tubule development 0.0066 -5.0 26 3 Pkd2, Stat1, Sox9 negative regulation of endothelial cell GO:0001937 proliferation 0.0066 -5.0 26 3 Vash1, Stat1, Tgfbr1 GO:0018149 peptide cross-linking 0.0066 -5.0 26 3 Sprr1a, Tgm1, Anxa1

Sparc, Pkdcc, Grhl2, Pdgfa, GO:0030324 lung development 0.0069 -5.0 186 8 Sav1, Tgfb3, Sim2, Ctsh Itga2, Tnni2, Nedd4l, Atp2a1, GO:0006937 regulation of muscle contraction 0.0069 -5.0 113 6 Cald1, Gper1

Nupr1, Cd40, Rhob, Sav1, Gper1, Id3, Casp7, Cdkn1a, GO:0043068 positive regulation of programmed cell death 0.0069 -5.0 353 12 Tgfb3, Hmgcr, Anxa1, Apbb2 Rgl1, Cdon, Ocrl, Fgd6, Cd40, Arap2, Mcf2l, Epha1, regulation of small GTPase mediated signal Tbc1d2, Dennd2a, Tnk1, GO:0051056 transduction 0.0071 -5.0 354 12 Arhgap8

Sparc, Pkdcc, Grhl2, Pdgfa, GO:0030323 respiratory tube development 0.0073 -4.9 188 8 Sav1, Tgfb3, Sim2, Ctsh GO:0007044 cell-substrate junction assembly 0.0073 -4.9 27 3 Lama3, Itgb4, Fermt2 GO:0006695 cholesterol biosynthetic process 0.0073 -4.9 27 3 Mvd, Hmgcr, Pmvk

GO:0090183 regulation of kidney development 0.0074 -4.9 52 4 Tacstd2, Sox9, Pdgfa, Gata3 Thbs3, Tmem79, Stxbp1, Fam20c, Palm, Ntn4, Gata3, GO:0021700 developmental maturation 0.0075 -4.9 189 8 Tdrd5 GO:0031099 regeneration 0.0079 -4.8 53 4 Bcl9, Tnc, Plau, Wnt7a positive regulation of nucleotide metabolic Ramp2, Fzd2, Cxcl10, Ece1, GO:0045981 process 0.0080 -4.8 83 5 Gper1 Prss8, Sox9, Apcdd1, Pdgfa, GO:0001942 hair follicle development 0.0080 -4.8 83 5 Sav1 regulation of striated muscle cell Cdon, Ehd2, Sox9, Id3, GO:0051153 differentiation 0.0080 -4.8 83 5 Maml1 positive regulation of purine nucleotide Ramp2, Fzd2, Cxcl10, Ece1, GO:1900544 metabolic process 0.0080 -4.8 83 5 Gper1 Fzd2, Serpinb6b, Nupr1, development of primary sexual Pcyt1b, Map7, Sox9, Foxc1, GO:0045137 characteristics 0.0082 -4.8 192 8 Gata3 Cdon, Bcl9, Krt19, Met, GO:0051146 striated muscle cell differentiation 0.0084 -4.8 154 7 Rcan1, Chrnb1, Maml1 Prss8, Sox9, Pdgfa, Apcdd1, GO:0022405 hair cycle process 0.0084 -4.8 84 5 Sav1

33 Target Genes in Genes in TermID Term p-value logP Term Term Gene Symbols Prss8, Sox9, Pdgfa, Apcdd1, GO:0022404 molting cycle process 0.0084 -4.8 84 5 Sav1

Cdon, Met, Nupr1, Id3, Klf5, GO:0007517 muscle organ development 0.0084 -4.8 233 9 Wnt2, Bcl9, Rcan1, Foxc1 Sparc, Ddit4, Fermt2, Smad6, Ramp2, Tgfb3, GO:0070848 response to growth factor 0.0084 -4.8 233 9 Sox9, Tgfbr1, Gata3 Sema5a, Met, Cdh1, Grhl2, Zeb1, Ovol2, Pkd2, Ddr1, GO:0035239 tube morphogenesis 0.0084 -4.8 318 11 Sox9, Ctsh, Gata3

GO:0060322 head development 0.0085 -4.8 54 4 Tgfb3, Csrnp1, Grhl2, Twist2 negative regulation of canonical Wnt Cdh1, Sox9, Apc2, Mcc, GO:0090090 signaling pathway 0.0088 -4.7 85 5 Dkk3 negative regulation of cell projection Klk8, Tacstd2, Fgf13, Vim, GO:0031345 organization 0.0088 -4.7 85 5 Ntm Sprr2g, Cdh1, Aacs, Gper1, GO:0097305 response to alcohol 0.0088 -4.7 119 6 Tgfbr1, Ramp3 Cdon, Bcl9, Met, Rcan1, GO:0007519 skeletal muscle tissue development 0.0088 -4.7 119 6 Nupr1, Klf5 negative regulation of epithelial cell GO:0030857 differentiation 0.0089 -4.7 29 3 Ovol2, Zeb1, Tgfbr1 GO:0071333 cellular response to glucose stimulus 0.0089 -4.7 29 3 Aacs, Rab11fip5, Gper1

Fam101b, Ovol2, Wnt2, Bcl9, GO:0048864 stem cell development 0.0090 -4.7 195 8 Sox9, Wnt7a, Foxc1, Tgfbr1

GO:0009880 embryonic pattern specification 0.0090 -4.7 55 4 Sim2, Wnt7a, Smad6, Tdrd5 Cdon, Grhl2, Zeb1, Meis2, Sox9, Twist2, Foxc1, Vim, GO:0001654 eye development 0.0092 -4.7 322 11 Gata3, Abi2, Tgfbr1 Wnt2, Smarcd3, Sox9, GO:0072091 regulation of stem cell proliferation 0.0092 -4.7 86 5 Pdgfa, Zeb1 Cxcl10, Ddit4, Cd40, Ifnlr1, GO:0009615 response to virus 0.0092 -4.7 157 7 Isg15, Ifit3, Batf3 Cdon, Grhl2, Zeb1, Sox9, Twist2, Foxc1, Vim, Gata3, GO:0043010 camera-type eye development 0.0095 -4.7 280 10 Abi2, Tgfbr1

Btn1a1, Pkia, Pkdcc, Gsto1, Apc2, Ppif, Pkd2, Hmgcr, GO:0051051 negative regulation of transport 0.0096 -4.6 324 11 Nedd4l, Grb10, Anxa1 Sfn, Ddit4, Sema5a, Purb, Met, Klk8, Fgf13, Figf, Col4a3, Zbtb32, Map7, Sox9, GO:0008283 cell proliferation 0.0098 -4.6 462 14 Wnt7a, Foxc1 intrinsic apoptotic signaling pathway in response to DNA damage by p53 class GO:0042771 mediator 0.0098 -4.6 30 3 Cdkn1a, Ddit4, Nupr1 Dlx1, Sema5a, Cdon, Smarcd3, Zeb1, Smad6, Ovol2, Sim2, Pkd2, Wnt7a, GO:0007389 pattern specification process 0.0099 -4.6 416 13 Foxc1, Tgfbr1, Tdrd5

34 Supplemental Table 3 Grhl2 core target module. Table explaining the generation of the Grhl2 core target module.

The following table contains genes that fulfill the major criteria for our Grhl2 core target set. This includes: no peak in adult kidney OR no rescue in Grhl-Res to be differentially expressed (control vs. KD), to have a differential H3K4me3 ChIP peak in IMCD-3 cells intersecting with the gene body or within +-500bp from TSS, no peak center related Grhl2 binding motif to have a Grhl2 ChIP peak in IMCD3 cells intersecting with the mentioned diff. H3K4me3 peak, to have a ChIP peak in adult kidney intersecting with the Grhl2 ChIP peak from IMCD-3 cells. final core target set

Grhl2 ChIP peak occurrence consistent with Grhl2 ChIP peak in IMCD-3 Motif occurrence in 50bp window cells in: Associated Grhl2 ChIP peak in IMCD-3 cells Associated diff. H3K4me3 ChIP peak in IMCD-3 ce around peak summit (p-value<1e-03) Success- Down- fully or rescued Entrez upreg. in Grhl2- Adult E18 MACS peak MACS peak p-value Name ID in KD Res kidney kidney Start End score Start End score (FIMO) Sequence Prss22 70835 DOWN YES YES YES chr17 24134882 24135365 486.88 chr17 24132298 24135860 1155.62 YES 0.000128 GGACCAGTC Tmem54 66260 DOWN YES YES YES chr4 128782536 128782999 2512.06 chr4 128782297 128784223 312.3 YES 0.000370 GGACCGGCT 2310007B03Rik 71874 DOWN YES NO YES chr1 95054266 95054542 146.89 chr1 95053725 95057685 1390.51 YES 0.000119 AGACCAGCC Rab25 53868 DOWN YES YES YES chr3 88352030 88352452 347.13 chr3 88350717 88353006 177.1 YES 0.000133 TAACCTGTT Tmem40 94346 DOWN YES YES NO chr6 115708850 115709040 51.48 chr6 115707783 115710545 777.65 YES 0.000293 TGACCAGTC Lama3 16774 DOWN YES NO NO chr18 12662427 12662735 112.58 chr18 12660937 12664719 787.32 YES 0.000750 ATACCTGCC Il17re 57890 DOWN YES YES YES chr6 113408498 113408875 346.9 chr6 113408356 113409883 535.47 NO Tgm1 21816 DOWN YES YES NO chr14 56332795 56332985 144.56 chr14 56331144 56334628 235.74 YES 0.000263 GAGCCAGTT Prom2 192212 DOWN YES YES YES chr2 127366959 127367430 1558.07 chr2 127365786 127367795 900.57 YES 0.000018 AAACCTGTC Cdh1 12550 DOWN YES YES YES chr8 109134806 109135281 1215.01 chr8 109134232 109135862 276.33 YES 0.000005 AAACCAGTT Ctsh 13036 DOWN YES YES YES chr9 89964274 89964634 907.72 chr9 89963827 89965216 145.43 NO Syt8 55925 DOWN NO YES YES chr7 149620101 149620413 2161.46 chr7 149620003 149623228 838.76 YES 0.000240 TGACCAGTT Arhgap40 545481 DOWN YES YES NO chr2 158348878 158349223 1575.98 chr2 158348457 158349800 65 NO Sprr1a 20753 DOWN NO NO YES chr3 92290303 92290585 272.11 chr3 92289025 92291064 64.95 YES 0.000030 AAACCTGCT Mpzl3 319742 DOWN YES YES NO chr9 44868176 44868442 447.43 chr9 44867641 44870275 1013.82 YES 0.000387 GAACCAGAT Dcbld1 66686 DOWN YES YES NO chr10 51997822 51998226 59.46 chr10 51997215 51998581 71.25 NO Fam110b 242297 DOWN YES NO YES chr4 5695796 5696142 248.05 chr4 5695820 5697132 272.84 0.000010 AAACCTGTT Ppl 19041 DOWN YES YES YES chr16 5119594 5119928 687.32 chr16 5118800 5120523 102.2 YES 0.000260 CAACCTGCC Eps8l1 67425 DOWN YES YES YES chr7 4416901 4417184 66.19 chr7 4415317 4418035 298.33 NO

35 Grhl2 ChIP peak occurrence consistent with Grhl2 ChIP peak in IMCD-3 Motif occurrence in 50bp window cells in: Associated Grhl2 ChIP peak in IMCD-3 cells Associated diff. H3K4me3 ChIP peak in IMCD-3 ce around peak summit (p-value<1e-03) Success- Down- fully or rescued Entrez upreg. in Grhl2- Adult E18 MACS peak MACS peak p-value Name ID in KD Res kidney kidney Start End score Start End score (FIMO) Sequence AI661453 224833 DOWN YES YES YES chr17 47588025 47588457 1419.88 chr17 47587526 47589113 110.4 YES 0.000069 AGACCTGTC Rasef 242505 DOWN YES YES YES chr4 73436734 73437120 1092.34 chr4 73435069 73437509 112.83 YES 0.000195 GGACCTGCT Ovol2 107586 DOWN YES YES YES chr2 144157800 144158064 1438.74 chr2 144156034 144158556 986.16 YES 0.000122 GAACCGGTC Otud7b 229603 DOWN YES NO NO chr3 95937325 95937653 54.68 chr3 95936534 95939733 70.49 YES 0.000312 GAGCCAGTC Apbb2 11787 UP YES NO NO chr5 66995044 66995359 71.29 chr5 66994269 66995651 71.78 NO Nedd4l 83814 UP YES NO NO chr18 65196669 65197350 634.54 chr18 65196294 65197210 64.17 NO

36 Supplemental Table 4 Primers used for quantitative RT-PCR.

Supplemental Table 5 Primers used for quantitative ChIP-PCR.

37 Supplemental References

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