Supplemental Material ZNF750 Interacts with KLF4 and RCOR1, KDM1A, and CTBP1/2 Chromatin Regulators to Repress Epidermal Progenitor Genes and Induce Differentiation Genes Lisa D. Boxer, Brook Barajas, Shiying Tao, Jiajing Zhang, and Paul Khavari Supplemental Inventory Figure S1. This figure supports Figure 1 and shows the Gene Ontology terms for ZNF750-bound but unaffected genes, the genomic enrichment of ZNF750 ChIP-seq peaks, and the percentage of peaks that contain the ZNF750 motif. Figure S2. This figure supports Figure 2 and shows gene expression changes with depletion of ZNF750-interacting proteins, confirms the knock-down of ZNF750-interacting proteins, and shows the quantification of Ki67 in ZNF750-interacting protein depleted organotypic tissue. Figure S3. This figure supports Figure 3 and shows the quantification of ZNF750 and interacting protein co-IPs, and IPs and Far western blots demonstrating competition between KLF4 and KDM1A for binding to ZNF750. Figure S4. This figure supports Figure 4 and shows the expression of ZNF750 mutant proteins and the effects of full-length or mutant ZNF750 on differentiation in organotypic tissue and on clonogenic growth. Figure S5. This figure supports Figure 5 and shows the effects of mutagenesis of ZNF750 and KLF4 motifs on reporter activity, and the changes in histone marks with depletion of ZNF750 and interacting proteins. Figure S6. This figure supports Figure 6 and shows the changes in mRNA and protein expression of ZNF750-interacting proteins during keratinocyte differentiation, and the expression of ZNF750-interacting proteins with ZNF750 depletion. Table S1. Supports Figure 1 and shows the genomic coordinates of ZNF750 ChIP-seq peaks. Table S2. Supports Figure 5 and shows the genomic coordinates of KLF4 ChIP-seq peaks. Table S3. Supports Figure 5 and shows the genomic coordinates of RCOR1 ChIP-seq peaks. Table S4. Supports Figure 5 and shows the genomic coordinates of KDM1A ChIP-seq peaks. Table S5. Supports Figure 5 and shows the genomic coordinates of KLF4-enriched ChIP-seq peaks. Table S6. Supports Figure 5 and shows the genomic coordinates of KDM1A-enriched ChIP-seq peaks. Supplemental Materials and Methods: This includes information on lentiviral constructs, siRNAs, and primer sequences. Figure S1 A B ) Genomic enrichment of ZNF750 peaks s l GO terms for ZNF750-bound and unaffected genes a v r 2.0 e t Regulation of transcription n i l 1.5 Signal transduction o r t RNA metabolic process n o 1.0 c Cell communication / s k 0.5 Transport a e p Protein modification process 0.0 0 0.0 0.5 1.0 1.5 2.0 5 7 F -0.5 -log10(p-value) N Z ( -1.0 2 g o l TTS Intron Exon 3' UTR Intergenic C PromoterPromoter - TSS - distal f i t o 100 m 0 5 7 80 F N Z g 60 n i n i a t 40 n o c s 20 k a e P 0 % ZNF750-activated ZNF750-repressed Figure S2 A B CTLi ZNF750i CTLi KLF4i ZNF750i RCOR1i CTBP1+2i KDM1Ai KLF4i SSBP1i UBR5i DCAF7i PPP2R1Ai ZNF516i CBX3i TRAF7i ZNF185i RBBP7i ZNF750 KLF4 Knock-down LCE3D LCE3A -Actin -Actin ASPRV1 LOR SPRR2G AZGP1 CTLiRCOR1i CTLi KDM1Ai CRNN FLG RPTN RCOR1 KDM1A KLK6 KPRP SPRR3 CDSN -Actin -Actin AQP9 KCTD4 HOPX LYPD5 CNFN CTLi CTBP1i CTLi CTBP2i KRT80 TMEM86A NLRP10 CTBP1 CTBP2 LCE3B IVL SPRR1A ATP12A -Actin -Actin EGR3 SBSN SPINK5 AGPAT9 OVOL1 ATP10B NDRG2 PRDM1 ETS1 PPARD TGFA C i ALOXE3 e l GRHL1 c i DSG1 60 u 5 e l n ATP2C2 c l u CDCA7 a s n 50 CDC25B 4 l *** a ** DCTPP1 a b s SERPINH1 a 40 r ** RFC5 a * p B 3 FKBP5 * u e s HAUS1 v 30 i e t ZWINT i v i s 2 NPM3 t i o 20 DPP3 s p - MAD2L1 o 7 p MCM6 - 1 6 i 10 ARHGEF3 7 K 6 QDPR i K % RAD51C 0 0 TYMS % CDH3 COL5A2 CTLi CTLi KLF4i LXN KLF4i KDM1Ai RCOR1iKDM1Ai HAUS4 ZNF750i RCOR1i CTBP1/2i ZNF750i CTBP1/2i RPA3 MCM3 IGFBP2 PRKCDBP LGALS1 RFC3 TCEA3 LAMB1 CDKN3 COL7A1 EPCAM MATN2 RBBP8 ECM2 METTL7A VSNL1 WNT3 MMP28 TSPAN1 KRT8 Figure S3 A ZNF750 WB KLF4 WB RCOR1 WB 25 15 10 20 8 10 15 6 10 t 4 t t 5 u u u 5 2 p p p n n n I I I 1.5 1.5 1.0 % % % 1.0 1.0 0.5 0.5 0.5 0.0 0.0 0.0 IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG KLF4 IP KLF4 IP KLF4 IP CTBP1 IP CTBP2 IP CTBP1 IP CTBP2 IP ZNF750 IP RCOR1 IPKDM1A IP CTBP1 IP CTBP2 IP ZNF750 IP RCOR1 IPKDM1A IP ZNF750 IP RCOR1 IPKDM1A IP KDM1A WB CTBP1 WB CTBP2 WB 8 20 8 6 15 6 10 4 4 t t t 5 u u u 2 2 p p p n n n I I I 1.0 1.0 1.5 % % % 1.0 0.5 0.5 0.5 0.0 0.0 0.0 IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG KLF4 IP KLF4 IP KLF4 IP KDM1A IP CTBP1 IP CTBP2 IP KDM1A IP CTBP1 IP CTBP2 IP ZNF750 IP RCOR1 IPKDM1A IP CTBP1 IP CTBP2 IP ZNF750 IP RCOR1 IP ZNF750 IP RCOR1 IP B Inputs ZNF750 IPs Inputs ZNF750 IPs ZNF750 WB KLF4 WB KDM1A WB CTLi KLF4i CTLi KLF4i CTLi KDM1Ai CTLi KDM1Ai 25 3.0 2.5 20 2.5 2.0 ZNF750 t t t 2.0 u u 15 u 1.5 p p p n n n I I I 1.5 % 10 % 1.0 KLF4 % 1.0 5 0.5 0.5 KDM1A 0 0.0 0.0 CTLi CTLi CTLi CTLi KLF4i CTLi KLF4i CTLi KLF4i KDM1Ai KDM1Ai KDM1Ai C g ZNF750 KDM1A blot .25 .12 .06 .03 .016 125 KDM1A KDM1A l a KDM1A + 1X KLF4 n 100 g i KDM1A + 5X KLF4 s KDM1A + 1X KLF4 y l 75 KDM1A + 1X MBP n o KDM1A + 5X MBP A KDM1A + 5X KLF4 1 50 M D K 25 KDM1A + 1X MBP % 0 KDM1A + 5X MBP 0.25 0.12 0.06 0.03 0.015 Anti-KDM1A µg ZNF750 g ZNF750 KLF4 blot .25 .12 .06 .03 .016 150 KLF4 KLF4 l KLF4 + 1X KDM1A a n g KLF4 + 5X KDM1A i s 100 KLF4 + 1X KDM1A y KLF4 + 1X MBP l n o KLF4 + 5X MBP KLF4 + 5X KDM1A 4 F L 50 K KLF4 + 1X MBP % 0 KLF4 + 5X MBP 0.25 0.12 0.06 0.03 0.015 Anti-KLF4 µg ZNF750 Figure S4 A B C 1% ChIP Inputs CTL FL C2H2 PLNLS FLAG IP 8 C2H2+ CTL FL C2H2 PLNLS PLNLS ZNF750 HA 6 C2H2 HA t u PLNLS p n 4 I -Actin % 2 0 FLAG KLF4 RCOR1 KDM1A CTBP1 CTBP2 Western blot D CTL Full-length (HA) C2H2 mutant (HA) PLNLS mutant (HA) i e l i 50 c 2.0 e u l n c * * l u a n LOR s l 40 * a a 1.5 b s a HA a r b p 30 u e DAPI s v 1.0 i t i e s 20 v i t o i p s - o 0.5 7 p 6 10 - i 7 K 6 i % K 0 0.0 Ki67 % FLG CTL CTL Full-length Full-length DAPI C2H2 mutant C2H2 mutant PLNLS mutant PLNLS mutant E CTL Full-length C2H2 mutant PLNLS mutant ** 50 * 2 ** m 40 m 1 30 s e i n 20 o l o C 10 # 0 CTL Full-length C2H2 mutant PLNLS mutant Figure S5 A B C Differentiated keratinocytes Undifferentiated keratinocytes ) n 1.0 o i ** s 80 s 1.50 WT ** * CTL e r WT + ZNF750 + KLF4 p e e ZNF750 + KLF4 x * c c 1.25 e MUT + ZNF750 + KLF4 n n motif mutant 0.5 ** i e e 60 L c Differentiation c s s T 1.00 ** ** e e C n / n i i gene i 0 m m 5 0.75 40 u u 7 reporters L 0.0 L F e e N v 0.50 v i i t Z t ( a l a l 20 2 e e g 0.25 R o R l -0.5 0.00 ZNF750 + + 0 PPL -8kb PKP1 +70kb PPL -8kb PKP1 +70kb KLF4 + RCOR1 + + 1.25 * KDM1A + 8 ** CTL WT ** WT + ZNF750 e ** e ZNF750 motif c c MUT + ZNF750 * n 1.00 ** n mutant e e 6 c c s Progenitor s e e n 0.75 i n gene i m m 4 u u L reporters L 0.50 * e e v i v t i t a l a l 2 e 0.25 e R R 0.00 0 CTLi IgG CTLi IgG HOMER3 -11kb RBBP8 -75kb HOMER3 -11kb RBBP8 -75kb ZNFi IgG ZNFi IgG KLF4i IgG KLF4i IgG RCOR1i IgG RCOR1i IgG LSD1i IgG D LSD1i IgG CTBPi IgG CTBPi IgG ChIP-qPCR CTLi H3K4me1 ChIP-qPCR CTLi H3K27ac 3.0 15 ZNFi H3K27ac IgG H3K27acZNFi H3K4me1 IgG H3K4me1 CTLi CTLi KLF4i H3K27ac KLF4i H3K4me1 RCOZNF750iR1i H3K4me1 ZNF750i RCOR1i H3K27ac 2.5 KLF4i KLF4i LSD1i H3K4me1 * LSD1i H3K27ac RCOR1i RCOR1i CTBPi H3K4me1 CTBPi H3K27ac KDM1Ai * KDM1Ai 2.0 CTBP1/2i 10 CTBP1/2i t t u * u p p n n I 1.5 I % % * * * * 1.0 ** * 5 * * * * * * * * 0.5 0.0 0 PPL -8kb PPL -8kb PKP1 +70kb PKP1 +70kb Gene desert DLX5 +100kb RBBP8 -75kb Gene desert DLX5 +100kb RBBP8 -75kb LAMC1 +37kb LAMC1 +37kb HOMER3 -11kb HOMER3 -11kb Figure S6 A B Day 0 Day 3 n o 80 i 1500 Day 0 s ZNF750 s Day 0 e Day 3 r 1000 Day 3 60 p KLF4 x e 500 M n i K e 40 RCOR1 t P o 3 r R p 2 KDM1A 0 20 y a 1 D CTBP1 / 3 0 0 y CTBP2 a D KLF4 KLF4 CTBP1CTBP2 CTBP1CTBP2 ZNF750 RCOR1KDM1A ZNF750 RCOR1KDM1A Actin C ChIP inputs ZNF750 CTBP1 CTBP2 RCOR1 KDM1A HA-KLF4 Supplemental Figure Legends Figure S1.