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. Genomic enrichment of ZNF750 ChIP-seq peaks and percentage of peaks that contain ZNF750 motif. Related to Figure 1. (A) Gene Ontology (GO) terms for ZNF750-bound but unaffected genes. (B) Genomic location enrichment of ZNF750 ChIP-seq peaks compared to size-matched control genomic intervals. Promoter-TSS (transcription start site): +1 to -5kb of TSS; Promoter-distal: -5 to -100kb of TSS; TTS: transcription termination site. (C) Percentage of ZNF750 ChIP-seq peaks proximal to either ZNF750-activated or ZNF750-repressed genes that contain the ZNF750 motif.
Figure S2. Gene expression changes and protein expression with depletion of ZNF750- interacting proteins. Related to Figure 2. (A) Heatmap of gene expression changes with depletion of ZNF750-interacting proteins in differentiating keratinocytes, by qPCR. (B) Western blots confirming protein depletion with siRNAs to ZNF750, KLF4, RCOR1, CTBP1, or CTBP2, compared to control siRNA (CTLi). (C) Quantification of Ki67-positive cells in basal layer or suprabasal layers with depletion of ZNF750 and interacting proteins in organotypic epidermal tissue. 40 cells in 10 fields were counted per condition. (T-test: *: p<0.05, **: p<0.01, ***: p<0.001, compared to CTLi).
Figure S3. Quantification of ZNF750, KLF4, RCOR1, KDM1A, CTBP1, and CTBP2 co-IPs, and competition between KLF4 and KDM1A for binding to ZNF750. Related to Figure 3. (A) Quantification of co-immunoprecipitation western blots among ZNF750, KLF4, RCOR1, KDM1A, CTBP1, and CTBP2, shown as % of input. (B) Inputs and ZNF750 immunoprecipitation (IP) with control (CTLi) compared to KLF4 or KDM1A depletion, probed with antibodies to ZNF750, KLF4, or KDM1A. Right: quantification of western blots, shown as % of input. (C) Far western analysis with ZNF750 on membrane, probed with combinations of recombinant KDM1A, KLF4, or MBP as indicated on left of blots, followed by antibodies to KDM1A (top) or KLF4 (bottom). Blots are quantified as percentage of KDM1A-only or KLF4-only signal.
Figure S4. Expression of ZNF750 mutant proteins, and effects of ZNF750 and mutant expression on differentiation and clonogenic growth. Related to Figure 4. (A) Quantification of western blots from FLAG IPs for CTL, ZNF750, C2H2 mutant, or PLNLS mutant ZNF750, shown as % of input. (B) Western blot of 1% input used for chromatin immunoprecipitation of HA-tagged full-length (FL), C2H2-mutant, PLNLS-mutant, or C2H2+PLNLS-double mutant ZNF750. (C) Western blot of protein expression of control (CTL), FL, C2H2, or PLNLS mutant ZNF750 in progenitor keratinocytes, used to assay gene expression changes. (D) Effects on differentiation and proliferation with ectopic expression of HA-tagged Full-length, C2H2 mutant, or PLNLS mutant ZNF750 compared to empty vector control in regenerated organotypic epidermal tissue. Top panel: loricrin (LOR, red), HA tag (HA, green). Bottom panel: proliferation (Ki67, red); fillagrin (FLG, green). Dashed line denotes basement membrane; scale bar = 25µm. (E) Clonogenic assays of keratinocytes with ectopic expression of Full-length, C2H2 mutant, or PLNLS mutant ZNF750 compared to empty vector control. Right: quantification of colonies !1mm2. (T-test: *: p<0.05, **: p<0.01, ***: p<0.001).
Figure S5. Reporter assays with mutation of ZNF750 and KLF4 motifs, and effects of ZNF750 and interacting protein depletion on histone modifications. Related to Figure 5.
(A) Log2 fold expression change with ZNF750 depletion of genes near ZNF750, KLF4, and RCOR1 peaks, or ZNF750, RCOR1, and KDM1A peaks. (B) Changes in luciferase activity in differentiated keratinocytes of differentiation gene reporters with mutation of ZNF750 and KLF4 motifs (top), or progenitor gene reporters with mutation of ZNF750 motif (bottom). (T-test: *: p<0.05, **: p<0.01, ***: p<0.001). (C) Change in luciferase activity in undifferentiated keratinocytes of differentiation gene reporters with ectopic expression of ZNF750 and KLF4 with wildtype (WT) or ZNF750 and KLF4 motif mutant (MUT) reporters (top), or progenitor gene reporters with ectopic expression of ZNF750 with WT or ZNF750 motif mutant (MUT) reporters (bottom). (D) Changes in histone modifications H3K27ac (left) and H3K4me1 (right) in differentiated keratinocytes with depletion of ZNF750 and interacting proteins. (T-test: *: p<0.05).
Figure S6. Expression of ZNF750-interacting proteins during keratinocyte differentiation, and expression of ZNF750-interacting proteins with ZNF750 depletion. Related to Figure 6. (A) Reads per kilobase mapped (RPKM) of ZNF750 and interacting proteins on Day 0 and Day 3 of calcium-induced keratinocyte differentiation in vitro. (B) Left: western blots of ZNF750 and interacting protein expression on Day 0 and Day 3 of keratinocyte differentiation. Right: quantification of western blots, shown as Day 3/Day 0 expression. (C) Western blots for inputs used for chromatin immunoprecipitation assays with control (CTLi) or ZNF750 depletion, probed with antibodies to CTBP1, CTBP2, RCOR1, KDM1A, or HA-KLF4. Supplemental Materials and Methods
Lentiviral Constructs Constructs for overexpressing ZNF750 were made by cloning ZNF750 or C2H2 mutant (Sen et al., 2012) into pLEX (Open Biosystems) with C-terminal FLAG, HA, and 6XHIS tags with the following primers: ZNF750 F: ACGCAGGATCCGCCACCATGAGTCTCCTCAAAGAGCGGAAGCCAAAAA; ZNF750 R: ACGCAGCGGCCGCGGGGACACCCGGGCCCTCCTTCGTAGTGTG; ZNF750 uORF F (used in proximity ligation analysis): ACGCAGGATCCACCATGGCATAACGACTAAGGAAAATGAGTCTCCTCAAAGAGCGGAAGCC AAAAA. The PLNLS mutants were generated using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent) with the following primers: PLNLS 1 F: CTCCGGGATGGGCGCCCTCGCCGCCGCCAAGAAATCAGAG; PLNLS 1 R: CTCTGATTTCTTGGCGGCGGCGAGGGCGCCCATCCCGGAG; PLNLS 2 F: GCTGCAGGACCTTGCACTCGCTGCCGCGGTGAAGGACCCC; PLNLS 2 R: GGGGTCCTTCACCGCGGCAGCGAGTGCAAGGTCCTGCAGC. Constructs for reporter assays were made by cloning the following regions from genomic DNA into pGreenFire (Systems Biosciences): PPL -8 F: ACGCATCTAGATTGTAGAGTTGGGGTCTCGCTATGTT; PPL -8 R: ACGCAGAATTCCAGCACGTGGCCTGGTTTATAGTAAAT; PKP1 +70 F: ACGCAATCGATGCCCTGTATGACCTTGGATGAGTC; PKP1 +70 R: ACGCAGAATTCCCTCTGGTGATGCCTTAAGAGCC; HOMER3 -11 F: ACGCAGAATTCCCTCTGGAGCTTTGCTTACCC; HOMER3 -11 R: ACGCAATCGATGGTGAAAGCGTGAGACCTTGTCTA; RBBP8 -75 F: ACGCAATCGATTTCTTTATTGTGGCTCCCTACACC; RBBP8 -75 R: ACGCAATCGATCTTGGGCAAGTTACTTACCTCTT. Primers for mutagenesis of ZNF750 and KLF4 motifs in reporters are: PPL -8 ZNF750 F: CACTTGTCAAGCCTGCTCTCAGGGGTGGG; PPL -8 ZNF750 R: CCCACCCCTGAGAGCAGGCTTGACAAGTG; PPL -8 KLF4 F: CAGAAAGAGGGGCTCCGGACAGGCTC; PPL -8 KLF4 R: GAGCCTGTCCGGAGCCCCTCTTTCTG; PKP1 +70 ZNF750 F: CAGCCCCAGCCTAGTTCTTCAGGTTTGC; PKP1 +70 ZNF750 R: GCAAACCTGAAGAACTAGGCTGGGGCTG; PKP1 +70 KLF4 F: CACCACCTCTCCCACTGCGTTCCTGAAG; PKP1 +70 KLF4 R: CTTCAGGAACGCAGTGGGAGAGGTGGTG; HOMER3 -11 ZNF750 F: GTTAGGACACCCTGGACCTCATGTCTGG; HOMER3 -11 ZNF750 R: CCAGACATGAGGTCCAGGGTGTCCTAAC; RBBP8 -75 ZNF750 F: GCAGCTGTTGTCATCATTAGCAATACTTTTTCCTTTAGTG; RBBP8 -75 R: CACTAAAGGAAAAAGTATTGCTAATGATGACAACAGCTGC. siRNAs siRNA sequences for control, ZNF750, and KLF4 were as previously described (Sen et al., 2012). siRNAs for RCOR1, KDM1A, CTBP1, CTBP2, DCAF7, and ZNF516 were from Dharmacon as follows: RCOR1: CGACGCCGCUUCAACAUAG and CGCCAUGCCCGGAAACAAA; KDM1A: CCACCGAGUUCACAGUUAU and GGAAGUUGUCAUUCAGUUA; CTBP1: GGAUAGAGACCACGCCAGU and GAGCAGGCAUCCAUCGAGA; CTBP2: GCUCAAUGGUGCCACAUAC and UGAGAGUGAUCGUGCGGAU; DCAF7: GGAAGGAGAUCUACAAGUA and GCUCAUCGCCCAUGACAAA; ZNF516: GGAGAUAACGCCUCGGAAA and AGAGCCAGUUCGAGCGUAA. shRNAs for CBX3 are from OpenBiosystems as follows: GAATTGATGTTTCTCATGA and GTTACTTTGAACAAATAAA. siRNAs for SSBP1, UBR5, PPP2R1A, TRAF7, and ZNF185 were SMARTpools from Dharmacon.
Primers for Quantitative Reverse Transcriptase-PCR Analysis Primers for ZNF750, KLF4, LCE3D, LCE3A, LOR, FLG, SPRR3, LCE3B, SPRR1A, OVOL1, and PRDM1 were as previously described (Sen et al., 2012). New primer sequences are as follows: Gene name Forward primer Reverse primer 18S GCAATTATTCCCCATGAACG GGCCTCACTAAACCATCCAA AGPAT9 GAGGCCTGACTGAACTTCCC GCTGAGAGATCCATTGCCCA ALOXE3 ACTCCCCCACACTCGATACA GAGGTAGATGAGGCCTTGCC AQP9 CTCGAGACCTGAGTCCCAGA ATGAGGCCTCCAATGACAGC ARHGEF3 GATGGAGAAGTGAGGCTGGG AGCGAGTGGGTCTGACTTTG ASPRV1 CAGCCCTTTGAGAATGTGGT GGAACTGTGCCTTCAGCTTC ATP10B CCACTGACTGGCGAGATTGT TCCGGGCTCGGATTTTTCTC ATP12A CTCGGAAGTGTCACAGCCTT AAGAGCTTCCGCACCTCATC ATP2C2 TCGGCTTTCTCAGGAACCAC GGGGATGTAAATGACCGCCA AZGP1 CACGTATTGCAGGGAAGGTT GGCTGGGATTTCTTTGTTGA CBX3 GAAAAGAGATGCTGCTGACAAACCA CTTCTGGACAAGAATGCCAAGTTAG CDC25B GGAGTTCTTCCCTCAGCACC GAGTCTTGAGGCGGAAGGTC CDCA7 CTGCAGAAACCCAGACTGCT TGCAGATTCCTCGACAAGGC CDH3 CATCCCGACACCCATGTACC GAACACCAAGAGGGTGTCGT CDKN3 GAGCAAGCCATAGACAGCCT TGTGCAGCTAATTTGTCCCG CDSN CCTTGAGCTGCCATCAGTCAG GTCTGAGAAGGTGCCAATGCT CNFN GCACTTTTGCTCCTCTGTGC AGGGCAGACAAAAGGTGAGG COL5A2 TTCTCAAAGGGGCAAATGAC ACAGTCTTGCCCACATTTCC COL7A1 TTGTCTATGGTGGCTGTGGA CAGTCCTGGGCAGTACCTGT CRNN GGGTCACACACCGAGACT TCTCCCTTGAGTGGTCATC CTBP1 GCATCCATCGAGATGCGAGA ATTGAGCTCAGGGTGCACGA CTBP2 TCAAGGAGGGCAGGATACGA TACCAGGCAGTGTGAGGAGT DCAF7 GGCGTCTATCCAGACCTA GATGGTGCATGTCGTATCAAT DCTPP1 AACAGTTCCATCAGCCTCGG AGGACGTCACTAAGCTCCTCT DPP3 GCTGCGTAAGGAATCTCGGA GAAGGATCGGATGAGGCCAG DSG1 AGCCTGTCGTGAAGGTGAAG TCCTACTCCAGAGATGCGGT ECM2 ACGTCCCGTCCTATCTACCC CATGTGGCCAAACACATAGC EGR3 GTCTGACCAACGAGAAGCCC GTCCTGGCACCAGTTGGAAG EPCAM ATGCAGGGTCTAAAAGCTGGT AGTTCCCTATGCATCTCACCC ETS1 GCCAGGAGATGGGGAAAGAG CTGCAGGTCACACACAAAGC FKBP5 AACCAGAATATGCATATGGC AGAAAAGGAATCGAAAGCTTC GRHL1 GCCTACCCACTCCATCAAGA GAGTCTGGAGTTCGCCTTTG HAUS1 ACAGAAAGGGCCAAAGTTGA CTCCTCTGCAGCCTTGATTC HAUS4 GAGGGAGCCATTCACCTACA CACCAGCCTGTCAAACTCCT HOPX CTTGCCTTTGCCTCTTCCAC ACAGCCCAGGAAAATGAGCA IGFBP2 GGCAAGGGTGGCAAGCATC GGTTGCAGACAATGGCGATGA IVL AAAGCACCTAGAGCACCC GGTTGAATGTCTTGGACCT KCTD4 TCGCATTGTTCTGGTGTCCA AGTCGAGTGCCATTTTCAGACT KDM1A CAGCCCAAAGAAACTGTGGT TGATCCTGCAGCAACATAGG KLK6 GTTGTGTGCTGGGGATGAGA CGTTGGTGTAGACTCCTGGC KPRP TAGTCCACACCGCCTAGACA CCTCCGCTCTGGAAACACAT KRT8 GCGGAGCTTGGCAACAT GAAGTTGATCTCGTCGGTCAG KRT80 AAAGCCTGGAGAGCTTCGTG CATACTGGGCCTTCACCTCC LAMB1 TTGCCAGAGCTGAGATGTTG CTGCTTCTTCCAGAGCTTCC LGALS1 GGAGGCTGTCTTTCCCTTCC GTTGATGGCCTCCAGGTTGA LXN AACGGGACAAGAAACTGCAC GCGGTTCCTTCATGGACTTA LYPD5 GACGAGTCCAGTGTCACCAG CGGTGGCAGGTTCTGATGTA MAD2L1 TGACAGTGCACCCAGAGAAA TTCCAACAGTGGCAGAAATG MATN2 AAGCTCTAGAAGACTCCGATG ATGGGAAAGGGCTCTATGACT MCM3 TCAAGCCTGTCCTGACACAG CAGGTCCACAGTCTTGCTCA MCM6 CAAGAGTCTGCTCCCAAAGC CCACCTTTCTGAGGTGAAGC METTL7A GCAGCTGAGTGTTCGACTTG GCTCTCTCTGGTCAGGTTGC MMP28 AGAGCGTTTCAGTGGGTGTC GGCCGCATAACTGTTGGTAT NDRG2 TACTTCCTGCAAGGCATGGG AAGAGTTCCAGACTCGCTGC NLRP10 CCTCAAGGGCTTGAAGGTCA ATTGACTCCTGCTTCCTGCC NPM3 TGACTTCCAGCTCCAACCAC CTCTTCCTCGCTCTCCTCCT PPARD GTGTGGAAGCAGTTGGTGAA TGCACGCCATACTTGAGAAG PPP2R1A CTACATGGTGGCTGACAAGTT GGCAAGATCTGGGACATGAT PRKCDBP AGCTCCACGTTCTGCTCTTC CTCTCTCCAACTTCGGCCTC QDPR ACCCTGGATACCCCGATGAA CTCGGTCGGTTTTTCCCTGT RAD51C AAACCCTCCGAGCTTAGCAA CTGGGTATGCTCCTGCTCAA RBBP7 CGAAGGAGAAGTAAACCGTG AATTCCAGGAGAGACCATAGC RBBP8 TCCGCTACATTCCACCCAAC GGCTGACGTCTTTTTGGACG RCOR1 AGCAGCTTCTCGCCGTACA GGTTACTGGGCCCATTGGTCT RFC3 GCCTGCAGAGTGCAACAATA GAGCCTTTGTGGAGTTTGCT RFC5 CTGGAACATGTCGTGGAAGA GTTCAGAGCCCTACGCATGT RPA3 CTGTCTGCTTCGTAGGGAGG ACTCTTCCAACCACTTCCACA RPTN TGGCTGAGTTTGGAGACATC CCAACTGGAACACCAACAAG SBSN TCGGTCAACACGCCTTTCAT TGATGTGACAACGGCGACAT SERPINH1 GAAGGCTGTTGCCATCTCCT CAGGTCCTTCTTGCCTGACA SPINK5 GACTACCGAGTATTGCCCAGG TAGGTTTGGCCATCGTCACC SPRR2G TGCCAGGATAAATGCCCTCC ACTTGCTCTTGGGTGGATACT SSBP1 AGGGGATAGTGAAGTTTACCA TGTTGCTTGTCGCCTCA TCEA3 GAGTTGAGGAATGCCATGACC TGATGAACTGAGGGAGTTGAG TGFA CAGAAGAAGCAGGCCATCAC TCACAGTGTTTTCGGACCTG TMEM86A GGGCTTGTCTTCTCTGCTGT GGCGTAGAACATGTGGGTCA TRAF7 CGTGGTGGTGAACAACATCGC CGCACAGGCCTGTAGTCACA TSPAN1 CAGCCAATGAAACCTGCACC ACCCACGGTGACTGCATTAG TYMS CCAAAGCTCAGGATTCTTCG AGTTGGATGCGGATTGTACC UBR5 TCTGTCTCTAATGCGGTCTCA AAGAGTTCTCGCGTCCTT VSNL1 TCTACAAAATGGTAGGCACTG GTTCTTTCCTTATGGAGACGC WNT3 AGGAAAAATGCCACTGCATC CAGGTGTGCACGTCGTAGAT ZNF185 AAGGGATTCTCTTCGTGAAGG CAGTCTCGGATCTCACGGTTG ZNF516 GCCGCTAGCATGCCTAAGAAT TCCTGGCCGTGACAAGGTTT ZWINT CAGGAGCTTGACAGGGTGTT TCAGCCTCAGGGAACAACAG
ChIP-qPCR primer sequences Genomic location Forward primer Reverse primer PPL -8kb CTGCTACACATCTTGGATCCC TGCCCTGACAGTGTGACT PKP1 +70kb CAGGCTTCTTCAGGTTTGCAT CACACCTGCAGCACGC PGLYRP4 -0.1kb TCCTCAGCCCCAACCTG CTCCTGGAGCAGAACTTCACA DLX5 +100kb GAGGACACAGAAAAGGTAGGGA TGCTGGCTAGTCTGTTTACTTGA RBBP8 -75kb GGCAGCTGTTGTCATCATTAG GTTCCTCCCTATTTAATGAGCTT HOMER3 -11kb GATTCAGGGCAATAGCACGC CCCACTCTGCCCAACTCACTT LAMC1 +37kb CCCTGGGGACAAGAGCTAGAT TCACCCATTCATTCGGTCA COL5A2 +15kb CACAGGGCCAGTTGCTATCTG CACCTTGCCTGAGCAGTCAAT Gene desert AAGAGGCCCTTCCTCTATGC TGTGATTAATCTCGACTCCAAGA