Supplementary Fig. 1 a c Control PBS WT1 KD ADR Nphs2-2 Synpo-1 Nphs2-2 Synpo-1

100 100 **** 150 250 **** **** **** 80 **** 80 200 100 60 60 **** 150 **** **** 40 100 40 **** **** 50 ** ** ** * ** ** 20 20 50 * **** Relative Enrichment Relative Relative Enrichment 0 0 0 0

H4K8Ac H3K9m3 H4K8Ac H4K8Ac H3K9m3 H4K8Ac H3K9m3 H3K4me3 H4K12Ac H4K12Ac H3K9m3 H3K4me3 H4K12Ac H3K4me3 H4K12Ac H3K4me3 H3K27me3 H3K27me3 b d ** ** *** * 60 25 50 30

20 40 40 20 15 30

10 20 20 10

Enrichment 5 Enrichment 10 WT1 Relative Relative WT1 Relative WT1

0 0 0 0 Control WT1 KD Control WT1 KD PBS ADR PBS ADR Supplementary Fig. 1. WT1 controls chromatin remodeling at Nphs2 and

Synpo genes in murine immortalized podocytes

(a, c) Histones direct ChIP-qPCR using active histones marks (H3K4m3, H4K8ac) and repressive histones marks (H3K9me3 and H3K27me3) at Nphs2-2 and

Synpo-1 peaks from immortalized mouse podocytes treated with PBS or 1µg/mL of ADR during 16 hours (a) or podocytes transfected with siRNA scramble or siRNA WT1 (c). **** P<0.0001, *** P<0.001, ** P<0.01, * P<0.05 (Multiple t-test with FDR determined using the two-stage linear step-up procedure of Benjamini,

Krieger and Yekutieli) compared to control. (b, d) WT1 direct ChIP-qPCR at

Nphs2-2 and Synpo-1 peaks from immortalized mouse podocytes treated with

PBS or 1µg/mL of ADR during 16 hours at Nphs2-2 and Synpo-1 peaks (b) or podocytes transfected with siRNA scramble or siRNA WT1 (d). Bars represent means and error bars ± SEMs. *** P<0.001; **P<0.01; *P<0.05 (n=3).

39 Supplementary Fig. 2

a PBS b Wt1 Nphs2 Synpo BALB/cJ ADR 800 5 * 80 15 ** * *** ** ** *** ** * ** 600 4 60 ** ** 10 3

400 GAPDH 40

ratio 2 ** 5 20 BALB/cJ 1

200 *** Normalized Gene/ 0 0 0 PBS D3 D4 D5 D6 D7 PBS D3 D4 D5 D6 D7 PBS D3 D4 D5 D6 D7

Albumin/creatinine(mg/g) 0 D0 D3 D4 D5 D7

c Saline D3 D5 D7 WT1 BALB/cJ Merge

Nphs2 Synpo d 24kb 33kb

[0-300] [0-300] seq - WT1 WT1 ChIP

Nphs2-1 Nphs2-2 Nphs2-3 Synpo-1 Synpo-2 Synpo-3 Nphs2-1 Nphs2-2 Nphs2-3 Synpo-1 Synpo-2 Synpo-3 40 30 80 20 40 50 * * **** *** * * ** ** ** **** 30 * 40 60 **** 15 30 20 30 40 10 20 20 WT1 20 10 Relative Relative 10 20 5 10 10 Enrichment 0 0 0 0 0 0 PBS D3 D4 D5 D6 D7 PBS D3 D4 D5 D6 D7 PBS D3 D4 D5 D6 D7 PBS D3 D4 D5 D6 D7 PBS D3 D4 D5 D6 D7 PBS D3 D4 D5 D6 D7 Supplementary Fig. 2. Transient increase in the expression of key podocyte genes in ADR-injured BALB/cJ mice

(a) Quantification of albumin/creatinine level during the course of ADR injury from

BALB/cJ mice injected with 10.5mg/kg of ADR (grey bars) or PBS (black bars).

Bars represent means and error bars ± SEMs. ***P<0.001; **P<0.01 (n=3 replicates). (b) RT-qPCR of Wt1, Nphs2 and Synpo from isolated glomeruli from

BALB/cJ during injury. One-way ANOVA with Tukey’s multiple comparisons test were used. ***P<0.001; **P<0.01; *P<0.05 (n=3 replicates). (c) Immunofluorescent staining of WT1 (red) in BALB/cJ mice in glomeruli. Scale bar 50µM. (d) Upper panels representing IGV plots of Nphs2 and Synpo genes for WT1 ChIP-seq showing WT1 binding sites (gray highlighted boxes) in uninjured podocytes:

Nphs2-1, Nphs2-2, Nphs2-3, Synpo-1, Synpo-2 and Synpo-3. Lower panels: WT1 dynamic binding at Nphs2 and Synpo genes measured by WT1 direct ChIP-qPCR from isolated glomeruli from BALB/cJ mice (n=3). ANOVA with Tukey’s multiple comparisons test were used. **** P<0.001; ***P<0.001; **P<0.01; *P<0.05.

40 Supplementary Fig. 3 a WT1 bound genes with new binding sites at D9 b New WT1 bound genes at D9 22kb 77kb [0-200] [0-200] PBS PBS [0-200] [0-200] D9 D9 [0-200] [0-200] D14 D14

Rhpn1 Kdr 77kb 25kb [0-400] [0-200] PBS PBS [0-400] [0-200] D9 D9 [0-400] [0-200] D14 D14

Ildr2 Cryab 205kb 88kb [0-200] [0-200] PBS PBS [0-200] [0-200]

D9 D9 [0-200] [0-200]

D14 D14

Zhx2 Itga6 Supplementary Fig. 3. Identification of new WT1 bound sites at the onset of proteinuria in mTmG-Nphs2cre mice

(a and b) Identification of new WT1 binding sites at D9 within genes that were already bound (a) or unbound (b) in uninjured podocytes. WT1 ChIP-seq IGV plots of Rhpn1, Ildr2 and Zhx2 genes (a) or Kdr, Cryab and Itga6 genes (b) showing

WT1 binding sites during injury (uninjured/PBS: blue, D9: orange, D14: red). Red arrows show TSSs and transcription direction. Green arrows show new WT1 binding sites present at D9.

41 Supplementary fig 4

15546 Percent (%) a 15000 13459 b c Percent (%) 0 20 40 60 80 100 10000 0 20 40 60 80 100 promoter PBS codingExon (n=29,005) intron Up ADR 5000 PBS (n=2,485)

Intersection size 2890 5'UTR 3'UTR 0 ADR intergenic Up Down 16,349 ADR 29,005 PBS Down ADR (n=14,109)

30000 0 WT1 binding changes Number of peaks d f 2500 Increased WT1 binding at ADR

class1 class1 negative regulation of phosphorylation(63) ● negative regulation of protein phosphorylation(60) ● negative regulation of phosphate metabolic process(68) ● class2 negative regulation of protein metabolic process(95) ● class2 regulation of cytokine production(94) ● logP negative regulation of protein modification process(67) ● 25 class3 negative regulation of cellular protein metabolic process(82) ● ● class3 cell junction organization(37) 20 class4 intrinsic apoptotic signaling pathway in response to DNA damage(25) ● apoptotic signaling pathway(55) ● 15 Genes class4 positive regulation of cell death(79) ● negative regulation of kinase activity(42) ● negative regulation of protein kinase activity(40) ● negative regulation of transferase activity(43) ● cell junction assembly(26) ● cellular response to organonitrogen compound(70) ● Unbound modulation by virus of host morphology or physiology(14) ● Unbound intrinsic apoptotic signaling pathway(36) ● actin cytoskeleton organization(83) ● cell−cell junction organization(31) ●

Decreased WT1 binding at ADR 0 paraxial mesoderm development(14) ● g ● PBS ADR paraxial mesoderm morphogenesis(9) glomerulus development(37) ● class1 class4 regulation of cell shape(58) ● mesoderm development(61) ● logP class2 Unbound organ growth(26) ● class3 lymph vessel morphogenesis(10) ● 60 cell−cell junction organization(62) ● 50 glomerular visceral epithelial cell differentiation(14) ● insulin receptor signaling pathway(33) ● 40 ● glomerular epithelial cell differentiation(14) 30 e cell junction organization(68) ● epithelial cell differentiation involved in kidney development(19) ● cellular response to peptide hormone stimulus(67) ● muscle cell proliferation(14) ● Percent (%) transforming growth factor beta receptor signaling pathway(44) ● ● 0 20 40 60 80 100 cardiac muscle cell proliferation(13) lymphangiogenesis(9) ● embryonic cranial skeleton morphogenesis(27) ● n=23968 Background cellular response to insulin stimulus(53) ●

Up ADR n=1816

Up Down

Down ADR n=5248

Genes with differential WT1 binding (Class defined in uninjured state) Supplementary Fig. 4. Dynamics of WT1 binding during the injury response in BALB/cJ mice

(a) Number of WT1 binding sites in control and after ADR. Grey bars represent the number of peaks common to each condition. Blue bar plot shows total binding site number. (b and c) Genomic distribution of (b) all WT1 binding sites or (c) WT1 binding sites that significantly changed during injury. (d) Alluvial diagram showing gene class changes after injury: class 1 (pink), class 2 (blue), class 3 (green), class

4 (purple) and unbound class (orange). Y-axis represents the number of genes per class, and X-axis the injury time points. (e) Proportion of each gene class for the genes associated with significant changes in WT1 binding intensity after ADR injury. Gene classes are based on the WT1 binding status in uninjured podocytes.

Background indicates the distribution of gene classes for all bound genes (green).

White represents unbound genes. The number on the top of the last two columns represents the number of genes with WT1 binding sites that significantly changed during the course of injury. (f) GO terms representing genes at which WT1 binding increased after ADR (upper panel) or decreased (lower panel).

42 Supplementary Fig. 5

a promoter intron 3'UTR c 8 codingExon 5'UTR intergenic 6 Percent (%) 0 20 40 60 80 100 4 Genome PBS n=23,163 2 D9 n=31,639 D14 n=6,567

Expression level (Log2 FPKM+1) 0 PBS D9

b Percent (%) 0 20 40 60 80 100 Differentially expressed genes PBS d

Up n=6,243 significant WT1 binding changes no significant WT1 binding changes

D9 Up 93% Down

Down n=468 D9 D14

Up n=93 38.6%

D14 Up Down 99% 64.3% WT1 binding changes Down n=17,440 Supplementary Fig. 5. Effect of ADR on WT1 binding and gene expression in in mTmG-Nphs2cre mice

(a and b) Genomic distribution of all WT1 binding sites (a) or WT1 binding sites that significantly changed during injury (b). (c) Expression levels of the 223 genes with more than 2 fold increase of expression at D9 compared to control. The majority of genes were silent in uninjured podocytes. (d) Portions of differentially expressed genes with significant changes in WT1 binding at D9 (38.6%) and D14

(64.3%).

43

49kb

21kb 19kb 45kb

ly(22) b

lamellipodium assem lamellipodium

ly(30) b

ization(24) r

regulation of actin filament depolyme filament actin of regulation Prpf39

response to laminar fluid shear stress(9) shear fluid laminar to response

Elavl1

regulation of mRNA processing(52) mRNA of regulation

ation(11) r mig cell uscle

Snrpa1 m Rbm17

a w path signaling receptor actor f wth o gr ed v i r

y(23)

a n deade , s proces catabolic RNA y(17) 15

ylation(34) h demet or ylation h DNA met DNA

phogenesis(11) r mo essel v lymph 20

ial changes(41) ial r apoptotic mitochond apoptotic

25 ylation(27) h DNA met DNA

xpression, epigenetic(90) xpression, e regulation of gene gene of regulation

RNA catabolic process(67) catabolic RNA

ly(51) b

cell junction assem junction cell

200]

200] 200]

- - -

200] 200] 200] 200] 200] 200] 200] 200] 200] ibed mRNA catabolic process(53) catabolic mRNA ibed r ansc r - n ------[0 [0

[0

[0 [0 [0 [0 [0 [0 [0 [0 [0 logP

mRNA catabolic process(58) catabolic mRNA mTmG-Nphs2cre ADR D14 down mTmG-Nphs2cre regulation of RNA stability(29) RNA of regulation PBS D9 D14

PBS D9 D14 PBS D9 D14 PBS D9 D14

regulation of mRNA stability(28) mRNA of regulation

BALB/C ADR down c mTmG-Nphs2cre ADR D9 up mTmG-Nphs2cre ADR D14 down E E BALB/C ADR down mTmG-Nphs2cre ADR D9 up mTmG-Nphs2cre ADR D14 down

y(44)

Ras protein signal t signal protein Ras ansduction(35) r ly(22) b

y(23) assem lamellipodium

a y(17)

y(44)

a

a y(23) a y(17) w ly(30) b e regulation of t of regulation e v negati ase activity(59) ase r e f ans

r a

w a

w

w

ization(24) r depolyme filament actin of regulation e regulation of actin filament depolyme filament actin of regulation e v negati ization(14)

r

elopment(16)

elopment(19)

v elopment(16) response to laminar fluid shear stress(9) shear fluid laminar to response xylic acid biosynthetic process(54) biosynthetic acid xylic o v monocarb

elopment(19)

e

v

ization(14) e v

e r

ization(14) e regulation of mRNA processing(52) mRNA of regulation e regulation of kinase activity(59) kinase of regulation e v

r negati y d

y d

y d e y d y e ation(11) r mig cell uscle m

e organization(48) filament actin

ulus(67) e

ulus(67)

actor receptor signaling path signaling receptor actor f wth o gr ed v i r y(23) a m w elopment(56) v e

placenta d placenta

m 15

ization(24)

r n deade , s proces catabolic RNA y(17) a ization(24) al epithelial cell dif cell epithelial al r visce ular r glome erentiation(12) f

erentiation(12) 15

r erentiation(14)

f

erentiation(12)

f

erentiation(14) Elavl1 f

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els(67) f

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v

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v 25 v y(33) e phogenesis(11) r mo essel v mone sti mone lymph v a 20 ate adhesion(42) ate ulus(53) r phogenesis(27) ol n mone sti mone

r a

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phogenesis(27) ol r ol n ase activity(59) r

v w

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e

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f erentiation(13) f

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f

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f e

b w o f

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e f b elopment(11) phogenesis(9) w

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v ly(22) v

s v

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b e ial changes(41)

e 39 Prpf b e ial changes(41) e actor beta receptoractor beta path signaling

e ly(51)

r e actor beta receptor beta actor path signaling ansduction(35) eleton mo ly(51) f xpression, epigenetic(90) r wth ansduction(35) process(67) catabolic RNA eleton mo eleton f xpression, epigenetic(90) wth response to decreased decreased to response els(67) v e l xygen o r

b r k

b

e

o

k e o

ation(14)

ation(14) phogenesis(11)

phogenesis(11) m d r al epithelial cell dif m d r phogenesis(10) al epithelial cell dif ly(51) b r assem junction cell m d m mo phogenesis(10) al epithelial cell dif cell epithelial al r m d m mo r erentiation i organization(55) junction cell r al epithelial cell dif cell al epithelial r erentiation i r ation(11) r

e r r r ation(11)

erentiation i erentiation r f

wth e r r erentiation i erentiation r f ibed mRNA catabolic process(53) wth wth

f r

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elopment(34)

r

r elopment(37)

o

elopment(37) o elopment(61) elopment(61) v

ibed mRNA catabolic process(53) catabolic mRNA ibed r ansc r v n xygen l xygen o to response els(69) v v e xygen l ed gr Snrpa1 v xygen l

ed gr v v logP e

e

elopment(56)

elopment(56) v e o anial s v e o anial s e i e i logP

eleton organization(131)

Increased WT1 binding at D9 at binding WT1 Increased r eleton organization(131) v r v mTmG-Nphs2cre ADR D9 up mTmG-Nphs2cre 1 0 2 xylic acid biosynthetic process(54) DecreasedWT1 bindingat D14 process(58) catabolic mRNA r xylic acid biosynthetic process(54) r

ate adhesion(42) m d m r mesode axial r pa elopment(11) v ate adhesion(42) e k k ylation(27) ylation or demet e ylation(27) ylation or demet e

ansc r ansc r

o mTmG-Nphs2cre ADR D14 down mTmG-Nphs2cre o

r r

1. 5 0. 5 h h uscle cell proli uscle cell

h h uscle cell proli uscle cell wth(26) wth(26)

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ming gr

ulus d ular epithelial cell dif ular visce

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r r

axial mesode axial mesode axial mesode axial mesode r axial mesode axial mesode r stability(28) mRNA of regulation r r uscle cell mig r r uscle cell mig uscle cell proli cell uscle uscle cell proli uscle cell ans d ans r apoptotic mitochond lymph cell junction assem RNA catabolic process(67) regulation of gene DNA met DNA met RNA catabolic proces n lamellipodium assem m regulation of mRNA processing(52) response to laminar fluid shear stress(9) regulation of actin filament depolyme regulation of mRNA stability(28) regulation of RNA stability(29) mRNA catabolic process(58) actin cytos glome pa epithelial cell dif apoptotic signaling path response to decreased response to organization(55) cell junction glome glome placenta d actin filament organization(48) negati monocarb negati Ras protein signal t negati r apoptotic mitochond lymph DNA met DNA met RNA catabolic proces cell junction assem RNA catabolic process(67) regulation of gene n lamellipodium assem m regulation of mRNA processing(52) response to laminar fluid shear stress(9) regulation of actin filament depolyme regulation of mRNA stability(28) regulation of RNA stability(29) mRNA catabolic process(58) actin cytos glome pa epithelial cell dif apoptotic signaling path response to decreased response to cell junction organization(55) glome negati glome placenta d actin filament organization(48) monocarb negati Ras protein signal t negati pa pa cellular response to insulin sti response to cellular emb cardiac cardiac lymphangiogenesis(9) regulation of cell shape(58) regulation mesode glome organ gr organ lymph insulin receptor signaling path signaling receptor insulin cellular response to peptide ho peptide to response cellular m t epithelial cell dif cell epithelial glome glome organization(68) junction cell pa pa cellular response to insulin sti cellular response emb cardiac cardiac lymphangiogenesis(9) regulation of cell shape(58) regulation mesode glome organ gr organ lymph insulin receptor signaling path signaling receptor insulin cellular response to peptide ho response to cellular m t epithelial cell dif epithelial glome glome organization(68) junction cell

Ras protein signal t signal protein Ras ansduction(35)

r

cellular response to insulin sti insulin to response cellular ulus(53)

m

e regulation of t of regulation e v negati ase activity(59) ase r e f ans 15 r 35 25 60 50 40 30

20 15 25

emb phogenesis(27) r mo eleton k s anial r c onic y

r 35 25 15 30 60 50 40 15 20 25

e regulation of actin filament depolyme filament actin of regulation e v negati ization(14) r

logP logP logP logP logP logP lymphangiogenesis(9)

b a xylic acid biosynthetic process(54) biosynthetic acid xylic o monocarb cardiac cardiac ation(13) r e f proli cell uscle

m

Supplementary6 Fig. r t actor beta receptor signaling path signaling receptor beta actor f wth o gr ming r o f ans y(44) a w e regulation of kinase activity(59) kinase of regulation e v

negati

m ation(14) r e f proli cell uscle

organization(48) filament actin

cellular response to peptide ho peptide to response cellular mone sti mone r ulus(67) m

elopment(56) v e

d placenta

30

15

epithelial cell dif cell epithelial ed in kidn in ed v ol v n i erentiation f elopment(19) v e d y

e r visce ular r glome erentiation(12) f

al epithelial cell dif cell epithelial al cell junction organization(68) junction cell

40

ular epithelial cell dif cell epithelial ular r glome erentiation(13)

f

glome erentiation(14) f dif cell epithelial ular r

25

ate adhesion(42) ate r 50

insulin receptor signaling path signaling receptor insulin y(33) a

w

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f

60

35 apoptotic signaling path signaling apoptotic y(72) a

w

essel mo essel v lymph phogenesis(10) r erentiation i erentiation f dif cell epithelial elopment(16) v e d y e kidn in ed v ol v

n

wth(26) o gr organ response to decreased decreased to response els(67) v e l xygen

BALB/C ADR down BALB/C o

m d m r mesode elopment(61) v e

organization(55) junction cell

logP

regulation of cell shape(58) cell of regulation

xygen l xygen o to response els(69) v

e

logP

ulus d ulus r glome elopment(37) v

mTmG-Nphs2cre ADR D9 up mTmG-Nphs2cre e r mesode axial r pa elopment(11) v e

m d m axial mesode axial r pa phogenesis(9) r mo m

r

elopment(34) v e d ulus r

glome axial mesode axial r pa elopment(14) v e d m

r

eleton organization(131) eleton k

actin cytos actin

E

cellular response to insulin sti insulin to response cellular ulus(53)

m

emb phogenesis(27) r mo eleton k s anial r c onic y

r

lymphangiogenesis(9)

cardiac cardiac ation(13) r e f proli cell uscle

m

t actor beta receptor signaling path signaling receptor beta actor f wth o gr ming r o f ans r y(44) a

w

m ation(14) r e f

uscle cell proli cell uscle

cellular response to peptide ho peptide to response cellular mone sti mone r ulus(67)

m

30

epithelial cell dif cell epithelial ed in kidn in ed v ol v n i erentiation f elopment(19) v e d y

e

cell junction organization(68) junction cell

40

glome erentiation(14) f dif cell epithelial ular

r

50

y(33) a w

insulin receptor signaling path signaling receptor insulin

ular visce ular r glome erentiation(14) f dif cell epithelial al r

60

phogenesis(10) r mo essel v

lymph lymph

wth(26) o

BALB/C ADR down BALB/C gr organ

m d m r mesode elopment(61) v

e logP

regulation of cell shape(58) cell of regulation

ulus d ulus r glome elopment(37) v

e

axial mesode axial r pa phogenesis(9) r mo m

r

axial mesode axial r pa elopment(14) v e d m r E Supplementary Fig. 6. Gene ontology terms enriched for significant WT1 binding after ADR in mTmG-Nphs2cre mice

(a) GO terms enriched for the genes associated with significantly increased WT1 binding at D9. (b) GO terms enriched for the genes associated with significantly decreased WT1 binding at D14. (c) WT1 ChIP-seq IGV plots showing examples of increased expressed genes associated with a decrease of WT1 binding at D14.

Uninjured/PBS: blue, ADR-D9: orange, ADR-D14: red. Red arrows show TSSs and transcription direction. (d) Log2 expression fold change of genes shown in (c).

FC: fold change.

44 Supplementary Fig. 7

A B

Expression Log2 Fold Change 2 1 2 6 4 2 0 − 1 0 − − d14 d9 ctr d9 d14 d14 d9 ctr d9 d14 Tmsb4x Clu Nupr1 Mt2 Cryab Egf Ctsl Wnk1 Adm Gadd45a Gabarapl1 Cdkn1c Atp1b1 Rasl11a Oxct1 Igfbp7 Kl Sdc4 Slc12a3 Iqgap2 Golim4 Slc25a5 Tcf21 S100a1 Npr3 Atp1a1 Col4a3 Mt1 Tmem150c Pard3b Chchd10 Nphs1 Pgam2 Podxl Acat1 Pak1 Wfdc2 Htra1 Epcam Sept11 Eif3m Defb1 Arhgap24 Klk1 Rab3b Nudt4 Nes Tmem52b Cd59a Shisa3 Sfrp1 WT1 Slc16a7 Nap1l1 Pvalb Hoxc8 Fabp3 Enpep Kng2 Mertk Robo2 Ppp1r1a Clic3 Mal Dpp4 S100g Tdrd5 Sostdc1 Synpo Pth1r Clcnkb Rhpn1 Kcnj1 Nphs2 Expression fold change compared to control for tubule genes tubule for control to compared change fold Expression Umod

Expression fold change compared to control for podocytes genes podocytes for control to compared change fold Expression Vegfa Wfdc15b Mafb Tmem213 Npnt Sema3g Spp1 Gas1 Calb1 Ildr2 Ldhb Thsd7a Fxyd2 Unbound class4 class3 class1 class2 Supplementary Fig. 7. Podocyte specific gene expression during ADR- induced injury in mTmG-Nphs2cre mice

(a and b) Heatmap showing the expression changes and WT1 gene classes for podocyte specific genes (a, reproduced from Figure 5 for comparision) or for tubule specific genes (b). Red: increased expression in injured podocytes compared to uninjured podocytes. Blue: decreased expression. Colors on the top indicate gene classes based on WT1 binding status.

45 Supplementary Fig. 8 a 12kb b [0-200] FoxC2 40 PBS [0-200] 30

D9 20 [0-200]

Relative 10 D14 Enrichment 0 PBS ADR D9 ADR D14 FoxC2 93kb [0-400] Lmx1b 60 PBS [0-400] 40 D9 [0-400] 20 Relative D14 Enrichment 0 PBS ADR D9 ADR D14

Lmx1b 48kb [0-150] 60 Tcf21 PBS [0-150] 40

D9 20 [0-150] Relative Enrichment

D14 0 PBS ADR D9 ADR D14

Tcf21

84kb Mafb [0[0--200]400] 50

PBS 40 [0[0--200]400] 30

D9 20 [0[0--200]400] Relative 10 D14 Enrichment 0 PBS ADR D9 ADR D14 Mafb

c Expression changes during injury Log2(FPKM+1) group 10 group PBS Foxc2 D9 Lmx1b D14

Tead1 5 Zhx2

Tcf21

Mafb 0 Wt1

Klf6 Supplementary Fig. 8. WT1 binding and gene expression during ADR- induced injury in mTmG-Nphs2cre mice at podocyte TFs genes

(a) WT1 ChIP-seq IGV plots of FoxC2, Lmx1b, Tcf21 and Mafb genes showing

WT1 binding sites during injury (uninjured/PBS: blue, D9: orange, D14: red). Red arrows show TSS. Green lines show amplicon for direct ChIP-qPCR. (b) WT1 direct ChIP-qPCR after ADR injury from mTmG-Nphs2cre isolated glomeruli (n=3 replicates). (c) Heatmap representing the expression level of transcription factors that were found enriched at peaks with significant binding changes during injury.

46 Unbound class4 class3 class1 class2 Unbound class4 class3 class1 class2

Supplementary Fig. d14 9 d9 6 5 4 3 2 1 0

Expression for integrind14 d9 genesctr ctr d9 d14 Itga1 Itga2b Itgam 6 Itgb7 d14 d14 d9 Unbound Itga6 5 ctr class4 Itgb2 4 class3 ctr Itgb8 class1 Itga2 3 class2 Itgb2l d9 2 d9 Itgad Unbound Itga10 d14 1 class4 class3 Itga3 Itgb5 Itgb1 Itga5 Itgav Itga7 Itga8 Itgal Itga9 Itgae Itgb6 Itga4 Itga11 Itgb3 Itgax Itgb4 Itgbl1 Itga10 Itgad Itgb2l Itga2 Itgb8 Itgb2 Itga6 Itgb7 Itgbl1Itgam Itga2b Itga1 0 class1 Itgb4 class2 Itgax Itgb3 Itga11 Itga4 Itgb6 Itgae

Expression for integrin genes Expression for Itga9 Itgal Itga8 Itga7 Itgav Itga5 Itgb1 Itgb5 Itga3

Supplementary Fig. 9. Integrins gene expression during ADR-induced injury in mTmG-Nphs2cre mice

Heatmap showing the expression changes and WT1 gene classes for integrin genes. The red/blue range represents the expression fold change between control and D9, D14 podocytes; red-increased, blue-decreased at D9 or D14 compared to control. Green colors above heatmap indicate gene classes based on WT1 binding at each time point.

47 Supplementary Table S1. Primers sequences used for genotyping

Sequence Primer forward (5’-3’) Sequence Primer reverse (5’-3’) Alleles

WT-rev: CGA GGC GGA TCA CAA GCA ATA

R26-mTmG CTC TGC TGC CTC CTG GCT TCT Mutant-rev: TCA ATG GGC GGG GGT CGT T

Nphs2:Cre GGA CAT GTT CAG GGA TCG CCA GGC G GCA TAA CCA GTG AAA CAG CAT TGC TG

WT1 flox CCT TTT ACT TGG ACC GTT TG GGG GAG CCT GTT AGG GTA

Nphs2:iCre TCA ACA TGC TGC ACA GGA GAT ACC ATA GAT CAG GCG GTG GGT

WT-fwd: AAG GGA GCT GCA GTG GAG TA WT-rev: CCG AAA ATC TGT GGG AAG TC R 26- TdTomato Mutant-fwd: CTG TTC CTG TAC GGC ATG Mutant-rev: GGC ATT AAA GCA GCG TAT CC G

Supplementary Table S2. Primers sequences used for RT-qPCR

Sequence Primer forward (5’-3’) Sequence Primer reverse (5’-3’) Genes

Nphs2 (m) TGC TAC TAC CGC ATG GAA AAT G GCA CAA CCT TTA TGC AGA ACC AG

Synpo (m) CTG CAT CCG TGG TCA ACA G GGG ACT CCT ATC CGC CAT AC

WT1 (m) GAG AGC CAG CCT ACC ATC C CCC TGC TGT CCA TTC TCA AT

Gapdh (m) GGT GAA GGT CGG TGT GAA CAA TGA AGG GGT CGT TGA T

Nphs2 (h) AAG AGT AAT TGG ACA T TGG TCA CGA TCT CAT GAA AAG G

Synpo (h) CCC AAG GTG ACC CCG AAT CTG CCG CCG CTT CTC A

WT1 (h) GTG ACT TCA AGG ACT GTG AAC G CGG GAG AAC TTT CGC TGA CAA

Nphs1 (h) CTG CCT GAA AAC CTG ACG GT CGA CCT GGC ACT CAT ACT CC

Gapdh (h) GGC TCT CCA GAA CAT CAT CCC TGC GGG TGT CGC TGT TGA AGT CAG AGG

(m): mouse; (h): human Supplementary Table S3. Primers sequences used for ChIP

Sequence Primer forward (5’-3’) Sequence Primer reverse (5’-3’) Genes

Nphs2-1 ACC TGG TCT CTT CAC AGC AC TCC GCA GTG ACC TGG TAT TTG

Nphs2-2 TAT CCG TAA CCC CAA CCA AC TGA GGG GGC AAA CAT TTA AG

Nphs2-3 ATC CAG ACC CAA GAA GGA AC CCC TGC TGT CCA TTC TCA A’

Synpo-1 CCT GCC TTG AGT CCT TTC TG CTG TTA GGG CAG AGC AGA CC

Synpo-2 TGC TGG CAC TCT GGC TAC TC TGT GTG GGC AGC TAC TTG AG

Synpo-3 CCG ACG AAG AGA GAG GAA AA CCG GTG AAT CTG GTG AAT CT

FoxC2 ATG TTC GAG AAT GGC AGC TT GAC TTT CTT CTC GGC CTC CT

Lmx1b GGC CAG AGA AGT GGG TAA CA CTG CAA ACA CCA AGG GAA CT

Tcf21 AAA GGG TGG AGA GGG TGA GT TGT TTC GGG GTT CCA GTT AG

Mafb AAG GTC GAA GTC GTT GAC GTA GTC CCC AGA CAA AGG CTT G

CAG GAG CCC AGG GAA GAT ACA AAT A ACG CAT ACA CAT ATA CAA CCA GTC A Gapdh Supplementary Table S4. WT1 binding status and gene expression at mutated nephropathy genes

Expression change WT1 binding compared to REFs control* Gene Protein Control D9 D14 D9 D14 symbol ACTN4 α-actinin 4 class2 class2 class2 down down 1 ADAMTS9 ADAM class2 class2 class2 down down 2 Metallopeptidase with Thrombospondin Type 1 Motif 9 ADCK4 AarF Domain Unbound Unbound Unbound down down 3 Containing Kinase 4 ALG1 Asparagine-linked class2 class2 Unbound up down 4 glycosylation 1 ANLN Anillin actin binding Unbound Unbound Unbound up up 5 protein APOL1 Apolipoprotein L1 (no mouse homolog) 6 ARHGAP24 Rho GTPase- class3 class3 class3 no down 7 activating protein 24 change ARHGDIA Rho GDP class1 class1 Unbound down down 8 dissociation inhibitor α ARHGEF17 Rho Guanine class2 class2 class3 up down 9 Nucleotide Exchange Factor 17 BPTF Bromodomain PHD class2 class2 Unbound up up 9 Finger Transcription Factor CD151 CD151 antigen class2 class2 class1 down up 10 CD2AP CD2-associated class2 class2 class1 up down 11 protein CDK20 Cyclin-dependent class2 class2 Unbound no down 12 kinase change CFH Complement factor H Unbound Unbound Unbound up down 13 COL4A3 Type IV collagen α3 class2 class2 class2 no down 14 change COL4A4 Type IV collagen α4 class2 class2 class2 no down 14 change COQ2 Coenzyme Q2 class4 class4 Unbound up up 15 COQ6 Coenzyme Q6 Unbound class4 Unbound down up 16 CRB2 Crumbs family class3 class3 class3 up no 17 member2 change CUBN Cubilin class3 class3 class3 up up 18 DGKE Diacylglycerol kinase ε class3 class3 Unbound up down 19,20 DLC1 DLC1 Rho GTPase- class3 class3 class3 down down 12 activating protein DLG5 Discs Large MAGUK class2 class2 class2 down up 9 Scaffold Protein 5 E2F3 E2F transcription class2 class2 class3 up down 21 factor EMP2 Epithelial membrane class1 class2 Unbound up up 22 protein 2 FAT1 FAT atypical cadherin class2 class2 class4 up down 23 1 GAPVD1 GTPase activating class3 class3 Unbound up down 24 protein and VPS9 domains 1 GCC1 GRIP And Coiled-Coil class4 class4 Unbound down down 9 Domain Containing 1 GPC5 Glypican 5 class3 class3 Unbound down up 25 INF2 Inverted formin 2 class2 class2 class2 down up 26 ITGA3 Integrin α3 class2 class2 class3 no down 27 change ITGB4 Integrin β4 class4 class4 Unbound up up 28 ITSN1 Intersectin protein class2 class2 class2 down up 12 ITSN2 Intersectin protein class2 class2 Unbound up no 12 change KANK1 Kidney ankyrin class2 class2 class3 down down 9 repeat-containing protein KAT2B Lysine class2 class2 class4 up down 9 Acetyltransferase 2B LAMB2 Laminin subunit β2 class2 class2 class1 no down 29 change LMNA Lamin A and C class3 class2 class3 down down 30 LMX1B LIM homeobox class2 class2 class3 up down 31 transcription factor 1β MAGI2 Membrane class3 class3 class3 up down 12 Associated Guanylate Kinase, inverted 2 MEFV Pyrin Unbound Unbound Unbound up up 32 MYH9 Myosin heavy chain class2 class2 class2 down down 33,34 9, non-muscle MYO1E Myosin 1E class3 class3 class3 up down 35 NEIL1 Nei endonuclease Unbound Unbound Unbound up down 36 VIII-like 1 NPHS1 Nephrin class2 class2 class2 up down 37 NPHS2 Podocin class2 class3 class4 up down 38 NUP107 Nuclear pore class3 class3 Unbound no down 39 complex protein change NUP133 Nuclear pore class1 class1 Unbound no down 39 complex protein change NUP160 Nuclear pore class3 class3 class3 down down 39 complex protein NUP85 Nuclear pore class1 class1 Unbound up up 39 complex protein NXF5 Nuclear RNA export class4 class4 Unbound down up 40 Factor 5 OCRL1 Oculocerebrorenal class2 class2 class2 up down 41 syndrome of Lowe OSGEP KEOPS complex Unbound Unbound Unbound no up 42 protein change PAX2 Paired box protein 2 class3 class3 Unbound up up 43 PDSS2 Decaprenyl class2 class2 Unbound no up 44 Diphosphate change Synthase Subunit 2 PLCE1 Phospholipase C class3 class3 class3 no down 45,46 epsilon 1 change PMM2 Phosphomannomuta class3 class3 Unbound up up 47 se 2 PODXL Podocalyxin class2 class2 class2 up down 48 PTPRO Protein-tyrosine class2 class2 class3 up down 49 phosphatase- RO SCARB2 Scavenger receptor Unbound class2 Unbound down down 50,51 class B, member 2 SMARCAL1 SMARCA-like protein class4 class2 Unbound up up 52,53 SYNPO Synaptopodin class3 class3 class3 up down 54 TBC1D8B TBC1 domain family class1 class2 class1 up down 55 member 8B TNS2 Tensin-2 class2 Unbound Unbound down down 12 TP53RK KEOPS complex Unbound Unbound Unbound down down 42 protein TPRKB KEOPS complex class2 Unbound Unbound down up 42 protein TRPC6 Transient receptor unbound class2 unbound up down 56,57 potential channel C6 TTC21B Tetratricopeptide class1 class1 Unbound up down 58 repeat protein 21B WDR73 WD repeat domain class3 Unbound Unbound up down 59 73 WNK4 WNK Lysine Deficient class2 class2 class4 up down 9 Protein Kinase 4 WT1 Wilms Tumor 1 class2 class2 class2 up down 60,61 XPO5 Exportin 5 Unbound Unbound Unbound down down 62 XYLT1 Xylosyltransferase 1 class2 class2 class1 up up 9 ZMPSTE24 Zinc Unbound class4 Unbound down down 63 metallopeptidase STE24

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Supplementary Table S5. WT1 binding status at human eQTL genes with increased expression at D9 and decreased expression at D14

n=140 eQTL genes Class1 PBS Class2 n=27 n=60 n=10 Class3 Class4 D9 n=69 n=27 Unbound

D14 n=13 n=31 n=13 n=43

Distribution of eQTL genes by class in WT1 ChIP-seq in control (PBS), day 9 and day 14 after Adriamycin

WT1 binding Gene Protein Control D9 D14 symbol AKAP13 A-Kinase Anchoring Protein 13 class3 class3 class3 ANXA5 Annexin A5 class2 class2 class4 APP Amyloid Beta Precursor Protein class3 class2 class3 ARHGAP21 Rho GTPase Activating Protein 21 class3 class3 class3 ART3 ADP-Ribosyltransferase 3 class2 class3 Unbound ATP1B3 ATPase Na+/K+ Transporting Subunit Beta 3 class2 class2 class4 ATP9B ATPase Phospholipid Transporting 9B class2 class2 Unbound BOK BCL2 Family Apoptosis Regulator BOK class2 class2 Unbound CC2D2A Coiled-Coil And C2 Domain Containing 2A class2 class2 class4 CDK5RAP2 CDK5 Regulatory Subunit Associated Protein 2 class2 class2 class3 CEP120 Centrosomal Protein 120 class2 class2 class2 CEP68 Centrosomal Protein 68 class1 class2 Unbound CNKSR3 CNKSR Family Member 3 class2 class2 class4 CNPY4 Canopy FGF Signaling Regulator 4 class1 class1 Unbound CREB3L2 CAMP Responsive Element Binding Protein 3 Like class2 class2 class3 2 CTNNAL1 Catenin Alpha Like 1 class3 class2 class3 DAD1 Defender Against Cell Death 1 class2 class2 Unbound DCBLD2 Discoidin, CUB And LCCL Domain Containing 2 class2 class2 class2 DHDH Dihydrodiol Dehydrogenase class1 class2 Unbound DOCK8 Dedicator Of Cytokinesis 8 class2 class3 class3 ENOX1 Ecto-NOX Disulfide-Thiol Exchanger 1 class1 class1 Unbound ERAP1 Endoplasmic Reticulum Aminopeptidase 1 class1 class2 Unbound F2R Coagulation Factor II Thrombin Receptor class3 class3 class3 FAM180A Family With Sequence Similarity 180 Member A class3 class2 Unbound FAM43A Family With Sequence Similarity 43 Member A class2 class2 class3 FAM81A Family With Sequence Similarity 81 Member A class3 class3 class3 FDX1 Ferredoxin 1 class2 class2 Unbound FKTN FKTN class2 class2 class4 FMO2 Flavin Containing Dimethylaniline Monoxygenase class1 class3 Unbound 2 GAB1 GRB2 Associated Binding Protein 1 class2 class2 class3 GALC Galactosylceramidase class2 class2 class3 GAS7 Growth Arrest Specific 7 class3 class3 class3 GMEB2 Glucocorticoid Modulatory Element Binding class1 class2 Unbound Protein 2 HIBCH 3-Hydroxyisobutyryl-CoA Hydrolase class1 class2 Unbound HTRA1 HtrA Serine Peptidase 1 class3 class3 class3 IFITM2 Interferon Induced Transmembrane Protein 2 Unboun class1 Unbound d IL1R1 Interleukin 1 Receptor Type 1 class2 class2 Unbound IQGAP1 IQ Motif Containing GTPase Activating Protein 1 class2 class2 class2 IQSEC2 IQ Motif And Sec7 Domain ArfGEF 2 class4 class3 Unbound ITGAV Integrin Subunit Alpha V class2 class2 class2 ITGB5 Integrin Subunit Beta 5 class2 class2 class3 ITPR1 Inositol 1,4,5-Trisphosphate Receptor Type 1 class2 class2 class2 KANSL1 KAT8 Regulatory NSL Complex Subunit 1 class2 class2 class1 KCDN3 Potassium Voltage-Gated Channel Subfamily D class2 class2 Unbound Member 3 KDM2B Lysine Demethylase 2B class2 class2 class4 KIF16B Kinesin Family Member 16B class2 class2 Unbound KIF5C Kinesin Family Member 5C class3 class3 Unbound LYN1 LYN Proto-Oncogene, Src Family Tyrosine Kinase class2 class2 class4 LZTS2 Leucine Zipper Tumor Suppressor 2 class2 class2 Unbound MAP3K5 Mitogen-Activated Protein Kinase Kinase Kinase 5 class2 class2 class3 MCC MCC Regulator Of WNT Signaling Pathway class3 class3 class3 MGAT4A Alpha-1,3-Mannosyl-Glycoprotein 4-Beta-N- class3 class3 class4 Acetylglucosaminyltransferase A MICAL3 Microtubule Associated Monooxygenase, Calponin class3 class3 class3 And LIM Domain Containing 3 MPI Mannose Phosphate Isomerase class3 class3 Unbound MRVI1 Murine Retrovirus Integration Site 1 Homolog class3 class3 class3 MYH9 Myosin Heavy Chain 9 class2 class2 class2 MYOF Myoferlin class2 class2 class2 N6AMT1 N-6 Adenine-Specific DNA Methyltransferase 1 class2 class2 Unbound NMNAT3 Nicotinamide Nucleotide Adenylyltransferase 3 class1 class4 Unbound PAK1 P21 (RAC1) Activated Kinase 1 class2 class2 class3 PAPSS2 3'-Phosphoadenosine 5'-Phosphosulfate Synthase class4 class4 Unbound 2 PCSK6 Proprotein Convertase Subtilisin/Kexin Type 6 class2 class2 class3 PDCD1LG2 Programmed Cell Death 1 Ligand 2 class3 class3 Unbound PHTF1 Putative Homeodomain Transcription Factor 1 class2 class3 Unbound PIH1D1 PIH1 Domain Containing 1 class4 class2 Unbound PIP5K1B Phosphatidylinositol-4-Phosphate 5-Kinase Type class4 class2 Unbound 1 Beta PKN2 Protein Kinase N2 class2 class2 Unbound PLA2R1 Phospholipase A2 Receptor 1 class3 class3 Unbound PNRC2 Proline Rich Nuclear Receptor Coactivator 2 class2 class2 class2 PPAT Phosphoribosyl Pyrophosphate Amidotransferase class1 class1 class1 PRKCI Protein Kinase C Iota class2 class2 class2 PRKG2 Protein Kinase CGMP-Dependent 2 class2 class2 class3 PTH1R Parathyroid Hormone 1 Receptor class2 class3 Unbound RAB31 RAB31, Member RAS Oncogene Family class2 class2 Unbound RABGAP1L RAB GTPase Activating Protein 1 Like class2 class2 Unbound RIN2 Ras And Rab Interactor 2 class3 class3 class3 RNF111 Ring Finger Protein 111 class2 class2 Unbound RNF150 Ring Finger Protein 150 class2 class2 class3 RSU1 Ras Suppressor Protein 1 class3 class2 class3 SASH1 SAM And SH3 Domain Containing 1 class2 class2 class3 SCML4 Scm Polycomb Group Protein Like 4 class3 class3 Unbound SDC4 Syndecan 4 class2 class2 class3 SFT2D1 SFT2 Domain Containing 1 class2 class2 Unbound SIDT2 SID1 Transmembrane Family Member 2 class2 class2 Unbound SKAP1 Src Kinase Associated Phosphoprotein 1 class4 class3 Unbound SLC24A3 Solute Carrier Family 24 Member 3 class2 class2 Unbound SLC25A39 Solute Carrier Family 25 Member 39 class2 class2 class1 SLC44A2 Solute Carrier Family 44 Member 2 class4 class4 Unbound SLC5A26 Solute Carrier Family 5 Member 26 class3 class3 Unbound SLCA12 Solute Carrier Family class2 class2 class2 SORCS2 Sortilin Related VPS10 Domain Containing class3 class3 class4 Receptor 2 SPINT2 Serine Peptidase Inhibitor, Kunitz Type 2 class2 class2 class4 ST6GALNAC3 ST6 N-Acetylgalactosaminide Alpha-2,6- class3 class3 class3 Sialyltransferase 3 SUCLG2 Succinate-CoA Ligase GDP-Forming Subunit Beta class2 class2 class3 SULF2 Sulfatase 2 class2 class2 class2 TDRD5 Tudor Domain Containing 5 class3 Unboun Unbound d THSD7A Thrombospondin Type 1 Domain Containing 7A class3 class2 class3 TMEM135 Transmembrane Protein 135 class3 class2 class4 TMEM178 Transmembrane Protein 178A class2 class2 class4 TNIK TRAF2 And NCK Interacting Kinase class3 class2 class4 UCP2 Uncoupling Protein 2 class2 class2 class1 VAMP8 Vesicle Associated Membrane Protein 8 class2 class2 class2 VEGFA Vascular Endothelial Growth Factor A class2 class3 class3 WIPF3 WAS/WASL Interacting Protein Family Member 3 class2 class2 class2