Identifying collagen VI as a target of fibrotic diseasesregulated by CREBBP/EP300Lynn M. Williamsa,1, Fiona E. McCanna,1, Marisa A. Cabritaa, Thomas Laytona, Adam Cribbsb, Bogdan Knezevicc,Hai Fangc

, Julian Knightc, Mingjun Zhangd,2, Roman Fischere, Sarah Bonhame, Leenart M. Steenbeekf, Nan Yanga,

Manu Soodg, Chris Bainbridgeh, David Warwicki, Lorraine Harryj, Dominique Davidsonk, Weilin Xied,2,

Michael Sundstrӧml, Marc Feldmanna,3, and Jagdeep Nanchahala,3

aKennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Science, University of Oxford, Oxford OX37FY, United Kingdom; bBotnar Research Centre, National Institute for Health Research Oxford Biomedical Research Unit, Nuffield Department ofOrthopaedics, Rheumatology and Musculoskeletal Science, University of Oxford, Oxford OX3 7LD, United Kingdom; cWellcome Trust Centre for HumanGenetics, University of Oxford, Oxford OX3 7BN, United Kingdom; dBiotherapeutics Department, Celgene Corporation, San Diego, CA 92121; eTargetDiscovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7FZ, United Kingdom; fDepartment of Plastic Surgery, GeertGrooteplein Zuid 10, 6525 GA Nijmegen, The Netherlands; gDepartment of Plastic and Reconstructive Surgery, Broomfield Hospital, Mid and South EssexNational Health Service Foundation Trust, Chelmsford CM1 4ET, Essex, United Kingdom; hPulvertaft Hand Surgery Centre, Royal Derby Hospital, UniversityHospitals of Derby and Burton National Health Service Foundation Trust, Derby DE22 3NE, United Kingdom; iDepartment of Trauma and OrthopaedicSurgery, University Hospital Southampton National Health Service Foundation Trust, Southampton SO16 6YD, United Kingdom; jDepartment of Plastic andReconstructive Surgery, Queen Victoria Hospital National Health Service Foundation Trust, East Grinstead RH19 3DZ, United Kingdom; kDepartment ofPlastic and Reconstructive Surgery, St. John’s Hospital, Livingston, West Lothian EH54 6PP, United Kingdom; and lStructural Genomics Consortium,Karolinska Centre for Molecular Medicine, Karolinska University Hospital, 171 76 Stockholm, Sweden

Contributed by Marc Feldmann, June 16, 2020 (sent for review March 6, 2020; reviewed by John J. O’Shea and Georg Wick)

Fibrotic diseases remain a major cause of morbidity and mortality,yet there are few effective therapies. The underlying pathology ofall fibrotic conditions is the activity of myofibroblasts. Using cellsfrom freshly excised disease tissue from patients with Dupuytren’sdisease (DD), a localized fibrotic disorder of the palm, we soughtto identify new therapeutic targets for fibrotic disease. We hy-pothesized that the persistent activity of myofibroblasts in fibroticdiseases might involve epigenetic modifications. Using a validatedgenetics-led target prioritization algorithm (Pi) of genome wideassociation studies (GWAS) data and a broad screen of epigeneticinhibitors, we found that the acetyltransferase CREBBP/EP300 is amajor regulator of contractility and extracellular matrix produc-tion via control of H3K27 acetylation at the profibrotic genes,ACTA2 and COL1A1. Genomic analysis revealed that EP300 ishighly enriched at enhancers associated with genes involved inmultiple profibrotic pathways, and broad transcriptomic and pro-teomic profiling of CREBBP/EP300 inhibition by the chemical probeSGC-CBP30 identified collagen VI (Col VI) as a prominent down-stream regulator of myofibroblast activity. Targeted Col VI knock-down results in significant decrease in profibrotic functions,including myofibroblast contractile force, extracellular matrix(ECM) production, chemotaxis, and wound healing. Further evi-dence for Col VI as a major determinant of fibrosis is its abundantexpression within Dupuytren’s nodules and also in the fibrotic fociof idiopathic pulmonary fibrosis (IPF). Thus, Col VI may represent atractable therapeutic target across a range of fibrotic disorders.

EP300 | fibrosis | collagen VI | epigenetic

Fibrosis of visceral organs such as the lungs, heart, kidneys, andliver is a major cause of morbidity and mortality (1). How-

ever, despite intense research efforts, little progress has beenmade in the identification of effective therapeutic targets. Po-tential reasons for this include late presentation of patients withfibrotic diseases involving internal organs, lack of good diseasebiomarkers, and poor translation through to clinical practice oftargets identified in murine models (2). A greater understandingof the mechanisms controlling the fibrotic pathways in humantissues is required to facilitate the development of next-generationtherapies.Dupuytren’s disease (DD) is a debilitating fibroproliferative

disorder restricted to the palms of genetically susceptible indi-viduals (3). The initial clinical presentation is the appearance ofa firm cellular nodule in the hand, which expands into fibrouscollagenous cords that extend into the digits. As the disease

progresses, the relatively acellular cords mature, thicken, andcontract, leading to permanent flexion deformities of the fingers.Unlike fibrotic diseases of visceral organs such as the lung andliver which are diagnosed late, DD tissue can be diagnosed earlyand is readily accessible for research as the disease is oftentreated by surgical excision. Using the relatively early-stagenodules from patients with Dupuytren’s disease enables us toanalyze signaling and regulatory pathways and hence has the

Significance

Fibrosis remains a major unmet medical need as therapeutictargets discovered in animal models have failed to translate. Amajor challenge for identifying novel targets is the limitedavailability of early-stage human disease tissue. Here, we uti-lize Dupuytren’s disease (DD), a common localized fibroticdisorder, to evaluate the impact of epigenetic regulation ofmyofibroblasts and identify potential tractable targets in hu-man fibrosis. We demonstrate that the epigenetic regulatorCREBBP/EP300 is a critical determinant of the profibrotic phe-notype. Furthermore, we identify collagen VI to be a keydownstream target of CREBBP/EP300 and reveal valuable in-sights in the role it plays in key profibrotic functions, includingcontractile force, chemotaxis, and wound healing, and henceits potential as a therapeutic target.

Author contributions: L.M.W., F.E.M., J.K., W.X., M. Sundstrӧm, M.F., and J.N. designedresearch; L.M.W., F.E.M., M.A.C., T.L., B.K., H.F., M.Z., R.F., S.B., L.M.S., and N.Y. performedresearch; H.F., J.K., M. Sood, C.B., D.W., L.H., and D.D. contributed new reagents/analytictools; L.M.W., F.E.M., T.L., A.C., B.K., H.F., R.F., S.B., and J.N. analyzed data; and L.M.W.,F.E.M., T.L., A.C., B.K., H.F., J.K., N.Y., W.X., M. Sundstrӧm, M.F., and J.N. wrote the paper.

Reviewers: J.J.O., NIH; and G.W., Medical University of Innsbruck.

Competing interest statement: J.N. and M.F. have received consulting fees and hold eq-uity in 180 Therapeutics, which is an exclusively licensed intellectual property from theUniversity of Oxford to treat Dupuytren’s disease with anti-TNF.

This open access article is distributed under Creative Commons Attribution License 4.0(CC BY).1L.M.W. and F.E.M. contributed equally to this work.2Present address: Department of Biological Sciences, Institute of Materia Medica, Shan-dong First Medical University and Shandong, Academy of Medical Sciences, Jinan250062, Shandong Province, People’s Republic of China.

3To whom correspondence may be addressed. Email: marc.feldmann@kennedy.ox.ac.ukor jagdeep.nanchahal@kennedy.ox.ac.uk.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2004281117/-/DCSupplemental.

First published August 5, 2020.

www.pnas.org/cgi/doi/10.1073/pnas.2004281117 PNAS | August 25, 2020 | vol. 117 | no. 34 | 20753–20763

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potential for the identification of novel therapeutic targets.Dupuytren’s nodules represent a complex disease system, hous-ing densely packed myofibroblasts (MFs) alongside other lessabundant stromal and immune cells (4). Myofibroblasts arecharacterized by the expression of α-smooth muscle actin(α-SMA) and excessive production of extracellular matrix(ECM) proteins such as collagens, glycoproteins, and proteo-glycans, which enhances their ability to contract tissue (5, 6).Matrix stiffness and mechanical stress in severely damaged tis-sues initiates a positive feedback loop between myofibroblastsand their surrounding microenvironment that perpetuates fi-brosis (7, 8). Therefore, Dupuytren’s nodules provide an idealmodel system to study primary human myofibroblasts, the celltype responsible for deposition and contraction of excessivematrix in all forms of fibrosis (9, 10).Despite the substantial heritability of 80% (11) in DD and

multiple risk loci identified by genome wide association studies(GWAS) (12), why this disease is restricted to the palmar fasciaof the hand is still poorly understood. The distinct anatomicallocation and proposed role of environmental factors such asexcessive alcohol consumption (13), heavy manual labor, andexposure to vibrations (14) suggest epigenetic regulation is likelyto be important in the pathogenesis of the disease. Epigeneticmechanisms have been shown to be crucial mediators of fibroticgene expression in a wide range of fibrotic disorders (15), in-cluding cardiac fibrosis (16), liver fibrosis (17), and systemicsclerosis (18), and probably account for the persistence ofmyofibroblasts in disease (19).We hypothesized that specific targeting of transcriptional

coactivators could provide valuable insight into epigeneticmechanisms controlling the myofibroblast phenotype in fibrosis.Here we show that the histone acetyltransferases CREBBP andEP300 (20) are key regulators of profibrotic phenotype andfunction of myofibroblasts and act through control of collagenVI. Furthermore, collagen VIα3 is highly expressed in bothDupuytren’s nodules and in the fibroblastic foci in idiopathicpulmonary fibrosis (IPF), with a distribution distinct from col-lagen I but closely aligned with α-SMA positive regions, thusemerging as a signature marker of myofibroblasts and a tractabletherapeutic target for DD and potentially also other fibrotic diseases.

ResultsComputational Target Prioritization and Screening UsingPharmacological Inhibitors Both Identify CREBBP/EP300 as a CentralRegulatory Network in Human Fibrosis. To leverage the power ofgenetic variance in target discovery, we applied our recentlydeveloped prioritization “Pi” approach (21) to risk loci in DDGWAS data (12). This validated approach allows us to prioritizegenes from GWAS hits, not simply based on genomic proximitybut also including evidence from chromatin conformation andexpression quantative trait loci mapping in immune cells, plusgene networks defined by the Search Tool for the Retrieval ofInteracting Genes/Proteins database. This unsupervised networkconnectivity analysis identified FoxO and WNT signaling ashighly ranked pathways that share CREBBP and EP300 membergenes, as shown at the intersection of delineated pathways inblue and green, respectively (Fig. 1A and SI Appendix, Table S1),providing unbiased support for our hypothesis that epigeneticmodulators are important in controlling disease processes in DD.For functional validation, we screened a focused library of 39

epigenetic inhibitors (SI Appendix, Table S2) to study their effecton the expression of key profibrotic genes (ACTA2, COL1A1,COL3A1, and TGFB1) on early passage myofibroblasts derivedfrom nodules in patients with DD (Fig. 1B and SI Appendix, Fig.S1). This revealed the pan bromodomain inhibitor bromosporine(BrSp), the BET bromodomain inhibitors, PFI-1 and JQ1, andthe two structurally distinct CREBBP/EP300 bromodomainsinhibitors SGC-CBP30 (referred to as CBP30 throughout) and

I-CBP112, as the most potent and significant inhibitors ofACTA2 and COL1A1 gene expression (Fig. 1B), with a strongdose-dependent effect (SI Appendix, Fig. S1). We also employeda recently developed, highly specific CBP/EP300 HAT inhibitorA485 (22), to confirm the role of CBP/EP300-mediated histoneacetylation regulating ACTA2 and COL1A1 gene expression (SIAppendix, Fig. S1 G–I).To establish whether pharmacologic inhibition of CBP/EP300

bromodomains would impact histone acetylation at the tran-scription starts sites of ACTA2 and COL1A1, we employedchromatin immunoprecipitation (ChIP) qPCR with antibodiesfor the H3K27ac mark. We also included the BET inhibitor JQ1as it also reduced expression of ACTA2 and COL1A1 in ourprimary qPCR screen. Both CBP30 and JQ1 treatment reducedH3K27 acetylation at the transcriptional start sites of ACTA2and COL1A1, confirming a direct role in regulating gene tran-scription (Fig. 1 E and F).Since excessive myofibroblast contractility is a key feature in

disease pathogenicity in vivo, we measured the effects of JQ1and CBP30 on myofibroblast force generation as a function ofcontractility using traction force microscopy (23). This demon-strated that both compounds significantly inhibited cell con-tractility, with both peak and average traction force reduced by∼50% (Fig. 1 G–I). In addition, both inhibitors reduced cellproliferation (SI Appendix, Fig. S1A), with JQ1, but not CBP30,also reducing cell viability and confluency (SI Appendix, Fig. S1 Band C). Myofibroblasts display spindle or stellate-cell morphol-ogy, and while treatment with CBP30 had no clear effect onmyofibroblast morphology, JQ1 treatment also altered cellshape, resulting in an elongated neuron-like morphology, indic-ative of broad effects not shared with CBP30 (SI Appendix, Fig.S2 D–G). Collectively, these data indicate CREBBP/EP300 is ahighly associated pathway in localized human fibrosis that con-trols key aspects of myofibroblast phenotype and function, includingACTA2 and COL1A1 gene expression and cell contractility.

EP300 Associates with Active Enhancers in Human Myofibroblasts.Transcriptional regulation of gene expression is achievedthrough cooperation between promoter and enhancer elements.Specific histone modifications function as binding elements foreffector proteins that serve to regulate transcription throughmanipulation of the chromatin environment or assembly oftranscription machinery. Acetylation at lysine 27 of histone H3serves as a signature mark of active enhancers (24) in all celltypes, where monomethylation of histone H3 at lysine 4(H3K4me1) marks both primed and active enhancers in acell-type-specific manner (25, 26). To further delineate the mo-lecular mechanisms underlying the functional impact of thebromodomain inhibitors CBP30 and JQ1, we performed ChIPanalysis coupled with deep sequencing using H3K27ac-,H3K4me1-, EP300-, and BRD4-specific antibodies (27). Geno-mic distribution of H3K27ac and BRD4 were strikingly similar(Fig. 2A) in many genomic regions, in accordance with previousdata (28). In contrast, H3K4me1 and EP300 displayed similargenomic distribution, with genomic occupancy assigned pre-dominantly to intronic and intergenic regions. However, therewas also significant overlap of genomic occupancy between thedatasets; EP300 shared 91% of sites with H3K27ac, 87% withH3K4me1, and 78% with BRD4 (Fig. 2B).We next probed the ChIP sequencing (ChIP-Seq) datasets to

verify EP300 binding within the genomic loci of all of the genesidentified within the FoxO (blue) and WNT (green) pathways,previously identified from the Pi pathway analysis in Fig. 1A. Allgenomic loci, with the exception of RELA and AKT1 identifiedEP300 binding within 50 kB of the annotated genes. We haveshown two genes each, FOXO3 and EGFR from the FoxOpathway and JUNB and CSNK1A1 from the WNT pathway in SIAppendix, Fig. S3, as an illustration of these findings.

20754 | www.pnas.org/cgi/doi/10.1073/pnas.2004281117 Williams et al.

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CFTR[4.61@5]

Pi rank dGenepGenefGenenGene eGenecGene

0 51 2 3 4

numeric value and shading of outer rim

/~16,000 genes

Seed gene(colored segments)

Non-seed gene(no colored segments)

Pi rating

FDR=7.2e-3FDR=9.3e-3FDR=7.3e-3FDR=4.3e-3

FDR=1.8e-3FDR=2.3e-4

FDR=4.4e-7

FDR=7.2e-33FDR=9.3e-33FDR=7.3e-33FDR=4.3e-33

FDR=1.8e-33FDR=2.3e-44

FDR=4.4e-77

cAMP signaling pathwayHIF-1 signaling pathway

PI3K-Akt signaling pathwayErbB signaling pathway

MAPK signaling pathway

0.0 2.5 5.0 7.5 10.0

Z-scores

Pathways enriched (FDR < 0.01)

MAPK3, AKt1, EGFR, GRB2, CREBBP, EP300, SIRT1, FOXO3, CDK2, GABARAP, MDM2, PLK1, GABAPAPL1, GABAPAPL2, RBL2

PRKACA, JUN, MYC, CTNNB1, CSNK1A1 CREBBP, EP300, RUVBL1, TPS3, CTFR, RELA

RELA[3.8@57]

MAPK3APMAMA 3M[2.96@445][[2[ @445

PRKKAACAC[3.28@203]203]

GRB2RBGRRBGRRBGRB2RBGRB2RB[3.66@82]@[[

EGFREEEEE[3.94@39].99.9

NTRK1[4.44@8]TP533

[4.08@25]][ 5]

AATF2TAAAA[4.33@15][4.3 ]]5]5]

AKT1[3.01@403]][3.[3.0

TRAF2[3.38@162]

CDC42C4C4C4242[3.6@94]3.6@

HSPA1APP[3.34@175][

HSPA1BBPP[3.33@17[3 79]179]

HSHSPPA8PP[3.34@174][3[3.34@174]

JUNJUJUN[4.42@10][4.4

MYC

ERBB2[3.66@81]

SRCRC[3.95@37]95[3.[3.9

RALBP1[3.32@185]5

ACTB[3.97@32]

CTNNB1[3.93@42]

PPP1CAP1PP[3.55@106]@1055

PPP1CB[3.61@92]

PPP1CC[3.33@182]

CFTR[4.61@5]

CREBBPB[3.57@102]

EP300300E 00[3.61@90][3.61[3.6 @@@9@90

FFFFOOOOXO3X 3OOOO[2.41@1145]@12.4 4

MDM2MDM2DMD 222[3.51@115]11[3.5 @ 11 ]

SISIRRT1R3.68@75]3.6[3.63.[

CDK2DDK[3.44@138]84@@

RBL2[4.33@16]6]

PLK1[3.73@66]

GABARAPG A[3.8@54]

GABARAPL22[5@1]

GABARAPL1[4.05@28]

YWHAEY HW AEAA[3.53@111][ @5 1111

YWHAZ[4.01@31]]

HSP90AA1[3.77@62]@62]

HSP90AB1A[3.84@49]@4

RUVBL1[3.81@52]

CSNK1A1SNK1NK1ANK A1[3.49@122][3.4[3.49[3.49@12

SP1[4.23@19]]

E2F4[4.33@14]

SQSTM1[4.01@30]

POLR2E[3.3@193]

ESR1[4.31@17]@17]

PKMMMM[3.46@132]3 32.3. 13132

CPSF1[3.97@34]

POLR2A[4.44@9]@

POLR2C[3.38@159]

ACTA2 TGFB1COL1A1 COL3A1

***

******** **** ****

ACTA2

COL1A1

B

A

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FoxO signaling pathway

-) JQ1(+)

DMSO

I-CBP1

12 PF1

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JQ1

0.5

1.0

****

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****

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0.0

0

5

10

15

20

%in

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5

10

15

20

25

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COL1A1 promotor H3K27Ac ChIP

ACTA2 promotor H3K27Ac ChIP

D + + - - CBP30 - - + + JQ1 3 24 3 24 hrs

JQ1(+)))))) JJJJJJJQQQQQQQQQ11111111((((((((++++++++))))))

JJJJJQQQQQ111111(((++++)))

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HG

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DMSOJQ

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500

1000

1500

Peak

Trac

tion

(Pa)

***

DMSOJQ

1

CBP300

50

100

150

200

Aver

age

Trac

tion

(Pa)

***

DMSO JQ1 CBP30

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30819118515315214313613112412111811511511010910310110095919089868585828181797472676763514020886

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PFI-3 TP-064

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SGC707TP-472

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GSK-J4A-196

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A-366GSK-LSD1

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100

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I-BRD9 PFI-1 BrSpJQ1

100

200

3001311271231161151091091061041009088868483818079797978737373727169696463625554463231251072

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OF-1 A-196 A-366

LLY-507 UNC0638

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100

200

300

Fig. 1. CREBBP/EP300 regulates profibrotic gene expression and contractile phenotype in myofibroblasts. (A) Pi analysis identifies CREBBP and EP300 ashighly ranked central network genes in DD. Target pathway crosstalk for DD is shown, maximizing numbers of highly prioritized interconnecting genes. (B)Epigenetic probe library screening of ACTA2, COL1A1, COL3A1, and TGFB1 gene expression in DD patient-derived myofibroblasts. Cells were incubated withprobes for 3 d. Gene expression was analyzed by Taqman qPCR using the ΔΔCt method, normalized to GAPDH. Data are means ± SEM of five independentexperiments from five individual donors, represented as a heatmap where values are normalized to DMSO control value; 100% represented in white, redrepresents inhibition and blue represents augmentation of gene expression relative to control. (C and D) Data from heatmap where the most potent in-hibitory probes are depicted graphically; mean ± SEM from five donors. P value was determined by one-sample t test to normalized control (DMSO) sample ofvalue of 1. *P ≤ 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ChIP-qPCR analysis of JQ1 (0.5 μM) and SGC-CBP30 (2.5 μM) inhibition of the H3K27ac mark atpromotor proximal regions of ACTA2 (E) and COL1A1 (F) in patient-derived myofibroblasts. Data are representative of three independent experiments fromthree individual donors, mean ± SD of technical triplicates. Myofibroblasts were plated onto 2.55-kPa hydrogels for traction force microscopy in the presenceof the indicated bromodomain inhibitors for 72 h. Color scale in stiffness maps indicates shear modulus in kilopascals. (G) Representative traction vector maps,(H) peak traction, and (I) average traction are depicted (mean ± SEM; n = 4 independent experiments with a minimum of 20 independent cells per condition;**P < 0.01 and *P < 0.05 versus controls).

Williams et al. PNAS | August 25, 2020 | vol. 117 | no. 34 | 20755

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TT SExonIntronIntergenicPromoter5UTR3UTR

H3K27ac H3K4me1

BRD4 EP300

A B

H3K4me1EP300

BRD4H3K27ac

453 464

1577

178

95

64

873 3497

1625

583

5273

449

1355147

6516

ACTA2-AS1 ACTA2

COL1A1

FN1

EP300

EP300

BRD4

10kb

FDR=1E−10

FDR=1E−10

FDR=1E−10

FDR=1E−10

FDR=1E−10

FDR=1E−10

FDR=1.5E−09

FDR=1.5E−06

FDR=3E−06

FDR=3E−06

mTOR signaling pathway

VEGFR1 and VEGFR2 signiling pathways

FAK signalling pathways

ErbB1 downstream signaling

PDGFR−beta signaling pathway

Arf6 downstream pathway

Circadian rhythm pathway

p38 MAPK signaling pathway

PTEN is signlling pathays

Ret tyrosine kinase signaling pathways

0.0 2.5 5.0 7.5 10.0

Enrichment significance -log10(FDR)

C

E BRD4

G

F

H3K4me1

H3K27ac

BRD4

EP300

H3K4me1

H3K27ac

BRD4

EP300

H3K4me1

H3K27ac

BRD4

Rank Motif Sequence Log Pvalue

% sites

1 Fra1 -1.25e+04 48.4%

2 TEAD4 -4.23e+03 33.8%

3 MF0010 -1.07e+03 31.1%

4 RUNX1 -1.07e+03 13.3%

5 NFIX -6.30e+02 28.8%

Rank Motif Sequence Log P value % sites

1 Fra1 -1.98e+03 13.9%

2 Elk1(ETS) - 5.98e+02 19.3%

3 CEBPG -5.59e+02 23.5%

4 YY1 -2.59e+02 3.6%

5 KLF6 -2.33e+02 6.75%

FDR= 5.1E-06

FDR=6.1E−06

FDR=2.4E−04

FDR=5.5E−4

FDR=5.6E−04

FDR=5.7E−04

FDR=1.2E−03

FDR=1.2E−03

FDR=1.4E−03

FDR=1.4E−03Glypican 1 network

Signaling events mediated by PTP1B

FAK signalling pathways

Beta1 integrin cell surface interactions

AP−1 transcription factor network

HIF−1−alpha transcription factor network

ECM glycoproteins, collagens and proteoglycans

Beta3 integrin cell surface interactions

SHP2 signaling

PDGFR−beta signaling pathway

0 2 4 6 8Enrichment significance -log10(FDR)

EP300D

RUNX1FOSL1

EP300

SFRP4COL6A3CCL26

CCL2CXCL8.

FN1HAS2POSTN

TEAD4

H

Fig. 2. EP300 associates with active enhancers. (A) Genomic distribution of H3K27ac, H3K4me1, BRD4, and EP300 peaks. (B) Venn diagram of overlap ofH3K27ac, H3K4me1, BRD4, and EP300 peaks as determined by Homer AnnotatePeaks. (C) Visualization in IGV of ChIP-Seq signal at the ACTA2, COL1A1, andFN1 loci; for scaling, group autoscaling was used; all files were normalized to input. (D) XGR gene enrichment analysis of canonical pathway analysis of EP300and (E) BRD4 peaks. De novo motif analysis of (F) EP300 and (G) BRD4-bound genomic regions showing top enriched sequence motifs. P values and fre-quencies are indicated. (H) Schematic representing transcription factor-dependent gene expression. FDR, false discovery rate.

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We next sought to establish the direct binding of EP300 andBRD4 to key profibrotic genes. We found strong evidence ofactive transcription at the loci of ACTA2, COL1A1, and FN1(Fig. 2C), with H3K27ac and BRD4 highly enriched at thetranscriptional start site of all three genes. In contrast, EP300and H3K4me1 exhibited binding at more distal locations, in-dicative of enhancer activity. Taken together these data confirmthe role of EP300 as an enhancer regulating the expression ofACTA2 and COL1A1.Several groups have shown that much of disease-associated

DNA sequence variation occurs in transcriptional regulatoryregions defined by DNase hypersensitivity (29, 30). It has nowbeen demonstrated using multiple GWAS datasets that a subsetof (super) enhancers are especially important for genes associ-ated with cell identity and genetic risk of disease (31, 32). Giventhat EP300 loading identifies regions of the genome bearingsuper enhancer architecture in all cell types, we sought to de-termine whether EP300 binding was associated with the singlepolynucleotide polymorphisms (SNPs) we have previously iden-tified in Dupuytren’s disease GWAS datasets. We analyzed thegenomic location of EP300 at the 22 SNPs significantly associ-ated with Dupuytren’s disease (12). As shown in SI Appendix,Fig. S4 and summarized in SI Appendix, Table S2, EP300 is veryclosely associated with genomic regions of 8 of the 22 regions,independent of H3K27ac or H3K4me1 binding.Pathway analysis of annotated ChIP-Seq peaks demonstrated

that the EP300 binding (Fig. 2D) sites are most significantlyenriched for PDGFRβ, integrin, and ECM interaction pathways.In contrast, BRD4 peaks displayed enrichment for a myriad ofdiverse signaling pathways. Notably, we observed few commonpathways enriched across annotated BRD4 and EP300 peaks,suggesting a distinction of biological themes between the twoepigenetic regulators, with the exception of PDGFRβ and FAKsignaling pathways (Fig. 2E; Dataset S1), both of which arehighly associated with fibrosis in multiple organs (33). Next, weperformed de novo motif analysis of the EP300-enriched loci(Fig. 2F). Ranking the motifs by enrichment, the top five cor-responded to the known consensus binding sequences for thetranscription factors FRA1, TEAD4, MF0010, RUNX1, andNFIX. Finally, motif analysis of the BRD4 dataset also identifiedFRA1 as the highest number of sites, with ELK1 (ETS),CEBPG, YY1, and KLF6 also significantly enriched (Fig. 2G).To investigate a putative role for the EP300-regulated tran-

scription factors in profibrotic gene expression, we performedsiRNA-mediated gene knockdown of Fra1/FOSL1, RUNX1, andTEAD4 in DD myofibroblasts and measured the expression of apanel of profibrotic genes (SI Appendix, Table S3). This dem-onstrated that knockdown of FOSL1 significantly increased ex-pression of several genes, including CXCL8, IL6, and CTGF (SIAppendix, Fig. S6 A–C) and inhibited CCL26, SFRP4, and CO-L6A3 expression (SI Appendix, Fig. S5 D–G). In contrast,TEAD4 knockdown inhibited mRNA expression of CXCL8 andCCL2 (SI Appendix, Fig. S5 H–J), while RUNX1 knockdownsignificantly down-regulated mRNA expression of genes in-volved in ECM remodeling, including fibronectin-1 (FN1) andhyaluronan synthase 2 (HAS2) (SI Appendix, Fig. S5 K–M). To-gether, these data support the role of EP300 in modulating di-verse profibrotic pathways and identified RUNX1, TEAD4, andFra1 as EP300-associated transcription factors regulating dis-crete gene modules in myofibroblasts as summarized in Fig. 2H.

Inhibition with CBP30 Identifies Collagen VI as a DownstreamTherapeutic Target of Multiple CBP30-Regulated Core ECM Genes inFibrosis. After establishing that EP300 interacts directly withenhancer sites on key profibrotic genes, we evaluated changes inthe transcriptomic profile following CBP/EP300 inhibition usingCBP30 in human DD myofibroblasts and compared with JQ1treatment as a bromodomain inhibitor (Fig. 3 A and B) (34). JQ1

resulted in extensive transcriptional changes, 1,441 down-regulated, 624 up-regulated (Dataset S2). In contrast, bromo-domain inhibition with CBP30 affected a much smaller set ofgenes (225 down-regulated versus 48 up-regulated genes,Fig. 3 A and B). Gene enrichment (canonical) pathway analysis(Dataset S3) indicated that CBP30 down-regulated genes weresignificantly enriched in ECM and proteoglycan-associated pro-tein pathways, integrin signaling, and syndecan-1 pathways(Fig. 3C and Dataset S3), in common with JQ1. Pathways uniqueto JQ1 included the Aurora A/B, PLK1, and FOXM1 pathways(Fig. 3D), consistent with the potent antiproliferative propertiesof JQ1 (35, 36).Focusing on the most significantly enriched pathways, ECM

and ECM associated, we performed cluster analysis of the datafrom six donors following bromodomain inhibitor treatment. Theheatmap in Fig. 3E clearly identifies that each donor falls con-sistently within each treatment group within the dendrogram,with JQ1 treatment resulting in more potent inhibition thanCBP30. We confirmed several of the most significant hits fromtranscriptomic data using siRNA-mediated knockdown ofCREBBP, EP300, and BRD4, also including combinedCREBBP/EP300 knockdown to represent CBP30 targeting bothbromodomains (SI Appendix, Fig. S6 A–L).Having established the profound transcriptional effect of

bromodomain inhibition on profibrotic gene expression, we ex-tended our analysis to include the effects on global protein ex-pression following bromodomain inhibition. Shotgun proteomeanalysis identified a limited number of significantly regulatedproteins (SI Appendix, Table S5), most notable being the threecollagen VI (collagen VIα1/2/3) isoforms in CBP30-treated cells(Dataset S4). We integrated the pathways generated by thetranscriptomic dataset of CBP30-treated myofibroblasts with theproteome analysis to systematically explore the broader molec-ular landscape directed by CBP30 in myofibroblasts. This en-abled a comprehensive CBP30 pathway analysis, which wasfound to be dominated by pathways associated with extracellularmatrix organization (Fig. 3F). Focused correlation analysis ofindividual gene/protein expression levels showed collagen VIα1/2/3 and fibronectin were core targets driving the enriched ECMpathways (Fig. 3G). To validate our findings, we performedWestern blotting using the bromodomain inhibitors on steady-state expression of key ECM components. We observed markedinhibition of collagen Iα1, collagen VIα1, EDA-FN, and α-SMAafter 6 d of treatment with the bromodomain inhibitors(Fig. 3H).

Collagen VI Drives a Profibrotic Phenotype in Myofibroblasts. Ourmultiomics profiling converged on collagen VI as a principaldownstream target regulated by CBP/EP300. Immunostainingconfirmed expression of the α3 chain of collagen VI throughoutthe nodules of DD, colocalizing with α-SMA positive myofibro-blast foci (Fig. 4A). Similarly, collagen VI α3 was also associatedwith α-SMA positive myofibroblasts localized to the fibrotic fociof samples from patients with IPF (SI Appendix, Fig. S7). Wealso confirmed the presence of abundant levels of the full-lengthα3 chain of collagen VI in tissue culture supernatants of freshlyisolated Dupuytren’s nodular cells and passaged MFs (Fig. 4B).As collagen VI was highly associated with the myofibroblast

populations in human fibrosis, we further explored its function.We performed RNA-Seq on DD myofibroblasts treated withCOL6A1/A2/A3 siRNA for either 4 or 6 d (Fig. 4C) (37). Thedifferentially expressed genes are displayed as a volcano plot(Fig. 4D). This revealed a discrete number of differentiallyexpressed genes, highlighting that more genes were targetedfollowing longer exposure to COL6 depletion. These genes in-cluded HGF, SDC1, CCL2, CCL7, ADAMT4/8, and HAS2(Dataset S5). These differentially expressed genes were associ-ated with ECM remodeling, FOXM1 transcription network,

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Enrichment significance -log10(FDR)

ransductionBeta1 integ face interactions

ECMECM proteins

Transcr forms

Transcr ra1 and Fra2Do ve CD8+ T cells

Strevents

eventsProteoglycans

factorsble factors

Wnt−mediated signal trin cell sur

Regulators of remodeling of the Proteins affiliated structurally to

Integrins in angiogenesisiptional targets of deltaNp63 iso

Genes encoding collagen proteinsiptional targets of F

wnstream signaling in naiuctural ECM glycoproteins

Syndecan−1−mediated signaling BMP receptor signaling

Alpha9 beta1 integrin signaling

ECM glycoproteins and proteoglycansECM regulators and secreted

Secreted solu ECM and ECM−associated proteins

0 3 6 9 12Integrins in angiogenesis

Cytokines can induce activation of MMPsAurora A signaling

Alpha9 beta1 integrin signaling eventsIL27−mediated signaling events

Beta1 integrin cell surface interactionsRegulation of RAC1 activity

Structural ECM glycoproteinsProteins affiliated structurally to ECM proteins

Wnt signaling networkSyndecan−1−mediated signaling events

FOXM1 transcription factor networkRegulators of the ECM remodellling

Aurora B signalingPLK1 signaling events

ECM glycoproteins and proteoglycansGenes encoding collagen proteins

Secreted soluble factors ECM regulators and secreted factors

ECM and ECM−associated proteins

0 5 10 15 20Enrichment significance -log10(FDR)

A

−6 −4 −2 0 2 4

050

100

150

log2FoldChange

−log

10(F

DR

) PNPLA2GDF11HAS2

TRIB3FST

PHGDHPYCR1

SPATA18

MTHFD1L

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MAZPEG10

ADM2ELN

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log2FoldChange

−log

10(F

DR

)

−3 −2 −1 0 1 2

05

1015

2025

3035

ADAMTS8

SYT7

KCNN4

ASPN

CRABP2

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LPAR1CBP30 log2FC > 1.5FDR < 0.05

CD

CBP30 JQ1

E

−1

0

1

2 CBP30 ProteomicsCBP30 RNA−Seq

Extracellular matrix organisation

OR = 3.33

OR = 2.46

OR = 5.59

OR = 4.34

OR = 2.03

OR = 5.44

OR = 4.83

OR = 18.6

OR = 6.47

OR = 13.0

OR = 10.8

OR = 17.3

OR = 27.1

OR = 4.34

OR = 9.08

OR = 5.03

OR = 9.08

Elastin fibre formation

Degradation of extracellular matrix

Collagen formation

Collagen degradation

Collagen chain trimerization

-log (FDR)

Integrin surface interactions

0 1 2 3 4

Col6A1

- + - - - - - + - - - - vehicle

Col1A1

Actin

-SMA

EDA-FN

- - 0.1 0.5 - - - - 0.1 0.5 - - JQ1 uM- - - - 0.5 2.5 - - - - 0.5 2.5 CBP30 uM

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DMSO JQ1 CBP30

D1 D2 D3 D4 D5 D6 D1 D2 D3 D4 D5 D6D1 D2 D3 D4 D5 D6

CRISPLD2 SEMA4GBMP6

IGFBP5SCUBE3

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COL15A1ASPN

POSTNESMOMDADAMTS8

FGF1VEGFC

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WISP1INHBALTBP1PLXDC1ABI3BPCOL6A3MMP1GDF10

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JQ1

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24

600 24

JQ1CBP30

321248 193down-regulated

up-regulated

Pearson’s correlation=0.56p< 0.002

CTNNB1

COL6A1

COL6A3 COL6A2FN1

LOXPYCR1

CTHRC1

−1

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CBP to DMSO Protein (LogFC)

CB

P to

DM

SO R

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(Log

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peptides255075

CRABP2

B

4 days 6 days

−2

F

Fig. 3. CBP30 treatment targets a limited subset of genes enriched in ECM-mediated regulated pathways. (A) Venn diagram showing the number of geneswith significantly different expression (log2 FC > 1, FDR < 0.05) following JQ1 (0.5 μM) and CBP30 (2.5 μM) treatment. (B) Volcano plots of differentiallyexpressed genes; data are generated for six donors. (C) Gene enrichment analysis of canonical pathways of significantly differentially expressed genes treatedwith CBP30 or (D) JQ1 treatment for 3 d. (E) Heatmap of genes identified within the ECM and ECM-associated proteins following JQ1 and CBP30 treatmentfrom six donors. (F) Pathway analysis of RNA-Seq and proteomic analysis datasets following 3 d of drug treatment; data are mean of six donors with sig-nificantly different expression (log2 FC > 1.5, FDR < 0.05). (G) Correlation between RNA-Seq and proteomic datasets. (H) Myofibroblasts were treated witheither JQ1 (0.1 to 0.5 μM) or CBP30 (0.5 to 2.5 μM) for either 4 or 6 d; protein levels of proteomic hits collagen I α1, EDA-fibronectin, and collagen VI α1 andα-SMA were determined via Western blotting by SDS/PAGE on 4 to 20% Tris-glycine polyacrylamide gradient; data are representative of three donors.

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2030

40

HIST1H2BK

PLXNC1

RRAGD

HS3ST2

HRKCNGA3DMRTA1

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COL6A1

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PALM2

BCL2A1CCL8

FAIM2

SELLJAKMIP2

MILR1ESPNL

log2FoldChange

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JDP2

MKI67

HAS2

ENPP1

SHROOM2

ATP6V1D

CELF2

FDR=8.0E−06

FDR=7.4E−05

FDR=9.7E−05

FDR=9.3E−05

FDR=6.4E−04

FDR=6.4E−04

FDR=7.6E−04

FDR=2.3E−03

FDR=6.0E−03

FDR=6.1E−03

FDR=6.1E−03

FDR=7.5E−03

FDR=8.7E−02

FDR=1.8−E02

FDR=1.8−E02

FDR=1.8−E02

FDR=1.9E−02

FDR=1.9E−02

FDR=2.3E−02

FDR=2.6E−02Integrin Signaling PathwayuPAR−mediated signaling

ALK1 signaling eventsIntegrins in angiogenesis

α9 β2 integrin signaling eventsATR signaling pathway

ECM glycoproteins, collagens and proteoglycans

p73 transcription factor networkECM and ECM−associated proteins

β2 integrin cell surface interactionsE2F transcription factor network

amb2 Integrin signaling Syndecan−1 signaling events

Aurora B signaling Androgen receptor activity

Integrin cell surface interactionsIntegrin family cell surface interactions

PLK1 signaling eventsβ2 integrin cell surface interactionsFOXM1 transcription factor network

0 1 2 3 4 5

2345

enrichment

enrichment -log10FDR

4 days 6 days

10111 26

control COL6A1/2/30

20

40

60

80

Ave

rage

Trac

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(Pa)

p < 0.0001

chemotaxis migration

control RNAi Col6A1/2/3 RNAi

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5000

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CO

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3(p

g/m

l)

αSMA (Cyan) Collagen VI α3 (red) Collagen I α1 (green) σSMA (Cyan) / Collagen VI (red)

DD

IPF

nil

contro

l

BMP1

PCSK1

PCSK5

PCSK6

PCSK7

Col6A3

0.0

0.5

1.0

1.5

HGF

siRNA

relHGFmRNA/GAPDH

*****

nil

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l

BMP1

PCSK1

PCSK5

PCSK6

PCSK7

Col6A3

0.0

0.5

1.0

1.5

2.0

SHROOM2

siRNA

relSHROOM2mRNA/ GAPDH

***

nil

contro

l

BMP1

PCSK1

PCSK5

PCSK6

PCSK7

Col6A3

0.0

0.5

1.0

1.5

SDC1

siRNA

rel SDC1mRNA/GAPDH

*****

* *

nil

contro

l

BMP1

PCSK1

PCSK5

PCSK6

PCSK7

Col6A3

0.0

0.5

1.0

1.5

PCSK7

siRNA

rel PCSK7

mRN

A/GAPDH

****

siRNA siRNA siRNA siRNA siRNA siRNA

RNA-Seq following Col6A1/2/3

traction force microscopy

siRNA depletion

A B

C D E

F G H I

J K L M

Fig. 4. Collagen VI drives a profibrotic phenotype in myofibroblasts. (A) Confocal microscopy after immunostaining using anti-αSMA, anti-collagen VI α3, orcollagen I α1-specific antibodies in either (Top) DD nodule or IPF (Bottom) sections; representative of three donors, three slides per donor. (Scale bar, 25 or 50μM for higher magnification slides.) (B) Freshly dissociated DD nodule cells or passaged (p2) DD myofibroblasts were cultured for 3 d and secreted collagen VIα3 levels were detected by ELISA. (C) Venn diagram showing the number of genes with significant differential expression (log2 FC > 1.5, FDR < 0.05) followingeither nontargeting control siRNA (20 nM) or COL6A1/2/3 siRNA (20 nM)-mediated depletion over 4 or 6 d. (D) Volcano plots of differentially expressed genes,at day 6; data are generated from six donors. (E) Gene enrichment analysis of canonical pathways of significantly differently expressed genes at day 6. (F) THP-1 chemotaxis to myofibroblast-conditioned media generated from control siRNA (20 nM) or COL6A1/2/3 siRNA-treated cells for 6 d; migrated cells werequantified by Hoechst 3342 fluorescent stain for 1 h using the Celigo Imaging Cytometer. (G) Scratch assay measuring migration of myofibroblasts followingCOL6A1/2/3 depletion; migrated cells were quantified by staining with calcein fluorescent stain for 1 h using the Celigo Imaging Cytometer in eight donors. Pvalue was determined by paired sample t test in eight donors. (H) Effect of COL6A1/2/3 depletion as measured by traction force microscopy; cells weredetached after 6 d in presence of 20 nM siRNA. Color scale in stiffness maps indicates shear modulus in kilopascals. Representative (I) traction vector maps,average traction is depicted (mean ± SEM; n = 4 independent experiments with a minimum of 20 independent cells per condition; **P < 0.01 and *P ≤ 0.05versus controls). Six-day siRNA-mediated PCSK (20 nM) depletion in DD myofibroblasts inhibits (J) HGF, (K) SHROOM2, (L) SDC1, and (M) PCSK7 gene ex-pression as measured by Taqman PCR using the ΔΔCt method normalized to GAPDH; mean ± SEM from six donors. P value was determined by one-samplet test to normalized control (nontargeting oligo) sample of value of 1. *P ≤ 0.05, **P < 0.01, ***P < 0.001.

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integrin signaling, Syndecan-1, and the androgen receptor ac-tivity pathways (Fig. 4E and Dataset S6). This latter finding is ofparticular interest, given the male predominance of Dupuytren’sdisease in younger age groups (38). Syndecan-1 has been shownto regulate ECM fiber alignment and the activities of severalintegrins, including αvβ3, αvβ5, and α2β1 which control stromalcell migration (39). When comparing our EP300 ChIP-Seqdataset, we found 45% of the differentially regulated genes(log2 fold change [FC] > 1) were also enriched for EP300binding sites. Notably, we found siRNA depletion of COL6A3alone was sufficient to significantly suppress the expression ofmultiple target genes reported in the RNA-Seq (SI Appendix,Fig. S8), suggesting this may be the dominant isoform driving theprofibrotic function of collagen VI.We asked whether the downstream targets of collagen VI are

functional in human myofibroblasts. As COL6A1/2/3 depletionmarkedly reduces expression of multiple chemokines, we con-firmed that COL6A1/2/3 siRNA treatment significantly inhibitedCCL2 and CCL7 protein secretion by myofibroblasts (SI Ap-pendix, Fig. S9). We confirmed that COL6A1/2/3 depletion inmyofibroblasts significantly reduced chemotaxis of the myeloidcell line THP-1 toward myofibroblast conditioned media, sug-gesting collagen VI may play an important role in promotingimmune cell recruitment in localized fibrosis (Fig. 4F). As theCOL6A1/2/3 targets HGF and SDC1 are known mediators of cellmigration (39), we next sought to determine whether collagen VIregulates myofibroblast migration using an in vitro wound heal-ing assay. We found that myofibroblast migration was signifi-cantly impaired following COL6A1/2/3 depletion (Fig. 4G).Finally, as many genes regulated by collagen VI are associatedwith ECM interaction and integrin pathways, we investigated theeffect of COL6A1/2/3 depletion on contractility of myofibro-blasts on collagen-coated hydrogels using traction force micros-copy. We found that COL6A1/2/3 depletion resulted in apronounced reduction of contraction (Fig. 4 H and I). Collec-tively, these data demonstrate the collagen VI pathway, regu-lated by EP300, orchestrates multiple important profibroticfunctions, including chemoattraction, migration, and contractil-ity of human myofibroblasts.Proprotein convertases process and modulate proteins within

the secretory compartment, at the plasma membrane and inthe extracellular space (40) and, along with the procollagenC-proteinase BMP-1, have recently been described as key regu-lators in the proteolytic cleavage of collagen VI α3 (41). Ana-lyzing our RNA-Seq datasets we identified all of the proproteinconvertase subtilisin/kexin (PCSKs) expressed by the DD myo-fibroblasts and systematically depleted them to determine theirimportance in the regulation of the COL6A3-dependent geneexpression. We found that several genes identified from our geneenrichment pathway analysis following the COL6A1/2/3 RNA-Seq analysis within the Syndecan-1 pathway (HGF and SDC1)were also regulated by PCSK7 to a level similar to that observedby COL6A3 depletion alone (Figs. 4 J–M). PCSK7 depletion alsoregulated the chemokine CCL26 and its family member PCSK1inhibited ADAMTS8 (SI Appendix, Fig. S10).

DiscussionClinical trials in human fibrosis have repeatedly failed, probablydue to the poor predictive accuracy of targets identified usingmurine models of fibrosis. DD represents an important platformto study mechanisms of human fibrosis because of the readyavailability of tissue from patients undergoing surgery as atherapeutic intervention. We combined multiomic profiling withfunctional validation using these well-characterized patientsamples and identified collagen VI as a major regulator of themyofibroblast phenotype.Our genetics-led network connectivity algorithm (Pi) proposed

CREBBP/EP300 as key epigenetic regulators of the myofibroblast

phenotype in DD. Screening of a panel of high-quality epigeneticprobes highlighted bromodomain inhibitors as potent regulatorsof profibrotic gene expression in DD myofibroblasts. While re-lying on chemical probes alone has pitfalls due to potential off-target effects, here we provide multiple lines of evidence ofsupporting a direct role of CBP/EP300 in profibrotic gene reg-ulation. First, two structurally distinct bromodomain inhibitorsCBP30 and I-CBP112, and the EP300 HAT inhibitor A485,display similar activity profiles in suppressing gene expression,with an IC50 of 1 μM, consistent with reported activity on theCBP-bromodomain (42, 43). Second, siRNA-mediated suppres-sion of CBP/EP300 also confirmed a role of these epigeneticmodifiers in the regulation of numerous genes identified in theRNA-Seq dataset. Critically, using paired epigenetic and tran-scriptomic profiling, we provide a mechanistic insight and con-firm bromodomain inhibition directly blocks gene transcriptionthrough inhibiting the recruitment of CREBBP/EP300 to theirbinding sites, thereby reducing levels of H3K27ac at the ACTA2and COL1A1 loci.H3K27ac and H3K4me1 occupancy are chromatin features

used to identify cell-type-specific enhancers (44, 45). Signifi-cantly, our data show a high degree of genomic co-occupancy ofEP300 with H3K27Ac and H3K4me1, indicating that super en-hancer sites are indeed regulating coordinated transcriptionalcontrol of the highly enriched ECM pathways dominating theEP300 pathway analysis in myofibroblasts. Interestingly, EP300showed a distinct binding profile closely localizing with 8 of the22 most significant SNPs associated with Dupuytren’s disease(12). Since both H3K27ac and EP300 (32, 46) demark the ar-chitecture of super enhancers in all cell types, this warrantsfurther investigation using coactivators such as MED1 and othermaster transcription factors. Of particular note is the SFRP4variant, as this was shown to be the most strongly associatedvariant (rs16879765) in our recent DD GWAS study (12) and wehave shown here that SFRP4 is regulated by FOSL1 depletion.Furthermore, the study by Ng et al. (12) suggested that a subtleimbalance of WNT signaling contributes to the fibrotic pheno-type, allowing an increase in WNT3A signaling through thenoncanonical pathway. Wnt/β-catenin signaling has been shownto activate Fra1 expression, and Fra1 has been shown to play arole in epithelial–mesenchymal transition (EMT) activated byWnt/β-catenin signal pathway (47).Our epigenetic profiling and motif analysis revealed several

putative EP300 coregulators of gene transcription, includingFra1, RUNX1, and TEAD4. Functional analysis of these tran-scription factors demonstrated discrete functional properties ofeach in myofibroblasts. Fra1 (FOSL1) has previously beenreported as a negative regulator of AP-1 (48–50) and its deple-tion enhanced bleomycin-induced lung fibrosis. Our data con-firm Fra-1 acts as a transcriptional repressor of CXCL8, IL6, andCTGF in DD myofibroblasts but conversely acts to positivelyregulate SPFR4, CCL26, and COL6A3 expression, suggesting ahighly complex level of regulation of the transcriptional ma-chinery at these genes. TEAD factors act as mediators of theHippo signaling pathway interacting with the YAP and WWTR1(TAZ) transcriptional coactivators (51) and EP300 has recentlybeen shown to activate YAP/TAZ (52). We observed TEAD4depletion resulted in decreased expression of two key chemo-kines CCL2 and CXCL8 (IL-8), important recruiters of mono-cytes and neutrophils. Depletion of RUNX1 highlighted a roleof this transcription factor in the regulation of two key ECMregulators, fibronectin-1 (FN-1) and Hyaluronan Synthase 2(HAS2). Together, these data describe a transcriptional networkdownstream of EP300 that orchestrates diverse cellular profi-brotic functions in myofibroblasts associated with pathogenicity,including enhanced matrix remodeling, development of a con-tractile phenotype, and immunomodulatory properties, therebyproviding a framework by which these transcription factors link

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EP300 gene regulation with the profibrotic phenotype typicallyassociated with myofibroblasts.Collagen VI is an interstitial collagen that functions as a

structural component of the matrix in addition to influencing celladhesion. Levels are elevated in fibrotic conditions such as IPF(53), chronic liver disease (54), and chronic kidney disease (55).Fragments from the C-terminal part of the α3 chain have beenshown to have signaling effects, including profibrotic featuresand macrophage chemoattractant properties. One such fragmentPRO-COL6/endotrophin is now commonly used as a biomarkerin cancer (56) and fibrotic conditions (57, 58) and has been ableto distinguish individuals with IPF who have progressive diseasefrom those with more stable disease (59). We confirmed thepresence of high levels of collagen VIα3 in supernatants fromfreshly isolated Dupuytren’s nodules and in cultured myofibro-blasts. Our immunofluorescence studies highlighted that it ishighly expressed in both Dupuytren’s nodules and IPF patientsamples, localizing closely with α-SMA positive myofibroblastsregions. It is interesting to note that whereas collagen VIα3 waswidely distributed in DD nodules, in the IPF samples a morediscrete cellular localization was observed when compared tocollagen Iα1, suggesting discrete functions as dictated by thelocal cellular–stromal architecture. Alternatively, it may reflectdiffering disease stages as IPF is usually diagnosed late. Fur-thermore, we show that collagen VI regulates expression of thechemokines CCL2, CCL7, and CCL26, in accordance with pre-vious data suggesting a potential role of endotrophin (ETP) inchemotaxis (60), and we confirmed a role of collagen VI inmyofibroblast-induced chemotaxis of THP1 cells. Previously (4),we identified macrophages and mast cells as the predominantsource of TNF within Dupuytren’s nodule cultures and demon-strated that macrophage-derived TNF plays a key role in pro-moting the fibrotic phenotype in Dupuytren’s disease (4).Excessive production of collagen VI may contribute to thechronicity of the disease by promoting the recruitment ofmonocytic cells to the site of this disease and probably plays asimilar role in other fibrotic disorders (61). A central function ofmyofibroblasts in both health and disease is the generation oftraction force (62), which plays a key role in remodeling the matrixand also modulates the activities of the embedded stromal cells (63,64). Importantly for disease management, we also demonstratedthat collagen VI regulates myofibroblast contraction.Mutations in collagen VI genes is associated with Bethlem

myopathy (BM) and Ullrich congenital muscular dystrophy(UCMD) (65). A mouse model in which the Col6a1 gene wasinactivated has identified key functions of Col6a1, includingautophagic dysfunction, skeletal, heart, and tendon defects (66).COL6A3 protein deficiency in mice leads to muscle and tendondefects similar to those seen in human collagen VI congenitalmuscular dystrophy (67). Therefore, directly targeting collagenVI is likely to be deleterious. The C5 region of the α3 chain ofcollagen VI has been shown to undergo proteolysis in a tissue-specific manner by the proprotein convertase furin/PCSK3 andBMP1 (68), generating diverse fragment sizes depending on thetissue source (41). However, free endotrophin was shown to bescarce under physiological conditions, suggesting that the pro-teolysis of collagen VI is tightly regulated. Hence targeting thecollagen VI proteolysis cleavage machinery could represent amore tractable approach. Analysis of our RNA-Seq datasetsrevealed that PCSK3 is not expressed in the DD myofibroblasts,but we were able to detect mRNA expression of PCSK1, PCSK5,PCSK6, and PCSK7, with PCSK7 showing the highest basal levelof gene expression. Our siRNA studies identified PCSK7 as apotential protease regulating collagen VIα3 cleavage. Depletionof PCSK7 suppressed gene expression of a subset of the genesfrom the Syndecan-1 signaling pathway, a key pathway regulatingstromal cell migration and ECM fiber alignment (39); this wasalso regulated by COL6A3 depletion. Our future work will

explore the role of PCSK7 in collagen VI proteolysis and work isunderway to identify any PRO-C6 fragments in our model. It isnoteworthy that PCSK7 missense variants identified in GWASstudies are associated with liver fibrosis (69). More recently aPCSK7 variant has been shown to lead to increased intracellularPCSK7 expression and secretion from hepatocytes, potentiallylinking dyslipidemia with a tendency to more severe liver damagein high risk individuals (70). Given that PCSK7 can be secretedas well as localized on the plasma membrane, this raises thepotential for therapeutic development of monoclonal-basedPCSK7 inhibitors. The translational potential of the wider fam-ily of proprotein convertases has already been realized as thereare two FDA-approved drugs, evolocumab and alirocumab,targeting the plasma protein PCSK9 for the treatment of familialhypercholesterolemia and clinical atherosclerotic cardiovasculardisease (71). Further work is needed to explore the tissue-specific nature of PCSK7 expression and a wider understand-ing of its physiological role.A limitation of this study is the absence of appropriate control

tissue. Dupuytren’s disease is restricted to certain fibers of thepalmar fascia. Previous studies used cells from the fascia in theregion of the carpal tunnel or the transverse carpal ligamentfrom affected or normal individuals or uninvolved transversepalmar fibers from patients with Dupuytren’s disease as controls(72). However, this approach has significant limitations. Thepalmar fascia over the carpal tunnel is rarely affected byDupuytren’s disease and the transverse carpal ligament is alwaysunaffected; hence, it is possible that the constituent cells areinherently different. Furthermore, normal palmar fascia issparsely populated by cells and hence to obtain adequate num-bers requires repeating passaging of cells in vitro (73). Further-more, we and others have shown that at passage 5 thephenotypes of myofibroblasts and normal human dermal fibro-blasts tend to merge (74).In summary, by exploiting the availability of samples from

patients with DD and a combination of epigenetic and tran-scriptional profiling studies, we defined EP300 as an importantregulator of human fibrosis that directs diverse cellular processesin myofibroblasts. There are supporting data showing thatpharmacological inhibition of EP300 is effective in murinemodels of pulmonary fibrosis (75, 76). However, the many tar-gets of epigenetic regulators suggest their long-term global in-hibition may result in off-target effects. Here we reveal thatcollagen VI is a downstream target of EP300 and is widelyexpressed in DD nodules and IPF lung tissue and mediates an

Fig. 5. Schematic illustrating the epigenetic control by EP300 of the profi-brotic phenotype of myofibroblasts in Dupuytren’s disease. Persistent acti-vation of EP300 acetylates histones and transcription factors to promoteextracellular matrix production and myofibroblast contractility. Collagen VI,a key target of EP300, plays a dominant role in regulating contraction.Proteolytic cleavage by PCSK7 generates small bioactive collagen VI frag-ments which control recruitment of immune cells by chemokine production,thereby perpetuating the cycle of chronic inflammation and fibrosis by thesecretion of cytokines such as TNF.

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important role in regulating ECM production, chemotaxis, andcontractility as shown in Fig. 5. Crucially, our data with EP300regulation identify collagen VI and PCSK7 as previously un-identified therapeutic targets. The important translational po-tential of our findings is underpinned by using primary humansamples exclusively, including Dupuytren’s disease and IPF,diseases where therapeutic options are limited and patient out-comes are currently suboptimal.

MethodsIn brief, screening the effects of a panel of epigenetic inhibitors uponACTA2,COL1A1, COL3A1 and TGFB1 gene expression was performed using primaryhuman myofibroblasts isolated from DD patients by Taqman PCR. Inhibitionof acetylation at the promotors of ACTA2 and COL1A1 by the EP300 probeSGC-CBP30 (CBP30) was shown using ChIP PCR. Inhibition of myofibroblastcontraction was assessed using traction force microscopy (23). Evidence ofdirect recruitment of EP300 to a profibrotic gene landscape was shown usingChIP-Seq. Transcriptional profiling of CBP30 in myofibroblasts was performed

using RNA-Seq and hits confirmed using siRNA of CREBBP/EP300 depletionstudies. The effects of CBP30 on global protein expression in DD myofibroblastswas determined using shotgun proteomic profiling. The role of collagen VI incontrolling profibrotic gene expression was confirmed by siRNA-mediatedCOL6A1/2/3 depletion followed by RNA-Seq; subsequent roles in contraction,migration, and wound healing were confirmed using traction force microscopy,chemotaxis, and scratch assays, respectively. The methodology is describedin detail in SI Appendix.

Data Availability. RNA-Seq and ChIP-Seq datasets are deposited within theNCBI SRA database under accession numbers PRJNA625874, PRJNA624331,and PRJNA624119, respectively.

ACKNOWLEDGMENTS. We thank all the research nurses at Queen VictoriaHospital, Southampton General Hospital, Broomfield Hospital, Royal DerbyHospital, and St. John’s Hospital and Dr. Steve Nathan for kindly supplyingthe IPF samples. The work was funded by European Union ULTRA-InnovativeMedicines Initiative Grant 115766 and Oxford-Celgene Research FellowshipGrant AZR00610.

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