Identifying Collagen VI As a Target of Fibrotic Diseases Regulated by CREBBP/EP300

Home , ACTA2, Fibrosis, INHBA, POLR2A, POLR2C, POLR2E

Identifying collagen VI as a target of fibrotic diseases regulated by CREBBP/EP300

Lynn 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 OX3 7FY, United Kingdom; bBotnar Research Centre, National Institute for Health Research Oxford Biomedical Research Unit, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Science, University of Oxford, Oxford OX3 7LD, United Kingdom; cWellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom; dBiotherapeutics Department, Celgene Corporation, San Diego, CA 92121; eTarget Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7FZ, United Kingdom; fDepartment of Plastic Surgery, Geert Grooteplein Zuid 10, 6525 GA Nijmegen, The Netherlands; gDepartment of Plastic and Reconstructive Surgery, Broomfield Hospital, Mid and South Essex National Health Service Foundation Trust, Chelmsford CM1 4ET, Essex, United Kingdom; hPulvertaft Hand Surgery Centre, Royal Derby Hospital, University Hospitals of Derby and Burton National Health Service Foundation Trust, Derby DE22 3NE, United Kingdom; iDepartment of Trauma and Orthopaedic Surgery, University Hospital Southampton National Health Service Foundation Trust, Southampton SO16 6YD, United Kingdom; jDepartment of Plastic and Reconstructive Surgery, Queen Victoria Hospital National Health Service Foundation Trust, East Grinstead RH19 3DZ, United Kingdom; kDepartment of Plastic 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, progresses, the relatively acellular cords mature, thicken, and yet there are few effective therapies. The underlying pathology of contract, leading to permanent flexion deformities of the fingers. all fibrotic conditions is the activity of myofibroblasts. Using cells Unlike fibrotic diseases of visceral organs such as the lung and from freshly excised disease tissue from patients with Dupuytren’s liver which are diagnosed late, DD tissue can be diagnosed early disease (DD), a localized fibrotic disorder of the palm, we sought and is readily accessible for research as the disease is often to identify new therapeutic targets for fibrotic disease. We hy- treated by surgical excision. Using the relatively early-stage MEDICAL SCIENCES pothesized that the persistent activity of myofibroblasts in fibrotic nodules from patients with Dupuytren’s disease enables us to diseases might involve epigenetic modifications. Using a validated genetics-led target prioritization algorithm (Pi) of genome wide analyze signaling and regulatory pathways and hence has the association studies (GWAS) data and a broad screen of epigenetic inhibitors, we found that the acetyltransferase CREBBP/EP300 is a Significance major regulator of contractility and extracellular matrix production via control of H3K27 acetylation at the profibrotic genes, Fibrosis remains a major unmet medical need as therapeutic ACTA2 and COL1A1. Genomic analysis revealed that EP300 is targets discovered in animal models have failed to translate. A highly enriched at enhancers associated with genes involved in major challenge for identifying novel targets is the limited multiple profibrotic pathways, and broad transcriptomic and pro- availability of early-stage human disease tissue. Here, we uti- teomic profiling of CREBBP/EP300 inhibition by the chemical probe lize Dupuytren’s disease (DD), a common localized fibrotic SGC-CBP30 identified collagen VI (Col VI) as a prominent down- disorder, to evaluate the impact of epigenetic regulation of stream regulator of myofibroblast activity. Targeted Col VI knock- myofibroblasts and identify potential tractable targets in hu- down results in significant decrease in profibrotic functions, man fibrosis. We demonstrate that the epigenetic regulator including myofibroblast contractile force, extracellular matrix CREBBP/EP300 is a critical determinant of the profibrotic phe- (ECM) production, chemotaxis, and wound healing. Further evi- notype. Furthermore, we identify collagen VI to be a key dence for Col VI as a major determinant of fibrosis is its abundant downstream target of CREBBP/EP300 and reveal valuable in- expression within Dupuytren’s nodules and also in the fibrotic foci sights in the role it plays in key profibrotic functions, including of idiopathic pulmonary fibrosis (IPF). Thus, Col VI may represent a contractile force, chemotaxis, and wound healing, and hence tractable therapeutic target across a range of fibrotic disorders. its potential as a therapeutic target.

EP300 | fibrosis | collagen VI | epigenetic Author contributions: L.M.W., F.E.M., J.K., W.X., M. Sundstrӧm, M.F., and J.N. designed research; 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. performed research; H.F., J.K., M. Sood, C.B., D.W., L.H., and D.D. contributed new reagents/analytic ibrosis of visceral organs such as the lungs, heart, kidneys, and tools; 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., Fliver is a major cause of morbidity and mortality (1). How- 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. ever, despite intense research efforts, little progress has been Reviewers: J.J.O., NIH; and G.W., Medical University of Innsbruck. made in the identification of effective therapeutic targets. Po- Competing interest statement: J.N. and M.F. have received consulting fees and hold eq- tential reasons for this include late presentation of patients with uity in 180 Therapeutics, which is an exclusively licensed intellectual property from the ’ fibrotic diseases involving internal organs, lack of good disease University of Oxford to treat Dupuytren s disease with anti-TNF. biomarkers, and poor translation through to clinical practice of This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY). targets identified in murine models (2). A greater understanding 1 of the mechanisms controlling the fibrotic pathways in human L.M.W. and F.E.M. contributed equally to this work. 2 tissues is required to facilitate the development of next-generation Present address: Department of Biological Sciences, Institute of Materia Medica, Shan- dong First Medical University and Shandong, Academy of Medical Sciences, Jinan therapies. 250062, Shandong Province, People’s Republic of China. ’ Dupuytren s disease (DD) is a debilitating fibroproliferative 3To whom correspondence may be addressed. Email: [email protected] disorder restricted to the palms of genetically susceptible indi- or [email protected]. viduals (3). The initial clinical presentation is the appearance of This article contains supporting information online at https://www.pnas.org/lookup/suppl/ a firm cellular nodule in the hand, which expands into fibrous doi:10.1073/pnas.2004281117/-/DCSupplemental. collagenous cords that extend into the digits. As the disease First published August 5, 2020.

www.pnas.org/cgi/doi/10.1073/pnas.2004281117 PNAS | August 25, 2020 | vol. 117 | no. 34 | 20753–20763 Downloaded by guest on October 1, 2021 potential for the identification of novel therapeutic targets. I-CBP112, as the most potent and significant inhibitors of Dupuytren’s nodules represent a complex disease system, hous- ACTA2 and COL1A1 gene expression (Fig. 1B), with a strong ing densely packed myofibroblasts (MFs) alongside other less dose-dependent effect (SI Appendix, Fig. S1). We also employed abundant stromal and immune cells (4). Myofibroblasts are a recently developed, highly specific CBP/EP300 HAT inhibitor characterized by the expression of α-smooth muscle actin A485 (22), to confirm the role of CBP/EP300-mediated histone (α-SMA) and excessive production of extracellular matrix acetylation regulating ACTA2 and COL1A1 gene expression (SI (ECM) proteins such as collagens, glycoproteins, and proteo- Appendix, Fig. S1 G–I). glycans, which enhances their ability to contract tissue (5, 6). To establish whether pharmacologic inhibition of CBP/EP300 Matrix stiffness and mechanical stress in severely damaged tis- bromodomains would impact histone acetylation at the tran- sues initiates a positive feedback loop between myofibroblasts scription starts sites of ACTA2 and COL1A1, we employed and their surrounding microenvironment that perpetuates fi- chromatin immunoprecipitation (ChIP) qPCR with antibodies brosis (7, 8). Therefore, Dupuytren’s nodules provide an ideal for the H3K27ac mark. We also included the BET inhibitor JQ1 model system to study primary human myofibroblasts, the cell as it also reduced expression of ACTA2 and COL1A1 in our type responsible for deposition and contraction of excessive primary qPCR screen. Both CBP30 and JQ1 treatment reduced matrix in all forms of fibrosis (9, 10). H3K27 acetylation at the transcriptional start sites of ACTA2 Despite the substantial heritability of 80% (11) in DD and and COL1A1, confirming a direct role in regulating gene tran- multiple risk loci identified by genome wide association studies scription (Fig. 1 E and F). (GWAS) (12), why this disease is restricted to the palmar fascia Since excessive myofibroblast contractility is a key feature in of the hand is still poorly understood. The distinct anatomical disease pathogenicity in vivo, we measured the effects of JQ1 location and proposed role of environmental factors such as and CBP30 on myofibroblast force generation as a function of excessive alcohol consumption (13), heavy manual labor, and contractility using traction force microscopy (23). This demon- exposure to vibrations (14) suggest epigenetic regulation is likely strated that both compounds significantly inhibited cell con- to be important in the pathogenesis of the disease. Epigenetic tractility, with both peak and average traction force reduced by mechanisms have been shown to be crucial mediators of fibrotic ∼50% (Fig. 1 G–I). In addition, both inhibitors reduced cell gene expression in a wide range of fibrotic disorders (15), in- proliferation (SI Appendix, Fig. S1A), with JQ1, but not CBP30, cluding cardiac fibrosis (16), liver fibrosis (17), and systemic also reducing cell viability and confluency (SI Appendix, Fig. S1 B sclerosis (18), and probably account for the persistence of and C). Myofibroblasts display spindle or stellate-cell morphol- myofibroblasts in disease (19). ogy, and while treatment with CBP30 had no clear effect on We hypothesized that specific targeting of transcriptional myofibroblast morphology, JQ1 treatment also altered cell coactivators could provide valuable insight into epigenetic shape, resulting in an elongated neuron-like morphology, indic- mechanisms controlling the myofibroblast phenotype in fibrosis. ative of broad effects not shared with CBP30 (SI Appendix, Fig. Here we show that the histone acetyltransferases CREBBP and S2 D–G). Collectively, these data indicate CREBBP/EP300 is a EP300 (20) are key regulators of profibrotic phenotype and highly associated pathway in localized human fibrosis that con- function of myofibroblasts and act through control of collagen trols key aspects of myofibroblast phenotype and function, including VI. Furthermore, collagen VIα3 is highly expressed in both ACTA2andCOL1A1 gene expression and cell contractility. Dupuytren’s nodules and in the fibroblastic foci in idiopathic pulmonary fibrosis (IPF), with a distribution distinct from col- EP300 Associates with Active Enhancers in Human Myofibroblasts. lagen I but closely aligned with α-SMA positive regions, thus Transcriptional regulation of gene expression is achieved emerging as a signature marker of myofibroblasts and a tractable through cooperation between promoter and enhancer elements. therapeutic target for DD and potentially also other fibrotic diseases. Specific histone modifications function as binding elements for effector proteins that serve to regulate transcription through Results manipulation of the chromatin environment or assembly of Computational Target Prioritization and Screening Using transcription machinery. Acetylation at lysine 27 of histone H3 Pharmacological Inhibitors Both Identify CREBBP/EP300 as a Central serves as a signature mark of active enhancers (24) in all cell Regulatory Network in Human Fibrosis. To leverage the power of types, where monomethylation of histone H3 at lysine 4 genetic variance in target discovery, we applied our recently (H3K4me1) marks both primed and active enhancers in a developed prioritization “Pi” approach (21) to risk loci in DD cell-type-specific manner (25, 26). To further delineate the mo- GWAS data (12). This validated approach allows us to prioritize lecular mechanisms underlying the functional impact of the genes from GWAS hits, not simply based on genomic proximity bromodomain inhibitors CBP30 and JQ1, we performed ChIP but also including evidence from chromatin conformation and analysis coupled with deep sequencing using H3K27ac-, expression quantative trait loci mapping in immune cells, plus H3K4me1-, EP300-, and BRD4-specific antibodies (27). Geno- gene networks defined by the Search Tool for the Retrieval of mic distribution of H3K27ac and BRD4 were strikingly similar Interacting Genes/Proteins database. This unsupervised network (Fig. 2A) in many genomic regions, in accordance with previous connectivity analysis identified FoxO and WNT signaling as data (28). In contrast, H3K4me1 and EP300 displayed similar highly ranked pathways that share CREBBP and EP300 member genomic distribution, with genomic occupancy assigned pre- genes, as shown at the intersection of delineated pathways in dominantly to intronic and intergenic regions. However, there blue and green, respectively (Fig. 1A and SI Appendix, Table S1), was also significant overlap of genomic occupancy between the providing unbiased support for our hypothesis that epigenetic datasets; EP300 shared 91% of sites with H3K27ac, 87% with modulators are important in controlling disease processes in DD. H3K4me1, and 78% with BRD4 (Fig. 2B). For functional validation, we screened a focused library of 39 We next probed the ChIP sequencing (ChIP-Seq) datasets to epigenetic inhibitors (SI Appendix, Table S2) to study their effect verify EP300 binding within the genomic loci of all of the genes on the expression of key profibrotic genes (ACTA2, COL1A1, identified within the FoxO (blue) and WNT (green) pathways, COL3A1, and TGFB1) on early passage myofibroblasts derived previously identified from the Pi pathway analysis in Fig. 1A. All from nodules in patients with DD (Fig. 1B and SI Appendix, Fig. genomic loci, with the exception of RELA and AKT1 identified S1). This revealed the pan bromodomain inhibitor bromosporine EP300 binding within 50 kB of the annotated genes. We have (BrSp), the BET bromodomain inhibitors, PFI-1 and JQ1, and shown two genes each, FOXO3 and EGFR from the FoxO the two structurally distinct CREBBP/EP300 bromodomains pathway and JUNB and CSNK1A1 from the WNT pathway in SI inhibitors SGC-CBP30 (referred to as CBP30 throughout) and Appendix, Fig. S3, as an illustration of these findings.

20754 | www.pnas.org/cgi/doi/10.1073/pnas.2004281117 Williams et al. Downloaded by guest on October 1, 2021 RALBP1 A [3.32@185]5

E2F4 ACTB [4.33@14] [3.97@32] Seed gene CFTR (colored segments) [4.61@5] CDC42C4422 CPSF1 SP1 [3.6@94]3.6@ nGenecGene eGene [3.97@34] [4.23@19]] PPP1CB HSP90AB1A SRCRC Pi rating Pi rank fGene pGene dGene [3.61@92] [3.84@49]@4 [3.95@37][3.[3.995 /~16,000 genes ESR1 051234 [4.31@17]@17] Non-seed gene PPP1CAPPP1 numeric value and shading (no colored segments) [3.55@106]55@10 of outer rim MYC MDM2MDD 2 TP533 NTRK1 [3.51@115][3.5 @111 ] [4.08@25][ 5]] [4.44@8] PPP1CC [3.33@182] EGFRE MAPK3, AKt1, EGFR, GRB2, CREBBP, EP300, AKT1 [3.94@39].99 [3.01@403][3.[3.0 ] MAPK3MAMAP 3 JUNJU GRB2GRRB [2.96@445][[2 @445 [4.42@10][4.4 [3.66@82][ @ SIRT1, FOXO3, CDK2, GABARAP, MDM2, ERBB2 [3.66@81] PLK1, GABAPAPL1, GABAPAPL2, RBL2 ATF2T SQSTM1 HSP90AA1 HSPA1BB [3.77@62]@62] [4.33@15][4.3 5]] HSPA1A [4.01@30] [3.33@17[3 179]79] [3.34@175][ FOXO3X 3 PRKACA, JUN, MYC, CTNNB1, CSNK1A1 [2.41@1145]2.4 @1 4 RELA HSPA8 RBL2 TRAF2 [3.34@174][3 [4.33@16]6] CREBBP, EP300, RUVBL1, TPS3, CTFR, RELA [3.38@162] [3.8@57] PRKKACAC SIRT1 [3.28@203]203] [3[3.68@75]3.63..6 YWHAEYWHAEA YWHAZ CFTR [3.53@111][ 5 @1111 [4.61@5] CSNK1A1SNK1NK1ANK A1 [4.01@31]] Pathways enriched [3.49@122][3.4[3.49@122] (FDR < 0.01) CDK2DDK CTNNB1 [3.44@138]3 4@@ 8 GABARAPL22 FoxO signaling pathway FDR=4.4e-7FDR=4.4e-7 [3.93@42] [5@1] GABARAPG A MAPK signaling pathway FFDR=2.3e-4DR=2.3e-4 [3.8@54] GABARAPL1 FDR=1.FDR=1.8e-38e-3 EP300E 3000 [4.05@28] ErbB signaling pathway CREBBPB FFDR=4.3e-3DR=4.3e-3 [3.57@102] [3.61@90][3.61[3.6 @@9@90 PLK1 PI3K-Akt signaling pathway RUVBL1 [3.73@66] WNT signaling pathway FDR=7.3e-3FDR=7.3e-3 [3.81@52] HIF-1 signaling pathway FDR=9.3e-3FDR=9.3e-3 POLR2C PKMM FFDR=7.2e-3DR=7.2e-3 [3.38@159] [3.46@132]33.. 1313232 POLR2E cAMP signaling pathway [3.3@193] 0.0 2.5 5.0 7.5 10.0 POLR2A Z-scores [4.44@9]@ B CE ACTA2 ACTA2 promotor ACTA2 COL1A1 COL3A1 TGFB1 20 1.0 H3K27Ac ChIP

SGC0946 308 TP-472 156 TP-064 131 BrSp 217 300 300 300 300 UNC0642 191 GSK343 155 BAY-299 127 JQ1 153 15

LLY-507 185 UNC1999 145 A-395 123 PFI-4 135 t GSK591 153 BAY-299 132 TP-472 116 MS023 127 MEDICAL SCIENCES A-395 152 UNC1215 122 SGC707 115 TP-064 122 ***

UNC0638 143 (R)-PFI-2 119 UNC1999 109 LP99 120 0.5 npu 10 PFI-3 136 PFI-4 119 L-Moses 109 PFI-1 118 * TP-064 131 TP-064 117 MS049 106 TP-472 114 %i ** GSK343 124 A-395 115 IOX1 104 NVS-CECR2 110 **** OICR-9429 121 NI-57 112 DMSO 100 BAZ2-ICR 107 5 ** LP99 118 MS023 112 OICR-9429 90 SGC-CBP30 106 **** **** **** ** BAY-299 115 GSK591 111 GSK591 88 GSK591 106 200 200 200 200 UNC1999 115 BI-9564 108 LP99 86 PFI-3 105

NI-57 110 L-Moses 103 UNC1215 84 SGC707 103 rel ACTA2 mRNA/ GAPDH 0.0 0 SGC707 109 IOX1 102 BI-9564 83 L-Moses 102 2 1 TP-472 103 GSK2801 101 GSK2801 81 DMSO 100 O 1 30 F D + + - - CBP30 1 P JQ1 101 DMSO 100 I-BRD9 80 99 MS BrSp PFI-4 A-395 D BP BP- - - + + JQ1 DMSO 100 A-196 97 BAZ2-ICR 79 GSK-LSD1 98 I-C C OF-1 95 OICR-9429 96 NI-57 79 GSK2801 96 3 24 3 24 hrs UNC1215 91 PFI-3 95 PFI-3 79 NI-57 96 D F (R)-PFI-2 90 BAY-598 95 PFI-4 78 BAY-299 95 GSK-J4 89 SGC0946 93 SGC0946 73 IOX1 85 COL1A1 COL1A1 promotor A-196 86 100 LP99 90 100 UNC0642 73 100 I-BRD9 85 100 1.0 25 I-BRD9 85 GSK-LSD1 86 MS023 73 I-CBP112 85 IOX1 85 BAZ2-ICR 79 (R)-PFI-2 72 OICR-9429 84 H3K27Ac ChIP L-Moses 82 MS049 78 GSK-LSD1 71 MS049 84 20 BAZ2-ICR 81 OF-1 75 GSK343 69 OF-1 82 GAPDH MS049 81 UNC0642 74 NVS-CECR2 69 BI-9564 74 BAY-598 79 LLY-507 73 BAY-598 64 GSK-J4 72 t GSK2801 74 NVS-CECR2 71 OF-1 63 UNC0638 72 *** 15 pu NVS-CECR2 72 A-366 71 A-196 62 SGC0946 68 0.5 mRNA/ A-366 67 SGC707 70 A-366 55 A-366 68 in GSK-LSD1 67 UNC0638 67 LLY-507 54 UNC0642 65 **** 10

BI-9564 63 I-CBP112 57 UNC0638 46 LLY-507 64 % MS023 51 GSK-J4 48 GSK-J4 32 A-196 60 *** I-CBP112 40 SGC-CBP30 43 I-CBP112 31 BAY-598 58 **** **** **** 5 SGC-CBP30 20 I-BRD9 35 SGC-CBP30 25 UNC1215 53 *** COL1A1 PFI-1 8 PFI-1 32 PFI-1 10 (R)-PFI-2 49 *** *** JQ1 8 BrSp 16 JQ1 7 GSK343 44 0.0 rel 0 BrSp 6 JQ1 13 BrSp 2 UNC1999 44 0 1 1 112 3 F Q D + + - - CBP30 P P J DMSO B BrSp C - - + + JQ1 I- CBP 3 24 3 24 hrs

G H ** I ** DMSO JQ1 CBP30 * 200 * -) JQ1(+) 1500 JQ1(+)

150 1000 action (Pa)

r 100 raction (Pa)

500 ge T kT

ra 50 e Pea Av min max 0 0 traction magnitude (pa) O 1 O 1 0 Q 30 Q 3 J P S J P MS B M B D C D C

Fig. 1. CREBBP/EP300 regulates profibrotic gene expression and contractile phenotype in myofibroblasts. (A) Pi analysis identifies CREBBP and EP300 as highly 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 with probes for 3 d. Gene expression was analyzed by Taqman qPCR using the ΔΔCt method, normalized to GAPDH. Data are means ± SEM of five independent experiments from five individual donors, represented as a heatmap where values are normalized to DMSO control value; 100% represented in white, red represents 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 of value 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 at promotor proximal regions of ACTA2 (E) and COL1A1 (F) in patient-derived myofibroblasts. Data are representative of three independent experiments from three individual donors, mean ± SD of technical triplicates. Myofibroblasts were plated onto 2.55-kPa hydrogels for traction force microscopy in the presence of 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 Downloaded by guest on October 1, 2021 H3K27ac BRD4 ABH3K27ac H3K4me1

TT S Exon 453 464 H3K4me1 Intron EP300 Intergenic Promoter 5UTR 95 1577 583 3UTR

178 3497 BRD4 EP300 873 5273

6516 64 1625 147 1355

449

C 10kb D EP300 EP300 BRD4 PDGFR−beta signaling pathway FDR= 5.1E-06

H3K4me1 SHP2 signaling FDR=6.1E−06 H3K27ac Beta3 integrin cell surface interactions FDR=2.4E−04 ACTA2-AS1 ACTA2 EP300 ECM glycoproteins, collagens and proteoglycans FDR=5.5E−4 BRD4 HIF−1−alpha transcription factor network FDR=5.6E−04 H3K4me1 AP−1 transcription factor network FDR=5.7E−04 H3K27ac

Beta1 integrin cell surface interactions FDR=1.2E−03 COL1A1 FAK signalling pathways FDR=1.2E−03 EP300

BRD4 Signaling events mediated by PTP1B FDR=1.4E−03 H3K4me1 Glypican 1 network FDR=1.4E−03 H3K27ac 02468 FN1 Enrichment significance -log10(FDR) E BRD4 F

Ret tyrosine kinase signaling pathways FDR=1E−10 EP300

PTEN is signlling pathays FDR=1E−10 Rank Motif Sequence Log P %sites p38 MAPK signaling pathway FDR=1E−10 value

Circadian rhythm pathway FDR=1E−10 1 Fra1 -1.25e+04 48.4%

Arf6 downstream pathway FDR=1E−10 2 TEAD4 -4.23e+03 33.8%

PDGFR−beta signaling pathway FDR=1E−10 3 MF0010 -1.07e+03 31.1% ErbB1 downstream signaling FDR=1.5E−09 4 RUNX1 -1.07e+03 13.3% FAK signalling pathways FDR=1.5E−06 5 NFIX -6.30e+02 28.8% VEGFR1 and VEGFR2 signiling pathways FDR=3E−06

mTOR signaling pathway FDR=3E−06

0.0 2.5 5.0 7.5 10.0 Enrichment significance -log (FDR) G 10 H BRD4

Rank Motif Sequence Log P value % sites

1 Fra1 -1.98e+03 13.9% EP300 2 Elk1(ETS) - 5.98e+02 19.3% FOSL1 3 CEBPG -5.59e+02 23.5% TEAD4 RUNX1

4 YY1 -2.59e+02 3.6% SFRP4 CCL2 FN1 COL6A3 HAS2 5 KLF6 -2.33e+02 6.75% CXCL8. CCL26 POSTN

Fig. 2. EP300 associates with active enhancers. (A) Genomic distribution of H3K27ac, H3K4me1, BRD4, and EP300 peaks. (B) Venn diagram of overlap of H3K27ac, H3K4me1, BRD4, and EP300 peaks as determined by Homer AnnotatePeaks. (C) Visualization in IGV of ChIP-Seq signal at the ACTA2, COL1A1,and FN1 loci; for scaling, group autoscaling was used; all files were normalized to input. (D) XGR gene enrichment analysis of canonical pathway analysis of EP300 and (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.

20756 | www.pnas.org/cgi/doi/10.1073/pnas.2004281117 Williams et al. Downloaded by guest on October 1, 2021 We next sought to establish the direct binding of EP300 and resulted in extensive transcriptional changes, 1,441 down- BRD4 to key profibrotic genes. We found strong evidence of regulated, 624 up-regulated (Dataset S2). In contrast, bromo- active transcription at the loci of ACTA2, COL1A1, and FN1 domain inhibition with CBP30 affected a much smaller set of (Fig. 2C), with H3K27ac and BRD4 highly enriched at the genes (225 down-regulated versus 48 up-regulated genes, transcriptional start site of all three genes. In contrast, EP300 Fig. 3 A and B). Gene enrichment (canonical) pathway analysis and H3K4me1 exhibited binding at more distal locations, in- (Dataset S3) indicated that CBP30 down-regulated genes were dicative of enhancer activity. Taken together these data confirm significantly enriched in ECM and proteoglycan-associated pro- the role of EP300 as an enhancer regulating the expression of tein pathways, integrin signaling, and syndecan-1 pathways ACTA2 and COL1A1. (Fig. 3C and Dataset S3), in common with JQ1. Pathways unique Several groups have shown that much of disease-associated to JQ1 included the Aurora A/B, PLK1, and FOXM1 pathways DNA sequence variation occurs in transcriptional regulatory (Fig. 3D), consistent with the potent antiproliferative properties regions defined by DNase hypersensitivity (29, 30). It has now of JQ1 (35, 36). been demonstrated using multiple GWAS datasets that a subset Focusing on the most significantly enriched pathways, ECM of (super) enhancers are especially important for genes associ- and ECM associated, we performed cluster analysis of the data ated with cell identity and genetic risk of disease (31, 32). Given from six donors following bromodomain inhibitor treatment. The that EP300 loading identifies regions of the genome bearing heatmap in Fig. 3E clearly identifies that each donor falls con- super enhancer architecture in all cell types, we sought to de- sistently within each treatment group within the dendrogram, termine whether EP300 binding was associated with the single with JQ1 treatment resulting in more potent inhibition than polynucleotide polymorphisms (SNPs) we have previously iden- CBP30. We confirmed several of the most significant hits from tified in Dupuytren’s disease GWAS datasets. We analyzed the transcriptomic data using siRNA-mediated knockdown of genomic location of EP300 at the 22 SNPs significantly associ- CREBBP, EP300, and BRD4, also including combined ated with Dupuytren’s disease (12). As shown in SI Appendix, CREBBP/EP300 knockdown to represent CBP30 targeting both Fig. S4 and summarized in SI Appendix, Table S2, EP300 is very bromodomains (SI Appendix, Fig. S6 A–L). closely associated with genomic regions of 8 of the 22 regions, Having established the profound transcriptional effect of independent of H3K27ac or H3K4me1 binding. bromodomain inhibition on profibrotic gene expression, we ex- Pathway analysis of annotated ChIP-Seq peaks demonstrated tended our analysis to include the effects on global protein ex- that the EP300 binding (Fig. 2D) sites are most significantly pression following bromodomain inhibition. Shotgun proteome enriched for PDGFRβ, integrin, and ECM interaction pathways. analysis identified a limited number of significantly regulated In contrast, BRD4 peaks displayed enrichment for a myriad of proteins (SI Appendix, Table S5), most notable being the three MEDICAL SCIENCES diverse signaling pathways. Notably, we observed few common collagen VI (collagen VIα1/2/3) isoforms in CBP30-treated cells pathways enriched across annotated BRD4 and EP300 peaks, (Dataset S4). We integrated the pathways generated by the suggesting a distinction of biological themes between the two transcriptomic dataset of CBP30-treated myofibroblasts with the epigenetic regulators, with the exception of PDGFRβ and FAK proteome analysis to systematically explore the broader molec- signaling pathways (Fig. 2E; Dataset S1), both of which are ular landscape directed by CBP30 in myofibroblasts. This en- highly associated with fibrosis in multiple organs (33). Next, we abled a comprehensive CBP30 pathway analysis, which was performed de novo motif analysis of the EP300-enriched loci found to be dominated by pathways associated with extracellular (Fig. 2F). Ranking the motifs by enrichment, the top five cor- matrix organization (Fig. 3F). Focused correlation analysis of responded to the known consensus binding sequences for the individual gene/protein expression levels showed collagen VIα1/ transcription factors FRA1, TEAD4, MF0010, RUNX1, and 2/3 and fibronectin were core targets driving the enriched ECM NFIX. Finally, motif analysis of the BRD4 dataset also identified pathways (Fig. 3G). To validate our findings, we performed FRA1 as the highest number of sites, with ELK1 (ETS), Western blotting using the bromodomain inhibitors on steady- CEBPG, YY1, and KLF6 also significantly enriched (Fig. 2G). state expression of key ECM components. We observed marked To investigate a putative role for the EP300-regulated tran- inhibition of collagen Iα1, collagen VIα1, EDA-FN, and α-SMA scription factors in profibrotic gene expression, we performed after 6 d of treatment with the bromodomain inhibitors siRNA-mediated gene knockdown of Fra1/FOSL1, RUNX1, and (Fig. 3H). TEAD4 in DD myofibroblasts and measured the expression of a panel of profibrotic genes (SI Appendix, Table S3). This dem- Collagen VI Drives a Profibrotic Phenotype in Myofibroblasts. Our onstrated that knockdown of FOSL1 significantly increased ex- multiomics profiling converged on collagen VI as a principal pression of several genes, including CXCL8, IL6, and CTGF (SI downstream target regulated by CBP/EP300. Immunostaining Appendix, Fig. S6 A–C) and inhibited CCL26, SFRP4, and CO- confirmed expression of the α3 chain of collagen VI throughout L6A3 expression (SI Appendix, Fig. S5 D–G). In contrast, the nodules of DD, colocalizing with α-SMA positive myofibro- TEAD4 knockdown inhibited mRNA expression of CXCL8 and blast foci (Fig. 4A). Similarly, collagen VI α3 was also associated CCL2 (SI Appendix, Fig. S5 H –J), while RUNX1 knockdown with α-SMA positive myofibroblasts localized to the fibrotic foci significantly down-regulated mRNA expression of genes in- of samples from patients with IPF (SI Appendix, Fig. S7). We volved in ECM remodeling, including fibronectin-1 (FN1) and also confirmed the presence of abundant levels of the full-length hyaluronan synthase 2 (HAS2)(SI Appendix, Fig. S5 K–M). To- α3 chain of collagen VI in tissue culture supernatants of freshly gether, these data support the role of EP300 in modulating di- isolated Dupuytren’s nodular cells and passaged MFs (Fig. 4B). verse profibrotic pathways and identified RUNX1, TEAD4, and As collagen VI was highly associated with the myofibroblast Fra1 as EP300-associated transcription factors regulating dis- populations in human fibrosis, we further explored its function. crete gene modules in myofibroblasts as summarized in Fig. 2H. We performed RNA-Seq on DD myofibroblasts treated with COL6A1/A2/A3 siRNA for either 4 or 6 d (Fig. 4C) (37). The Inhibition with CBP30 Identifies Collagen VI as a Downstream differentially expressed genes are displayed as a volcano plot Therapeutic Target of Multiple CBP30-Regulated Core ECM Genes in (Fig. 4D). This revealed a discrete number of differentially Fibrosis. After establishing that EP300 interacts directly with expressed genes, highlighting that more genes were targeted enhancer sites on key profibrotic genes, we evaluated changes in following longer exposure to COL6 depletion. These genes in- the transcriptomic profile following CBP/EP300 inhibition using cluded HGF, SDC1, CCL2, CCL7, ADAMT4/8, and HAS2 CBP30 in human DD myofibroblasts and compared with JQ1 (Dataset S5). These differentially expressed genes were associ- treatment as a bromodomain inhibitor (Fig. 3 A and B) (34). JQ1 ated with ECM remodeling, FOXM1 transcription network,

Williams et al. PNAS | August 25, 2020 | vol. 117 | no. 34 | 20757 Downloaded by guest on October 1, 2021 A JQ1 B log2FC > 1.5 JQ1 CBP30 LPAR1 150 MTHFD1L CBP30 FDR < 0.05 LRRC2 down-regulated 1248 193 XPOT 32 FST TRIB3 PYCR1 PNPLA2

100 HAS2 PHGDH GDF11 ADAMTS8 MAZ SPATA18 PEG10 SYT7 ADM2 HAS2 ELN −log10(FDR)

JQ1 −log10(FDR) KCNN4 CRABP2 50 up-regulated CBP30 ASPN CDS1 600 24 CPVL 24 0 0 5 10 15 20 25 30 35 −3 −2 −1 0 1 2 −6 −4 −2 0 2 4 C log2FoldChange log2FoldChange

ECM and ECM−associated proteins D ECM and ECM−associated proteins Secreted soluble factors ECM regulators and secreted factors ECM regulators and secreted factors Secreted soluble factors ECM glycoproteins and proteoglycans Genes encoding collagen proteins Proteoglycans ECM glycoproteins and proteoglycans PLK1 signaling events Alpha9 beta1 integrin signaling events Aurora B signaling BMP receptor signaling Regulators of the ECM remodellling Syndecan−1−mediated signaling events FOXM1 transcription factor network Structural ECM glycoproteins Syndecan−1−mediated signaling events Downstream signaling in nai ve CD8+ T cells Wnt signaling network Transcriptional targets of F ra1 and Fra2 Proteins affiliated structurally to ECM proteins Genes encoding collagen proteins Structural ECM glycoproteins Transcriptional targets of deltaNp63 iso forms Regulation of RAC1 activity Integrins in angiogenesis Beta1 integrin cell surface interactions Proteins affiliated structurally to ECM proteins IL27−mediated signaling events Regulators of remodeling of the ECM Alpha9 beta1 integrin signaling events Aurora A signaling Beta1 integrin cell sur face interactions CBP30 Wnt−mediated signal transduction Cytokines can induce activation of MMPs JQ1 Integrins in angiogenesis 03 6 9 12 0 5 10 15 20

Enrichment significance -log10(FDR) Enrichment significance -log10(FDR) E F

CRISPLD2 2 CBP30 RNA−Seq CBP30 Proteomics SEMA4G BMP6 IGFBP5 SCUBE3 GDF5 1 OR = 3.33 FST Integrin surface interactions SERPINB7 OR = 18.6 PAPPA ELN 0 OR = 4.34 COL15A1 ASPN Extracellular matrix organisation OR = 2.46 POSTN OR = 6.47 ESM OMD −1 ADAMTS8 OR = 9.08 FGF1 Elastin fibre formation OR = 5.59 VEGFC ADAMTS1 −2 SEMA3A IL34 OR = 5.03 INHB WISP1 Degradation of extracellular matrix OR = 4.34 INHBA LTBP1 OR = 13.0 PLXDC1 ABI3BP COL6A 3 MMP1 Collagen formation OR = 2.03 GDF10 OR = 10.8 FGF5 COL13A1 PRG4 OR = 9.08 C1QTNF3 Collagen degradation OR = 5.44 FN1 GDNF OR = 17.3 C1QTNF9B S100A5 TGM5 PGF Collagen chain trimerization OR = 4.83 INHBE S100A3 OR = 27.1

D1 D2 D3 D4 D5 D6 D1 D2 D3 D4 D5 D6 D1 D2 D3 D4 D5 D6 0 1 2 3 4 -log (FDR) DMSO JQ1 CBP30 GH Pearson’s correlation=0.56 peptides 4 days 6 days p< 0.002 25 50 Col6A1 1 75 Col1A1

CTNNB1 EDA-FN

0 -SMA

COL6A1

CBP to DMSO RNA (LogFC) Actin

COL6A3 COL6A2 - + - - - - - + - - - - vehicle −1 FN1 PYCR1 - - 0.1 0.5 - - - - 0.1 0.5 - - JQ1 uM LOX - - - - 0.5 2.5 - - - - 0.5 2.5 CBP30 uM CTHRC1 CRABP2

−2.5 −2.0 −1.5 −1.0 −0.5 0.0

CBP to DMSO Protein (LogFC)

Fig. 3. CBP30 treatment targets a limited subset of genes enriched in ECM-mediated regulated pathways. (A) Venn diagram showing the number of genes with significantly different expression (log2 FC > 1, FDR < 0.05) following JQ1 (0.5 μM) and CBP30 (2.5 μM) treatment. (B) Volcano plots of differentially expressed genes; data are generated for six donors. (C) Gene enrichment analysis of canonical pathways of significantly differentially expressed genes treated with CBP30 or (D) JQ1 treatment for 3 d. (E) Heatmap of genes identified within the ECM and ECM-associated proteins following JQ1 and CBP30 treatment from 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 significantly different expression (log2 FC > 1.5, FDR < 0.05). (G) Correlation between RNA-Seq and proteomic datasets. (H) Myofibroblasts were treated with either 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 α1and α-SMA were determined via Western blotting by SDS/PAGE on 4 to 20% Tris-glycine polyacrylamide gradient; data are representative of three donors.

20758 | www.pnas.org/cgi/doi/10.1073/pnas.2004281117 Williams et al. Downloaded by guest on October 1, 2021 ABαSMA (Cyan) Collagen VI α3 (red) Collagen I α1 (green) σSMA (Cyan) / Collagen VI (red) Collagen VI α3 ELISA

3000 DD ) /ml g 2000 a3(p h p l a

- 1000

IPF lVI O C 0 le u od MF n D D CD E FOXM1 transcription factor network FDR=8.0E−06

RNA-Seq following Col6A1/2/3 40 COL6A3 β siRNA depletion HIST1H2BK 2 integrin cell surface interactions FDR=7.4E−05 PLK1 signaling events FDR=9.7E−05 Integrin family cell surface interactions FDR=9.3E−05 4 days 6 days Integrin cell surface interactions FDR=6.4E−04

30 Androgen receptor activity FDR=6.4E−04 Aurora B signaling FDR=7.6E−04 COL6A1 Syndecan−1 signaling events FDR=2.3E−03 amb2 Integrin signaling FDR=6.0E−03 PLXNC1 MKI67 E2F transcription factor network FDR=6.1E−03 ATP6V1D β HAS2 2 integrin cell surface interactions FDR=6.1E−03 COL6A2 ECM and ECM−associated proteins ENPP1 FDR=7.5E−03 −log10(padj) JDP2 p73 transcription factor network FDR=8.7E−02 ECM glycoproteins, collagens and 11 26 101 SHROOM2 RRAGD FDR=1.8−E02 CELF2 proteoglycans ATR signaling pathway FDR=1.8−E02 FAIM2 α9 β2 integrin signaling events FDR=1.8−E02 enrichment LGI2 5 PALM2 HS3ST2 Integrins in angiogenesis FDR=1.9E−02 CCL7 4 CCL8 HRK ALK1 signaling events FDR=1.9E−02 JAKMIP2 BCL2A1 CNGA3 3 MILR1 uPAR−mediated signaling FDR=2.3E−02 SELL DMRTA1 01020 2 ESPNL Integrin Signaling Pathway FDR=2.6E−02

−4 −2 0 2 4 0123 45 enrichment -log10FDR

log2FoldChange MEDICAL SCIENCES

FGHIchemotaxis migration traction force microscopy

p < 0.0001 p = 0.0017 p < 0.0001 80 25000 1000 control RNAi Col6A1/2/3 RNAi

20000 800 60 max ls on (Pa) l i t e

15000 c 600

ac 40 r .of o

10000 400 eT n no. of cells g a

r 20 min 5000 200 Ave 0 0 0 control COL6A1/2/3 control COL6A1/2/3 control COL6A1/2/3 siRNA siRNA siRNA siRNA siRNA siRNA

JKLM HGF SHROOM2 SDC1 PCSK7

1.5 2.0 1.5 DH 1.5 H P H DH A PD G 1.5 APD GA

G 1.0 1.0 NA/ 1.0 A/ * NA/ mR 1.0 * 2 ** ** mR *** M * 0.5 FmRN 0.5 ** *** 0.5 SK7 G OO 0.5 C H SDC1 mRNA/GAP P l

HR **** el l r S re l 0.0 0.0 0.0 re

e 0.0 r il l 1 1 7 3 il l 1 5 6 7 3 il l 1 1 5 7 3 il l 1 o n o o n tr K5 A n tro P1 K K K A tr P K K6 K A n tr P1 K K5 K6 A3 n SK S l6 n SK S S l6 M S SK S l6 n M S S l6 o BMP C C CSK6 CSK o o BM C CS C C o on B C C C CS o B C C CS CSK7 c P P P P C c P P P P C c P P P P Co c P P P P Co

siRNA siRNA siRNA siRNA

Fig. 4. Collagen VI drives a profibrotic phenotype in myofibroblasts. (A) Confocal microscopy after immunostaining using anti-αSMA, anti-collagen VI α3, or collagen 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 50 μM for higher magnification slides.) (B) Freshly dissociated DD nodule cells or passaged (p2) DD myofibroblasts were cultured for 3 d and secreted collagen VI α3 levels were detected by ELISA. (C) Venn diagram showing the number of genes with significant differential expression (log2 FC > 1.5, FDR < 0.05) following either 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 were quantified by Hoechst 3342 fluorescent stain for 1 h using the Celigo Imaging Cytometer. (G) Scratch assay measuring migration of myofibroblasts following COL6A1/2/3 depletion; migrated cells were quantified by staining with calcein fluorescent stain for 1 h using the Celigo Imaging Cytometer in eight donors. P value was determined by paired sample t test in eight donors. (H) Effect of COL6A1/2/3 depletion as measured by traction force microscopy; cells were detached 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 ≤ 0.05 versus controls). Six-day siRNA-mediated PCSK (20 nM) depletion in DD myofibroblasts inhibits (J) HGF,(K) SHROOM2,(L) SDC1, and (M) PCSK7 gene expression as measured by Taqman PCR using the ΔΔCt method normalized to GAPDH; mean ± SEM from six donors. P value was determined by one-sample t test to normalized control (nontargeting oligo) sample of value of 1. *P ≤ 0.05, **P < 0.01, ***P < 0.001.

Williams et al. PNAS | August 25, 2020 | vol. 117 | no. 34 | 20759 Downloaded by guest on October 1, 2021 integrin signaling, Syndecan-1, and the androgen receptor ac- phenotype in DD. Screening of a panel of high-quality epigenetic tivity pathways (Fig. 4E and Dataset S6). This latter finding is of probes highlighted bromodomain inhibitors as potent regulators particular interest, given the male predominance of Dupuytren’s of profibrotic gene expression in DD myofibroblasts. While re- disease in younger age groups (38). Syndecan-1 has been shown lying on chemical probes alone has pitfalls due to potential off- to regulate ECM fiber alignment and the activities of several target effects, here we provide multiple lines of evidence of integrins, including αvβ3, αvβ5, and α2β1 which control stromal supporting a direct role of CBP/EP300 in profibrotic gene reg- cell migration (39). When comparing our EP300 ChIP-Seq ulation. First, two structurally distinct bromodomain inhibitors dataset, we found 45% of the differentially regulated genes CBP30 and I-CBP112, and the EP300 HAT inhibitor A485, (log2 fold change [FC] > 1) were also enriched for EP300 display similar activity profiles in suppressing gene expression, binding sites. Notably, we found siRNA depletion of COL6A3 with an IC50 of 1 μM, consistent with reported activity on the alone was sufficient to significantly suppress the expression of CBP-bromodomain (42, 43). Second, siRNA-mediated suppres- multiple target genes reported in the RNA-Seq (SI Appendix, sion of CBP/EP300 also confirmed a role of these epigenetic Fig. S8), suggesting this may be the dominant isoform driving the modifiers in the regulation of numerous genes identified in the profibrotic function of collagen VI. RNA-Seq dataset. Critically, using paired epigenetic and tran- We asked whether the downstream targets of collagen VI are scriptomic profiling, we provide a mechanistic insight and con- functional in human myofibroblasts. As COL6A1/2/3 depletion firm bromodomain inhibition directly blocks gene transcription markedly reduces expression of multiple chemokines, we con- through inhibiting the recruitment of CREBBP/EP300 to their firmed that COL6A1/2/3 siRNA treatment significantly inhibited binding sites, thereby reducing levels of H3K27ac at the ACTA2 CCL2 and CCL7 protein secretion by myofibroblasts (SI Ap- and COL1A1 loci. pendix, Fig. S9). We confirmed that COL6A1/2/3 depletion in H3K27ac and H3K4me1 occupancy are chromatin features myofibroblasts significantly reduced chemotaxis of the myeloid used to identify cell-type-specific enhancers (44, 45). Signifi- cell line THP-1 toward myofibroblast conditioned media, sug- cantly, our data show a high degree of genomic co-occupancy of gesting collagen VI may play an important role in promoting EP300 with H3K27Ac and H3K4me1, indicating that super en- immune cell recruitment in localized fibrosis (Fig. 4F). As the hancer sites are indeed regulating coordinated transcriptional COL6A1/2/3 targets HGF and SDC1 are known mediators of cell control of the highly enriched ECM pathways dominating the migration (39), we next sought to determine whether collagen VI EP300 pathway analysis in myofibroblasts. Interestingly, EP300 regulates myofibroblast migration using an in vitro wound heal- showed a distinct binding profile closely localizing with 8 of the ing assay. We found that myofibroblast migration was signifi- 22 most significant SNPs associated with Dupuytren’s disease cantly impaired following COL6A1/2/3 depletion (Fig. 4G). (12). Since both H3K27ac and EP300 (32, 46) demark the ar- Finally, as many genes regulated by collagen VI are associated chitecture of super enhancers in all cell types, this warrants with ECM interaction and integrin pathways, we investigated the further investigation using coactivators such as MED1 and other effect of COL6A1/2/3 depletion on contractility of myofibro- master transcription factors. Of particular note is the SFRP4 blasts on collagen-coated hydrogels using traction force micros- variant, as this was shown to be the most strongly associated copy. We found that COL6A1/2/3 depletion resulted in a variant (rs16879765) in our recent DD GWAS study (12) and we pronounced reduction of contraction (Fig. 4 H and I). Collec- have shown here that SFRP4 is regulated by FOSL1 depletion. tively, these data demonstrate the collagen VI pathway, regu- Furthermore, the study by Ng et al. (12) suggested that a subtle lated by EP300, orchestrates multiple important profibrotic imbalance of WNT signaling contributes to the fibrotic pheno- functions, including chemoattraction, migration, and contractil- type, allowing an increase in WNT3A signaling through the ity of human myofibroblasts. noncanonical pathway. Wnt/β-catenin signaling has been shown Proprotein convertases process and modulate proteins within to activate Fra1 expression, and Fra1 has been shown to play a the secretory compartment, at the plasma membrane and in role in epithelial–mesenchymal transition (EMT) activated by the extracellular space (40) and, along with the procollagen Wnt/β-catenin signal pathway (47). C-proteinase BMP-1, have recently been described as key regu- Our epigenetic profiling and motif analysis revealed several lators in the proteolytic cleavage of collagen VI α3 (41). Ana- putative EP300 coregulators of gene transcription, including lyzing our RNA-Seq datasets we identified all of the proprotein Fra1, RUNX1, and TEAD4. Functional analysis of these tran- convertase subtilisin/kexin (PCSKs) expressed by the DD myo- scription factors demonstrated discrete functional properties of fibroblasts and systematically depleted them to determine their each in myofibroblasts. Fra1 (FOSL1) has previously been importance in the regulation of the COL6A3-dependent gene reported as a negative regulator of AP-1 (48–50) and its deple- expression. We found that several genes identified from our gene tion enhanced bleomycin-induced lung fibrosis. Our data con- enrichment pathway analysis following the COL6A1/2/3 RNA- firm Fra-1 acts as a transcriptional repressor of CXCL8, IL6, and Seq analysis within the Syndecan-1 pathway (HGF and SDC1) CTGF in DD myofibroblasts but conversely acts to positively were also regulated by PCSK7 to a level similar to that observed regulate SPFR4, CCL26, and COL6A3 expression, suggesting a by COL6A3 depletion alone (Figs. 4 J–M). PCSK7 depletion also highly complex level of regulation of the transcriptional ma- regulated the chemokine CCL26 and its family member PCSK1 chinery at these genes. TEAD factors act as mediators of the inhibited ADAMTS8 (SI Appendix, Fig. S10). Hippo signaling pathway interacting with the YAP and WWTR1 (TAZ) transcriptional coactivators (51) and EP300 has recently Discussion been shown to activate YAP/TAZ (52). We observed TEAD4 Clinical trials in human fibrosis have repeatedly failed, probably depletion resulted in decreased expression of two key chemo- due to the poor predictive accuracy of targets identified using kines CCL2 and CXCL8 (IL-8), important recruiters of mono- murine models of fibrosis. DD represents an important platform cytes and neutrophils. Depletion of RUNX1 highlighted a role to study mechanisms of human fibrosis because of the ready of this transcription factor in the regulation of two key ECM availability of tissue from patients undergoing surgery as a regulators, fibronectin-1 (FN-1) and Hyaluronan Synthase 2 therapeutic intervention. We combined multiomic profiling with (HAS2). Together, these data describe a transcriptional network functional validation using these well-characterized patient downstream of EP300 that orchestrates diverse cellular profi- samples and identified collagen VI as a major regulator of the brotic functions in myofibroblasts associated with pathogenicity, myofibroblast phenotype. including enhanced matrix remodeling, development of a con- Our genetics-led network connectivity algorithm (Pi) proposed tractile phenotype, and immunomodulatory properties, thereby CREBBP/EP300 as key epigenetic regulators of the myofibroblast providing a framework by which these transcription factors link

20760 | www.pnas.org/cgi/doi/10.1073/pnas.2004281117 Williams et al. Downloaded by guest on October 1, 2021 EP300 gene regulation with the profibrotic phenotype typically explore the role of PCSK7 in collagen VI proteolysis and work is associated with myofibroblasts. underway to identify any PRO-C6 fragments in our model. It is Collagen VI is an interstitial collagen that functions as a noteworthy that PCSK7 missense variants identified in GWAS structural component of the matrix in addition to influencing cell studies are associated with liver fibrosis (69). More recently a adhesion. Levels are elevated in fibrotic conditions such as IPF PCSK7 variant has been shown to lead to increased intracellular (53), chronic liver disease (54), and chronic kidney disease (55). PCSK7 expression and secretion from hepatocytes, potentially Fragments from the C-terminal part of the α3 chain have been linking dyslipidemia with a tendency to more severe liver damage shown to have signaling effects, including profibrotic features in high risk individuals (70). Given that PCSK7 can be secreted and macrophage chemoattractant properties. One such fragment as well as localized on the plasma membrane, this raises the PRO-COL6/endotrophin is now commonly used as a biomarker potential for therapeutic development of monoclonal-based in cancer (56) and fibrotic conditions (57, 58) and has been able PCSK7 inhibitors. The translational potential of the wider fam- to distinguish individuals with IPF who have progressive disease ily of proprotein convertases has already been realized as there from those with more stable disease (59). We confirmed the are two FDA-approved drugs, evolocumab and alirocumab, presence of high levels of collagen VIα3 in supernatants from targeting the plasma protein PCSK9 for the treatment of familial freshly isolated Dupuytren’s nodules and in cultured myofibro- hypercholesterolemia and clinical atherosclerotic cardiovascular blasts. Our immunofluorescence studies highlighted that it is disease (71). Further work is needed to explore the tissue- highly expressed in both Dupuytren’s nodules and IPF patient specific nature of PCSK7 expression and a wider understand- samples, localizing closely with α-SMA positive myofibroblasts ing of its physiological role. regions. It is interesting to note that whereas collagen VIα3 was A limitation of this study is the absence of appropriate control widely distributed in DD nodules, in the IPF samples a more tissue. Dupuytren’s disease is restricted to certain fibers of the discrete cellular localization was observed when compared to palmar fascia. Previous studies used cells from the fascia in the collagen Iα1, suggesting discrete functions as dictated by the region of the carpal tunnel or the transverse carpal ligament local cellular–stromal architecture. Alternatively, it may reflect from affected or normal individuals or uninvolved transverse differing disease stages as IPF is usually diagnosed late. Fur- palmar fibers from patients with Dupuytren’s disease as controls thermore, we show that collagen VI regulates expression of the (72). However, this approach has significant limitations. The CCL2 CCL7 CCL26 chemokines , , and , in accordance with pre- palmar fascia over the carpal tunnel is rarely affected by vious data suggesting a potential role of endotrophin (ETP) in Dupuytren’s disease and the transverse carpal ligament is always chemotaxis (60), and we confirmed a role of collagen VI in unaffected; hence, it is possible that the constituent cells are myofibroblast-induced chemotaxis of THP1 cells. Previously (4), inherently different. Furthermore, normal palmar fascia is MEDICAL SCIENCES we identified macrophages and mast cells as the predominant sparsely populated by cells and hence to obtain adequate num- ’ source of TNF within Dupuytren s nodule cultures and demon- bers requires repeating passaging of cells in vitro (73). Further- strated that macrophage-derived TNF plays a key role in pro- ’ more, we and others have shown that at passage 5 the moting the fibrotic phenotype in Dupuytren s disease (4). phenotypes of myofibroblasts and normal human dermal fibro- Excessive production of collagen VI may contribute to the blasts tend to merge (74). chronicity of the disease by promoting the recruitment of In summary, by exploiting the availability of samples from monocytic cells to the site of this disease and probably plays a patients with DD and a combination of epigenetic and tran- similar role in other fibrotic disorders (61). A central function of scriptional profiling studies, we defined EP300 as an important myofibroblasts in both health and disease is the generation of regulator of human fibrosis that directs diverse cellular processes traction force (62), which plays a key role in remodeling the matrix in myofibroblasts. There are supporting data showing that and also modulates the activities of the embedded stromal cells (63, pharmacological inhibition of EP300 is effective in murine 64). Importantly for disease management, we also demonstrated models of pulmonary fibrosis (75, 76). However, the many tar- that collagen VI regulates myofibroblast contraction. gets of epigenetic regulators suggest their long-term global in- Mutations in collagen VI genes is associated with Bethlem hibition may result in off-target effects. Here we reveal that myopathy (BM) and Ullrich congenital muscular dystrophy collagen VI is a downstream target of EP300 and is widely (UCMD) (65). A mouse model in which the Col6a1 gene was expressed in DD nodules and IPF lung tissue and mediates an inactivated has identified key functions of Col6a1, including autophagic dysfunction, skeletal, heart, and tendon defects (66). COL6A3 protein deficiency in mice leads to muscle and tendon defects similar to those seen in human collagen VI congenital muscular dystrophy (67). Therefore, directly targeting collagen VI is likely to be deleterious. The C5 region of the α3 chain of collagen VI has been shown to undergo proteolysis in a tissue- specific manner by the proprotein convertase furin/PCSK3 and BMP1 (68), generating diverse fragment sizes depending on the tissue source (41). However, free endotrophin was shown to be scarce under physiological conditions, suggesting that the proteolysis of collagen VI is tightly regulated. Hence targeting the collagen VI proteolysis cleavage machinery could represent a more tractable approach. Analysis of our RNA-Seq datasets revealed that PCSK3 is not expressed in the DD myofibroblasts, but we were able to detect mRNA expression of PCSK1, PCSK5, Fig. 5. Schematic illustrating the epigenetic control by EP300 of the profi- PCSK6, and PCSK7, with PCSK7 showing the highest basal level brotic phenotype of myofibroblasts in Dupuytren’s disease. Persistent acti- of gene expression. Our siRNA studies identified PCSK7 as a vation of EP300 acetylates histones and transcription factors to promote α extracellular matrix production and myofibroblast contractility. Collagen VI, potential protease regulating collagen VI 3 cleavage. Depletion a key target of EP300, plays a dominant role in regulating contraction. of PCSK7 suppressed gene expression of a subset of the genes Proteolytic cleavage by PCSK7 generates small bioactive collagen VI frag- from the Syndecan-1 signaling pathway, a key pathway regulating ments which control recruitment of immune cells by chemokine production, stromal cell migration and ECM fiber alignment (39); this was thereby perpetuating the cycle of chronic inflammation and fibrosis by the also regulated by COL6A3 depletion. Our future work will secretion of cytokines such as TNF.

Williams et al. PNAS | August 25, 2020 | vol. 117 | no. 34 | 20761 Downloaded by guest on October 1, 2021 important role in regulating ECM production, chemotaxis, and using RNA-Seq and hits confirmed using siRNA of CREBBP/EP300 depletion contractility as shown in Fig. 5. Crucially, our data with EP300 studies. The effects of CBP30 on global protein expression in DD myofibroblasts regulation identify collagen VI and PCSK7 as previously un- was determined using shotgun proteomic profiling. The role of collagen VI in identified therapeutic targets. The important translational po- controlling profibrotic gene expression was confirmed by siRNA-mediated tential of our findings is underpinned by using primary human COL6A1/2/3 depletion followed by RNA-Seq; subsequent roles in contraction, samples exclusively, including Dupuytren’s disease and IPF, migration, and wound healing were confirmed using traction force microscopy, chemotaxis, and scratch assays, respectively. The methodology is described diseases where therapeutic options are limited and patient out- in detail in SI Appendix. comes are currently suboptimal. Data Availability. RNA-Seq and ChIP-Seq datasets are deposited within the Methods NCBI SRA database under accession numbers PRJNA625874, PRJNA624331, In brief, screening the effects of a panel of epigenetic inhibitors upon ACTA2, and PRJNA624119, respectively. COL1A1, COL3A1 and TGFB1 gene expression was performed using primary human myofibroblasts isolated from DD patients by Taqman PCR. Inhibition ACKNOWLEDGMENTS. We thank all the research nurses at Queen Victoria of acetylation at the promotors of ACTA2 and COL1A1 by the EP300 probe Hospital, Southampton General Hospital, Broomfield Hospital, Royal Derby SGC-CBP30 (CBP30) was shown using ChIP PCR. Inhibition of myofibroblast Hospital, and St. John’s Hospital and Dr. Steve Nathan for kindly supplying contraction was assessed using traction force microscopy (23). Evidence of the IPF samples. The work was funded by European Union ULTRA-Innovative direct recruitment of EP300 to a profibrotic gene landscape was shown using Medicines Initiative Grant 115766 and Oxford-Celgene Research Fellowship ChIP-Seq. Transcriptional profiling of CBP30 in myofibroblasts was performed Grant AZR00610.

1. S. L. Friedman, D. Sheppard, J. S. Duffield, S. Violette, Therapy for fibrotic diseases: 28. Z. Najafova et al., BRD4 localization to lineage-specific enhancers is associated with a Nearing the starting line. Sci. Transl. Med. 5, 167sr1 (2013). distinct transcription factor repertoire. Nucleic Acids Res. 45, 127–141 (2017). 2. J. Nanchahal, B. Hinz, Strategies to overcome the hurdles to treat fibrosis, a major 29. B. Vernot et al., Personal and population genomics of human regulatory variation. unmet clinical need. Proc. Natl. Acad. Sci. U.S.A. 113, 7291–7293 (2016). Genome Res. 22, 1689–1697 (2012). 3. L. S. Verjee et al., Myofibroblast distribution in Dupuytren’s cords: Correlation with 30. M. T. Maurano et al., Systematic localization of common disease-associated variation digital contracture. J. Hand Surg. Am. 34, 1785–1794 (2009). in regulatory DNA. Science 337, 1190–1195 (2012). 4. D. Izadi et al., Identification of TNFR2 and IL-33 as therapeutic targets in localized 31. G. Vahedi et al., Super-enhancers delineate disease-associated regulatory nodes in fibrosis. Sci. Adv. 5, eaay0370 (2019). T cells. Nature 520, 558–562 (2015). 5. B. Hinz et al., The myofibroblast: One function, multiple origins. Am. J. Pathol. 170, 32. D. Hnisz et al., Super-enhancers in the control of cell identity and disease. Cell 155, 1807–1816 (2007). 934–947 (2013). 6. J. J. Tomasek, G. Gabbiani, B. Hinz, C. Chaponnier, R. A. Brown, Myofibroblasts and 33. X. K. Zhao et al., Focal adhesion kinase regulates fibroblast migration via integrin mechano-regulation of connective tissue remodelling. Nat. Rev. Mol. Cell Biol. 3, beta-1 and plays a central role in fibrosis. Sci. Rep. 6, 19276 (2016). 349–363 (2002). 34. L. Williams, Transcriptional profiling of JQ1 and SGC-CBP30 treated human myofi- 7. M. W. Parker et al., Fibrotic extracellular matrix activates a profibrotic positive broblasts. NCBI, SRA. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA625874. Depos- feedback loop. J. Clin. Invest. 124, 1622–1635 (2014). ited 17 April 2020. 8. F. Liu et al., Feedback amplification of fibrosis through matrix stiffening and COX-2 35. D. R. Wonsey, M. T. Follettie, Loss of the forkhead transcription factor FoxM1 causes suppression. J. Cell Biol. 190, 693–706 (2010). centrosome amplification and mitotic catastrophe. Cancer Res. 65, 5181–5189 (2005). 9. I. A. Darby, N. Zakuan, F. Billet, A. Desmoulière, The myofibroblast, a key cell in 36. P. D. Andrews, E. Knatko, W. J. Moore, J. R. Swedlow, Mitotic mechanics: The auroras normal and pathological tissue repair. Cell. Mol. Life Sci. 73, 1145–1157 (2016). come into view. Curr. Opin. Cell Biol. 15, 672–683 (2003). 10. T. A. Wynn, T. R. Ramalingam, Mechanisms of fibrosis: Therapeutic translation for 37. L. Williams, Transcriptional profiling of COL6A1/2/3 depleted human myofibroblasts. fibrotic disease. Nat. Med. 18, 1028–1040 (2012). NCBI, SRA. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA624331. Deposited 10 April 11. S. Larsen et al., Genetic and environmental influences in Dupuytren’s disease: A study 2020. of 30,330 Danish twin pairs. J. Hand Surg. Eur. Vol. 40, 171–176 (2015). 38. R. Lanting, D. C. Broekstra, P. M. Werker, E. R. van den Heuvel, A systematic review 12. M. Ng et al., A genome-wide association study of Dupuytren disease reveals 17 ad- and meta-analysis on the prevalence of Dupuytren disease in the general population ditional variants implicated in fibrosis. Am. J. Hum. Genet. 101, 417–427 (2017). of Western countries. Plast. Reconstr. Surg. 133, 593–603 (2014). 13. N. S. Godtfredsen, H. Lucht, E. Prescott, T. I. Sørensen, M. Grønbaek, A prospective 39. N. Yang, A. Friedl, Syndecan-1-induced ECM fiber alignment requires integrin αvβ3 study linked both alcohol and tobacco to Dupuytren’ s disease. J. Clin. Epidemiol. 57, and syndecan-1 ectodomain and heparan sulfate chains. PLoS One 11, e0150132 858–863 (2004). (2016). 14. A. Descatha, P. Jauffret, J. F. Chastang, Y. Roquelaure, A. Leclerc, Should we consider 40. N. G. Seidah, A. Prat, The biology and therapeutic targeting of the proprotein con- Dupuytren’s contracture as work-related? A review and meta-analysis of an old de- vertases. Nat. Rev. Drug Discov. 11, 367–383 (2012). bate. BMC Musculoskelet. Disord. 12, 96 (2011). 41. S. E. Heumüller et al., C-terminal proteolysis of the collagen VI α3 chain by BMP-1 and 15. S. O’Reilly, Epigenetics in fibrosis. Mol. Aspects Med. 54,89–102 (2017). proprotein convertase(s) releases endotrophin in fragments of different sizes. J. Biol. 16. A. K. Ghosh, R. Rai, P. Flevaris, D. E. Vaughan, Epigenetics in reactive and reparative Chem. 294, 13769–13780 (2019). cardiac fibrogenesis: The promise of epigenetic therapy. J. Cell. Physiol. 232, 42. D. A. Hay et al., Discovery and optimization of small-molecule ligands for the CBP/ 1941–1956 (2017). p300 bromodomains. J. Am. Chem. Soc. 136, 9308–9319 (2014). 17. V. Massey, J. Cabezas, R. Bataller, Epigenetics in liver fibrosis. Semin. Liver Dis. 37, 43. A. R. Conery et al., Bromodomain inhibition of the transcriptional coactivators CBP/ 219–230 (2017). EP300 as a therapeutic strategy to target the IRF4 network in multiple myeloma. eLife 18. S. Aslani, S. Sobhani, F. Gharibdoost, A. Jamshidi, M. Mahmoudi, Epigenetics and 5, e10483 (2016). pathogenesis of systemic sclerosis; the ins and outs. Hum. Immunol. 79, 178–187 44. N. Dogan et al., Occupancy by key transcription factors is a more accurate predictor of (2018). enhancer activity than histone modifications or chromatin accessibility. Epigenetics 19. J. H. W. Distler et al., Shared and distinct mechanisms of fibrosis. Nat. Rev. Rheumatol. Chromatin 8, 16 (2015). 15, 705–730 (2019). 45. A. S. Nord et al., Rapid and pervasive changes in genome-wide enhancer usage during 20. E. T. Manning, T. Ikehara, T. Ito, J. T. Kadonaga, W. L. Kraus, p300 forms a stable, mammalian development. Cell 155, 1521–1531 (2013). template-committed complex with chromatin: Role for the bromodomain. Mol. Cell. 46. W. A. Whyte et al., Master transcription factors and mediator establish super- Biol. 21, 3876–3887 (2001). enhancers at key cell identity genes. Cell 153, 307–319 (2013). 21. H. Fang et al.; ULTRA-DD Consortium, A genetics-led approach defines the drug 47. L. Zhang, H. Liu, X. Mu, J. Cui, Z. Peng, Dysregulation of Fra1 expression by Wnt/ target landscape of 30 immune-related traits. Nat. Genet. 51, 1082–1091 (2019). β-catenin signalling promotes glioma aggressiveness through epithelial-mesenchymal 22. A. Ebrahimi et al., Bromodomain inhibition of the coactivators CBP/EP300 facilitate transition. Biosci. Rep. 37, BSR20160643 (2017). cellular reprogramming. Nat. Chem. Biol. 15, 519–528 (2019). 48. H. Morishita et al., Fra-1 negatively regulates lipopolysaccharide-mediated inflam- 23. T. B. Layton et al., Single cell force profiling of human myofibroblasts reveals a bio- matory responses. Int. Immunol. 21, 457–465 (2009). physical spectrum of cell states. Biol. Open 9, bio049809 (2020). 49. S. Rajasekaran, N. M. Reddy, W. Zhang, S. P. Reddy, Expression profiling of genes 24. M. P. Creyghton et al., Histone H3K27ac separates active from poised enhancers and regulated by Fra-1/AP-1 transcription factor during bleomycin-induced pulmonary predicts developmental state. Proc. Natl. Acad. Sci. U.S.A. 107, 21931–21936 (2010). fibrosis. BMC Genomics 14, 381 (2013). 25. N. D. Heintzman et al., Histone modifications at human enhancers reflect global 50. S. Rajasekaran, M. Vaz, S. P. Reddy, Fra-1/AP-1 transcription factor negatively regu- cell-type-specific gene expression. Nature 459, 108–112 (2009). lates pulmonary fibrosis in vivo. PLoS One 7, e41611 (2012). 26. A. Visel et al., ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 51. A. V. Pobbati, S. W. Chan, I. Lee, H. Song, W. Hong, Structural and functional similarity 457, 854–858 (2009). between the Vgll1-TEAD and the YAP-TEAD complexes. Structure 20, 1135–1140 27. L. Williams, Identifying collagen VI as a target of fibrotic diseases regulated by (2012). CREBBP/EP300. NCBI, SRA. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA624119. 52. N. R. Zemke, D. Gou, A. J. Berk, Dedifferentiation by adenovirus E1A due to inacti- Deposited 9 April 2020. vation of Hippo pathway effectors YAP and TAZ. Genes Dev. 33, 828–843 (2019).

20762 | www.pnas.org/cgi/doi/10.1073/pnas.2004281117 Williams et al. Downloaded by guest on October 1, 2021 53. J. Herrera et al., Registration of the extracellular matrix components constituting the 67. T. C. Pan et al., COL6A3 protein deficiency in mice leads to muscle and tendon defects fibroblastic focus in idiopathic pulmonary fibrosis. JCI Insight 4, e125185 (2019). similar to human collagen VI congenital muscular dystrophy. J. Biol. Chem. 288, 54. C. Lee et al., COL6A3-derived endotrophin links reciprocal interactions among hepatic 14320–14331 (2013). – cells in the pathology of chronic liver disease. J. Pathol. 247,99 109 (2019). 68. T. Aigner, L. Hambach, S. Söder, U. Schlötzer-Schrehardt, E. Pöschl, The C5 domain of 55. A. G. Nerlich, E. D. Schleicher, I. Wiest, U. Specks, R. Timpl, Immunohistochemical Col6A3 is cleaved off from the Col6 fibrils immediately after secretion. Biochem. localization of collagen VI in diabetic glomeruli. Kidney Int. 45, 1648–1656 (1994). Biophys. Res. Commun. 290, 743–748 (2002). 56. N. Willumsen, C. Bager, M. A. Karsdal, Matrix metalloprotease generated fragments 69. S. Pelucchi et al., Proprotein convertase 7 rs236918 associated with liver fibrosis in of type VI collagen have serum biomarker potential in cancer–A proof of concept Italian patients with HFE-related hemochromatosis. J. Gastroenterol. Hepatol. 31, study. Transl. Oncol. 12, 693–698 (2019). – 57. P. Juhl et al., Serum biomarkers of collagen turnover as potential diagnostic tools in 1342 1348 (2016). diffuse systemic sclerosis: A cross-sectional study. PLoS One 13, e0207324 (2018). 70. P. Dongiovanni et al., PCSK7 gene variation bridges atherogenic dyslipidemia with – 58. J. M. Sand et al., MMP mediated degradation of type IV collagen alpha 1 and alpha 3 hepatic inflammation in NAFLD patients. J. Lipid Res. 60, 1144 1153 (2019). chains reflects basement membrane remodeling in experimental and clinical 71. B. A. Warden, S. Fazio, M. D. Shapiro, The PCSK9 revolution: Current status, contro- fibrosis–Validation of two novel biomarker assays. PLoS One 8, e84934 (2013). versies, and future directions. Trends Cardiovasc. Med. 30, 179–185 (2019). 59. L. A. Organ et al., Biomarkers of collagen synthesis predict progression in the PROFILE 72. L. Satish et al., Fibroblasts from phenotypically normal palmar fascia exhibit molecular idiopathic pulmonary fibrosis cohort. Respir. Res. 20, 148 (2019). profiles highly similar to fibroblasts from active disease in Dupuytren’s Contracture. 60. K. Sun et al., Endotrophin triggers adipose tissue fibrosis and metabolic dysfunction. BMC Med. Genomics 5, 15 (2012). Nat. Commun. 5, 3485 (2014). 73. M. A. Bisson, D. A. McGrouther, V. Mudera, A. O. Grobbelaar, The different charac- – 61. G. Wick et al., The immunology of fibrosis. Annu. Rev. Immunol. 31, 107 135 (2013). teristics of Dupuytren’s disease fibroblasts derived from either nodule or cord: Ex- 62. H. Colin-York et al., Cytoskeletal control of antigen-dependent T cell activation. Cell pression of alpha-smooth muscle actin and the response to stimulation by TGF-beta1. Rep. 26, 3369–3379.e5 (2019). J. Hand Surg. [Br.] 28, 351–356 (2003). 63. F. Liu et al., Mechanosignaling through YAP and TAZ drives fibroblast activation and 74. L. S. Verjee, K. Midwood, D. Davidson, M. Eastwood, J. Nanchahal, Post- fibrosis. Am. J. Physiol. Lung Cell. Mol. Physiol. 308, L344–L357 (2015). transcriptional regulation of alpha-smooth muscle actin determines the contractile 64. Z. Sun et al., Kank2 activates talin, reduces force transduction across integrins and ’ – induces central adhesion formation. Nat. Cell Biol. 18, 941–953 (2016). phenotype of Dupuytren s nodular cells. J. Cell. Physiol. 224, 681 690 (2010). 65. A. K. Lampe, K. M. Bushby, Collagen VI related muscle disorders. J. Med. Genet. 42, 75. K. Rubio et al., Inactivation of nuclear histone deacetylases by EP300 disrupts 673–685 (2005). the MiCEE complex in idiopathic pulmonary fibrosis. Nat. Commun. 10, 2229 66. S. Lettmann et al., Col6a1 null mice as a model to study skin phenotypes in patients (2019). with collagen VI related myopathies: Expression of classical and novel collagen VI 76. J. Tao et al., Inhibition of EP300 and DDR1 synergistically alleviates pulmonary fibrosis variants during wound healing. PLoS One 9, e105686 (2014). in vitro and in vivo. Biomed. Pharmacother. 106, 1727–1733 (2018). MEDICAL SCIENCES

Williams et al. PNAS | August 25, 2020 | vol. 117 | no. 34 | 20763 Downloaded by guest on October 1, 2021