Theleadingcauseofdeathinsystemicsclerosis(SSc)isthefibroticcomplication,interstitiallung disease (ILD), but skin fibrosis is a leading cause for morbidity resulting in disfiguring, painful and itchy skin,andjointcontractures.Theemergenceandexpansionofmyofibroblastsasthemainprofibroticcell underlies the pathogenesis of both SSc skin and ILD. Although much is understood about myofibroblast biology in vitro and in murine models of fibrosis, the cell source and molecular signals driving myofibroblast differentiation in SSc remain obscure. We have recently identified SSc dermal myofibroblasts, SFRP2-?expressing myofibroblast progenitors and the associated altered transcriptome by single cell RNA-?sequencing (scRNA-?seq). In order to understand the underlying drivers of myofibroblast differentiation we will examine the epigenetic and transcriptional control of genes regulatedinSScskinmyofibroblasts.WehaveanalyzedourscRNA-?seqtranscriptomedatausingSCENIC, a computational method developed for detecting transcription factor (TF)-?associated regulatory networks (regulons). In scRNA-?seq analysis of SSc myofibroblasts, we saw upregulated regulons associated with the TFs: FOXP1, NPDC1, IRF7, ZEB1, HSF1 and FOSL2. In the first aim of the R61 phase we will test the role of predicted TFs in regulating myofibroblast transcriptome in dermal fibroblasts. PrimaryfibroblastculturesfromSScandhealthyskinbiopsieswillbetransfectedwithdCas9-?CRISPRaor dCas9-?CRISPRiandsingleguideRNAs(sgRNAs)targetingFOXP1,NPDC1,IRF7,ZEB1,HSF1orFOSL2,or SMAD2 or SMAD3 as positive controls for the canonical TGF? regulated pathway. Cells will then be analyzed by scRNA-?seq, cDNA libraries prepared, sequenced, and analyzed for alterations in gene expression (PERTURB-?seq). Gene expression by TF-?perturbed cells will be compared to unperturbed cells of the same SFRP2+ fibroblast phenotype. Recent technological advances have also provided methodology for single cell Assay for Transposase Accessible Chromatin by Sequencing (scATAC-?seq) permitting assessment of epigenetic changes in DNA that reflect regions of TF binding to DNA. In the second aim of the R61 phase we will analyze open chromatin in cells from skin biopsies from healthy and SSc patients by scATAC-?seq. Peaks of open chromatin in myofibroblasts will be identified and comparedtoopenchromatininmyofibroblastprogenitor,SFRP2-?expressingfibroblasts.Wewillfocuson analyzing chromatin remodeling of genes, such as SFRP4 and WIF1 that show altered expression by SSc myofibroblasts. We will correlate predicted TF binding sites in open chromatin with TF regulation of myofibroblast associated genes identified in aim 1. In the R33 phase we will confirm the results in the R61 phase by overexpressing TFs shown to regulate myofibroblast genes, singly or in combination, and analyzingtheeffectsonchromatinremodeling,andonmyofibroblastdifferentiation.
Systemicsclerosisisasystemicautoimmunediseasethatleadstoscarringoftheskinandinternal organs.Skinscarringisdrivenbyspecializedfibroblastsknownasmyofibroblasts,buttheoriginsof thesecellsandthemolecularpathwaysactivatingthemtodifferentiateintomyofibroblastsare unknown.Wewillexaminetheroleoftranscriptionfactorspredictedtoregulatealteredgene expressioninmyofibroblastsbyactivatingorinhibitingtheirexpressionusing,respectively, CRISPRaandCRISPRitechnology.Further,wewillinvestigatechromatinremodelingin myofibroblastsbysinglecellAssay for Transposase Accessible Chromatin (ATAC)-sequencing. Through these complementary methodologies we will identify the key transcriptional regulation of genes leading to myofibroblast differentiation.
