Single-cell epigenomic dysregulation of Systemic Sclerosis fibroblasts via CREB1/EGR1 axis in self-assembled human skin equivalents

Abstract

Systemic sclerosis (SSc) is an autoimmune disease characterized by skin fibrosis, internal organ involvement and vascular dropout. We previously developed and phenotypically characterized an in vitro 3D skin-like tissue model of SSc, and now analyze the transcriptomic (scRNA-seq) and epigenetic (scATAC-seq) characteristics of this model at single-cell resolution. SSc 3D skin-like tissues were fabricated using autologous fibroblasts, macrophages, and plasma from SSc patients or healthy control (HC) donors. SSc tissues displayed increased dermal thickness and contractility, as well as increased alpha-SMA staining. Single-cell transcriptomic and epigenomic analyses identified keratinocytes, macrophages, and five populations of fibroblasts (labeled FB1 - 5). Notably, FB1 APOE-expressing fibroblasts were 12-fold enriched in SSc tissues and were characterized by high EGR1 motif accessibility. Pseudotime analysis suggests that FB1 fibroblasts differentiate from a TGF-beta-responsive fibroblast population and ligand-receptor analysis indicates that the FB1 fibroblasts are active in macrophage crosstalk via soluble ligands including FGF2 and APP. These findings provide characterization of the 3D skin-like model at single cell resolution and establish that it recapitulates subsets of fibroblasts and macrophage phenotypes observed in skin biopsies.

RUNX1 is Expressed in a Subpopulation of Dermal Fibroblasts and Higher RUNX1 Levels are Associated with the Severity of Systemic Sclerosis

“…We have also demonstrated that RUNX1 is hypomethylated in the SSc-derived fibroblast genome, indicating that the increased RUNX1 expression observed in SSc patients is likely due to dysregulation at the epigenetic level. Using a single-cell multiomic analysis, in a separate study, we showed that RUNX1 is consistently enriched in a subpopulation of fibroblasts in an SSc-derived self-assembled skin-equivalent (saSE) tissue model at levels similar to those observed in SSc skin biopsies 45 . In an analysis of single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq), a fibroblast subpopulation was identified by elevated TF motifs for RUNX1 and RUNX2 , together with SMAD3 , SMAD5 , and NFKB1 .…”