A scATAC-seq atlas of stasis zone in rat skin burn injury wound process

Abstract

Burn injuries often leave behind a "stasis zone", a region of tissue critically important for determining both the severity of the injury and the potential for recovery. To understand the intricate cellular and epigenetic changes occurring within this critical zone, we utilized single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) to profile over 31,500 cells from both healthy rat skin and the stasis zone at nine different time points after a burn injury. This comprehensive approach revealed 26 distinct cell types and the dynamic shifts in the proportions of these cell types over time. We observed distinct gene activation patterns in different cell types at various stages post-burn, highlighting key players in immune activation, tissue regeneration, and blood vessel repair. Importantly, our analysis uncovered the regulatory networks governing these genes, offering valuable insights into the intricate mechanisms orchestrating burn wound healing. This comprehensive cellular and molecular atlas of the stasis zone provides a powerful resource for developing targeted therapies aimed at improving burn injury recovery and minimizing long-term consequences.

Keywords: chromatin accessibility; rat burn injuries; stasis zone; transcription factor; wound healing.

FIGURE 1

Identification of scATAC data cell types in burn stasis area of rats (A) Images of rat burn samples at each time point. (B) Experimental workflow: Rat skin samples were collected at nine time points (ctrl, 0h, 12h, D1, D3, D7, D11, D15, D19) for scATAC-seq profiling. (C) Diagram of skin burn pattern, including the structure and state of the necrotic area, the stasis area, and the normal skin. (D) The analysis workflow for scATAC-seq profiles. (E) UMAP visualization of 26 cell types identified by gene score in scATAC-seq data. aHFSC, active hair follicle stem cell; bENDO, burn specific endothelial; bHFSC, burn specific hair follicle stem cells; DP, dermal papilla; DS, dermal sheath; fFB, fascial fibroblast; HS, hair shaft; IFEB, interfollicular epidermis basal; IFEG, interfollicular epidermis granular; IFESP, interfollicular epidermis spinous; ILC, innate lymphoid cell; INF, infundibular; IRS, Inner root sheath; lENDO, lymphatic endothelial; LGS, langerhans; MACRO, macrophage; MELA, melanocyte; MUSCLE, muscle; ORS, outter root sheath; PCT, pericyte; qHFSC, quiescent hair follicle stem cell; rFB, reticular fibroblast; SG, sebaceous gland; TAC, transit amplifying cell; vENDO, vascular endothelium. (F) Cell type-specific genome browser views of scATAC-seq signal for the dynamically identified peaks. (G) Subpopulations of skin fibroblasts, marker genes, and their proportions over time. (H) UMAP shows cell clusters from two datasets. (I) Integration of scRNA data from rat skin with radiation injury by Tao Y et al. and distribution of cell types in the two datasets. (J) Correlation analysis between cell types in this study and public datasets, with red indicating high correlation and blue indicating low correlation. (K) FeaturePlot of marker gene expression for bHFSC in the integrated dataset, with darker colors indicating higher expression.

FIGURE 2

Temporal and pseudotime analyses of gene regulation, cell type dynamics, and the integration of single-cell RNA data. (A) Cell type-specific peaks. (B) Cell type-specific genes. (C) Cell type-specific motifs. (D) Enriched Gene Ontology (GO) Terms for aHFSC, bHFSC, bFB and bENDO. (E) Standardized fuzzy clustering of scATAC signals for bHFSC and IFEB, with blue lines representing individual loci and orange lines representing cluster center values. (F) Genomic browser view of dynamic peaks of bHFSC and IFEB signals. (G) GO enrichment of clusters for bHFSC and IFEB, with dot size representing the proportion of genes in the GO term and color representing the -Log10 (P-value). (H) Motif enrichment of clusters for bHFSC and IFEB, with dot size representing the fold. enrichment and color representing the -Log10 (P-value). (I) UMAP of cell types constructed in pseudotime. (J) Pseudotime analysis of post-injury epithelial lineage of HFSC and normal hair follicle differentiation using Monocle2. (K) Peaks associated with burn-specific epithelial temporal changes will be annotated to genes, with red coloration indicating high scores and blue coloration indicating low scores.