Abstract:
An extreme chronic wound tissue microenvironment causes epigenetic gene silencing. An unbiased whole-genome
methylome was studied in the wound-edge tissue of patients with chronic wounds. A total of 4,689 differentially methylated
regions (DMRs) were identified in chronic wound-edge skin compared with unwounded human skin. Hypermethylation was
more frequently observed (3,661 DMRs) in the chronic wound-edge tissue compared with hypomethylation (1,028 DMRs).
Twenty-six hypermethylated DMRs were involved in epithelial-mesenchymal transition (EMT). Bisulfite sequencing validated
hypermethylation of a predicted specific upstream regulator TP53. RNA-Seq analysis was performed to qualify findings
from methylome analysis. Analysis of the downregulated genes identified the TP53 signaling pathway as being significantly
silenced. Direct comparison of hypermethylation and downregulated genes identified 4 genes, ADAM17, NOTCH, TWIST1,
and SMURF1, that functionally represent the EMT pathway. Single-cell RNA-Seq studies revealed that these effects on
gene expression were limited to the keratinocyte cell compartment. Experimental murine studies established that tissue
ischemia potently induces wound-edge gene methylation and that 5′-azacytidine, inhibitor of methylation, improved wound
closure. To specifically address the significance of TP53 methylation, keratinocyte-specific editing of TP53 methylation at the
wound edge was achieved by a tissue nanotransfection-based CRISPR/dCas9 approach. This work identified that reversal of
methylation-dependent keratinocyte gene silencing represents a productive therapeutic strategy to improve wound closure.
