Genome-wide DNA hypermethylation opposes healing in patients with chronic wounds by impairing epithelial-mesenchymal transition
Kanhaiya Singh, … , Sashwati Roy, Chandan K. Sen
Kanhaiya Singh, … , Sashwati Roy, Chandan K. Sen
Published July 12, 2022
Citation Information: J Clin Invest. 2022;132(17):e157279. https://doi.org/10.1172/JCI157279.
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Research Article Dermatology

Genome-wide DNA hypermethylation opposes healing in patients with chronic wounds by impairing epithelial-mesenchymal transition

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.

Authors

Kanhaiya Singh, Yashika Rustagi, Ahmed S. Abouhashem, Saba Tabasum, Priyanka Verma, Edward Hernandez, Durba Pal, Dolly K. Khona, Sujit K. Mohanty, Manishekhar Kumar, Rajneesh Srivastava, Poornachander R. Guda, Sumit S. Verma, Sanskruti Mahajan, Jackson A. Killian, Logan A. Walker, Subhadip Ghatak, Shomita S. Mathew-Steiner, Kristen E. Wanczyk, Sheng Liu, Jun Wan, Pearlly Yan, Ralf Bundschuh, Savita Khanna, Gayle M. Gordillo, Michael P. Murphy, Sashwati Roy, Chandan K. Sen

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Figure 6

Correction of P53 hypermethylation improves ischemic wound closure.

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Correction of P53 hypermethylation improves ischemic wound closure. (A) ...
(A) Topical delivery of 5-azacytidine to murine ischemic bipedicle wounds. (B) Representative IHC and intensity analysis (right panel) of 5-methylcytosine in ischemic wounds treated with either vehicle control or 5-azacytidine. (Scale bar: 50 μm; n = 5; *P < 0.05, Student’s t test). (C) Wound closure determined by digital planimetry (top). Data presented as percentage of wound area (bottom). n = 7, 8, *P < 0.05 (Student’s t test). Data represented as mean ± SEM. (D) Vector components used for targeted demethylation of P53 promoter in keratinocytes using CRISPR/dCas9 approach. Keratinocyte targeting was achieved by KRT14 promoter–driven guide RNAs. (E) The mouse P53 promoter locus used for demethylation events. The locations of the targets (1–3) for sgRNAs are indicated by red pointers. (F) Topical delivery of TET1 CD and targeted sgRNAs to the ischemic wound employing tissue nanotransfection (TNT2.0) technology. (G) Schematic diagram of the TNT process. (H) Western blot analysis showing the expression of P53 in bipedicle ischemic wounds of mice nanotransfected with TET1CD and peptide repeat in presence or absence of KRT14 promoter–driven P53 gRNA targets. Data expressed as fold-change using β-actin as loading control. (n = 6; *P < 0.05, Student’s t test). (I) Demethylation activity was measured by bisulfite sequencing of murine P53 promoter region (mm10_chr11:69,578,954-69,579,215). (J) Wound closure was monitored at different days after wounding in bipedicle ischemic wounds of mice subjected to TNT by digital planimetry (left). Data presented as percentage of wound area (right). n = 8, *P < 0.05 (Student’s t test). (K) Representative IHC analysis of P53 in ischemic wounds subjected to TNT. (L) Intensity analysis of the images. (Scale bar: 100 μm; n = 6; *P < 0.05, Student’s t test). nairing, hair-removal technique using a depilatory agent (Nair, Church and Dwight).