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 Therapeutics

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 5

TP53 methylation hinders human keratinocyte migration.

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TP53 methylation hinders human keratinocyte migration. (A) Immunofluores...
(A) Immunofluorescence analysis of 5-methylcytosine (5mc) in keratinocytes (HaCaT cells) exposed to vehicle control or S-adenosylmethionine (SAM) (80 μM, 48 h) and (B) their intensity analysis. DAPI intensity was used for normalization. (Scale bar: 20 μm; n = 6–8; *P < 0.001, Student’s t test). (C) Quantitation and (D) representative images of scratch-wound migration assay of keratinocytes. Scratch wound migration was performed after 48 hours of pretreatment with vehicle control or SAM (80 μM) and followed for 44 hours after wounding. (Scale bar: 200 μm; n = 6; *P < 0.05, Student’s t test). (E) Schematic diagram showing DNA methylation strategy of TP53 promoter mediated by dCas9-DNMT3A. Transfection efficiency of GFP-labeled plasmids shown in Supplemental Figure 9K through flow cytometry. (F) Left: schematic diagram of TP53 promoter (–1069 bp to –821 bp) analyzed through bisulfite genomic sequencing of DNA. Diagrammatic representation of the promoter methylation status shown (methylated CpG, black; unmethylated CpG, white). Right: Venn diagram showing the distribution of methylated and unmethylated TP53 promoter in HaCaT cells transfected with control (dCas-9-DNMT3A-ANV) (top) and in dCas-9-DNMT3A (bottom) in presence of TP53-guide RNAs. (G) Western blot analysis showing the expression of TP53, (H) activated NOTCH1, and (I) ADAM17 in HaCaT cells transfected with dCas-9-DNMT3A-EGFP or control (dCas-9-DNMT3A-EGFP-ANV) in presence of TP53-guide RNAs. Data expressed as fold change; β-actin used as loading control. (n = 6, 7; *P < 0.05, Student’s t test). Data represented as mean ± SEM. (J) Representative images and (K) quantitation of scratch-wound migration assay of HaCaT keratinocytes. Scratch-wound assay was performed after 48 hours of transfection with dCas-9-DNMT3A or control (dCas-9-DNMT3A-ANV) in presence of TP53-guide RNAs and followed for 44 hours after wounding. (n = 6; *P < 0.05, Student’s t test). Data represented as mean ± SEM.