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 1

Increased global DNA methylation is strongly associated with human chronic WE tissue.

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Increased global DNA methylation is strongly associated with human chron...
(A) Representative IHC analysis of DNMT1 (top), DNMT3A (middle), and DNMT3B (bottom) in paraffin sections from human unwounded (UW) skin and chronic wound-edge (WE) tissue. Bottom panel represent the intensity analysis of the images. (Scale bar: 50 μm; n = 5; *P < 0.05, Student’s t test). (B) Dot blot analysis (left) and its intensity analysis (right) of 5-methylcytosine (5mc) in human chronic WE compared with UW skin (n = 5; *P < 0.05, Student’s t test). (C) Circos plot demonstrating the distribution of significant differentially methylated regions (DMRs) associated with 1 kb upstream and 1 kb downstream of Ref-Seq genes in human chronic WE. Chromosome number marked in the periphery. Red lines and dots represent hypermethylated loci, and green lines and dots represent hypomethylated loci in chronic WE. (D) Hierarchical clustering analysis of 4689 significant DMRs associated with Ref-Seq genes in chronic WE. (n = 3; FDR adjusted P < 0.05; 3661 hypermethylated and 1028 hypomethylated in chronic WE tissue) were obtained. (E) IPA upstream regulator analysis of methylation data identified TP53 to be the most significant hypermethylated upstream regulator in chronic WE. (F) Methylation status of a region of TP53 promoter (–1069 bp to –821 bp) analyzed through bisulfite sequencing (methylated CpG, black; unmethylated CpG, white) (n = 10 clones). (G) Distribution of methylated and unmethylated CpGs in TP53 promoter (human UW skin (top); chronic WE (bottom). (H) qRT-PCR analysis of TP53 expression in human chronic WE and skin. (n = 5, 7; *P < 0.05, Student’s t test). (I) Representative IHC analysis of TP53 in sections from human UW skin and chronic WE and (J) intensity analysis of the images. (Scale bar: 50 μm; n = 6, 7; *P < 0.05, by Student’s t test). Data are presented as the mean ± SEM.