Epigenetic Mapping Of Wound Edge From Chronic Wound Patients Using Next Generation Sequencing
Kanhaiya Singh1, Sashwati Roy1, Durba Pal2, Subhadip Ghatak1, Dolly Khona1, Saba Tabasum1, Shomita Steiner1, Devleena Das1, Pearlly Yan2, Ralf Bundschuh2, Savita Khanna1, Chandan K. Sen1.
1Indiana University School of Medicine, Indianapolis, IN, USA, 2The Ohio State University, Columbus, OH, USA.
Background- The loud biochemical microenvironment of the chronic wound sharply departs from that of the skin under homeostatic conditions and is likely to induce epigenetic changes thus influencing wound healing outcomes.
Methods- Unbiased whole-genome DNA methylation (methylome) was studied in normal skin (NS) and in wound edge tissue of chronic wound (CW) patients. DNA (1 µg) isolated from CW or NS was enriched for the study of methylated DNA. Single-end 50 bp sequencing was performed using Illumina HiSeq 2500. Methylation status of proximal promoter (1 Kb) was calculated using MethylCap-Seq data analysis and PrEMeR-CG analyses. Whole genome RNA sequencing and microRNA nanoString analysis over CW and NS samples was performed to qualify the methylation data.
Results- In proximal promoter, genes involved in epithelial to mesenchymal transition (EMT) were hypermethylated in CW compared to NS (n=3, p<0.0001). Bisulfite sequencing was used to validate hypermethylation of predicted upstream regulators (TP53 and BRCA1). microRNA promoters were differentially methylated in CW compared to NS (71 hyper methylated; 20 hypomethylated in CW, n = 3, p<0.05). A sum total of 1281 genes were found to be differentially expressed in CW compared to NS (n=3, p<0.05). Comparison between hypermethylated and downregulated expressed genes identified common candidate genes of EMT pathway like ADAM17, TWIST1 and SMURF1 obtained through two independent global screening approaches. Topical application of classical DNA demethylation agent 5’-azacytidine successfully rescued ischemic wounds of mice by increasing the expression of ADAM17. Lentiviral overexpression of ADAM17 alone rescued ischemic wounds by enabling the P53-ADAM17 cascade.
Conclusion- Epigenetic activity is high at the chronic wound site. Such activity has significant impact on gene expression at the wound-edge and is therefore likely to influence healing outcomes.
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