Epigenetic Mapping Of Wound Edge From Chronic Wound Patients Using Next Generation Sequencing
Kanhaiya Singh, Sashwati Roy, Durba Pal, Subhadip Ghatak, Shomita Steiner, Devleena Das, Pearlly Yan, Ralf Bundschuh, Savita Khanna, Chandan K. Sen.
The 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 sonicated to generate 100-300 bp size fragments followed by methylated DNA fragment enrichment and Illumina-compatible sequencing library generation. Single-end 50 bp sequencing was done using Illumina HiSeq 2500. Separate methylation status of proximal promoters (1 Kb), distal promoters (10 kb), non-CpG promoters and within exons of genes were calculated using MethylCap-Seq data analysis and PrEMeR-CG analysis. Differential methylation analysis was performed using mean vector test. Results- In proximal promoter, genes of ERK, mTOR and Notch signaling were hypomethylated. In contrast, genes involved in epithelial to mesenchymal transition were hypermethylated in CW compared to NS (p<0.0001). Hypomethylation of mTOR and ERK genes was also observed in non-CpG promoters and within exons (p<0.0001). Bisulfite sequencing was used to validate hypermethylation of candidate genes (TP53, BRCA1, and ESR1). microRNA promoters were differentially methylated in CW compared to NS (71 hyper methylated; 20 hypomethylated in CW, p<0.05). The significance of these epigenetic changes on the transcriptome was studied. A sum total of 1281 genes were found to be differentially expressed in CW compared to NS (p<0.05). Comparison between differentially methylated and differentially expressed genes identified specific genes the methylation of which downregulated expression. Examples include Potassium Voltage-Gated Channel Subfamily A Member-3, protein tyrosine phosphatase, receptor type D, microRNA-31, Glucoside Xylosyltransferase, Protocadherin-17 and Transcription factor Spi-B. 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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