Role of TET-mediated DNA demethylation in wound healing

Summary: Abstract Body: DNA methylation and the subsequent oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) are key epigenetic mechanisms that regulate organ development, stem cell maintenance, and differentiation in mammals. TET enzyme family (TET1, TET2, and TET3) catalyzes the oxidation of 5mC, an essential step in DNA demethylation. Accumulating evidence shows that Tet proteins are essential in diverse biological processes, including development, regeneration, and cellular reprogramming. However, the role of Tet-mediated DNA demethylation in controlling gene expression in keratinocytes during wound healing remains unknown. In this study, we assess the dynamics of gene expression and chromatin accessibility in epidermal keratinocytes during wound healing using a mouse tail model. Our results show that the epigenetic landscape in keratinocytes is altered during regeneration, with DNA methylation changes occurring in gene regulatory regions. RNA-seq of wounded keratinocytes reveals dysregulation of genes involved in epidermal development, proliferation, motility and cytokine production. Similar to unwounded skin, we found that TET2 and TET3 protein expression predominated over TET1 in keratinocytes isolated from the wound epithelium. Both TET2, TET3, and 5hmC were expressed in proliferating and migrating keratinocytes of the regenerating epithelium. To study the role of TET2 and TET3 in epidermal regeneration, we generated conditional K14-Cre-driven Tet2/Tet3 double-knockout (DKO) mice. DKO mice exhibited significant delays in wound healing, which were associated with reduction of expression of genes that control the cell cycle, cytoskeleton organization, and cell migration in regenerating wound epithelium. These data provide a platform for further research towards pharmacological modulation of wound healing by targeting DNA methylation/demethylation enzymes in regenerating tissues. Elena Rozhkova², Iqra Fatima², Guodong Chen², Georgios Theocharidis¹, Daniel Roh², Aristidis Veves¹, Vladimir A. Botchkarev², Andrey Sharov² 1. Beth Israel Deaconess Medical Center, Boston, MA, United States. 2. Dermatology, Boston University, Boston, MA, United States. Stem Cell Biology, Tissue Regeneration and Wound Healing