PROJECT SUMMARY/ABSTRACT Non-healing wounds in patients with Type 2 Diabetes (T2D) are a major cause of morbidity and mortality and are increasing at an alarming rate. Failure of wound healing in T2D patients represents the most common cause of amputation in the US with a 5-year mortality rate of nearly 50%. Thus, a critical need exists for understanding the wound healing defects in T2D in order to develop targeted therapies. We will utilize both genetic (db/db) and dietary (diet-induced obese) murine models of T2D as well as human wound tissue and blood samples collected from T2D patients to explore mechanisms of impaired wound healing. We present data using human single cell RNA sequencing and murine diabetic wound models, that the repressive histone methyltransferase, SETDB2, is decreased in diabetic wound keratinocytes resulting in increased production of inflammatory genes, namely TNFα. Further, we have identified that interferon-beta (IFNꞵ), via a janus kinase (JAK1) / signal transducer and activator of transcription (STAT) mechanism induces SETDB2 in wound keratinocytes. Our preliminary data also identified that IL-17A is increased in diabetic wounds and may, in addition to reduced levels of IFNꞵ in diabetic wounds, suppress SETDB2 in diabetic wound keratinocytes. These results have led to our hypothesis that induction of SETDB2 in wound keratinocytes represses expression of NFkB-mediated genes (i.e., TNFα) that maintain wound inflammation and directly impair keratinocyte migration, thereby promoting tissue repair. We postulate that in diabetic wounds, it is the failure to induce SETDB2 and repress NFκB-mediated inflammatory (Tnfα) genes in keratinocytes that prevents resolution of inflammation and impairs keratinocyte migration and results in poor wound healing. We will test our hypotheses through three specific aims: Aim 1: To define the in vivo regulation of NFκB-mediated gene expression by SETDB2 in normal and diabetic wound keratinocytes. Aim 2: To determine the JAK/STAT-mediated mechanism(s) that regulate keratinocyte-specific SETDB2 expression in normal and diabetic wound tissue. Aim 3: To examine the therapeutic efficacy and timing of SETDB2-regulated TNFα inhibition on keratinocyte migration and inflammatory wound Mφ phenotype. In this translational approach, our data will pave the way for the development of promising therapeutic agents aimed at the targeting of epigenetic pathways that mediate diabetic wound keratinocyte function and thereby promote diabetic wound repair.