Impaired Epigenetic Reprogramming prevents Fibroblast to Myofibroblast Transition in Diabetic Wounds

Following tissue injury, a coordinated process of tissue repair occurs where immune cells regulate structural cell function, a process that is critical for wound healing. The transition of fibroblasts to myofibroblasts is necessary for tissue repair; however, the processes that regulate this plasticity are unclear. Further, in disease states, such as type 2 diabetes (T2D), fibroblasts fail to transition to myofibroblasts, preventing tissue repair. Fibroblasts were magnetically isolated from wounded mice on days seven and ten following punch biopsy creation. Isolated fibroblasts were treated with TGF-β and subjected to reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and chromatin immunoprecipitation (ChIP) for trimethylation at lysine four on histone H3 (H3K4me3, an activating mark) with a focus on myofibroblast-associated genes (Acta2, Myl9, Tagln). Important findings were translated to human tissue by utilizing single cell RNA sequencing (scRNAseq) from normal and T2D wounds. We identified that following injury, TGF-β, produced by anti-inflammatory macrophages, promotes fibroblast to myofibroblast transition (as evidenced by increased gene expression of myofibroblast-associated genes) in our murine model of would repair. Furthermore, H3K4me3 was increased at the promoters of these genes following TGF-β stimulation. In order to determine the epigenetic enzyme regulating H3K4me3 at these promoters, we examined several H3K4 methyltransferases and demethylases and found that MLL1 was upregulated in wound fibroblasts following TGF-β stimulation. When wound fibroblasts were treated with TGF-β and siRNA to MLL1, myofibroblasts gene expression was decreased. Importantly, when we examined fibroblasts isolated from murine diabetic wounds, MLL1 was not upregulated in response to TGF-β stimulation, nor was there an associated increase in H3K4me3 at the myofibroblast gene promoters by ChIP analysis. To determine if MLL1 may regulate the fibroblast to myofibroblast transition in human tissue, we performed single cell RNA sequencing on human wounds from control patients and found an upregulation in MLL1 in fibroblasts as compared to fibroblasts from wounds of patients with T2D. This work identifies that TGF-β may regulate the fibroblast to myofibroblast transition in wounds via an MLL1/H3K4me3 mechanism which is perturbed in the setting of T2D.