Title: KMT2D acts through both enzyme-dependent and -independent mechanisms to regulate cell fate changes during muscle regeneration
Abstract: Muscle regeneration is a multifaceted process reliant on the rapid response of muscle stem cells (MuSCs) to environmental stimuli. As MuSCs advance through regeneration, the expression of muscle-specific genes is tightly regulated to control cell fate transitions, which likely occurs through the commissioning of enhancer regions. The methyltransferase KMT2D (also known as MLL4) is responsible for commissioning enhancer regions through mono-methylation of H3K4. While KMT2D has been shown to be essential for cell differentiation in embryonic stem cells, its role in the regeneration of adult tissue is not well understood. As such, we are investigating the role of KMT2D in adult stem cell function using muscle regeneration as a model system. An inducible conditional knockout mouse model allowed us to specifically ablate KMT2D in MuSCs (KMT2DscKO) to determine its role in muscle repair. Following acute cardiotoxin-induced injury, KMT2DscKO mice showed impaired muscle regeneration. Ex vivo analysis of the distinct stages of muscle regeneration showed that deletion of KMT2D impaired the differentiation capabilities of isolated myoblasts, preventing the formation of multinucleated myotubes. To identify genes whose expression might contribute to this phenotype, we performed RNAseq analysis while profiling genomic enrichment of KMT2D and the enhancer-associated post-translational modification H3K4me1 using CUT&Tag. RNA-seq results identified ~1100 dysregulated genes at the differentiation time point, with the majority of these being downregulated. The integration of downregulated genes with KMT2D binding identified direct targets, including genes known to be essential for fusion. Comparing KMT2DscKO to an enzymatically dead KMT2DKI model (expresses a stable protein unable to methylate histone H3), we show that regulation of some KMT2D target genes occurs in an enzyme-dependent manner while others occur in an enzyme-independent manner. Together, these results suggest that KMT2D plays a crucial role in the expression of genes involved in cell fate changes that occur during the transition from proliferation to differentiation, and that KMT2D employs multiple mechanisms to regulate these changes in gene expression.
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