B-003 Assessing the role of KMT2D in muscle regeneration to understand hypotonia symptoms associated with Kabuki Syndrome

Abstract Background Kabuki syndrome (KS) is a rare multi-systemic genetic condition affecting 1 in 32,000 individuals globally. It is characterized by symptoms such as dysmorphic facial features, developmental delays, intellectual disabilities, and hypotonia. Muscle weakness as a result of hypotonia is a concern among patients and their families, causing significant impact on their quality of life. Genetic testing has shown that 80% of KS cases are caused by pathogenic mutations in the histone methyltransferase KMT2D. KMT2D is an epigenetic factor responsible for commissioning enhancer regions through the mono-methylation of histone 3 lysine 4 (H3K4me1). The muscle regeneration pathway requires the expression of tissue-specific genes that are tightly regulated to control cell fate transitions, which likely occurs through the commissioning of enhancer regions. Thus, it is our goal to identify the function of KMT2D in muscle stem cells (MuSCs) and explore whether KMT2D mutations in MuSCs contribute to the hypotonia phenotype that is observed in KS patients. Methods We used a mouse model with a MuSC-specific inducible deletion of KMT2D. Heterozygous deletion mice (KMT2DscKO/wt) represent a KS-like state and are compared to homozygous deletion mice (KMT2DscKO/scKO) and wildtype mice (KMT2Dwt/wt). To specifically investigate the enzymatic function of KMT2D, we used a knock-in mutant KMT2D (KMT2DKI) that is catalytically dead. We have performed in vivo and in vitro studies to characterize the muscle regeneration capabilities of MuSCs, looking at overall regeneration and the effects on distinct pathway stages. Additionally, we have performed RNAseq and CUT&Tag analyses to identify and characterize genes directly regulated by KMT2D. Results Using the outlined mouse models, we ablated KMT2D expression in MuSCs and tested muscle regeneration capabilities. Following acute cardiotoxin-induced injury, the KMT2DscKO/wt, KMT2DscKO/scKO, and KMT2DKI mice had impaired muscle regeneration with phenotypic defects observed in a protein concentration dependent manner. In vitro analysis of the distinct stages of muscle regeneration showed that both deletion of KMT2D and expression of enzyme mutant KMT2D resulted in impaired differentiation capabilities. RNA-seq analysis at the differentiation time point identified ∼1200 dysregulated genes, the majority being downregulated. Integration of KMT2D binding results from CUT&Tag identified direct targets including essential fusion genes. Comparing KMT2DscKO to KMT2DKI, we show that regulation of KMT2D target genes occurs in either an enzyme-dependent or enzyme-independent manner. Conclusion Our results show KMT2D to be important for the expression of genes involved in muscle cell fate changes, with gene dysregulation leading to impaired regenerative capabilities. We find that KMT2D functions in an enzyme-dependent and enzyme-independent manner. Together, these results provide the first mechanistic insight into the role of KMT2D in muscle regeneration and thus initial insights into KS pathology. Future experiments will work with patient samples to further understand the connection between KMT2D function and KS symptoms. Overall, our findings from these muscle studies will form the basis of future studies looking at altered stem cell function in other KS-related tissues which are less amenable to study mechanisms of action and aim to advance research into the development of new therapeutics.