The Dlk1-Dio3 noncoding RNA cluster coordinately regulates mitochondrial respiration and chromatin structure to establish proper cell state for muscle differentiation
To investigate the collective role of the Dlk1-Dio3 ncRNA cluster in myogenesis, we developed two stable CRISPR-Cas9 gene-edited C2C12 skeletal muscle cell lines, Meg3-proximal cis-acting sequence (Meg3-CAS or 2-4) to over-express and Meg3-transcription start site (Meg3-TSS or 2-7) to under-express the entire mega-cluster of Dlk1-Dio3 ncRNAs. Mutations affecting the dosage of all ncRNAs expressed from the Dlk1-Dio3 locus cause profound changes in myoblast fusion and differentiation. To evaluate genome-wide perturbations in chromatin accessibility we evaluated specific histone lysine modifications, inducing the repressive histone mark, H3K27me3, and the activating mark, H3K4me3 Overall design: CUT&RUN for H3K27me3 and H3K4me3 in gene-edited (Dlk1-Dio3 ncRNA locus) C2C12 cells compared to C2C12 control cells