Neuromuscular diseases are a broad range of disorders affecting muscle functions which may lead to muscle weakness, muscle wasting, paralysis or even death. Most of these diseases are genetically inherited. For precise treatment of the conditions and pre-natal screening, the causative mutations need to be identified. Screening the exomes of known neuromuscular disease-associated genes (~464 genes currently) yields a molecular diagnosis for approximately 60% of patients. For the remaining undiagnosed patients, we hypothesize that a significant fraction of the mutations may be due to mutations in cis regulatory elements such as promoters and enhancers. To screen for mutations in these regions we first need to build a map of which distal regulatory elements control each neuromuscular disease gene.
In this study, we have used promoter capture in situ DNase Hi-C to generate a high-resolution map of genomic interactions (up to 1kb bin size) involving the promoters of 952 skeletal muscle-enriched genes. This map has been generated using primary human myoblasts and in vitro differentiated myotubes from multiple donors. On average ~10,000 significant interactions were found in each sample. Of note, the regions interacting with these muscle promoters are significantly enriched for skeletal-muscle-enhancer-like regions (marked with H3K27Ac, H3K4Me1 and eRNA transcripts in skeletal muscle). Significantly, this data identifies human orthologs of several known skeletal muscle specific enhancer-promoter pairs from mouse. Furthermore, we have gone on to demonstrate that CRISPR-mediated deletion of these elements in primary human myoblasts significantly reduces expression of the interacting target gene. In addition to enhancing our understanding of muscle gene regulation, ultimately, this map will be used to interpret whole genome data from the currently unknown fraction of patients with mutations in regulatory regions.