Characterization of the Three Dimensional Genome in Human Skeletal Muscle Progenitor Cells.

Duchenne Muscular Dystrophy (DMD) is a devastating disease with no cure affecting approximately 1 in 3,500-5,000 boys. Stem cell treatments using skeletal muscle stem cells (or satellite cells, SCs) provide great potential for regenerating new muscle and we have developed directed differentiation strategies to generate skeletal muscle cells from human induced pluripotent stem cells (hiPSCs). Our work has shown that hiPSCs generate PAX7 skeletal muscle progenitor cells (SMPCs) resembling early myogenic cells that align closer to week 7-12 in human development and are not equivalent to adult SCs. We are interested in understanding the key molecular and functional differences that control SMPC versus SC cell states. Recently, there has been an intense interest in the three dimensional (3D) organization of the genome and its involvement in cell specific gene regulation. This has led to the discovery of chromatin loops between gene enhancers and promoters as well as self-interacting domains termed topologically associating domains (TADs). Recently published data support a role for the 3D genome in cellular differentiation, however, the differences between the 3D genome in human SMPCs compared to adult SCs is unknown. High throughput chromosome conformation capture (Hi-C) was used to characterize the 3D genome of SMPCs. We found genome-wide 3D configurations were different between hPSCs and SMPCs as was the number of TADs. Interestingly, TAD size was also different between cell types, suggesting dynamic control of TADs during differentiation. When focusing on the PAX7 locus, TAD boundaries differ between cell types further highlighting the dynamic nature of TADs with respect to cell specific gene expression. Not all muscle specific loci were different between cell types, however, suggesting that some TADs may be pre-established early in the differentiation process while others are established de-novo. With respect to chromatin looping at the PAX7 locus, we found SMPC specific looping between the PAX7 promoter and downstream sequences that were unique in SMPCs. Considering that PAX7 enhancer sequences have yet to be determined, these sequences may serve as candidate enhancers for PAX7. These data, for the first time, characterize the 3D genome of human SMPCs using Hi-C. Moreover, these data provide candidate enhancer sequences for that could provide unique candidates for support of PAX7 SMPCs.