Genetic Manipulation of Rad21 Gene Locus Compromises Differentiation of Skeletal Muscle Stem Cells
LIU Qinyao1, GAO Qing2, ZHANG Qianying1, WANG Ruiting1, ZHANG Yong1,2*, ZHU Dahai1,2*
Eukaryotic genome is dynamically organized to three-dimensional structure through hierarchical folding, which is important for regulating cell lineage differentiation during development. The cohesin complex is an essential regulator for chromatin looping and topological associated domain formation and consists of 4 subunits, one of which is the kleisin protein RAD21. To investigate whether cohesin-mediated high order chromatin structure regulates adult stem cell differentiation, an inducible Rad21 degron knockin mouse line was generated using AID (auxin-inducible degron) (Rad21-Loxp-Stop-Loxp-AID-EGFP-P2A-OsTIR1, Rad21Aid/+). By crossing the Rad21Aid/+mice with skeletal muscle stem cell specific Cre mice (Pax7-Cre), the RAD21 protein would be specifically degraded in skeletal muscle stem cells once treated with auxin. However, protein levels of RAD21 were significantly decreased in skeletal muscle stem cells from the both homozygotes (Hom) of Rad21Aid/Aid and Pax7-Cre; Rad21Aid/Aid even in absence of auxin, compared to wild-type littermates (WT), suggesting genetic manipulation of the Rad21 gene locus leads to dysregulation of its expression. Phenotypically, the homozygous mice had lower birth rate, less body weight and muscle mass, and smaller myofiber diameters, compared with WT littermates. Moreover, the significant reduction of RAD21 protein in skeletal muscle stem cells of the homozygous mice lead to compromised myogenic cell differentiation, which might account for their abnormal muscle growth and development. Together, the findings suggest RAD21 is critical regulator of development and stem cell differentiation in mice.
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