Cysteine Rich Protein 2 is a copper-responsive regulator of skeletal muscle differentiation. [CUT&Run]

Copper (Cu) is an essential trace element for diverse biological reactions such as respiration, neurotransmitter synthesis, oxidative stress and transcriptional regulation. If Cu homeostasis is disrupted, several pathological conditions can develop, leading to alterations of neuronal, cognitive, and muscular development. Known Cu+-binding transcription factors in mammalian cells are Atox1, Mtf1 and Sp1. We identified Crip2 as a novel Cu+-responsive transcriptional regulator that is required for the differentiation of primary myoblasts derived from mouse satellite cells. Functional characterization of CRISPR/Cas9-mediated deletion of Crip2 showed that myoblasts fail to differentiate and manifest a decrease in expression of the differentiation markers Myogenin and Myosin heavy chain. RNA-seq and CUT&RUN analysis were performed to understand the effect on Crip2 in the regulation of gene expression in proliferating and differentiating primary myoblasts stimulated with Cu. CUT&RUN targeting CRIP2 binding in differentiating (Diff) and proliferating (Prol) myoblasts, each paired with an untargeted IgG control experiment and completed in triplicate. Differentiating myoblasts were treated with Cu, Insulin, or no treatment, while proliferating myoblasts were treated with either Cu or no treatment.