Cysteine Rich Protein 2 is a copper-responsive regulator of skeletal muscle differentiation. [RNA-Seq]. Cysteine Rich Protein 2 is a copper-responsive regulator of skeletal muscle differentiation. [RNA-Seq]

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. Overall design: Total strand-specific RNA-seq libraries isolated from differentiating (Diff) and proliferating (Prol) myoblasts. CRIP2 knockout myoblasts were generated via lentiviral transduction. Each CRIP2 KO library was paired with a scrambled (SCR) control experiment and completed in duplicate.

铜（Cu）是参与呼吸作用、神经递质合成、氧化应激及转录调控等多种生物反应的必需微量元素。若铜稳态发生紊乱，可引发多种病理状态，进而导致神经元、认知及肌肉发育异常。目前已知的哺乳动物细胞内结合一价铜离子（Cu+）的转录因子包括Atox1、Mtf1及Sp1。我们鉴定出Crip2是一种新型的一价铜离子响应型转录调控因子，其是小鼠卫星细胞来源的原代成肌细胞分化所必需的。通过对CRISPR/Cas9介导的Crip2敲除进行功能表征，发现成肌细胞无法完成分化，且分化标志物肌生成素（Myogenin）与肌球蛋白重链（Myosin heavy chain）的表达水平出现下调。本研究开展了转录组测序（RNA-seq）与切割与释放测序（CUT&RUN）分析，以探究铜离子刺激下，Crip2对增殖与分化状态的原代成肌细胞中基因表达调控的影响。实验设计概述：从分化态（Diff）与增殖态（Prol）成肌细胞中提取链特异性RNA-seq文库；CRIP2敲除成肌细胞通过慢病毒转导构建；每一组CRIP2敲除（KO）文库均搭配乱序序列（SCR）对照实验，并设置两次生物学重复。