Summary of quantitative data used in the paper

Copper (Cu) is essential for respiration, neurotransmitter synthesis, oxidative stress response, and transcription regulation, with imbalances leading to neurological, cognitive, and muscular disorders. Here we show the role of a novel Cu-binding protein (Cu-BP) in mammalian transcriptional regulation, specifically on skeletal muscle differentiation using murine primary myoblasts. Utilizing synchrotron X-ray fluorescence-mass spectrometry, we identified murine cysteine-rich intestinal protein 2 (mCrip2) as a key Cu-BP abundant in both nuclear and cytosolic fractions. mCrip2 binds two to four Cu⁺ ions with high affinity and presents limited redox potential. CRISPR/Cas9-mediated deletion of <i>mCrip2</i> impaired myogenesis, likely due to Cu accumulation in cells. CUT&amp;RUN and transcriptome analyses revealed its association with gene promoters, including <i>MyoD1</i> and <i>metallothioneins</i>, suggesting a novel Cu-responsive regulatory role for mCrip2. Our work describes the significance of mCrip2 in skeletal muscle differentiation and metal homeostasis, expanding understanding of the Cu-network in myoblasts.

铜（Copper，Cu）是呼吸作用、神经递质合成、氧化应激应答以及转录调控所必需的微量元素，其稳态失衡会引发神经系统、认知功能障碍及肌肉系统相关疾病。本研究揭示了一种新型铜结合蛋白（Cu-binding protein，Cu-BP）在哺乳动物转录调控中的功能，并以小鼠原代成肌细胞为模型，重点探究其在骨骼肌分化过程中的作用。本研究利用同步辐射X射线荧光-质谱联用技术（synchrotron X-ray fluorescence-mass spectrometry），将小鼠富含半胱氨酸肠蛋白2（murine cysteine-rich intestinal protein 2，mCrip2）鉴定为一类关键的铜结合蛋白，该蛋白在细胞核与细胞质组分中均大量富集。mCrip2可与2~4个一价铜离子（Cu⁺）以高亲和力结合，且仅具备有限的氧化还原活性。通过CRISPR/Cas9介导的mCrip2基因敲除会损害成肌分化过程，这一表型可能与细胞内铜离子蓄积有关。CUT&RUN与转录组测序分析结果显示，mCrip2可与包括*MyoD1*与金属硫蛋白家族（metallothioneins）在内的诸多基因启动子区域相结合，这表明mCrip2具备一种全新的铜离子应答调控功能。本研究阐明了mCrip2在骨骼肌分化与金属稳态调控中的重要意义，拓展了人们对成肌细胞内铜离子调控网络的认知。