Metal-regulatory transcription factor 1 (MTF1) is a conserved metal-binding transcription factor in eukaryotes that binds to conserved DNA sequence motifs, termed metal response elements. MTF1 responds to both metal excess and deprivation, protects cells from oxidative and hypoxic stresses, and is required for embryonic development in vertebrates. To examine the role for MTF1 in cell differentiation, we use multiple experimental strategies [including gene knockdown (KD) mediated by small hairpin RNA and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), immunofluorescence, chromatin immunopreciptation sequencing, subcellular fractionation, and atomic absorbance spectroscopy] and report a previously unappreciated role for MTF1 and copper (Cu) in cell differentiation. Upon initiation of myogenesis from primary myoblasts, both MTF1 expression and nuclear localization increased. Mtf1 KD impaired differentiation, whereas addition of nontoxic concentrations of Cu(+)-enhanced MTF1 expression and promoted myogenesis. Furthermore, we observed that Cu(+) binds stoichiometrically to a C terminus tetra-cysteine of MTF1. MTF1 bound to chromatin at the promoter regions of myogenic genes, and Cu addition stimulated this binding. Of note, MTF1 formed a complex with myogenic differentiation (MYOD)1, the master transcriptional regulator of the myogenic lineage, at myogenic promoters. These findings uncover unexpected mechanisms by which Cu and MTF1 regulate gene expression during myoblast differentiation.

MTF1 is a conserved metal-binding transcription factor in eukaryotes that binds to conserved DNA sequence motifs, termed metal response elements (MREs). MTF1 responds to metal excess and deprivation, protects cells from oxidative and hypoxic stresses, and is required for embryonic development in vertebrates. We used multiple strategies to identify an unappreciated role for MTF1 and copper (Cu) in cell differentiation. Upon initiation of myogenesis from primary myoblasts, MTF1 expression increased, as did nuclear localization. Mtf1 knockdown impaired differentiation, while addition of non-toxic concentrations of Cu+ enhanced MTF1 expression and promoted myogenesis. Cu+ bound stoichiometrically to a C-terminus tetra-cysteine of MTF1. MTF1 bound to chromatin at the promoter regions of myogenic genes and binding was stimulated by copper. MTF1 formed a complex with MyoD at myogenic promoters, the master transcriptional regulator of the myogenic lineage. These studies establish novel mechanisms by which copper and MTF1 regulate gene expression in myoblast differentiation.

Zinc transporters facilitate metal mobilization and compartmentalization, playing a key role in cellular development. Little is known about the mechanisms and pathways of Zn movement between Zn transporters and metalloproteins during myoblast differentiation. We analyzed the differential expression of ZIP and ZnT transporters during C2C12 myoblast differentiation. Zn transporters account for a transient decrease of intracellular Zn upon myogenesis induction followed by a gradual increase of Zn in myotubes. Considering the subcellular localization and function of each of the Zn transporters, our findings indicate that a fine regulation is necessary to maintain correct metal concentrations in the cytosol and subcellular compartments to avoid toxicity, maintain homeostasis, and for loading metalloproteins needed during myogenesis. This study advances our basic understanding of the complex Zn transport network during muscle differentiation.