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Updated: Jul 18, 2026

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry
Published on: May 17, 2016
Activation of the beta myosin heavy chain promoter by MEF-2D, MyoD, p300, and the calcineurin/NFATc1 pathway
Joachim D Meissner1, Patrick K Umeda, Kin-Chow Chang
1Department of Physiology, Hannover Medical School, Hannover, Germany. meissner.joachim@mh-hannover.de
Abstract:
Calcium is a key element in intracellular signaling in skeletal muscle. Changes in intracellular calcium levels are thought to mediate the fast-to-slow transformation of muscle fiber type. One factor implicated in gene regulation in adult muscle is the nuclear factor of activated T-cells (NFAT) isoform c1, whose dephosphorylation by the calcium/calmodulin-dependent phosphatase calcineurin facilitates its nuclear translocation. Here, we report that differentiated C2C12 myotubes predominantly expressing fast-type MyHCII protein undergo fast-to-slow transformation following calcium-ionophore treatment, with several transcription factors and a transcriptional coactivator acting in concert to upregulate the slow myosin heavy chain (MyHC) beta promoter. Transient transfection assays demonstrated that the calcineurin/NFATc1 signaling pathway is essential for MyHCbeta promoter activation during transformation of C2C12 myotubes but is not sufficient for complete fast MyHCIId/x promoter inhibition. Along with NFATc1, myocyte enhancer factor-2D (MEF-2D) and the myogenic transcription factor MyoD transactivated the MyHCbeta promoter in calcium-ionophore-treated myotubes in a calcineurin-dependent manner. To elucidate the mechanism involved in regulating MyHCbeta gene expression, we analyzed the -2.4-kb MyHCbeta promoter construct for cis-regulatory elements. Using electrophoretic mobility shift assays (EMSAs), chromatin immunoprecipitation assays (ChIP), and nuclear complex coimmunoprecipitation (NCcoIP) assays, we demonstrated calcium-ionophore-induced binding of NFATc1 to a NFAT consensus site adjacent to a MyoD-binding E-box. At their respective binding sites, both NFATc1 and MyoD recruited the transcriptional coactivator p300, and in turn, MEF-2D bound to the MyoD complex. The calcium-ionophore-induced effects on the MyHCbeta promoter were shown to be calcineurin-dependent. Together, our findings demonstrate calcium-ionophore-induced activation of the beta MyHC promoter by NFATc1, MyoD, MEF-2D, and p300 in a calcineurin-dependent manner.
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