Activation of the β myosin heavy chain promoter by MEF-2D, MyoD, p300, and the calcineurin/NFATc1 pathway
✍ Scribed by Joachim D. Meissner; Patrick K. Umeda; Kin-Chow Chang; Gerolf Gros; Renate J. Scheibe
- Publisher
- John Wiley and Sons
- Year
- 2007
- Tongue
- English
- Weight
- 347 KB
- Volume
- 211
- Category
- Article
- ISSN
- 0021-9541
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✦ Synopsis
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) β promoter. Transient transfection assays demonstrated that the calcineurin/NFATc1 signaling pathway is essential for MyHCβ 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 MyHCβ promoter in calcium‐ionophore‐treated myotubes in a calcineurin‐dependent manner. To elucidate the mechanism involved in regulating MyHCβ gene expression, we analyzed the −2.4‐kb MyHCβ 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 MyHCβ promoter were shown to be calcineurin‐dependent. Together, our findings demonstrate calcium‐ionophore‐induced activation of the β MyHC promoter by NFATc1, MyoD, MEF‐2D, and p300 in a calcineurin‐dependent manner. J. Cell. Physiol. 211: 138–148, 2007. © 2006 Wiley‐Liss, Inc.