Calcineurin Broadly Regulates the Initiation of Skeletal Muscle-Specific Gene Expression by Binding Target Promoters and Facilitating the Interaction of the SWI/SNF Chromatin Remodeling Enzyme.

Hanna Witwicka,Kazumitsu Maehara,Sabriya A Syed,Yasuyuki Ohkawa,Anthony N Imbalzano,Jumpei Nogami,Teresita Padilla-Benavides

doi:10.1128/mcb.00063-19

Abstract

Calcineurin (Cn) is a calcium-activated serine/threonine protein phosphatase that is broadly implicated in diverse cellular processes, including the regulation of gene expression. During skeletal muscle differentiation, Cn activates the nuclear factor of activated T-cell (NFAT) transcription factor but also promotes differentiation by counteracting the negative influences of protein kinase C beta (PKCβ) via dephosphorylation and activation of Brg1, an enzymatic subunit of the mammalian SWI/SNF ATP-dependent chromatin remodeling enzyme. Here we identified four major temporal patterns of Cn-dependent gene expression in differentiating myoblasts and determined that Cn is broadly required for the activation of the myogenic gene expression program. Mechanistically, Cn promotes gene expression through direct binding to myogenic promoter sequences and facilitating the binding of Brg1, other SWI/SNF subunit proteins, and MyoD, a critical lineage determinant for skeletal muscle differentiation. We conclude that the Cn phosphatase directly impacts the expression of myogenic genes by promoting ATP-dependent chromatin remodeling and formation of transcription-competent promoters.

Highlights

Calcineurin (Cn) is a calcium-activated serine/threonine protein phosphatase that is broadly implicated in diverse cellular processes, including the regulation of gene expression
To better understand the involvement of Cn in the gene regulation program underlying myogenesis, we compared the transcriptomes of C2C12 myoblasts at three time points of differentiation (0, 24, and 72 h after induction of differentiation) in cells treated with the Cn inhibitor FK506 to those of control cells
C2C12 myoblasts treated with FK506 showed significant inhibition of myotube formation at the 48-h and 72-h time points compared to the dimethyl sulfoxide (DMSO) control, confirming prior results demonstrating that inhibition of Cn activity blocked myoblast differentiation [39,40,41] (Fig. 1A)

Summary

Introduction

Calcineurin (Cn) is a calcium-activated serine/threonine protein phosphatase that is broadly implicated in diverse cellular processes, including the regulation of gene expression. Cn activates the nuclear factor of activated T-cell (NFAT) transcription factor and promotes differentiation by counteracting the negative influences of protein kinase C beta (PKC␤) via dephosphorylation and activation of Brg, an enzymatic subunit of the mammalian SWI/SNF ATP-dependent chromatin remodeling enzyme. Myoblast differentiation is an essential process during skeletal muscle development where mononuclear myoblasts withdraw from the cell cycle and undergo fusion and other morphological changes to form multinucleated myotubes This process is coordinated by the family of myogenic regulatory factors (MRFs), whose members include MyoD, myogenin, Myf, and Mrf, in cooperation with the mouse embryonic fibroblast (MEF) family of transcription factors and other auxiliary transcriptional regulators. P38 mitogen-activated protein kinase (MAPK) phosphorylates the Baf60c subunit, which allows the recruitment of the rest of SWI/SNF remodeling complex to myogenic promoters [24]. We showed that Cn is bound to Brg at the myogenin promoter and that it dephosphorylates Brg shortly after cells start the differentiation process to positively promote differentiation [20]

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