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

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.

The Bromodomains of the mammalian SWI/SNF (mSWI/SNF) ATPases Brahma (BRM) and Brahma Related Gene 1 (BRG1) promote chromatin interaction and are critical for skeletal muscle differentiation

“…Chromatin immunoprecipitation assays were performed as described previously ( 73 ). Quantification was performed using the fold enrichment method (2 –(Ct sample – Ct IgG) ) and shown as relative to a control region.…”

Regulation of the Mammalian SWI/SNF Family of Chromatin Remodeling Enzymes by Phosphorylation during Myogenesis

“…In vitro work identified serine residues N-and C-terminal to the bromodomain of BRG1 as targets of PKCβ 1 and calcineurin [49]. Site directed mutagenesis studies demonstrated that mutation of these sites to phosphomimetic residues prevented myogenesis because the phosphomimetic BRG1 mutant was unable to bind to myogenic promoters while mutation of PKCβ 1 target sites to non-phosphorylatable (alanine) residues had no effect on chromatin binding or differentiation [49,188]. Calcineurin inhibition also blocked the interaction of other subunits of the mSWI/SNF complex with myogenic promoters [188].…”

“…Site directed mutagenesis studies demonstrated that mutation of these sites to phosphomimetic residues prevented myogenesis because the phosphomimetic BRG1 mutant was unable to bind to myogenic promoters while mutation of PKCβ 1 target sites to non-phosphorylatable (alanine) residues had no effect on chromatin binding or differentiation [49,188]. Calcineurin inhibition also blocked the interaction of other subunits of the mSWI/SNF complex with myogenic promoters [188]. This raises the possibility of regulated dephosphorylation of other mSWI/SNF subunits or an indirect effect due to the failure to dephosphorylate BRG1.…”

CK2-Dependent Phosphorylation of the Brg1 Chromatin Remodeling Enzyme Occurs during Mitosis

“…We and others have shown that the activity of Brg1 and other components of the mSWI/SNF complex is post-translationally regulated by various signaling pathways [16][17][18][19][20][21][22]. For instance, in the liver, BAF60c phosphorylation via the insulin-signaling pathway enables the expression of lipogenic genes [23].…”

“…mSWI/SNF enzymes are required for myoblast proliferation and at multiple stages of differentiation [27][28][29][30][31][32]. Regulated phosphorylation and dephosphorylation of Brg1 is an essential part of both proliferation and differentiation [16,17,22]. Phosphorylation of Brg1 by protein kinase C β1 (PKCβ1) prior to the induction of differentiation signaling has a repressive effect on the function of Brg1 and leads to a block of myogenic differentiation [16].…”