Opposing calcium-dependent signalling pathways control skeletal muscle differentiation by regulating a chromatin remodelling enzyme.

Brian T Nasipak,Karin M Green,Teresita Padilla-Benavides,Saïd Sif,Scott A Shaffer,Silvana Konda,Wenjie Mao,John D Leszyk,Anthony N Imbalzano,Yasuyuki Ohkawa

doi:10.1038/ncomms8441

Abstract

Calcium signaling is important for differentiation-dependent gene expression, but is also involved in other cellular functions. Therefore mechanisms must exist to distinguish calcium signaling relevant to differentiation. Calcineurin is a calcium-regulated phosphatase that is required for myogenic gene expression and skeletal muscle differentiation. Here, we demonstrate that inhibition of calcineurin blocks chromatin remodeling and that the Brg1 ATPase of the SWI/SNF chromatin remodeling enzyme, which is required for the activation of myogenic gene expression, is a calcineurin substrate. Furthermore, we identify the calcium-regulated classical protein kinase C beta (PKCβ) as a repressor of myogenesis and as the enzyme that opposes calcineurin function. Replacement of endogenous Brg1 with a phosphomimetic mutant in primary myoblasts inhibits myogenesis, while replacement with a non-phosphorylatable mutant allows myogenesis despite inhibition of calcineurin signaling, demonstrating the functionality of calcineurin/PKC modified residues. Thus the Brg1 chromatin remodeling enzyme integrates two antagonistic calcium-dependent signaling pathways that control myogenic differentiation.

Highlights

Calcium signalling is important for differentiation-dependent gene expression, but is involved in other cellular functions
We identify protein kinase C bI (PKCbI) as a repressor of myogenesis and as the kinase that opposes calcineurin function
We verified that inhibition of calcineurin, either with the drug FK506 or by overexpression of the calcineurin inhibitory peptide (CAIN)[23], blocked differentiation of C2C12 myoblasts and induction of myogenin (Supplementary Fig. 1a) and Myosin Heavy Chain (MyHC, Supplementary Fig. 1b), without altering levels of MyoD (Supplementary Fig. 1c)

Summary

Introduction

Calcium signalling is important for differentiation-dependent gene expression, but is involved in other cellular functions. We examined the Brg1-dependent chromatin remodelling of the myogenin promoter in the presence of FK506, and found that inhibition of calcineurin activity by FK506 led to a loss of chromatin remodelling activity (Fig. 1a) as well as loss of Brg[1] binding at the myogenin promoter (Fig. 1b), suggesting that calcineurin activity is required for the interaction of the SWI/SNF ATPase with chromatin in differentiating cells.

Results

Conclusion