Sharp-1/DEC2 inhibits skeletal muscle differentiation through repression of myogenic transcription factors.

Skeletal muscle differentiation is regulated by the basic-helix-loop-helix (bHLH) family of transcription factors. The myogenic bHLH factors form heterodimers with the ubiquitously expressed bHLH E-proteins and bind E-box (CANNTG) sites present in the promoters of several muscle-specific genes. Our previous studies have shown that the bHLH factor Sharp-1 is expressed in skeletal muscle and interacts with MyoD and E-proteins. However, its role in regulation of myogenic differentiation remains unknown. We report here that endogenous Sharp-1 is expressed in proliferating C2C12 myoblasts and is down-regulated during myogenic differentiation. Constitutive expression of Sharp-1 in C2C12 myoblasts promotes cell cycle exit causing a decrease in cyclin D1 expression but blocks terminal differentiation. Although MyoD expression is not inhibited, the induction of differentiation-specific genes such as myogenin, MEF2C, and myosin heavy chain is impaired by Sharp-1 overexpression. We demonstrate that the interaction of Sharp-1 with MyoD and E-proteins results in reduced DNA binding and transactivation from MyoD-dependent E-box sites. Re-expression of MyoD approximately E47 rescues the differentiation defect imposed by Sharp-1, suggesting that myogenic bHLH factors function downstream of Sharp-1. Our data suggest that protein-protein interactions between Sharp-1, MyoD, and E47 resulting in interference with MyoD function underlies Sharp-1-mediated repression of myogenic differentiation.

mSharp-1/DEC2, a basic helix-loop-helix protein functions as a transcriptional repressor of E box activity and Stra13 expression.

Transcription factors belonging to the basic helix-loop-helix (bHLH) family play critical roles in the regulation of cellular differentiation of distinct cell types. In this study, we have characterized the DNA-binding and transcriptional properties of the bHLH factor mSharp-1/DEC2. mSharp-1 belongs to the Hairy/Enhancer of Split subfamily of bHLH factors and exhibits the highest structural and sequence identity with Stra13. We show that mSharp-1 specifically binds to the E box motif (CANNTG) as a homodimer and acts as a potent transcriptional repressor of MyoD- and E12-induced E box activity and differentiation. The inhibitory activity of mSharp-1 occurs through several mechanisms including occupancy of E box sites by mSharp-1 homodimers and by direct physical interaction with MyoD and E proteins. Furthermore, by using gel mobility shift assays and chromatin immunoprecipitation experiments, we have identified Stra13 as a target for mSharp-1-mediated repression. We demonstrate that transcriptional repression of Stra13 depends, in part, on binding of mSharp-1 to three conserved E box motifs in the Stra13 proximal promoter. Moreover, mSharp-1 directly interacts with the transcriptional activator Sp1 and impairs Sp1 induction of Stra13 promoter. Our results suggest that mSharp-1 functions as a transcriptional repressor by DNA binding dependent and independent mechanisms.

Basal transcription of the mouse sarco(endo)plasmic reticulum Ca2+-ATPase type 3 gene in endothelial cells is controlled by Ets-1 and Sp1.

We reported previously that the sarco(endo)plasmic reticulum Ca(2+)-ATPase type 3 (SERCA3) gene is expressed in many tissues and in a subset of cells such as endothelial, epithelial, and lymphoid lineages. Here we analyzed the mechanisms involved in the regulation of transcription of the SERCA3 gene in endothelial cells. The promoter of the murine SERCA3 gene was isolated, and a single transcription initiation site located 301 bp upstream of the translation initiation site was identified. Analysis of the transcriptional activity of fragments of the SERCA3 promoter showed the existence of a minimal promoter region located between bases -97 and +153 that contains one ETS-binding site (EBS) and two Sp1 elements that are essential for basal transcription. Mutation of the EBS or of the Sp1 sites abolished the basal activity of the promoter. We identified Ets-1 and Sp1 among endothelial nuclear factors that recognize the EBS and Sp1 sites on the promoter. Furthermore, transactivation of the -97/+301 promoter fragment by Ets-1 requires the presence of both the EBS and Sp1 sites, suggesting an interaction of the transcription factors on the gene promoter. Finally, overexpression of Ets-1 induced the expression of SERCA3 in endothelial cells and in fibroblasts.