Sharp-1/DEC2 Inhibits Skeletal Muscle Differentiation through Repression of Myogenic Transcription Factors

Abstract

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 MyoDϳE47 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.

BHLHB3: a candidate tumor suppressor in lung cancer

“…As an additional assay of apoptosis, we used an anti-PARP antibody (Cell Signaling Tech Inc., Danvers, MA, USA) by western blot in stable transfected cells; again, we found no evidence of PARP protein cleavage (not shown). Instead, quantitative PCR of NCI-H520 cells stably transfected to overexpress BHLHB3 (Figure 3) and using cyclin D1 as a marker of proliferation (Berthet and Kaldis, 2007) revealed a decrease (0.25 ± 0.04-fold, n ¼ 8) of cyclin D1 mRNA expression (measured using Hs00277039_m1 (CCND1) TaqMan Gene Expression Assays; Applied Biosystems, Foster City, CA, USA) in BHLHB3-transfected versus vector-transfected cells, consistent with western blot analysis ( Figure 3) and with a previous study (Azmi et al, 2004) showing that BHLHB3 overexpression in mouse myoblast cell line C2C12 promotes cell cycle exit in association with decreased levels of cyclin D1 protein expression. Indeed, inhibition of cyclin D1 protein expression is associated with cell cycle arrest in cancer cell lines (Berthet and Kaldis, 2007).…”

MyoD and E-protein heterodimers switch rhabdomyosarcoma cells from an arrested myoblast phase to a differentiated state

“…The Id proteins are HLH factors that lack the basic DNA-binding domain and competitively heterodimerize with E-proteins, preventing DNA binding to an E-box (Benezra et al 1990). In addition, Eproteins can heterodimerize with bHLH transcriptional repressors, such as HES proteins (Dec2) (Azmi et al 2004;Fujimoto et al 2007) or Twist (Kophengnavong et al 2000) or, as in this study, MSC (see also Lu et. al.…”

Differentiated Embryo Chondrocyte 1 (DEC1) Represses PPARγ2 Gene through Interacting with CCAAT/Enhancer Binding Protein β (C/EBPβ)

“…Although DEC1 prevents neither C/EBPβ nor RXR from binding to its target gene, DEC1 prevents bHLH domain containing transactivators such as BMAL1, MyoD and SREBP-1c, from binding to their target DNA by either protein-protein interaction or by directly binding to their responsive elements containing an E-box (Azmi et al, 2004;Choi et al, 2008;Li et al, 2004). Recently, Gulbagci et al demonstrated that DEC2, isoform of DEC1, also interacts with C/EBPβ and increases the recruitment of histone methyltransferase G9a to the promoter of PPARγ2 (Gulbagci et al, 2009).…”