Transcription factors belonging to the basic helixloop-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.Members of the basic helix-loop-helix (bHLH) 1 superfamily of transcription factors are expressed in a wide range of tissues during development and are involved in the regulation of cell fate determination, myogenesis, neurogenesis, and hematopoiesis (1, 2). The common structures shared among the members of this superfamily are the basic domain, which is required for DNA binding, and the helix-loop-helix domain, which is involved in dimerization (3, 4).Based on the dimerization properties, tissue distribution, and the transcriptional activities, bHLH proteins can be categorized into three classes (5). Class A bHLH factors contain the mammalian "E" proteins, which include the two E2A gene products E12 and E47 as well as E2-2 and HEB. E proteins are ubiquitously expressed and can form homodimers or heterodimers with bHLH factors of the same class as well as with other classes. Class B bHLH proteins tend to be expressed in a tissue or cell type-specific manner and function as heterodimers with the class A bHLH factors. bHLH factors involved in tissue-specific differentiation generally belong to the class B subfamily and include the myogenic factors MyoD and myogenin, the neurogenic factors Mash1, NeuroD, and neurogenins, as well as the bHLH proteins SCL/TAL which are important for hematopoiesis (6 -14). Both class A and class B bHLH factors bind to a common DNA sequence called the E box (CANNTG) commonly found in the promoter or enhancer regions of numerous developmentally regulated genes (15) and function as transcriptional activators. Class C bHLH factors (2) contain the Drosophila Hairy and Enhancer of Split [E(Spl)] proteins, the mammalian Hes protei...

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.

Human platelets express several sarco/endoplasmic reticulum Ca P+ ATPase (SERCA) isoenzymes: SERCA2b of 100 kDa apparent molecular mass and two distinct enzymes of 97 kDa, one of them identified as being the SERCA3a isoform. The molecular identity of the third enzyme specifically recognized by the PL/IM430 monoclonal antibody has remained elusive. First, the study of the 3P-end part of platelet SERCA3 mRNA, by means of RT-PCR amplification using sets of primers covering the N33 to N (ultimate) exons of the human SERCA3 sequence, revealed the presence of two distinct mRNA sequences, SERCA3a and a longer variant. Second, this additional sequence was identified as SERCA3b and found to refer to the insertion of a new exon of 73 bp, located at bp 349 from the beginning of the intronic sequence, linking the penultimate (N31) exon to the last exon (N) of the human SERCA3 gene. Third, a relationship between the expression of this SERCA3b mRNA and the PL/ IM430 recognizable SERCA protein was observed. SERCA3b mRNA was found to be absent in epithelial HeLa cells not recognized by the PL/IM430 antibody and the expression of this SERCA3b RNA species correlated with that of the SERCA protein recognized by PL/IM430 which was down-modulated in the platelet precursor megakaryocytic CHRF 288-11 cell line as well as upon in vitro lymphocyte activation. Taken together, these results strongly support the notion of the presence of the SERCA3b protein in human cells by showing SERCA3b mRNA in platelets and the fact that the protein corresponding to this mRNA species is very likely the 97 kDa protein recognized by the PL/IM430 antibody.z 1998 Federation of European Biochemical Societies.

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