mSharp-1/DEC2, a Basic Helix-Loop-Helix Protein Functions as a Transcriptional Repressor of E Box Activity and Stra13 Expression

Abstract

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...

Adaptive Myogenesis under Hypoxia

“…8C). Furthermore, we examined the expression of DEC2, a homolog of DEC1/Stra13 that can suppress the transcription activity of myoD protein (2). As shown by RT-PCR, the expression of DEC2 does not change significantly in C2C12 cells up to 3 days at 0.5 or 0.01% O 2 (Fig.…”

“…2 , 0.1 mM EDTA, 0.5% NP-40, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 320 mM sucrose]. After centrifugation, the cytoplasmic fractions were collected and stored at Ϫ80°C until use.…”

“…The regulation of myoD transcription by hypoxia is correlated with transient deacetylation of histones associated with the myoD promoter. Furthermore, myofibers formed under hypoxia appeared to be smaller than those that developed at 21% O 2 . This observation provides a molecular basis to explain the contribution of hypoxia to the morphological differences found in regenerated myofibers under chronic ischemia, as well as skeletal muscle from highland dwellers.…”

BHLHB3: a candidate tumor suppressor in lung cancer

“…In addition, we found a direct correlation between BHLHB3 and BHLHB2 transcript levels (Spearman's r ¼ 0.508; P ¼ 0.006), whereas (Li et al, 2003) described an inverse relationship between BHLHB3 and BHLHB2 expression. On the basis of a previous study indicating repression of mouse Bhlhb2 promoter activity by Bhlhb3 transfection in mouse fibroblast cell line 10T1/2 (Azmi et al, 2003), we tested the hypothesis that BHLHB3 directly affects mRNA levels of BHLHB2. Measurement of BHLHB2 mRNA levels in NCI-H520 lung cancer cells stably transfected to overexpress BHLHB3 (see Figure 1 legend for details of kinetic reverse transcriptase) revealed only a slight decrease (0.56±0.10-fold, mean±s.e., n ¼ 12) as compared to vector-transfected cells, arguing against any significant, if any, direct modulation of BHLHB2 expression by BHLHB3 and instead, suggesting that the observed correlation between BHLHB2 and BHLHB3 expression in the normal lung and lung ADCA tissues rests in common transcription regulation.…”

Small Ubiquitin-like Modifier (SUMO) Protein-specific Protease 1 De-SUMOylates Sharp-1 Protein and Controls Adipocyte Differentiation

“…Sharp-1, a basic helix-loop-helix transcription factor, is one of the transcriptional corepressors modulating C/EBPs and PPAR␥ activity. Sharp-1 binds to class B E-box sites with high affinity to repress the transcription of target genes and also associates with distinct corepressors, including HDAC1, SIRT1, and the histone methyltransferase G9a, to inhibit gene transcription (7). Taneja and co-workers (8) found that Sharp-1 interacts with and inhibits the transcriptional activity of both C/EBP␤ and C/EBP␣ by retaining HDAC1 and G9a at the C/EBP regulatory sites on the C/EBP␣ and PPAR␥2 promoters to inhibit their expression and, thus, adipogenesis, identifying Sharp-1 function as a negative regulator during adipogenesis.…”