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.

Brooke-Spiegler syndrome (BSS), familial cylindromatosis (FC), and multiple familial trichoepithelioma (MFT), originally described as distinct inherited disorders, are characterized by a variety of skin appendage neoplasms. Mutations in the CYLD gene are found in individuals with these syndromes. We describe a single family with affected members exhibiting either the FC or the MFT phenotypes associated with a mutation in the CYLD gene. These findings support the notion that BSS, FC, and MFT represent phenotypic variation of a single defect. Of interest, one of the affected individuals described in this report exhibits a severe phenotype illustrating the morbidity of the disorder.

Central and peripheral mammalian circadian clocks regulate a variety of behavioral and physiological processes through the rhythmic transcription of hundreds of clock-controlled genes. The circadian expression of many transcriptional regulators suggests that a major part of this circadian gene network is indirectly regulated by clock genes. Here we show that the basic helixloop-helix transcriptional repressor Stra13 is rhythmically expressed in mouse peripheral organs. The circadian transcription of Stra13 is mediated by a response element recognized by the CLOCK-BMAL1 heterodimer and located in the proximal promoter region. CLOCK-BMAL1-dependent activation of Stra13 is strongly repressed by CRY1 and also by STRA13 itself. To determine putative Stra13 output genes, we performed microarray analyses of differential gene expression in the liver between wild type and Stra13 ؊/؊ mice and identified 42 target genes including a subset of 20 previously known as clock-controlled genes. Importantly, we demonstrate that circadian gene expression of the serum protein insulin-like growth factor-binding protein 1 and of the NKG2D receptor ligand retinoic acid early transcript was suppressed in Stra13 ؊/؊ mice. These biochemical and genetic data establish a role for the basic helix-loop-helix repressor STRA13 as a circadian output regulator in the periphery.Circadian rhythms in physiology and behavior are observed in most organisms. They are generated by a self-sustained endogenous circadian clock that is reset by external time cues such as light and temperature (1). This mechanism is believed to provide organisms with an anticipatory adaptive mechanism to the daily predictable changes in their environment. Biochemical and genetic studies in various model systems have identified a molecular oscillator generated by transcriptional/ translational feedback loops. In mammals, the main loop involves the E box-mediated transcriptional activation of the Per1, Per2, Per3, Cry1, and Cry2 clock genes by the CLOCK-BMAL1 heterodimer. Then PER and CRY proteins form complexes that enter into the nucleus in a phosphorylation-dependent manner to repress the CLOCK-BMAL1-dependent transcription of their own genes, thereby generating a ϳ24-h period molecular oscillator (2). This loop controls also the rhythmic expression of the repressor REV-ERB␣, which is required for rhythmic Bmal1 transcription, comprising a second loop thought to be important for the overall robustness of the oscillator (3). In mammals, the master oscillator is present in the suprachiasmatic nuclei (SCN) 1 of the hypothalamus, which orchestrates autonomous oscillators in peripheral organs. Surprisingly, this oscillator can be observed in synchronized cultured cells ex vivo. The SCN oscillator is directly reset by light perceived and transmitted via the retinohypothalamic tract and is believed to entrain peripheral oscillators via ill-defined neurohormonal pathways. Peripheral oscillators also appear to be reset by hormonal signals and the feeding schedule (4, 5). The mechani...

We describe the expression pattern of mouse Sharp-1 (mSharp-1), a member of the basic helix-loop-helix (bHLH) family of transcription factors. mSharp-1 belongs to the Hairy/Enhancer of Split (E(Spl)) subfamily of bHLH factors that are key targets of the Notch signaling pathway and exhibits the highest sequence identity with Stra13. RNA in situ hybridization analysis from embryonic day 7.5 (E7.5) to E16.5 revealed specific expression of mSharp-1 in several developing organs during mouse embryogenesis. In early stage embryos (E8.5-E12.5), mSharp-1 is expressed in specific dorsal regions of the developing brain, the heart, the developing eye and olfactory system, as well as in the limb buds. At later stages (E12.5-E16.5), mSharp-1 is also expressed in the liver, prevertebrae, and the developing adrenal and thyroid glands. The diversity of its expression pattern suggests that mSharp-1 may regulate the differentiation of several cell types during vertebrate development.