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Basic helix-loop-helix (bHLH) proteins are instrumental in determining cell type during development. A bHLH protein, termed NeuroD, for neurogenic differentiation, has now been identified as a differentiation factor for neurogenesis because (i) it is expressed transiently in a subset of neurons in the central and peripheral nervous systems at the time of their terminal differentiation into mature neurons and (ii) ectopic expression of neuroD in Xenopus embryos causes premature differentiation of neuronal precursors. Furthermore, neuroD can convert presumptive epidermal cells into neurons and also act as a neuronal determination gene. However, unlike another previously identified proneural gene (XASH-3), neuroD seems competent to bypass the normal inhibitory influences that usually prevent neurogenesis in ventral and lateral ectoderm and is capable of converting most of the embryonic ectoderm into neurons. The data suggest that neuroD may participate in the terminal differentiation step during vertebrate neuronal development.

The myoD gene converts many differentiated cell types into muscle. MyoD is a member of the basic-helix-loop-helix family of proteins; this 68-amino acid domain in MyoD is necessary and sufficient for myogenesis. MyoD binds cooperatively to muscle-specific enhancers and activates transcription. The helix-loop-helix motif is responsible for dimerization, and, depending on its dimerization partner, MyoD activity can be controlled. MyoD senses and integrates many facets of cell state. MyoD is expressed only in skeletal muscle and its precursors; in nonmuscle cells myoD is repressed by specific genes. MyoD activates its own transcription; this may stabilize commitment to myogenesis.

With modified two-hybrid technology, we have isolated a member of a new family of basic helix-loop-helix (bHLH) transcription factors. Thing1 (Th1) was identified in a screen of a mouse embryo cDNA library as a partner for the Drosophila E protein daughterless. RNA in situ hybridization and reverse transcriptase-PCR demonstrate a stage-and tissue-specific distribution for the expression of Th1. Although tissue specific, the expression pattern of Th1 is fairly complex. During development, Th1 mRNA is widely expressed in extraembryonic tissues, portions of the heart, autonomic ganglia, the gut, and pharyngeal arches. At embryonic day 7.5 (E7.5), extraembryonic derivatives show robust Th1 expression. By E8.5, expression in the embryonic heart becomes detectable. During the next 2 days of development, the signal also includes gut and pharyngeal arches. Predominant expression at E13.5 is in neural crest derivatives, especially the autonomic nervous system and adrenal medulla. Expression of Th1 persists in the adult, in which it is localized to the smooth muscle cells of the gut. In vitro, Th1 protein recognizes a set of DNA sites that are more degenerate than has been determined for other bHLH factors, indicating a reduced binding specificity. Transient transfection of NIH 3T3 cells with GAL4-Th1 fusions reveals a repression activity mediated by the Th1 bHLH domain. In combination, these properties define Th1 as a new bHLH protein with a unique set of properties.During development, specific genetic programs are activated by transcription factors which are often stage and tissue specific in their distribution. Understanding the combinatorial code of factors which defines each cell fate during the differentiation process requires identification of new factors with restricted spatial activity. One group of factors with pivotal roles during development are the basic helix-loop-helix (bHLH) proteins. These factors are important in differentiation processes as diverse as skeletal myogenesis, neurogenesis, and hematopoiesis (for reviews, see references 2, 12, 13, 43, 59, and 63).bHLH proteins have been grouped into four classes based on partner choice, DNA-binding ability, and transcriptional activity. (i) E proteins, also called class A, are widely expressed and act as partners for at least two of the other classes (6,42,47). (ii) Tissue-specific bHLH proteins, or class B, pair with E proteins to produce heterodimers. Class A homodimers and A/B heterodimers are thought to function as transcriptional activators (9,46,62). (iii) Id proteins lack a basic region and form a non-DNA-binding complex with class A and some class B proteins (6, 55). (iv) Hairy and Enhancer-of-split [h/E(spl)] bHLH proteins form a fourth class whose interactions with the other three groups are not well characterized. The h/E(spl) family is structurally defined, in part, by a WRPW motif at the C terminus and an absolutely conserved proline in the basic region. These proteins act genetically as repressors of bHLHmediated functions and can bind to DNA v...

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We focus on the mechanism by which MyoD activates transcription. Previous experiments showed that when the 13-amino-acid basic region of El2 replaced the corresponding basic region of MyoD, the resulting MyoD-E12Basic chimeric protein could bind specifically to muscle-specific enhancers in vitro and form dimers with El2, but could not activate a cotransfected reporter gene or convert 10T1A cells to muscle. Here we show that back mutation of this chimeric protein (with the corresponding residues in MyoD) re-establishes activation, and we identify a specific alanine involved in increasing DNA binding and a specific threonine required for activation. Using a reporter gene containing MyoD-binding sites located downstream from the transcription start site, we show that MyoD-E12Basic can bind in vivo and thereby inhibit expression of the reporter. In vivo binding is also supported by the fact that the addition of the "constitutive" VPI6 activation domain to MyoD-E12Basic restores full trans-activation potential. The normal MyoD-activation domain maps within the amino-terminal 53 residues and can be functionally replaced by the activation domain of VP16. The activity of the MyoD-activation domain is dramatically elevated when deletions are made almost anywhere in the rest of the MyoD molecule, suggesting that the activation domain in MyoD is usually masked. Surprisingly, MyoD-E12Basic can activate transcription in CV1 and B78 cells (but not in 10T1A or 3T3 cells), suggesting that the activation function of the basic domain requires a specific factor present in CV1 and B78 cells. We propose that to function, the masked MyoD-activation domain requires the participation of a second factor that recognizes the basic region. We refer to such a factor as a recognition factor.

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