The helix-loop-helix (HLH) family of transcriptional regulatory proteins are key players in a wide array of developmental processes. Over 240 HLH proteins have been identified to date in organisms ranging from the yeast Saccharomyces cerevisiae to humans (6). Studies in Xenopus laevis, Drosophila melanogaster, and mice have convincingly demonstrated that HLH proteins are intimately involved in developmental events such as cellular differentiation, lineage commitment, and sex determination. In yeast, HLH proteins regulate several important metabolic pathways, including phosphate uptake and phospholipid biosynthesis (19,67,112). In multicellular organisms, HLH factors are required for a multitude of important developmental processes, including neurogenesis, myogenesis, hematopoiesis, and pancreatic development (12,86,127,179). The purpose of this review is to examine the structure and functional properties of HLH proteins.E-box sites: elements mediating cell-type-specific gene transcription. Gene transcription of the immunoglobulin heavychain (IgH) gene has long been known to be regulated, in part, by a cis-acting DNA element known as the IgH intronic enhancer (109,156). By in vivo methylation protection assays, a number of sites were identified in both the IgH and the kappa light-chain gene enhancers which were specifically protected in B cells but not in nonlymphoid cells (41). These elements shared a signature motif which consisted of the core hexanucleotide sequence, CANNTG, and were subsequently dubbed E boxes (41). A total of five E-box elements are present in the IgH gene enhancer: E1, E2, E3, E4, and E5. The Ig kappa enhancer also contains three cannonical E boxes, designated E1, E2, and E3. E-box sites have been subsequently found in B-cell-specific promoter and enhancer elements, including a subset of Ig light-chain gene promoters, the IgH and Ig light-chain 3Ј enhancers, and, more recently, the 5 promoter (110,118,156).E-box elements have also been identified in promoter and enhancer elements that regulate muscle-, neuron-, and pancreas-specific gene expression. For example, in muscle, the muscle creatine kinase gene, acetylcholine receptor genes ␣ and ␦, and the myosin light-chain gene all require E-box elements for full activity (27,51,85). A number of genes whose expression is limited to the pancreas also require E-box sites for proper expression. The insulin and somatostatin genes, for example, contain E-box sites that, when multimerized, are sufficient to regulate pancreatic ␤-cell-specific gene expression (168). More recently, E-box regulatory sites have been identified in a number of neuron-specific genes, including the opsin, hippocalcin, beta 2 subunit of the neuronal nicotinic acetylcholine receptor, and muscarinic acetylcholine receptor genes (1, 21, 52, 125).E-box sites: cognate recognition sequence for HLH proteins. Two proteins, termed E12 and E47, were originally identified as binding to the E2/E5 site (65, 102). They have a region of homology with the Drosophila Daughterless protein, the...

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Previous studies demonstrated that the amino-terminal portions of E2A and E2-2 are crucial for transactivation. Subsequent findings showed that the same amino-terminal region of E2A is involved in two different translocation events contributing to the induction of a pre-B-cell acute lymphoblastic leukemia and a pro-B-cell acute lymphoblastic leukemia. These results led us to focus on the amino-terminal region of E2A to better understand its normal role in transcriptional regulation and its aberrant involvement in the two leukemias. We report here the identification of two conserved boxes in the E2A amino-terminal domain that show extensive homology within the transactivation domains of E12, E47, E2-2, HEB, and daughterless, all members of the same class of helix-loop-helix proteins. Together, both boxes are crucial for transcriptional activation and have the potential to form a new activation motif, that of a loop adjacent to an amphipathic a-helix, designated the loop-helix (LH) motif. A minimal region containing the LH motif is sufficient for transcriptional activation. Point mutations in the amphipathic helix of the minimal region reduce its transactivation capabilities dramatically. The same constructs expressed in yeast cells show identical patterns of activation, suggesting that the LH motif and its target proteins are functionally conserved in yeast cells. We propose that the LH motif represents a novel transactivation domain that is distinct from the previously characterized acidic blob, proline-rich, and glutamine-rich activation motifs. In addition, the LH motif is the first activation motif restricted to one class of DNA binding proteins.The helix-loop-helix (HLH) proteins are a growing family of proteins containing a conserved structural motif (HLH) that mediates DNA binding and dimerization (7, 18a, 19, 27).These proteins play a major role in the control of cell type differentiation by forming homo-and heterodimers with themselves and other HLH proteins (7, 18a, 19, 27). The members of one particular class of HLH proteins (class I) are closely related (70 to 90% identity in the HLH region), are ubiquitously expressed, and include E12 (7, 18a, 19, 21, 27), E47 (7, 9, 18a, 19, 21, 27), E2-2 (9), HEB (10), and daughterless (3, 6). E12 and E47 are encoded by the same gene (E2A) but arise through differential splicing (26). The E2A gene products are involved in B-cell-, muscle-, and pancreas-specific gene expression (2,4,14,20). For example, the presence of the E2A gene products or other ubiquitous class I HLH molecules is necessary for the mediation of specific differentiation programs by the formation of heterodimers with tissue-specific HLH proteins, such as MyoD, myogenin, and members of the achaete-scute protein family.In contrast to its normal role in regulating transcription, the E2A gene has been found to be translocated in two acute lymphoblastoid leukemias (ALL). In pro-B-cell ALL, containing a t(17;19) translocation, the E2A N-terminal domain is fused to a leucine zipper-like domain (11). In ...

The class I helix-loop-helix (HLH) proteins, which include E2A, HEB, and E2-2, have been shown to be required for lineage-specific gene expression during T and B lymphocyte development. Additionally, the E2A proteins function to regulate V(D)J recombination, possibly by allowing access of variable region segments to the recombination machinery. The mechanisms by which E2A regulates transcription and recombination, however, are largely unknown. Here, we identify a novel motif, LDFS, present in the vertebrate class I HLH proteins as well as in a yeast HLH protein that is essential for transactivation. We provide both genetic and biochemical evidence that the highly conserved LDFS motif stimulates transcription by direct recruitment of the SAGA histone acetyltransferase complex.

A conserved region, designated the AD1 domain, is present in a class of helix-loop-helix (HLH) proteins, E proteins, that includes E12, E47, HEB, E2-2, and a Xenopus laevis HLH protein closely related to E12. We demonstrate that the AD1 domain in E2A and the conserved region of E2-2 activate transcription in both yeast and mammalian cells. The AD1 domain contains a highly conserved putative helix that is crucial for its transactivation properties. Circular dichroism spectroscopy data show that AD1 is structured and contains distinctive helical properties. In addition, we show that a synthetic peptide corresponding to the conserved region is unstructured in aqueous solution at neutral pH but can adopt an ␣-helical conformation in the presence of the hydrophobic solvent trifluoroethanol. Amino acid substitutions that destabilize the helix abolish the transactivation ability of the AD1 domain. Both structural and functional analyses of AD1 reveal striking similarities to the acidic class of activators. Remarkably, when wild-type and mutant proteins are expressed in mammalian cells and Saccharomyces cerevisiae, identical patterns of transactivation are observed, suggesting that the target molecule is conserved between S. cerevisiae and mammals. These data show that transactivation by E proteins is mediated, in part, by a strikingly conserved peptide that has the ability to form a helix in a hydrophobic solvent. We propose that the unstructured domain may become helical upon interaction with its cellular target molecule.