SMART MODE:

NORMAL GENOMIC

• Quattrocchio F, Verweij W, Kroon A, Spelt C, Mol J, Koes R
• PH4 of Petunia Is an R2R3 MYB Protein That Activates Vacuolar Acidification through Interactions with Basic-Helix-Loop-Helix Transcription Factors of the Anthocyanin Pathway.
• Plant Cell. 2006; 18: 1274-91
• Display abstract

• The Petunia hybrida genes ANTHOCYANIN1 (AN1) and AN2 encode transcription factors with a basic-helix-loop-helix (BHLH) and a MYB domain, respectively, that are required for anthocyanin synthesis and acidification of the vacuole in petal cells. Mutation of PH4 results in a bluer flower color, increased pH of petal extracts, and, in certain genetic backgrounds, the disappearance of anthocyanins and fading of the flower color. PH4 encodes a MYB domain protein that is expressed in the petal epidermis and that can interact, like AN2, with AN1 and the related BHLH protein JAF13 in yeast two-hybrid assays. Mutation of PH4 has little or no effect on the expression of structural anthocyanin genes but strongly downregulates the expression of CAC16.5, encoding a protease-like protein of unknown biological function. Constitutive expression of PH4 and AN1 in transgenic plants is sufficient to activate CAC16.5 ectopically. Together with the previous finding that AN1 domains required for anthocyanin synthesis and vacuolar acidification can be partially separated, this suggests that AN1 activates different pathways through interactions with distinct MYB proteins.

• Azmi S, Ozog A, Taneja R
• Sharp-1/DEC2 inhibits skeletal muscle differentiation through repression of myogenic transcription factors.
• J Biol Chem. 2004; 279: 52643-52
• Display abstract

• 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 MyoD approximately E47 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.

• Zang MX, Li Y, Xue LX, Jia HT, Jing H
• Cooperative activation of atrial naturetic peptide promoter by dHAND and MEF2C.
• J Cell Biochem. 2004; 93: 1255-66
• Display abstract

• An intricate array of cell-specific multiprotein complexes participate in programs of cell-specific gene expression through combinatorial interaction with different transcription factors and cofactors. The dHAND basic helix-loop-helix (bHLH) transcription factor, which is essential for heart development and extra embryonic structures, is thought to regulate cardiomyocyte-specific gene expression through combinatorial interactions with other cardiac-restricted transcription factors such as GATA4 and NKX2.5. Here, we determine that dHAND also interacts with the myocyte enhancer binding factor-2c (MEF2C) protein, which belongs to MADS-box transcription factors and is essential for heart development. dHAND and MEF2C synergistically activated expression of the atrial naturetic peptide gene (ANP) in transfected HeLa cells. GST-pulldown and immunoprecipitation assay demonstrate that full-length MEF2C protein is able to interact with dHAND in vitro and in vivo, just like MEF2A and bHLH transcription factors MyoD in skeletal muscle cells. In addition, electrophoretic mobility shift assays (EMSAs) demonstrate that MEF2C and dHAND do not influence each other's DNA binding activity. Using chromatin immunoprecipitation (ChIP) analysis in H9c2 cells we show that dHAND interact with MEF2C to form protein complex and bind A/T sequence in promoter of ANP. Taken together with previous observations, these results suggest the existence of large multiprotein transcriptional complex with core DNA binding proteins that physically interact with other transcriptional factors to form favorable conformation to potentiate transcription.

• Dodou E, Xu SM, Black BL
• mef2c is activated directly by myogenic basic helix-loop-helix proteins during skeletal muscle development in vivo.
• Mech Dev. 2003; 120: 1021-32
• Display abstract

• Skeletal muscle development requires the coordinated expression of numerous transcription factors to control the specification of mesodermal progenitor cells to a muscle fate and the differentiation of those committed myoblasts into functional, contractile muscle. Two families of transcription factors play key roles in these processes. The myogenic basic helix-loop-helix (bHLH) proteins, MyoD and Myf5, are required for myoblast specification, while two members of the same family, myogenin and MRF4, play key roles in myoblast differentiation in vivo. All four members of the myogenic bHLH family are sufficient to dominantly induce myogenesis when introduced into a variety of non-muscle cells in culture, however this function requires the activity of a second family of transcriptional regulators, the myocyte enhancer factor 2 (MEF2) family. MEF2 factors are essential for muscle differentiation, and previous studies have shown that MyoD and MEF2 family members function combinatorially to activate transcription and myogenesis. Consistent with these observations, the majority of skeletal muscle genes require both MyoD and MEF2 family members to activate their transcription. A possible exception to this combinatorial model for activation is suggested by the observation that myogenic bHLH factors may be able to independently activate the expression of MEF2. This raises the question as to how mef2 gene transcription is induced by MyoD factors without cooperative activation by MEF2. During skeletal muscle development, mef2c is the first member of the MEF2 family to be expressed. In this study, we have investigated the regulation of a skeletal muscle-specific enhancer from the mouse mef2c gene using a transgenic approach. We show that mef2c is a direct transcriptional target of the MyoD family in vivo via an essential E box in the skeletal muscle enhancer of mef2c, and we show that mef2c is not a direct target for autoregulation by MEF2.

• Toledo-Ortiz G, Huq E, Quail PH
• The Arabidopsis basic/helix-loop-helix transcription factor family.
• Plant Cell. 2003; 15: 1749-70
• Display abstract

• The basic/helix-loop-helix (bHLH) proteins are a superfamily of transcription factors that bind as dimers to specific DNA target sites and that have been well characterized in nonplant eukaryotes as important regulatory components in diverse biological processes. Based on evidence that the bHLH protein PIF3 is a direct phytochrome reaction partner in the photoreceptor's signaling network, we have undertaken a comprehensive computational analysis of the Arabidopsis genome sequence databases to define the scope and features of the bHLH family. Using a set of criteria derived from a previously defined consensus motif, we identified 147 bHLH protein-encoding genes, making this one of the largest transcription factor families in Arabidopsis. Phylogenetic analysis of the bHLH domain sequences permits classification of these genes into 21 subfamilies. The evolutionary and potential functional relationships implied by this analysis are supported by other criteria, including the chromosomal distribution of these genes relative to duplicated genome segments, the conservation of variant exon/intron structural patterns, and the predicted DNA binding activities within subfamilies. Considerable diversity in DNA binding site specificity among family members is predicted, and marked divergence in protein sequence outside of the conserved bHLH domain is observed. Together with the established propensity of bHLH factors to engage in varying degrees of homodimerization and heterodimerization, these observations suggest that the Arabidopsis bHLH proteins have the potential to participate in an extensive set of combinatorial interactions, endowing them with the capacity to be involved in the regulation of a multiplicity of transcriptional programs. We provide evidence from yeast two-hybrid and in vitro binding assays that two related phytochrome-interacting members in the Arabidopsis family, PIF3 and PIF4, can form both homodimers and heterodimers and that all three dimeric configurations can bind specifically to the G-box DNA sequence motif CACGTG. These data are consistent, in principle, with the operation of this combinatorial mechanism in Arabidopsis.

• Spinner DS, Liu S, Wang SW, Schmidt J
• Interaction of the myogenic determination factor myogenin with E12 and a DNA target: mechanism and kinetics.
• J Mol Biol. 2002; 317: 431-45
• Display abstract

• The myogenic determination factors MyoD, myogenin, myf5, and MRF4 are members of the basic helix-loop-helix (bHLH) family of transcription factors and crucial agents of myogenesis. The bHLH regions of these proteins enable them to dimerize with E proteins, another class of the bHLH family, and to bind a specific DNA element known as an E box (CANNTG consensus sequence), which results in the activation of muscle-specific gene expression. As a model for such assembly of the myogenic determination factor/E protein-DNA ternary complex, we have studied the physiologically relevant association of myogenin, E12, and the 3' E box of the acetylcholine receptor (AChR) alpha-subunit gene enhancer. Using the technique of electrophoretic mobility shift assay combined with order-of-addition and time-course experiments, we find that heterodimerization of myogenin with E12 occurs prior to DNA-binding. In addition, we deduce the dissociation (Kd) and rate (k) constants for each step in the formation of the myogenin/E12-DNA ternary complex. Kinetic simulations indicate that at 37 degrees C myogenin and E12 heterodimerize with a Kd of 36 microM (k(on) of 573 M(-1) x s(-1) and k(off )of 0.0205 x s(-1)), and that subsequently the heterodimer binds the AChR alpha-subunit gene enhancer 3' E box with a Kd of 8.8 nM (with possible k(on) and k(off) values ranging from 1.0x10(8) to 14.1x10(8) M(-1) x s(-1), and 0.875 to 12.3 s(-1), respectively).

• Verzi MP et al.
• N-twist, an evolutionarily conserved bHLH protein expressed in the developing CNS, functions as a transcriptional inhibitor.
• Dev Biol. 2002; 249: 174-90
• Display abstract

• Members of the basic helix-loop-helix (bHLH) transcription factor family play an essential role in multiple developmental processes. During neurogenesis, positive and negative regulation by bHLH proteins is essential for proper development. Here we report the identification and initial characterization of the bHLH gene, Neuronal twist (N-twist), named for its neural expression pattern and high sequence homology and physical linkage to the mesodermal inhibitor, M-twist. N-twist is expressed in the developing mouse central nervous system in the midbrain, hindbrain, and neural tube. This neural expression is conserved in invertebrates, as expression of the Drosophila ortholog of N-twist is also restricted to the central nervous system. Like other bHLH family members, N-Twist heterodimerizes with E protein and binds DNA at a consensus bHLH-binding site, the E box. We show that N-Twist inhibits MASH1-dependent transcriptional activation by sequestering E protein in a dominant negative fashion. Thus, these studies support the notion that N-Twist represents a novel negative regulator of neurogenesis.

• Roy K, de la Serna IL, Imbalzano AN
• The myogenic basic helix-loop-helix family of transcription factors shows similar requirements for SWI/SNF chromatin remodeling enzymes during muscle differentiation in culture.
• J Biol Chem. 2002; 277: 33818-24
• Display abstract

• The myogenic basic helix-loop-helix family of transcription factors, MyoD, Myf5, myogenin, and MRF4, can each activate the muscle differentiation program when ectopically expressed in non-muscle cells. SWI/SNF complexes are ATP-dependent chromatin remodeling enzymes. We demonstrated previously that SWI/SNF enzymes promote MyoD-mediated muscle differentiation. To ascertain the requirement for SWI/SNF enzymes in muscle differentiation mediated by different MyoD family members, we examined MyoD, Myf5, MRF4, and myogenin-mediated induction of muscle differentiation in cells expressing dominant negative versions of BRG1 or BRM-based SWI/SNF enzymes. We demonstrated that expression of dominant negative BRG1 or BRM inhibited the induction of muscle-specific gene expression by Myf5 and MRF4; however, myogenin failed to induce measurable quantities of muscle-specific mRNAs, even in cells not expressing dominant negative SWI/SNF. In contrast, all four myogenic regulators induced expression of the cell cycle regulators p21, Rb, and cyclin D3 and promoted cell cycle arrest independently of the SWI/SNF enzymes. We proposed that SWI/SNF enzymes are required for the induction of all muscle-specific gene expression by MyoD, Myf5, and MRF4, whereas induction of the cell cycle regulators, p21, Rb, and cyclin D3 occurred independently of SWI/SNF function.

• Gong XQ, Li L
• Dermo-1, a multifunctional basic helix-loop-helix protein, represses MyoD transactivation via the HLH domain, MEF2 interaction, and chromatin deacetylation.
• J Biol Chem. 2002; 277: 12310-7
• Display abstract

• Dermo-1 is a multifunctional basic helix-loop-helix (bHLH) transcription factor that has been shown to be a potent negative regulator for gene transcription and apoptosis. To understand the molecular mechanisms that mediate the function of Dermo-1, we generated a series of Dermo-1 mutants and used a MyoD-mediated transcriptional activation model to characterize the roles of its N-terminal, bHLH, and C-terminal structural domains in transcriptional repression. Both the C-terminal and HLH domains of Dermo-1 were essential for its repression of MyoD-mediated transactivation. Dermo-1 repressed, in a dose-dependent fashion, the transactivation activity of myocyte enhancer factor 2 (MEF2), a protein known to cooperate with MyoD in activating E-box-dependent gene expression. Both the N- and C-terminal domains of Dermo-1, but not the bHLH domain, were required for the inhibition of MEF2, suggesting that Dermo-1 inhibits both MyoD- and MEF2-dependent transactivation but through different mechanisms. Dermo-1 interacted directly with MEF2 and selectively repressed the MEF2 transactivation domain. An overall increase of histone acetylation induced by trichostatin A treatment reduced Dermo-1 transcriptional repression activity, suggesting that histone deacetylation is involved in Dermo-1-mediated transcriptional repression. Together, these results suggest that MEF2 is an important target in Dermo-1-mediated transcriptional repression and provide initial evidence of the involvement of histone acetylation in Dermo-1 transcriptional repression.

• Ghil SH, Jeon YJ, Suh-Kim H
• Inhibition of BETA2/NeuroD by Id2.
• Exp Mol Med. 2002; 34: 367-73
• Display abstract

• Id (Inhibitor of Differentiation) proteins belong to a family of transcriptional modulators that are characterized by a helix loop helix (HLH) region but lack the basic amino acid domain. Id proteins are known to interact with basic helix-loop-helix (bHLH) transcription factors and function as their negative regulators. The negative role of Id proteins has been well demonstrated in muscle development and some in neuronal cells. In this study, we investigated the effect of Id on the function of BETA2/NeuroD, a bHLH transcription factor responsible for neuron and endocrine cell specific gene expression. cDNAs of several Id isoforms were isolated by yeast two-hybrid system using the bHLH domain of E47, a ubiquitous bHLH partner as a bait. Id proteins expressed in COS M6 cells, were found in both cytosolic and nuclear fractions. Electrophoretic mobility shift assay showed that coexpression of Id2 proteins inhibited BETA2/ NeuroD binding to its target sequence, E-box. Id2 inhibited E-box mediated gene expression in a dose dependent manner in BETA2/NeuroD expressing HIT cells. Id coexpressed with BETA2/NeuroD in HeLa cells, inhibited the stimulatory activity of BETA2/NeuroD. These results suggest that Id proteins may negatively regulate tissue specific gene expression induced by BETA2/NeuroD in neuroendocrine cells and the inhibitory role of Id proteins during differentiation may be conserved in various tissues.

• McFadden DG, McAnally J, Richardson JA, Charite J, Olson EN
• Misexpression of dHAND induces ectopic digits in the developing limb bud in the absence of direct DNA binding.
• Development. 2002; 129: 3077-88
• Display abstract

• Basic helix-loop-helix (bHLH) transcription factors control developmental decisions in a wide range of embryonic cell types. The HLH motif mediates homo- and heterodimerization, which juxtaposes the basic regions within the dimeric complex to form a bipartite DNA binding domain that recognizes a DNA consensus sequence known as an E-box. eHAND and dHAND (also known as HAND1 and HAND2) are closely related bHLH proteins that control cardiac, craniofacial and limb development. Within the developing limb, dHAND expression encompasses the zone of polarizing activity in the posterior region, where it has been shown to be necessary and sufficient to induce the expression of the morphogen sonic hedgehog. Misexpression of dHAND in the anterior compartment of the limb bud induces ectopic expression of sonic hedgehog, with resulting preaxial polydactyly and mirror image duplications of posterior digits. To investigate the potential transcriptional mechanisms involved in limb patterning by dHAND, we have performed a structure-function analysis of the protein in cultured cells and ectopically expressed dHAND mutant proteins in the developing limbs of transgenic mice. We show that an N-terminal transcriptional activation domain, and the bHLH region, are required for E-box-dependent transcription in vitro. Remarkably, however, digit duplication by dHAND requires neither the transcriptional activation domain nor the basic region, but only the HLH motif. eHAND has a similar limb patterning activity to dHAND in these misexpression experiments, indicating a conserved function of the HLH regions of these proteins. These findings suggest that dHAND may act via novel transcriptional mechanisms mediated by protein-protein interactions independent of direct DNA binding.

• Wang DZ, Valdez MR, McAnally J, Richardson J, Olson EN
• The Mef2c gene is a direct transcriptional target of myogenic bHLH and MEF2 proteins during skeletal muscle development.
• Development. 2001; 128: 4623-33
• Display abstract

• Members of the MEF2 family of transcription factors are upregulated during skeletal muscle differentiation and cooperate with the MyoD family of myogenic basic helix-loop-helix (bHLH) transcription factors to control the expression of muscle-specific genes. To determine the mechanisms that regulate MEF2 gene expression during skeletal muscle development, we analyzed the mouse Mef2c gene for cis-regulatory elements that direct expression in the skeletal muscle lineage in vivo. We describe a skeletal muscle-specific control region for Mef2c that is sufficient to direct lacZ reporter gene expression in a pattern that recapitulates that of the endogenous Mef2c gene in skeletal muscle during pre- and postnatal development. This control region is a direct target for the binding of myogenic bHLH and MEF2 proteins. Mutagenesis of the Mef2c control region shows that a binding site for myogenic bHLH proteins is essential for expression at all stages of skeletal muscle development, whereas an adjacent MEF2 binding site is required for maintenance but not for initiation of Mef2c transcription. Our findings reveal the existence of a regulatory circuit between these two classes of transcription factors that induces, amplifies and maintains their expression during skeletal muscle development.

• Davis RL, Turner DL
• Vertebrate hairy and Enhancer of split related proteins: transcriptional repressors regulating cellular differentiation and embryonic patterning.
• Oncogene. 2001; 20: 8342-57
• Display abstract

• The basic-helix-loop-helix (bHLH) proteins are a superfamily of DNA-binding transcription factors that regulate numerous biological processes in both invertebrates and vertebrates. One family of bHLH transcriptional repressors is related to the Drosophila hairy and Enhancer-of-split proteins. These repressors contain a tandem arrangement of the bHLH domain and an adjacent sequence known as the Orange domain, so we refer to these proteins as bHLH-Orange or bHLH-O proteins. Phylogenetic analysis reveals the existence of four bHLH-O subfamilies, with distinct, evolutionarily conserved features. A principal function of bHLH-O proteins is to bind to specific DNA sequences and recruit transcriptional corepressors to inhibit target gene expression. However, it is likely that bHLH-O proteins repress transcription by additional mechanisms as well. Many vertebrate bHLH-O proteins are effectors of the Notch signaling pathway, and bHLH-O proteins are involved in regulating neurogenesis, vasculogenesis, mesoderm segmentation, myogenesis, and T lymphocyte development. In this review, we discuss mechanisms of action and biological roles for the vertebrate bHLH-O proteins, as well as some of the unresolved questions about the functions and regulation of these proteins during development and in human disease.

• Bergstrom DA, Tapscott SJ
• Molecular distinction between specification and differentiation in the myogenic basic helix-loop-helix transcription factor family.
• Mol Cell Biol. 2001; 21: 2404-12
• Display abstract

• The myogenic basic helix-loop-helix (bHLH) proteins regulate both skeletal muscle specification and differentiation: MyoD and Myf5 establish the muscle lineage, whereas myogenin mediates differentiation. Previously, we demonstrated that MyoD was more efficient than myogenin at initiating the expression of skeletal muscle genes, and in this study we present the molecular basis for this difference. A conserved amphipathic alpha-helix in the carboxy terminus of the myogenic bHLH proteins has distinct activities in MyoD and myogenin: the MyoD helix facilitates the initiation of endogenous gene expression, whereas the myogenin helix functions as a general transcriptional activation domain. Thus, the alternate use of a similar motif for gene initiation and activation provides a molecular basis for the distinction between specification and differentiation within the myogenic bHLH gene family.

• Becker JR, Dorman CM, McClafferty TM, Johnson SE
• Characterization of a dominant inhibitory E47 protein that suppresses C2C12 myogenesis.
• Exp Cell Res. 2001; 267: 135-43
• Display abstract

• Skeletal muscle formation is controlled through the coordinated actions of the muscle regulatory factors (MRFs). The activities of these basic helix-loop-helix proteins is mediated in part through heterodimer formation with a family of ubiquitous bHLH proteins, referred to as E-proteins. The primary E-protein in skeletal muscle is the E2A splice variant, E47. To further address the role of E47 during skeletal myogenesis, we created a chimeric E47 repressor protein by replacing the transcriptional activation domain with the Drosophila Engrailed transcriptional repressor domain. The dominant inhibitory E-protein (EnDeltaE47) formed homodimers capable of binding DNA and abolished E47-directed gene transcription. Stable expression of EnDeltaE47 in mouse C2C12 myoblasts effectively blocked the cells' ability to differentiate into mature myofibers. Closer examination of the molecular basis for the inhibition of myogenesis revealed that EnDeltaE47 preferentially forms heterodimers with myogenin. Interestingly, the chimeric repressor did not form DNA-binding heterodimers with MyoD in C2C12 myocytes. The failure to detect MyoD:EnDeltaE47 heterodimers in myoblasts was not due to protein conformational defects as both wild-type E47 and EnDeltaE47 readily formed DNA binding complexes with MyoD in vitro. These results indicate that E47 plays a crucial role in C2C12 myogenesis by serving as the preferred heterodimer partner of the myogenin protein.

• Kataoka H et al.
• A novel snail-related transcription factor Smuc regulates basic helix-loop-helix transcription factor activities via specific E-box motifs.
• Nucleic Acids Res. 2000; 28: 626-33
• Display abstract

• Snail family proteins are zinc finger transcriptional regulators first identified in Drosophila which play critical roles in cell fate determination. We identified a novel Snail -related gene from murine skeletalmusclecells designated Smuc. Northern blot analysis showed that Smuc was highly expressed in skeletal muscle and thymus. Smuc contains five putative DNA-binding zinc finger domains in its C-terminal half. In electrophoretic mobility shift assays, recombinant zinc finger domains of Smuc specifically bound to CAGGTG and CACCTG E-box motifs (CANNTG). Because basic helix-loop-helix transcription factors (bHLH) bind to the same E-box sequences, we examined whether Smuc competes with the myogenic bHLH factor MyoD for DNA binding. Smuc inhibited the binding of a MyoD-E12 complex to the CACCTG E-box sequence in a dose-dependent manner and suppressed the transcriptional activity of MyoD-E12. When heterologously targeted to the thymidine kinase promoter as fusion proteins with the GAL4 DNA-binding domain, the non-zinc finger domain of Smuc acted as a transcriptional repressor. Furthermore, overexpression of Smuc in myoblasts repressed transactivation of muscle differentiation marker Troponin T. Thus, Smuc might regulate bHLH transcription factors by zinc finger domains competing for E-box binding, and non-zinc finger repressor domains might also confer transcriptional repression to control differentiation processes.

• Valdez MR, Richardson JA, Klein WH, Olson EN
• Failure of Myf5 to support myogenic differentiation without myogenin, MyoD, and MRF4.
• Dev Biol. 2000; 219: 287-98
• Display abstract

• The basic helix-loop-helix (bHLH) transcription factors-MyoD, Myf5, myogenin, and MRF4-can each activate the skeletal muscle-differentiation program in transfection assays. However, their functions during embryogenesis, as revealed by gene-knockout studies in mice, are distinct. MyoD and Myf5 have redundant functions in myoblast specification, whereas myogenin and either MyoD or MRF4 are required for differentiation. Paradoxically, myoblasts from myogenin mutant or MyoD/MRF4 double-mutant neonates differentiate normally in vitro, despite their inability to differentiate in vivo, suggesting that the functions of the myogenic bHLH factors are influenced by the cellular environment and that the specific myogenic defects observed in mutant mice do not necessarily reflect essential functions of these factors. Understanding the individual roles of these factors is further complicated by their ability to cross-regulate one another's expression. To investigate the functions of Myf5 in the absence of contributions from other myogenic bHLH factors, we generated triple-mutant mice lacking myogenin, MyoD, and MRF4. These mice appear to contain a normal number of myoblasts, but in contrast to myogenin or MyoD/MRF4 mutants, differentiated muscle fibers fail to form in vivo and myoblasts from neonates of this triple-mutant genotype are unable to differentiate in vitro. These results suggest that physiological levels of Myf5 are insufficient to activate the myogenic program in the absence of other myogenic factors and suggest that specialized functions have evolved for the myogenic bHLH factors to switch on the complete program of muscle gene expression.

• Fraidenraich D, Iwahori A, Rudnicki M, Basilico C
• Activation of fgf4 gene expression in the myotomes is regulated by myogenic bHLH factors and by sonic hedgehog.
• Dev Biol. 2000; 225: 392-406
• Display abstract

• The Fgf4 gene encodes an important signaling molecule which is expressed in specific developmental stages, including the inner cell mass of the blastocyst, the myotomes, and the limb bud apical ectodermal ridge (AER). Using a transgenic approach, we previously identified overlapping but distinct enhancer elements in the Fgf4 3' untranslated region necessary and sufficient for myotome and AER expression. Here we have investigated the hypothesis that Fgf4 is a target of myogenic bHLH factors. We show by mutational analysis that a conserved E box located in the Fgf4 myotome enhancer is required for Fgf4-lacZ expression in the myotomes. A DNA probe containing the E box binds MYF5, MYOD, and bHLH-like activities from nuclear extracts of differentiating C2-7 myoblast cells, and both MYF5 and MYOD can activate gene expression of reporter plasmids containing the E-box element. Analyses of Myf5 and MyoD knockout mice harboring Fgf4-lacZ transgenes show that Myf5 is required for Fgf4 expression in the myotomes, while MyoD is not, but MyoD can sustain Fgf4 expression in the ventral myotomes in the absence of Myf5. Sonic hedgehog (Shh) signaling has been shown to have an essential inductive function in the expression of Myf5 and MyoD in the epaxial myotomes, but not in the hypaxial myotomes. We show here that expression of an Fgf4-lacZ transgene in Shh-/- embryos is suppressed not only in the epaxial but also in the hypaxial myotomes, while it is maintained in the AER. This suggests that Shh mediates Fgf4 activation in the myotomes through mechanisms independent of its role in the activation of myogenic factors. Thus, a cascade of events, involving Shh and bHLH factors, is responsible for activating Fgf4 expression in the myotomes in a spatial- and temporal-specific manner.

• Chen SL, Dowhan DH, Hosking BM, Muscat GE
• The steroid receptor coactivator, GRIP-1, is necessary for MEF-2C-dependent gene expression and skeletal muscle differentiation.
• Genes Dev. 2000; 14: 1209-28
• Display abstract

• Nuclear receptor-mediated activation of transcription involves coactivation by cofactors collectively denoted the steroid receptor coactivators (SRCs). The process also involves the subsequent recruitment of p300/CBP and PCAF to a complex that synergistically regulates transcription and remodels the chromatin. PCAF and p300 have also been demonstrated to function as critical coactivators for the muscle-specific basic helix-loop-helix (bHLH) protein MyoD during myogenic commitment. Skeletal muscle differentiation and the activation of muscle-specific gene expression is dependent on the concerted action of another bHLH factor, myogenin, and the MADS protein, MEF-2, which function in a cooperative manner. We examined the functional role of one SRC, GRIP-1, in muscle differentiation, an ideal paradigm for the analysis of the determinative events that govern the cell's decision to divide or differentiate. We observed that the mRNA encoding GRIP-1 is expressed in proliferating myoblasts and post-mitotic differentiated myotubes, and that protein levels increase during differentiation. Exogenous/ectopic expression studies with GRIP-1 sense and antisense vectors in myogenic C2C12 cells demonstrated that this SRC is necessary for (1) induction/activation of myogenin, MEF-2, and the crucial cell cycle regulator, p21, and (2) contractile protein expression and myotube formation. Furthermore, we demonstrate that the SRC GRIP-1 coactivates MEF-2C-mediated transcription. GRIP-1 also coactivates the synergistic transactivation of E box-dependent transcription by myogenin and MEF-2C. GST-pulldowns, mammalian two-hybrid analysis, and immunoprecipitation demonstrate that the mechanism involves direct interactions between MEF-2C and GRIP-1 and is associated with the ability of the SRC to interact with the MADS domain of MEF-2C. The HLH region of myogenin mediates the direct interaction of myogenin and GRIP-1. Interestingly, interaction with myogenic factors is mediated by two regions of GRIP-1, an amino-terminal bHLH-PAS region and the carboxy-terminal region between amino acids 1158 and 1423 (which encodes an activation domain, has HAT activity, and interacts with the coactivator-associated arginine methyltransferase). This work demonstrates that GRIP-1 potentiates skeletal muscle differentiation by acting as a critical coactivator for MEF-2C-mediated transactivation and is the first study to ascribe a function to the amino-terminal bHLH-PAS region of SRCs.

• Skerjanc IS, Wilton S
• Myocyte enhancer factor 2C upregulates MASH-1 expression and induces neurogenesis in P19 cells.
• FEBS Lett. 2000; 472: 53-6
• Display abstract

• MEF2C is a transcription factor expressed in neural lineages. After transient transfection, the MEF2 family of factors can act synergistically with the neural-specific transcription factor, MASH-1, and activate exogenous neural-specific promoters. To determine whether MEF2C is capable of modulating endogenous gene expression, P19 cell lines were analyzed that overexpressed MEF2C, termed P19[MEF2C] cells. Here we show that P19[MEF2C] cells differentiate into neurons when aggregated with ME(2)SO. MEF2C-induced neurons expressed neurofilament protein, the nuclear antigen NeuN, as well as MASH-1. Our results indicate that MEF2C can directly or indirectly activate the expression of MASH-1, leading to neurogenesis.

• Firulli BA, Hadzic DB, McDaid JR, Firulli AB
• The basic helix-loop-helix transcription factors dHAND and eHAND exhibit dimerization characteristics that suggest complex regulation of function.
• J Biol Chem. 2000; 275: 33567-73
• Display abstract

• dHAND and eHAND are basic helix-loop-helix (bHLH) transcription factors expressed during embryogenesis and are required for the proper development of cardiac and extraembryonic tissues. HAND genes, like the myogenic bHLH genes, are classified as class B bHLH genes, which are expressed in a tissue-restricted pattern and function by forming heterodimers with class A bHLH proteins. Myogenic bHLH genes are shown not to form homodimers efficiently, suggesting that their activity is dependent on their E-protein partners. To identify HIPs (HAND-interacting proteins) that regulate the activity of the HAND genes, we screened an 9.5-10.5-day-old mouse embryonic yeast two-hybrid library with eHAND. Several HIPs held high sequence identity to eHAND, indicating that eHAND could form and function as a homodimer. Based on the high degree of amino acid identity between eHAND and dHAND, it is possible that dHAND could also form homodimers and heterodimers with eHAND. We show using yeast and mammalian two-hybrid assays as well as biochemical pull-down assays that eHAND and dHAND are capable of forming both HAND homo- and heterodimers in vivo. To investigate whether HAND genes form heterodimers with other biologically relevant bHLH proteins, we tested and show HAND heterodimerization with the recently identified Hairy-related transcription factors, HRT1-3. This finding is exciting, because both HRT and HAND genes are coexpressed in the developing heart and limb and both have been implicated in establishing tissue boundaries and pattern formation. Moreover, competition gel shift analysis demonstrates that dHAND and eHAND can negatively regulate the DNA binding of MyoD/E12 heterodimers in a manner similar to MISTI and Id proteins, suggesting a possible transcriptional inhibitory role for HAND genes. Taken together, these results show that dHAND and eHAND can form homo- and heterodimer combinations with multiple bHLH partners and that this broad dimerization profile reflects the mechanisms by which HAND genes regulate transcription.

• Poulin G, Lebel M, Chamberland M, Paradis FW, Drouin J
• Specific protein-protein interaction between basic helix-loop-helix transcription factors and homeoproteins of the Pitx family.
• Mol Cell Biol. 2000; 20: 4826-37
• Display abstract

• Homeoproteins and basic helix-loop-helix (bHLH) transcription factors are known for their critical role in development and cellular differentiation. The pituitary pro-opiomelanocortin (POMC) gene is a target for factors of both families. Indeed, pituitary-specific transcription of POMC depends on the action of the homeodomain-containing transcription factor Pitx1 and of bHLH heterodimers containing NeuroD1. We now show lineage-restricted expression of NeuroD1 in pituitary corticotroph cells and a direct physical interaction between bHLH heterodimers and Pitx1 that results in transcriptional synergism. The interaction between the bHLH and homeodomains is restricted to ubiquitous (class A) bHLH and to the Pitx subfamily. Since bHLH heterodimers interact with Pitx factors through their ubiquitous moiety, this mechanism may be implicated in other developmental processes involving bHLH factors, such as neurogenesis and myogenesis.

• Johanson M, Meents H, Ragge K, Buchberger A, Arnold HH, Sandmoller A
• Transcriptional activation of the myogenin gene by MEF2-mediated recruitment of myf5 is inhibited by adenovirus E1A protein.
• Biochem Biophys Res Commun. 1999; 265: 222-32
• Display abstract

• The basic helix-loop-helix (bHLH) transcription factor myogenin plays a crucial role in terminal differentiation of committed myoblasts into mature myocytes. Transcriptional activation of the myogenin gene requires coordinate action of myocyte enhancer factor 2 (MEF2) proteins and the myogenic bHLH regulators, MyoD or Myf5. Here we show that transcription of the myogenin gene in differentiated cells correlates with MEF2 and NF1 binding to their cognate sites in the proximal myogenin promoter but not with binding of Myf5 or MyoD to the E-box. The importance of MEF2 activity was further demonstrated by expression of antisense MEF2 RNA which repressed MEF2 and Myf5-mediated MEF2 site-dependent reporter gene activation and the synergistic transactivation of a myogenin CAT reporter by Myf5 and MEF2. Adenovirus E1A which has previously been shown to specifically interfere with myogenin gene transcription also inhibited the cooperative transactivation by Myf5/MEF2 and MEF2. Consistently, coimmunoprecipitation studies revealed impaired MEF2/Myf5 protein-protein interactions. These results support a model of transcriptional activation and stabilization of myogenin expression in which DNA-bound MEF2 recruits myogenic bHLH factors into an active but E1A-sensitive transcription factor complex.

• Chaudhary J, Skinner MK
• Basic helix-loop-helix proteins can act at the E-box within the serum response element of the c-fos promoter to influence hormone-induced promoter activation in Sertoli cells.
• Mol Endocrinol. 1999; 13: 774-86
• Display abstract

• The Sertoli cell is a terminally differentiated testicular cell in the adult required to maintain the process of spermatogenesis. Previously basic helix-loop-helix (bHLH) factors and c-fos have been shown to influence Sertoli cell-differentiated functions. The induction of Sertoli cell differentiation appears to involve the serum response element (SRE) of the c-fos promoter to activate c-fos and intermediate bHLH factor(s) that regulate down-stream Sertoli cell-differentiated genes (e.g. transferrin expression). The SRE of the c-fos promoter is influenced through the serum response factor (SRF). Interestingly, an E-box nucleotide sequence is present within the SRE. bHLH proteins act through E-box elements, and the current study investigates the possibility that bHLH proteins may directly influence the SRE of the c-fos promoter. The activation of the c-fos promoter in Sertoli cells was found to be inhibited with the overexpression of the inhibitory HLH protein Id. Analysis of major response elements within the c-fos promoter demonstrated that the expression of Id specifically inhibited the activation of SRE in Sertoli cells and no other elements tested. Mutations in the E-box of the SRE also inhibited the activation of SRE, suggesting the direct role of bHLH proteins in regulating SRE activity in Sertoli cells. In contrast, the activation of SRE containing a mutated E-box was comparable to wild-type SRE in control stromal cells. Analysis of SRE oligonucleotide gel mobility shift assays with nuclear extracts from Sertoli cells demonstrated the presence of both the SRF and the ubiquitously expressed bHLH protein E12/E47. In contrast, no E12/E47 was detected in the SRE oligonucleotide gel shift using control stromal cell nuclear extracts. Observations suggest the binding of E12/E47 to SRE may be a cell-specific event. The SRF and bHLH proteins appear to bind to the SRE and activate the c-fos promoter in Sertoli cells. Observations provide evidence that a bHLH protein can interact with the SRE of the c-fos promoter to influence hormone-induced promoter activation. Cross-talk between these nuclear transcription factors appears to be instrumental in the control of Sertoli cell-differentiated functions.

• Lu J, Webb R, Richardson JA, Olson EN
• MyoR: a muscle-restricted basic helix-loop-helix transcription factor that antagonizes the actions of MyoD.
• Proc Natl Acad Sci U S A. 1999; 96: 552-7
• Display abstract

• Skeletal muscle development is controlled by a family of muscle-specific basic helix-loop-helix (bHLH) transcription factors that activate muscle genes by binding E-boxes (CANNTG) as heterodimers with ubiquitous bHLH proteins, called E proteins. Myogenic bHLH factors are expressed in proliferating undifferentiated myoblasts, but they do not initiate myogenesis until myoblasts exit the cell cycle. We describe a bHLH protein, MyoR (for myogenic repressor), that is expressed in undifferentiated myoblasts in culture and is down-regulated during differentiation. MyoR is also expressed specifically in the skeletal muscle lineage between days 10.5 and 16.5 of mouse embryogenesis and down-regulated thereafter during the period of secondary myogenesis. MyoR forms heterodimers with E proteins that bind the same DNA sequence as myogenic bHLH/E protein heterodimers, but MyoR acts as a potent transcriptional repressor that blocks myogenesis and activation of E-box-dependent muscle genes. These results suggest a role for MyoR as a lineage-restricted transcriptional repressor of the muscle differentiation program.

• Novitch BG, Spicer DB, Kim PS, Cheung WL, Lassar AB
• pRb is required for MEF2-dependent gene expression as well as cell-cycle arrest during skeletal muscle differentiation.
• Curr Biol. 1999; 9: 449-59
• Display abstract

• BACKGROUND: The onset of differentiation-specific gene expression in skeletal muscle is coupled to permanent withdrawal from the cell cycle. The retinoblastoma tumor-suppressor protein (pRb) is a critical regulator of this process, required for both cell-cycle arrest in G0 phase and high-level expression of late muscle-differentiation markers. Although the cell-cycle defects that are seen in pRb-deficient myocytes can be explained by the well-described function of pRb as a negative regulator of the transition from G1 to S phase, it remains unclear how pRb positively affects late muscle-gene expression. RESULTS: Here, we show that the myogenic defect in Rb-/- cells corresponds to a deficiency in the activity of the transcription factor MEF2. Without pRb, MyoD induces the accumulation of nuclear-localized MEF2 that is competent to bind DNA yet transcriptionally inert. When pRb is present, MyoD stimulates the function of the MEF2C transcriptional activation domain and the activity of endogenous MEF2-type factors. Co-transfection of MyoD together with an activated form of MEF2C containing the Herpesvirus VP16 transcriptional activation domain partially bypasses the requirement for pRb and induces late muscle-gene expression in replicating cells. This ectopic myogenesis is nevertheless significantly augmented by co-expression of an E2F1-pRb chimeric protein that blocks the cell cycle. CONCLUSION: These findings indicate that pRb promotes the expression of late-stage muscle-differentiation markers by both inhibiting cell-cycle progression and cooperating with MyoD to promote the transcriptional activation activity of MEF2.

• Biesiada E, Hamamori Y, Kedes L, Sartorelli V
• Myogenic basic helix-loop-helix proteins and Sp1 interact as components of a multiprotein transcriptional complex required for activity of the human cardiac alpha-actin promoter.
• Mol Cell Biol. 1999; 19: 2577-84
• Display abstract

• Activation of the human cardiac alpha-actin (HCA) promoter in skeletal muscle cells requires the integrity of DNA binding sites for the serum response factor (SRF), Sp1, and the myogenic basic helix-loop-helix (bHLH) family. In this study we report that activation of the HCA correlates with formation of a muscle-specific multiprotein complex on the promoter. We provide evidence that proteins eluted from the multiprotein complex specifically react with antibodies directed against myogenin, Sp1, and SRF and that the complex can be assembled in vitro by using the HCA promoter and purified MyoD, E12, SRF, and Sp1. In vitro and in vivo assays revealed a direct association of Sp1 and myogenin-MyoD mediated by the DNA-binding domain of Sp1 and the HLH motif of myogenin. The results obtained in this study indicate that protein-protein interactions and the cooperative DNA binding of transcriptional activators are critical steps in the formation of a transcriptionally productive multiprotein complex on the HCA promoter and suggest that the same mechanisms might be utilized to regulate the transcription of muscle-specific and other genes.

• Wilson-Rawls J, Molkentin JD, Black BL, Olson EN
• Activated notch inhibits myogenic activity of the MADS-Box transcription factor myocyte enhancer factor 2C.
• Mol Cell Biol. 1999; 19: 2853-62
• Display abstract

• Skeletal muscle gene expression is dependent on combinatorial associations between members of the MyoD family of basic helix-loop-helix (bHLH) transcription factors and the myocyte enhancer factor 2 (MEF2) family of MADS-box transcription factors. The transmembrane receptor Notch interferes with the muscle-inducing activity of myogenic bHLH proteins, and it has been suggested that this inhibitory activity of Notch is directed at an essential cofactor that recognizes the DNA binding domains of the myogenic bHLH proteins. Given that MEF2 proteins interact with the DNA binding domains of myogenic bHLH factors to cooperatively regulate myogenesis, we investigated whether members of the MEF2 family might serve as targets for the inhibitory effects of Notch on myogenesis. We show that a constitutively activated form of Notch specifically blocks DNA binding by MEF2C, as well as its ability to cooperate with MyoD and myogenin to activate myogenesis. Responsiveness to Notch requires a 12-amino-acid region of MEF2C immediately adjacent to the DNA binding domain that is unique to this MEF2 isoform. Two-hybrid assays and coimmunoprecipitations show that this region of MEF2C interacts directly with the ankyrin repeat region of Notch. These findings reveal a novel mechanism for Notch-mediated inhibition of myogenesis and demonstrate that the Notch signaling pathway can discriminate between different members of the MEF2 family.

• Knoepfler PS et al.
• A conserved motif N-terminal to the DNA-binding domains of myogenic bHLH transcription factors mediates cooperative DNA binding with pbx-Meis1/Prep1.
• Nucleic Acids Res. 1999; 27: 3752-61
• Display abstract

• The t(1;19) chromosomal translocation of pediatric pre-B cell leukemia produces chimeric oncoprotein E2a-Pbx1, which contains the N-terminal transactivation domain of the basic helix-loop-helix (bHLH) transcription factor, E2a, joined to the majority of the homeodomain protein, Pbx1. There are three Pbx family members, which bind DNA as heterodimers with both broadly expressed Meis/Prep1 homeo-domain proteins and specifically expressed Hox homeodomain proteins. These Pbx heterodimers can augment the function of transcriptional activators bound to adjacent elements. In heterodimers, a conserved tryptophan motif in Hox proteins binds a pocket on the surface of the Pbx homeodomain, while Meis/Prep1 proteins bind an N-terminal Pbx domain, raising the possibility that the tryptophan-interaction pocket of the Pbx component of a Pbx-Meis/Prep1 complex is still available to bind trypto-phan motifs of other transcription factors bound to flanking elements. Here, we report that Pbx-Meis1/Prep1 binds DNA cooperatively with heterodimers of E2a and MyoD, myogenin, Mrf-4 or Myf-5. As with Hox proteins, a highly conserved tryptophan motif N-terminal to the DNA-binding domains of each myogenic bHLH family protein is required for cooperative DNA binding with Pbx-Meis1/Prep1. In vivo, MyoD requires this tryptophan motif to evoke chromatin remodeling in the Myogenin promoter and to activate Myogenin transcription. Pbx-Meis/Prep1 complexes, therefore, have the potential to cooperate with the myogenic bHLH proteins in regulating gene transcription.

• Lemercier C, To RQ, Carrasco RA, Konieczny SF
• The basic helix-loop-helix transcription factor Mist1 functions as a transcriptional repressor of myoD.
• EMBO J. 1998; 17: 1412-22
• Display abstract

• A good model system to examine aspects of positive and negative transcriptional regulation is the muscle-specific regulatory factor, MyoD, which is a basic helix-loop-helix (bHLH) transcription factor. Although MyoD has the ability to induce skeletal muscle terminal differentiation in a variety of non-muscle cell types, MyoD activity itself is highly regulated through protein-protein interactions involving several different co-factors. Here we describe the characterization of a novel bHLH protein, Mist1, and how it influences MyoD function. We show that Mist1 accumulates in myogenic stem cells (myoblasts) and then decreases as myoblasts differentiate into myotubes. Mist1 functions as a negative regulator of MyoD activity, preventing muscle differentiation and the concomitant expression of muscle-specific genes. Mist1-induced inhibition occurs through a combination of mechanisms, including the formation of inactive MyoD-Mist1 heterodimers and occupancy of specific E-box target sites by Mist1 homodimers. Mist1 lacks a classic transcription activation domain and instead possesses an N-terminal repressor region capable of inhibiting heterologous activators. Thus, Mist1 may represent a new class of repressor molecules that play a role in controlling the transcriptional activity of MyoD, ensuring that expanding myoblast populations remain undifferentiated during early embryonic muscle formation.

• Dang W, Sun XH, Sen R
• ETS-mediated cooperation between basic helix-loop-helix motifs of the immunoglobulin mu heavy-chain gene enhancer.
• Mol Cell Biol. 1998; 18: 1477-88
• Display abstract

• The muE motifs of the immunoglobulin mu heavy-chain gene enhancer bind ubiquitously expressed proteins of the basic helix-loop-helix (bHLH) family. These elements work together with other, more tissue-restricted elements to produce B-cell-specific enhancer activity by presently undefined combinatorial mechanisms. We found that muE2 contributed to transcription activation in B cells only when the muE3 site was intact, providing the first evidence for functional interactions between bHLH proteins. In vitro assays showed that bHLH zipper proteins binding to muE3 enhanced Ets-1 binding to muA. One of the consequences of this protein-protein interaction was to facilitate binding of a second bHLH protein, E47, to the muE2 site, thereby generating a three-protein-DNA complex. Furthermore, transcriptional synergy between bHLH and bHLH zipper factors also required an intermediate ETS protein, which may bridge the transcription activation domains of the bHLH factors. Our observations define an unusual form of cooperation between bHLH and ETS proteins and suggest mechanisms by which tissue-restricted and ubiquitous factors combine to generate tissue-specific enhancer activity.

• Cripps RM, Black BL, Zhao B, Lien CL, Schulz RA, Olson EN
• The myogenic regulatory gene Mef2 is a direct target for transcriptional activation by Twist during Drosophila myogenesis.
• Genes Dev. 1998; 12: 422-34
• Display abstract

• MEF2 is a MADS-box transcription factor required for muscle development in Drosophila. Here, we show that the bHLH transcription factor Twist directly regulates Mef2 expression in adult somatic muscle precursor cells via a 175-bp enhancer located 2245 bp upstream of the transcriptional start site. Within this element, a single evolutionarily conserved E box is essential for enhancer activity. Twist protein can bind to this E box to activate Mef2 transcription, and ectopic expression of twist results in ectopic activation of the wild-type 175-bp enhancer. By use of a temperature-sensitive mutant of twist, we show that activation of Mef2 transcription via this enhancer by Twist is required for normal adult muscle development, and reduction in Twist function results in phenotypes similar to those observed previously in Mef2 mutant adults. The 175-bp enhancer is also active in the embryonic mesoderm, indicating that this enhancer functions at multiple times during development, and its function is dependent on the same conserved E box. In embryos, a reduction in Twist function also strongly reduced Mef2 expression. These findings define a novel transcriptional pathway required for skeletal muscle development and identify Twist as an essential and direct regulator of Mef2 expression in the somatic mesoderm.

• Erck C, Seidl K
• MyoD and MEF2A mediate activation and repression of the p75NGFR gene during muscle development.
• Biochem Biophys Res Commun. 1998; 245: 871-7
• Display abstract

• In an effort to clarify transient expression of the NGF low-affinity receptor p75NGFR during muscle development we have focused on the molecular mechanisms involved in the initiation and cessation of p75NGFR gene expression. Using quiescent C3H10T1/2 fibroblast as a tool, we observed that induction of differentiation competence in MyoD-transfected 10T1/2 fibroblasts was accompanied by the initiation of p75NGFR expression. Moreover, we could show that the bHLH transcription factor MyoD itself is a powerful candidate for transcriptional activation of the p75NGFR gene in muscle precursor cells. By means of MyoD-mutants we have found that both the amino terminus of the MyoD molecule as well as the bHLH-region are essential for transcriptional activity on the p75NGFR promoter. The fact that myocyte enhancer factor MEF2A inactivated MyoD-induced p75NGFR promoter activity strongly suggests that cell-specific regulation of the p75NGFR gene might be strictly dependent on the intracellular composition and balance of the appropriate bHLH-transcription factors and their modulators.

• Hermann S, Saarikettu J, Onions J, Hughes K, Grundstrom T
• Calcium regulation of basic helix-loop-helix transcription factors.
• Cell Calcium. 1998; 23: 135-42
• Display abstract

• The basic helix-loop-helix (bHLH) family of transcription factors is essential for numerous developmental and growth control processes. The regulation of bHLH proteins occurs at many levels, including tissue specific expression, differential oligomerization and DNA binding specificities, interaction with negatively acting HLH proteins and post-translational modifications. This review focuses on what is emerging as another level of bHLH protein regulation, calcium regulation through interaction with Ca2+ loaded calmodulin and S-100 proteins. The mechanism and implications of these Ca2+ regulated interactions are discussed.

• Black BL, Molkentin JD, Olson EN
• Multiple roles for the MyoD basic region in transmission of transcriptional activation signals and interaction with MEF2.
• Mol Cell Biol. 1998; 18: 69-77
• Display abstract

• Establishment of skeletal muscle lineages is controlled by the MyoD family of basic helix-loop-helix (bHLH) transcription factors. The ability of these factors to initiate myogenesis is dependent on two conserved amino acid residues, alanine and threonine, in the basic domains of these factors. It has been postulated that these two residues may be responsible for the initiation of myogenesis via interaction with an essential myogenic cofactor. The myogenic bHLH proteins cooperatively activate transcription and myogenesis through protein-protein interactions with members of the myocyte enhancer factor 2 (MEF2) family of MADS domain transcription factors. MyoD-E12 heterodimers interact with MEF2 proteins to synergistically activate myogenesis, while homodimers of E12, which lack the conserved alanine and threonine residues in the basic domain, do not interact with MEF2. We have examined whether the myogenic alanine and threonine in the MyoD basic region are required for interaction with MEF2. Here, we show that substitution of the MyoD basic domain with that of E12 does not prevent interaction with MEF2. Instead, the inability of alanine-threonine mutants of MyoD to initiate myogenesis is due to a failure to transmit transcriptional activation signals provided either from the MyoD or the MEF2 activation domain. This defect in transcriptional transmission can be overcome by substitution of the MyoD or the MEF2 activation domain with the VP16 activation domain. These results demonstrate that myogenic bHLH-MEF2 interaction can be uncoupled from transcriptional activation and support the idea that the myogenic residues in myogenic bHLH proteins are essential for transmission of a transcriptional activation signal.

• Lu J, Richardson JA, Olson EN
• Capsulin: a novel bHLH transcription factor expressed in epicardial progenitors and mesenchyme of visceral organs.
• Mech Dev. 1998; 73: 23-32
• Display abstract

• Members of the basic helix-loop-helix (bHLH) family of transcription factors have been shown to control development and differentiation of a variety of cell types. We describe a novel bHLH protein, called capsulin, which is expressed specifically in mesodermally-derived cells that surround the epithelium of the developing gastrointestinal, genitourinary and respiratory systems during mouse embryogenesis. Capsulin transcripts also mark the spiral septum of the heart and progenitor cells that give rise to the pericardium and coronary arteries. Capsulin shares high homology with a recently identified bHLH protein from Drosophila, called bHLH102C, which is expressed in visceral muscle cells that surround the midgut. Capsulin binds a specific E-box consensus sequence (CANNTG) as a heterodimer with the widely-expressed bHLH protein E12, but it does not activate transcription through that sequence on its own. Its restricted expression pattern and DNA binding activity suggest that capsulin regulates gene expression in specific subtypes of visceral mesodermal cells involved in organogenesis and in precursor cells that contribute to the pericardium, coronary arteries and regions of the heart.

• O'Mahoney JV et al.
• Identification of a novel slow-muscle-fiber enhancer binding protein, MusTRD1.
• Mol Cell Biol. 1998; 18: 6641-52
• Display abstract

• The molecular mechanisms which are responsible for restricting skeletal muscle gene expression to specific fiber types, either slow or fast twitch, are unknown. As a first step toward defining the components which direct slow-fiber-specific gene expression, we identified the sequence elements of the human troponin I slow upstream enhancer (USE) that bind muscle nuclear proteins. These include an E-box, a MEF2 element, and two other elements, USE B1 and USE C1. In vivo analysis of a mutation that disrupts USE B1 binding activity suggested that the USE B1 element is essential for high-level expression in slow-twitch muscles. This mutation does not, however, abolish slow-fiber specificity. A similar analysis indicated that the USE C1 element may play only a minor role. We report the cloning of a novel human USE B1 binding protein, MusTRD1 (muscle TFII-I repeat domain-containing protein 1), which is expressed predominantly in skeletal muscle. Significantly, MusTRD1 contains two repeat domains which show remarkable homology to the six repeat domains of the recently cloned transcription factor TFII-I. Furthermore, both TFII-I and MusTRD1 bind to similar but distinct sequences, which happen to conform with the initiator (Inr) consensus sequence. Given the roles of MEF2 and basic helix-loop-helix (bHLH) proteins in muscle gene expression, the similarity of TFII-I and MusTRD1 is intriguing, as TFII-I is believed to coordinate the interaction of MADS-box proteins, bHLH proteins, and the general transcription machinery.

• Arnold HH, Winter B
• Muscle differentiation: more complexity to the network of myogenic regulators.
• Curr Opin Genet Dev. 1998; 8: 539-44
• Display abstract

• Recent genetic and biochemical approaches have advanced our understanding of control mechanisms underlying myogenesis in vertebrate organisms. In particular, systematic combinations of targeted gene disruptions in mice have revealed unique and overlapping functions of members of the MyoD family of transcription factors within the regulatory network that establishes skeletal muscle cell lineages. Moreover, Pax3 has been identified as a key regulator of myogenesis which seems to act genetically upstream of MyoD. In addition, novel genes have been discovered that modulate myogenesis and the activity of myogenic basic helix-loop-helix (bHLH) proteins in positive or negative ways. The molecular mechanisms of these interactions and cooperativity are being elucidated, most notably between the myogenic bHLH factors and MEF2 transcription factors.

• Lemercier C, To RQ, Swanson BJ, Lyons GE, Konieczny SF
• Mist1: a novel basic helix-loop-helix transcription factor exhibits a developmentally regulated expression pattern.
• Dev Biol. 1997; 182: 101-13
• Display abstract

• Basic helix-loop-helix (bHLH) proteins often belong to a family of transcription factors that bind to the DNA target sequence -CANNTG- (E-box) that is present in the promoter or enhancer regions of numerous developmentally regulated genes. In this study, we report the isolation and initial characterization of a novel bHLH factor, termed Mist1, that was identified by virtue of its ability to interact with E-box regulatory elements in a yeast "one-hybrid" screening procedure. Northern analysis revealed that Mist1 transcripts are expressed in several adult tissues, including stomach, liver, lung, and spleen but no expression is detected in the heart, brain, kidney, or testis. During mouse embryogenesis, Mist1 mRNA is first observed at E10.5 in the primitive gut and in the developing lung bud. Expression persists through E16.5 and remains restricted primarily to the epithelial lining. Mist1 also is detected in skeletal muscle tissues beginning at E12.5, persisting throughout all embryonic stages examined although in older embryos and in the adult expression becomes severely reduced. At later developmental times, Mist1 transcripts also are found in the pancreas, submandibular gland, and adult spleen. As predicted, the Mist1 protein is nuclear and binds efficiently to E-box sites as a homodimer. Mist1 also is capable of binding to E-box elements when complexed as a heterodimer with the widely expressed E-proteins, E12 and E47. Surprisingly, although Mist1 binds to E-boxes in vivo, the Mist1 protein lacks a functional transcription activation domain. These observations suggest that Mist1 may function as a unique regulator of gene expression in several different embryonic and postnatal cell lineages.

• Meierhans D, Allemann RK
• High level expression in soluble form, one step purification, and characterization of the DNA binding domain of MEF-2C.
• Protein Expr Purif. 1997; 11: 297-303
• Display abstract

• Members of the MEF-2 family of transcription factors act as coregulators of basic helix-loop-helix (BHLH) proteins in the control of lineage specific gene expression in many cell types through direct interaction between the respective DNA binding domains. To make possible a thorough biochemical, biophysical, and structural characterization of the properties of myocyte enhancer factor (MEF) proteins and of their interactions with BHLH-proteins, a simple system for high level expression and rapid purification of myocyte enhancer factor-2C (MEF-2C) was developed. A T7 expression system was used to produce in high yield in Escherichia coli an N-terminal fragment of MEF-2C comprising both the MADS box and the MEF domain. Purification by a single round of cation-exchange chromatography on a Resource-S HPLC column at elevated pH afforded an essentially pure protein. Recombinant MEF-2C (1-117) bound with high affinity to the MEF consensus DNA binding site (CTATAAATAG). Mutations in this sequence that replaced adenines with thymine or vice versa did not significantly alter the affinity for MEF-2C(1-117). The introduction of G-C pairs into the core of the MEF-site, however, dramatically increased the concentration of MEF-2C(1-117) needed for half maximal DNA binding. We propose an explanation of the DNA binding specificity of MEF-2C based on the intrinsic bending properties of the unbound DNA.

• Sartorelli V, Huang J, Hamamori Y, Kedes L
• Molecular mechanisms of myogenic coactivation by p300: direct interaction with the activation domain of MyoD and with the MADS box of MEF2C.
• Mol Cell Biol. 1997; 17: 1010-26
• Display abstract

• By searching for molecules that assist MyoD in converting fibroblasts to muscle cells, we have found that p300 and CBP, two related molecules that act as transcriptional adapters, coactivate the myogenic basic-helix-loop-helix (bHLH) proteins. Coactivation by p300 involves novel physical interactions between p300 and the amino-terminal activation domain of MyoD. In particular, disruption of the FYD domain, a group of three amino acids conserved in the activation domains of other myogenic bHLH proteins, drastically diminishes the transactivation potential of MyoD and abolishes both p300-mediated coactivation and the physical interaction between MyoD and p300. Two domains of p300, at its amino and carboxy terminals, independently function to both mediate coactivation and physically interact with MyoD. A truncated segment of p300, unable to bind MyoD, acts as a dominant negative mutation and abrogates both myogenic conversion and transactivation by MyoD, suggesting that endogenous p300 is a required coactivator for MyoD function. The p300 dominant negative peptide forms multimers with intact p300. p300 and CBP serve as coactivators of another class of transcriptional activators critical for myogenesis, myocyte enhancer factor 2 (MEF2). In fact, transactivation mediated by the MEF2C protein is potentiated by the two coactivators, and this phenomenon is associated with the ability of p300 to interact with the MADS domain of MEF2C. Our results suggest that p300 and CBP may positively influence myogenesis by reinforcing the transcriptional autoregulatory loop established between the myogenic bHLH and the MEF2 factors.

• Lenormand JL, Benayoun B, Guillier M, Vandromme M, Leibovitch MP, Leibovitch SA
• Mos activates myogenic differentiation by promoting heterodimerization of MyoD and E12 proteins.
• Mol Cell Biol. 1997; 17: 584-93
• Display abstract

• The activities of myogenic basic helix-loop-helix (bHLH) factors are regulated by a number of different positive and negative signals. Extensive information has been published about the molecular mechanisms that interfere with the process of myogenic differentiation, but little is known about the positive signals. We previously showed that overexpression of rat Mos in C2C12 myoblasts increased the expression of myogenic markers whereas repression of Mos products by antisense RNAs inhibited myogenic differentiation. In the present work, our results show that the rat mos proto-oncogene activates transcriptional activity of MyoD protein. In transient transfection assays, Mos promotes transcriptional transactivation by MyoD of the muscle creatine kinase enhancer and/or a reporter gene linked to MyoD-DNA binding sites. Physical interaction between Mos and MyoD, but not with E12, is demonstrated in vivo by using the two-hybrid approach with C3H10T1/2 cells and in vitro by using the glutathione S-transferase (GST) pull-down assays. Unphosphorylated MyoD from myogenic cell lysates and/or bacterially expressed MyoD physically interacts with Mos. This interaction occurs via the helix 2 region of MyoD and a highly conserved region in Mos proteins with 40% similarity to the helix 2 domain of the E-protein class of bHLH factors. Phosphorylation of MyoD by activated GST-Mos protein inhibits the DNA-binding activity of MyoD homodimers and promotes MyoD-E12 heterodimer formation. These data support a novel function for Mos as a mediator (coregulator) of muscle-specific gene(s) expression.

• Kong Y, Flick MJ, Kudla AJ, Konieczny SF
• Muscle LIM protein promotes myogenesis by enhancing the activity of MyoD.
• Mol Cell Biol. 1997; 17: 4750-60
• Display abstract

• The muscle LIM protein (MLP) is a muscle-specific LIM-only factor that exhibits a dual subcellular localization, being present in both the nucleus and in the cytoplasm. Overexpression of MLP in C2C12 myoblasts enhances skeletal myogenesis, whereas inhibition of MLP activity blocks terminal differentiation. Thus, MLP functions as a positive developmental regulator, although the mechanism through which MLP promotes terminal differentiation events remains unknown. While examining the distinct roles associated with the nuclear and cytoplasmic forms of MLP, we found that nuclear MLP functions through a physical interaction with the muscle basic helix-loop-helix (bHLH) transcription factors MyoD, MRF4, and myogenin. This interaction is highly specific since MLP does not associate with nonmuscle bHLH proteins E12 or E47 or with the myocyte enhancer factor-2 (MEF2) protein, which acts cooperatively with the myogenic bHLH proteins to promote myogenesis. The first LIM motif in MLP and the highly conserved bHLH region of MyoD are responsible for mediating the association between these muscle-specific factors. MLP also interacts with MyoD-E47 heterodimers, leading to an increase in the DNA-binding activity associated with this active bHLH complex. Although MLP lacks a functional transcription activation domain, we propose that it serves as a cofactor for the myogenic bHLH proteins by increasing their interaction with specific DNA regulatory elements. Thus, the functional complex of MLP-MyoD-E protein reveals a novel mechanism for both initiating and maintaining the myogenic program and suggests a global strategy for how LIM-only proteins may control a variety of developmental pathways.

• Puri PL et al.
• p300 is required for MyoD-dependent cell cycle arrest and muscle-specific gene transcription.
• EMBO J. 1997; 16: 369-83
• Display abstract

• The nuclear phosphoprotein p300 is a new member of a family of 'co-activators' (which also includes the CREB binding protein CBP), that directly modulate transcription by interacting with components of the basal transcriptional machinery. Both p300 and CBP are targeted by the adenovirus E1A protein, and binding to p300 is required for E1A to inhibit terminal differentiation in both keratinocytes and myoblasts. Here we demonstrate that, in differentiating skeletal muscle cells, p300 physically interacts with the myogenic basic helix-loop-helix (bHLH) regulatory protein MyoD at its DNA binding sites. During muscle differentiation, MyoD plays a dual role: besides activating muscle-specific transcription, it induces permanent cell cycle arrest by up-regulating the cyclin-dependent kinase inhibitor p21. We show that p300 is involved in both these activities. Indeed, E1A mutants lacking the ability to bind p300 are greatly impaired in the repression of E-box-driven transcription, and p300 overexpression rescues the wild-type E1A-mediated repression. Moreover, p300 potentiates MyoD- and myogenin-dependent activation of transcription from E-box-containing reporter genes. We also provide evidence, obtained by microinjection of anti-p300 antibodies, that p300 is required for MyoD-dependent cell cycle arrest in either myogenic cells induced to differentiate or in MyoD-converted C3H10T1/2 fibroblasts, but is dispensable for maintenance of the postmitotic state of myotubes.

• Postigo AA, Dean DC
• ZEB, a vertebrate homolog of Drosophila Zfh-1, is a negative regulator of muscle differentiation.
• EMBO J. 1997; 16: 3935-43
• Display abstract

• A number of genes, spanning the evolutionary scale from yeast to mammals, that are involved in spatial and temporal patterning during development contain zinc finger and homeodomain motifs. One such zinc finger/homeodomain protein is Drosophila Zfh-1, a member of the zfh family of Drosophila genes, which is expressed in muscle precursors and is critical for the proper development of muscle. Here we demonstrate that a vertebrate homolog of Zfh-1 (ZEB) is a negative regulator of muscle differentiation. We show that ZEB binds to a subset of E boxes in muscle genes and functions by actively repressing transcription. One target of this repression is the members of the MEF-2 family, which synergize with proteins of the myogenic basic helix-loop-helix family (bHLH) (myoD, myf-5, myogenin and MRF-4) to induce myogenic differentiation. As muscle differentiation proceeds, myogenic bHLH proteins accumulate to levels sufficient to displace ZEB from the E boxes, releasing the repression and allowing bHLH proteins to further activate transcription. This mechanism of active transcriptional repression distinguishes ZEB from other negative regulators of myogenesis (Id, Twist and I-mfa) that inhibit muscle differentiation by simply binding and inactivating myogenic factors. The relative affinity of ZEB versus myogenic bHLH proteins varies for E boxes in different genes such that ZEB would be displaced from different genes at distinct times as myogenic bHLH proteins accumulate during myogenesis, thus providing a mechanism to regulate temporal order of gene expression.

• Brand NJ
• Myocyte enhancer factor 2 (MEF2).
• Int J Biochem Cell Biol. 1997; 29: 1467-70
• Display abstract

• The Myocyte Enhancer Factor 2 (MEF2) proteins are transcription factors expressed during development of all three muscle lineages. Of the four mammalian mef2 genes, three (A, C and D) can be alternatively spliced, producing transcripts and proteins which may have significant functional differences. Specific binding sites for MEF2 proteins have been characterized in many striated muscle genes and MEF2 proteins can trans-activate gene expression both as homo- and heterodimers. Loss-of-function mutants in Drosophila indicate that MEF2 is an essential co-factor, but not a primary determinent, in the development of all three muscle lineages in the fly. Recent data suggest an interaction between the DNA-binding domains of mammalian MEF2 proteins and those of tissue-specific basic helix-loop-helix (bHLH) factors and thyroid hormone receptor alpha 1 (TR alpha 1) in the expression of target genes and the development of specific cell phenotypes. Understanding how MEF2 proteins function in the three mammalian muscle types may allow the development of therapeutic strategies for manipulating muscle growth and characteristics.

• Chaudhary J, Cupp AS, Skinner MK
• Role of basic-helix-loop-helix transcription factors in Sertoli cell differentiation: identification of an E-box response element in the transferrin promoter.
• Endocrinology. 1997; 138: 667-75
• Display abstract

• Sertoli cells are critical for testicular function and maintenance of the spermatogenic process. The induction of Sertoli cell differentiation in the embryo promotes testicular development and male sex determination. The progression of Sertoli cell differentiation during puberty promotes the onset of spermatogenesis. The maintenance of optimal Sertoli cell differentiation in the adult is required for spermatogenesis to proceed. The current study was designed to investigate the transcriptional regulation of Sertoli cell differentiation through the analysis of a previously identified marker of differentiation, transferrin gene expression. Sertoli cells produce transferrin to transport iron to developing spermatogenic cells sequestered within the blood-testis barrier. The transferrin promoter was characterized and found to contain two critical response elements, designated Sertoli element 1 (SE1) and Sertoli element 2 (SE2). Through sequence analysis, SE2 was found to contain an E-box response element, which has been shown to respond to basic-helix-loop-helix (bHLH) transcription factors. The bHLH proteins are a class of transcription factors associated with the induction and progression of cell differentiation. bHLH proteins dimerize through the conserved helix-loop-helix region and bind DNA through the basic region. Nuclear extracts from Sertoli cells were found to cause an E-box gel shift when the cells were stimulated to differentiate in culture, but not under basal conditions. The SE2 gel shift of Sertoli nuclear extracts was competed with excess unlabeled SE2 or E-box DNA fragments. Several Sertoli nuclear proteins associate with the SE2 gel shifts, including 70-, 42-, and 25-kDa proteins. Therefore, the critical SE2 element in the transferrin promoter is an E-box element capable of binding bHLH transcription factors. The ubiquitously expressed E12 bHLH protein dimerizes with numerous cell-specific bHLH factors. A Western blot analysis demonstrated that E12 was present in Sertoli cell nuclear extracts and associated with the SE2 gel shift. A ligand blot of Sertoli cell nuclear extracts with radiolabeled E12 had apparent bHLH proteins when the cells were stimulated to differentiate. The E-box sequence in the SE2 fragment of the transferrin promoter was CATCTG and was similar in gel shifts to the consensus E-box elements (CANNTG) previously characterized. A bHLH inhibitory factor (Id) competed and inhibited formation of the Sertoli cell nuclear extract E-box gel shift. To extend this observation, Id protein was overexpressed in cultured Sertoli cells. A transferrin promoter chloramphenicol acetyltransferase construct was used to monitor Sertoli cell function. The presence of Id suppressed the activation of the promoter induced by Sertoli differentiation factors. Therefore, the inhibition of Sertoli bHLH factors by Id suppressed Sertoli cell differentiated function, as measured by transferrin expression. An E-box-chloramphenicol acetyltransferase construct was also found to be active in Sertoli cells when cells were induced to differentiate. Screening the computerized nucleotide data bases demonstrated that putative E-box response elements are present in the promoters of a large number of Sertoli cell differentiated genes. In summary, a critical E-box response element has been identified in the transferrin promoter that can be activated by bHLH factors (e.g. E12) present in Sertoli cells. Inhibition of Sertoli bHLH factors by Id suppresses Sertoli cell differentiated function (i.e. transferrin expression), suggesting that bHLH transcription factors may be important in regulating Sertoli cell differentiated functions.

• Lin SC, Lin MH, Horvath P, Reddy KL, Storti RV
• PDP1, a novel Drosophila PAR domain bZIP transcription factor expressed in developing mesoderm, endoderm and ectoderm, is a transcriptional regulator of somatic muscle genes.
• Development. 1997; 124: 4685-96
• Display abstract

• In vertebrates, transcriptional control of skeletal muscle genes during differentiation is regulated by enhancers that direct the combinatorial binding and/or interaction of MEF2 and the bHLH MyoD family of myogenic factors. We have shown that Drosophila MEF2 plays a role similar to its vertebrate counterpart in the regulation of the Tropomyosin I gene in the development of Drosophila somatic muscles, however, unlike vertebrates, Drosophila MEF2 interacts with a muscle activator region that does not have binding sites for myogenic bHLH-like factors or any other known Drosophila transcription factors. We describe here the isolation and characterization of a component of the muscle activator region that we have named PDP1 (PAR domain protein 1). PDP1 is a novel transcription factor that is highly homologous to the PAR subfamily of mammalian bZIP transcription factors HLF, DBP and VBP/TEF. This is the first member of the PAR subfamily of bZIP transcription factors to be identified in Drosophila. We show that PDP1 is involved in regulating expression of the Tropomyosin I gene in somatic body-wall and pharyngeal muscles by binding to DNA sequences within the muscle activator that are required for activator function. Mutations that eliminate PDP1 binding eliminate muscle activator function and severely reduce expression of a muscle activator plus MEF2 mini-enhancer. These and previous results suggest that PDP1 may function as part of a larger protein/DNA complex that interacts with MEF2 to regulate transcription of Drosophila muscle genes. Furthermore, in addition to being expressed in the mesoderm that gives rise to the somatic muscles, PDP1 is also expressed in the mesodermal fat body, the developing midgut endoderm, the hindgut and Malpighian tubules, and the epidermis and central nervous system, suggesting that PDP1 is also involved in the terminal differentiation of these tissues.

• Araki I, Terazawa K, Satoh N
• Duplication of an amphioxus myogenic bHLH gene is independent of vertebrate myogenic bHLH gene duplication.
• Gene. 1996; 171: 231-6
• Display abstract

• Gene duplication is thought to be a major genetic change that may have permitted the evolution of vertebrates from invertebrates. The myogenic genes encode basic helix-loop-helix (bHLH) transcriptional factors essential for the formation of skeletal muscle. The invertebrate genome contains only a single myogenic bHLH gene, whereas the vertebrate genome contains four (MyoD, Myf-5, myogenin and MRF4). Since the tunicate genome contains a single myogenic bHLH gene, its duplication might have occurred some time during chordate evolution. To determine whether the duplication of the myogenic bHLH gene occurred prior to, or after the divergence of vertebrates from the cephalochordate lineage, we amplified target fragments from the amphioxus, Branchiostoma floridae, by means of PCR. Sequence analysis and genomic Southern analysis revealed that the amphioxus genome contains two myogenic bHLH genes (BMD1 and BMD2). A comparison of the amino acid sequences in the bHLH domain between BMD1, BMD2 and four vertebrate myogenic bHLH gene products, however, showed that neither BMD1 nor BMD2 resembled any of the four genes. These results suggested that the duplication of amphioxus myogenic bHLH gene occurred independently of that leading to the four myogenic bHLH genes in vertebrates.

• Peyton M, Stellrecht CM, Naya FJ, Huang HP, Samora PJ, Tsai MJ
• BETA3, a novel helix-loop-helix protein, can act as a negative regulator of BETA2 and MyoD-responsive genes.
• Mol Cell Biol. 1996; 16: 626-33
• Display abstract

• Using degenerate PCR cloning we have identified a novel basic helix-loop-helix (bHLH) transcription factor, BETA3, from a hamster insulin tumor (HIT) cell cDNA library. Sequence analysis revealed that this factor belongs to the class B bHLH family and has the highest degree of homology with another bHLH transcription factor recently isolated in our laboratory, BETA2 (neuroD) (J. E. Lee, S. M. Hollenberg, L. Snider, D. L. Turner, N. Lipnick, and H. Weintraub, Science 268:836-844, 1995; F. J. Naya, C. M. M. Stellrecht, and M.-J. Tsai, Genes Dev. 8:1009-1019, 1995). BETA2 is a brain- and pancreatic-islet-specific bHLH transcription factor and is largely responsible for the tissue-specific expression of the insulin gene. BETA3 was found to be tissue restricted, with the highest levels of expression in HIT, lung, kidney, and brain cells. Surprisingly, despite the homology between BETA2 and BETA3 and its intact basic region, BETA3 is unable to bind the insulin E box in bandshift analysis as a homodimer or as a heterodimer with the class A bHLH factors E12, E47, or BETA1. Instead, BETA3 inhibited both the E47 homodimer and the E47/BETA2 heterodimer binding to the insulin E box. In addition, BETA3 greatly repressed the BETA2/E47 induction of the insulin enhancer in HIT cells as well as the MyoD/E47 induction of a muscle-specific E box in the myoblast cell line C2C12. In contrast, expression of BETA3 had no significant effect on the GAL4-VP16 transcriptional activity. Immunoprecipitation analysis demonstrates that the mechanism of repression is via direct protein-protein interaction, presumably by heterodimerization between BETA3 and class A bHLH factors.

• Kunne AG, Meierhans D, Allemann RK
• Basic helix-loop-helix protein MyoD displays modest DNA binding specificity.
• FEBS Lett. 1996; 391: 79-83
• Display abstract

• The expression of MyoD can activate muscle specific genes and myogenic differentiation in many cell types. The hypothesis that the DNA binding specificity of MyoD is responsible for its biological specificity was tested. Homodimers of MyoD bind to E-box containing DNA with high affinity, but do not form stable and well defined complexes with heterologous DNA sequences. The physiologically active heterodimer of MyoD and E12 binds an oligonucleotide containing an E-box sequence with an affinity only two orders of magnitude higher than a completely unrelated DNA sequence, stressing the importance of cooperative interactions with other proteins of the transcriptional machinery for specific gene activation.

• Groisman R et al.
• Physical interaction between the mitogen-responsive serum response factor and myogenic basic-helix-loop-helix proteins.
• J Biol Chem. 1996; 271: 5258-64
• Display abstract

• Terminal differentiation of muscle cells results in opposite effects on gene promoters: muscle-specific promoters, which are repressed during active proliferation of myoblasts, are turned on, whereas at least some proliferation-associated promoters, such as c-fos, which are active during cell division, are turned off. MyoD and myogenin, transcription factors from the basic-helix-loop-helix (bHLH) family, are involved in both processes, up-regulating muscle genes and down-regulating c-fos. On the other hand, the serum response factor (SRF) is involved in the activation of muscle-specific genes, such as c-fos, as well as in the up-regulation of a subset of genes that are responsive to mitogens. Upon terminal differentiation, the activity of these various transcription factors could be modulated by the formation of distinct protein-protein complexes. Here, we have investigated the hypothesis that the function of SRF and/or MyoD and myogenin could be modulated by a physical association between these transcription factors. We show that myogenin from differentiating myoblasts specifically binds to SRF. In vitro analysis, using the glutathione S-transferase pull-down assay, indicates that SRF-myogenin interactions occur only with myogenin-E12 heterodimers and not with isolated myogenin. A physical interaction between myogenin, E12, and SRF could also be demonstrated in vivo using a triple-hybrid approach in yeast. Glutathione S-transferase pull-down analysis of various mutants of the proteins demonstrated that the bHLH domain of myogenin and that of E12 were necessary and sufficient for the interaction to be observed. Specific binding to SRF was also seen with MyoD. In contrast, Id, a natural inhibitor of myogenic bHLH proteins, did not bind SRF in any of the situations tested. These data suggest that SRF, on one hand, and myogenic bHLH, on the other, could modulate each other's activity through the formation of a heterotrimeric complex.

• Mak KL, Longcor LC, Johnson SE, Lemercier C, To RQ, Konieczny SF
• Examination of mammalian basic helix-loop-helix transcription factors using a yeast one-hybrid system.
• DNA Cell Biol. 1996; 15: 1-8
• Display abstract

• Basic helix-loop-helix (bHLH) transcription factors play diverse roles in controlling many developmental events. Although a great deal is understood about how bHLH factors activate gene transcription via E-box DNA consensus sequences, studies of bHLH factor function in higher eukaryotes often have been hindered by the presence of multiple family members. As a first step in developing a simplified in vivo system to examine bHLH factor activities, we examined whether the bHLH muscle regulatory factors MRF4 and MyoD function appropriately in yeast. We show that Gal4-MRF4 fusion proteins, or native MRF4 proteins, activate expression of an E-box HIS3 reporter gene whereas MyoD proteins remain inactive. Deletion of the MRF4 transcription activation domain (TAD) or point mutations that abolish MRF4 DNA interactions inhibit HIS3 expression. Substitution of the MRF4 TAD with the Gal4 TAD also produces a functional protein, demonstrating that these transcription activation domains are functionally equivalent in yeast. Replacement of the MRF4 TAD with the related MyoD TAD, however, generates an inactive protein, suggesting that some specificity exists between bHLH family members. Using this experimental system, we also demonstrate that mammalian cDNA libraries can be screened successfully for cDNAs encoding novel bHLH proteins that interact with E-box targets. Thus, this in vivo yeast system provides a novel approach to facilitate functional studies of bHLH factor regulation.

• Hofmann TJ, Cole MD
• The TAL1/Scl basic helix-loop-helix protein blocks myogenic differentiation and E-box dependent transactivation.
• Oncogene. 1996; 13: 617-24
• Display abstract

• The TAL1 gene is transcriptionally activated by chromosomal translocation in the most common genetic lesion associated with T-cell acute lymphoblastic leukemia. TAL1 encodes a bHLH protein that exhibits sequence-specific DNA binding activity when it forms dimers with another bHLH protein such as E2A. We show that ectopic expression of TAL1 blocks the ability of the bHLH gene myogenin to induce myotube differentiation in C3H10T1/2 cells. Cotransfection of TAL1 with either myogenin or E2-5 suppresses the transcriptional activation function of each gene on its respective reporter constructs. TAL1 was as effective as Id in both transcriptional suppression and inhibition of differentiation. Deletion of the C-terminal domain of TAL1 reduces or eliminates its ability to suppress transcription while preserving the bHLH domain that determines the sequence-specificity of DNA binding. These data suggest that the C-terminal domain of TAL1 may directly mask the transactivation domain of E2A-related proteins. Since E2A-related genes are involved in lymphocyte differentiation, the dominant inhibition of E2A-related proteins may be the primary mechanism by which the TAL1 oncogene promotes leukemia.

• Black BL, Ligon KL, Zhang Y, Olson EN
• Cooperative transcriptional activation by the neurogenic basic helix-loop-helix protein MASH1 and members of the myocyte enhancer factor-2 (MEF2) family.
• J Biol Chem. 1996; 271: 26659-63
• Display abstract

• Establishment of skeletal muscle and neural cell types is controlled by families of myogenic and neurogenic basic helix-loop-helix (bHLH) proteins, respectively. Myogenic bHLH proteins have been shown to activate skeletal muscle transcription in collaboration with members of the myocyte enhancer factor-2 (MEF2) family of MCM1-agamous-deficiens-serum response factor (MADS)-box transcription factors, which are expressed in differentiated myocytes and neurons. Here, we show that the neurogenic bHLH protein MASH1 interacts with members of the MEF2 family and that this interaction, mediated by the DNA binding and dimerization domains of these factors, results in synergistic activation of transcription through either the MASH1 or the MEF2 DNA binding site. Consistent with their involvement in activation of neuronal gene expression, members of the MEF2 family are expressed in P19 embryonal carcinoma cells that have been induced to form neurons following treatment with retinoic acid. These results suggest that members of the MEF2 family perform similar roles in synergistic activation of transcription in myogenic and neurogenic lineages by serving as cofactors for cell type-specific bHLH proteins.

• Wang J, Huang Q, Tang W, Nadal-Ginard B
• E2F1 inhibition of transcription activation by myogenic basic helix-loop-helix regulators.
• J Cell Biochem. 1996; 62: 405-10
• Display abstract

• Cellular transcription factor E2F1 is thought to regulate the expression of genes important for cell cycle progression and cell proliferation. Deregulated E2F1 expression induces S-phase entry in quiescent cells and inhibits myogenic differentiation. We show here that E2F1 inhibits the activation of gene transcription by myogenic basic helix-loop-helix proteins myoD and myogenin. Transfection assay using different deletion constructs indicates that both the DNA binding and the transactivation domains of E2F1 are required for its inhibition of myoD transcription activation. However, the retinoblastoma protein (RB) binding domain is not required. Furthermore, co-transfection with the RB, which inhibits the transcription activity of E2F1, can also repress E2F1 inhibition of myoD transactivation. These results suggest an essential role of E2F1-mediated transcription in its inhibition of myogenesis.

• Spicer DB, Rhee J, Cheung WL, Lassar AB
• Inhibition of myogenic bHLH and MEF2 transcription factors by the bHLH protein Twist.
• Science. 1996; 272: 1476-80
• Display abstract

• The myogenic basic helix-loop-helix (bHLH) and MEF2 transcription factors are expressed in the myotome of developing somites and cooperatively activate skeletal muscle gene expression. The bHLH protein Twist is expressed throughout the epithelial somite and is subsequently excluded from the myotome. Ectopically expressed mouse Twist (Mtwist) was shown to inhibit myogenesis by blocking DNA binding by MyoD, by titrating E proteins, and by inhibiting trans-activation by MEF2. For inhibition of MEF2, Mtwist required heterodimerization with E proteins and an intact basic domain and carboxyl-terminus. Thus, Mtwist inhibits both families of myogenic regulators and may regulate myotome formation temporally or spatially.

• Gigliani F, Longo F, Gaddini L, Battaglia PA
• Interactions among the bHLH domains of the proteins encoded by the Enhancer of split and achaete-scute gene complexes of Drosophila.
• Mol Gen Genet. 1996; 251: 628-34
• Display abstract

• The Enhancer of split and achaete-scute gene complexes [E(spl)-C and AS-C] encode helix-loop-helix proteins required for neurogenesis in Drosophila. Using a heterologous bacterial system, we show that (i) the bHLH domains of the proteins encoded by the two gene complexes differ in their ability to form homo- and/or heterodimers; (ii) the bHLH domains of the E(spl)-C proteins m5, m7 and m8 interact with the bHLH domains of the Ac and Sc proteins. These bHLH domains form an interaction network which may represent the molecular mechanism whereby the competent state of the proneural cells is maintained until the terminal determination to neuroblast occurs. Also, the pattern of interactions of the bHLH domains of certain proteins encoded by the two gene complexes may explain their functional redundancy.

• Molkentin JD, Olson EN
• Defining the regulatory networks for muscle development.
• Curr Opin Genet Dev. 1996; 6: 445-53
• Display abstract

• The formation of skeletal muscle during vertebrate embryogenesis requires commitment of mesodermal precursor cells to the skeletal muscle lineage, withdrawal of myoblasts from the cell cycle, and transcriptional activation of dozens of muscle structural genes. The myogenic basic helix-loop-helix (bHLH) factors - MyoD, myogenin, Myf5, and MRF4 - act at multiple points in the myogenic lineage to establish myoblast identity and to control terminal differentiation. Recent studies have begun to define the inductive mechanisms that regulate myogenic bHLH gene expression and muscle cell determination in the embryo. Myogenic bHLH factors interact with components of the cell cycle machinery to control withdrawal from the cell cycle and act combinatorially with other transcription factors to induce skeletal muscle transcription. Elucidation of these aspects of the myogenic program is leading to a detailed understanding of the regulatory circuits controlling muscle development.

• Yun K, Wold B
• Skeletal muscle determination and differentiation: story of a core regulatory network and its context.
• Curr Opin Cell Biol. 1996; 8: 877-89
• Display abstract

• Regulation of skeletal muscle determination and differentiation in vertebrates centers on a core regulatory network which is composed of two families of transcription factors, the MyoD group basic helix-loop-helix (bHLH) muscle regulatory factors (MRFs) and the myocyte enhancer factor 2 (MEF2) group of MADS-box regulators. Members of this network interact with each other genetically and physically, and together they cooperate to positively regulate transcription of downstream muscle-specific differentiation genes. During development, the myogenic network can be activated or repressed in response to patterning signals, some of which have recently been identified. Once activated, the powerful myogenic activity of the core network can be modulated and held in check by a remarkably large group of negative regulators that operate on network components by diverse mechanisms. Recent discoveries highlight extensive parallels between myogenesis and peripheral neurogenesis in the structures of their respective regulatory networks and in the interaction of their bHLH networks with other regulatory circuits. Comparisons with Drosophila indicate that these ensembles of interacting molecular circuits have been highly conserved during evolution.

• Johnson SE, Wang X, Hardy S, Taparowsky EJ, Konieczny SF
• Casein kinase II increases the transcriptional activities of MRF4 and MyoD independently of their direct phosphorylation.
• Mol Cell Biol. 1996; 16: 1604-13
• Display abstract

• The myogenic regulatory factors (MRFs) are a subclass of a much larger group of basic helix-loop-helix transcription factors which includes members of the E protein such as E47, E2-2, and HEB. Although the MRFs are unique in their ability to confer a myogenic phenotype on nonmuscle cells, they require E protein partners to form a MRF-E protein heterodimer, which represents the functional myogenesis-inducing complex. The mechanisms controlling homodimer and heterodimer formation in vivo remain largely unknown, although it is likely that posttranslational modification of one or both basic helix-loop-helix partners is critical to this regulatory event. In this respect, MyoD and MRF4, both members of the MRF family, exist in vivo as phosphoproteins and contains multiple consensus phosphorylation sites, including sites for casein kinase II (CKII) phosphorylation. In this study, we demonstrate that overexpression of CKII increases the transcriptional activities of MRF4 and MyoD in vivo. Interestingly, mutation of the individual CKII sites within MRF4 and MyoF does not alter the ability of CKII to enhance MRF transcriptional activity, suggesting that the effect of CKII expression on the MRFs is indirect. Given that the MRFs require dimerization with E protein partners to activate muscle-specific transcription, the effects of CKII expression on E protein function also were examined. Our studies show that E47 serves as an in vitro substrate for CKII and that CKII-phosphorylated E-47 proteins no longer bind to DNA. These observations were confirmed by in vivo experiments showing that overexpressing of CKII produces a dramatic reduction in E47 homodimer-directed transcription. We conclude from these studies that CKII may act as a positive regulator of myogenesis by preventing E protein homodimers from binding to muscle gene regulatory elements.

• Olson EN, Arnold HH, Rigby PW, Wold BJ
• Know your neighbors: three phenotypes in null mutants of the myogenic bHLH gene MRF4.
• Cell. 1996; 85: 1-4

• Goldfarb AN, Lewandowska K, Shoham M
• Determinants of helix-loop-helix dimerization affinity. Random mutational analysis of SCL/tal.
• J Biol Chem. 1996; 271: 2683-8
• Display abstract

• Dimerization represents a key regulatory step in the function of basic helix-loop-helix transcriptional factors. In many instances tissue-specific basic helix-loop-helix proteins, such as the hematopoietic factor SCL/tal or the myogenic factor MyoD, interact with ubiquitously expressed basic helix-loop-helix proteins, such as E2A or E2-2. Such dimerization is necessary for high affinity, sequence-specific DNA binding. Previous biochemical and structural studies have shown the helix-loop-helix region to be necessary and sufficient for this interaction. In the present study, we analyzed the relative affinities of various helix-loop-helix interactions using the yeast two-hybrid system. The relative affinities of selected helix-loop-helix species for the partner protein E2-2 were as follows: Id2 > MyoD > SCL/tal. Mutants of SCL/tal with increased affinity for E2-2 were selected from a library of randomly mutated basic helix-loop-helix domains. The amino acid changes in these high affinity versions of SCL/tal introduced residues that resembled those in the corresponding positions of the Id proteins and MyoD. One of the mutants, SCL 12, also contained mutations in highly conserved residues previously thought to be necessary for dimerization. This mutant of SCL demonstrated diminished temperature sensitivity in in vitro interaction assays as compared with the wild type protein. Computational modeling of helix-loop-helix dimers provides an explanation for the increased dimerization affinity of SCL mutant 12.

• Sandmoller A, Meents H, Arnold HH
• A novel E1A domain mediates skeletal-muscle-specific enhancer repression independently of pRB and p300 binding.
• Mol Cell Biol. 1996; 16: 5846-56
• Display abstract

• The adenovirus E1A oncoprotein completely blocks muscle differentiation and specifically inhibits the transactivating function of myogenic basic helix-loop-helix (bHLH) transcription factors. This inhibition is dependent on the conserved region CR1 of E1A, which also constitutes part of the binding sites for the pocket proteins pRB, p107, and p130 and the transcriptional coactivators p300 and CBP. Here we report a detailed mutational analysis of E1A and the identification of a muscle inhibition motif within CR1. This motif encompasses amino acids 38 to 62 and inhibits Myf-5- or MyoD-mediated activation of myogenin and the muscle creatine kinase gene. Overexpression of this E1A region also inhibits the conversion of 10T1/2 fibroblasts to the myogenic lineage. The sequence motif EPDNEE (amino acids 55 to 60) within CR1 appears to be particularly important, because point mutations of this sequence diminish the E1A inhibitory activity. Interactions of E1A with pRB and with p300 do not seem to be necessary for the muscle-specific enhancer repression, because E1A mutants which lack these interactions still inhibit Myf-5- and MyoD-mediated transactivation. Moreover, overexpression of p300 fails to overcome muscle-specific inhibition by wild-type E1A and mutant E1A protein which lacks pRB binding. Since we have no evidence for direct E1A interaction with bHLH proteins, we propose that E1A may target a necessary cofactor of the muscle-specific bHLH transcription complex.

• Eckner R, Yao TP, Oldread E, Livingston DM
• Interaction and functional collaboration of p300/CBP and bHLH proteins in muscle and B-cell differentiation.
• Genes Dev. 1996; 10: 2478-90
• Display abstract

• Differentiation of skeletal muscle cells and B lymphocytes is regulated by basic helix-loop-helix (bHLH) proteins. Both differentiation programs are inhibited by the adenovirus E1A oncoprotein. Analysis of E1A mutants has implicated two of its cellular-binding proteins, p300 and CBP, in controlling certain aspects of differentiation. We find that p300 can cooperate with tissue-specific bHLH proteins in activating target genes and requires only the bHLH domain of such proteins to stimulate E box-directed transcription. Importantly, the ability of bHLH proteins to activate transcription correlates with the presence of p300/CBP in E box-dependent DNA-binding complexes, because both phenomena require at least two adjacent E-box motifs. Microinjection of p300/CBP antibodies into myoblasts blocks terminal differentiation, cell fusion, and transcriptional activity of myogenic bHLH proteins. These results suggest that the function of p300/CBP is essential for the execution of key aspects of cellular differentiation.

• Gerber AN, Tapscott SJ
• Tumor cell complementation groups based on myogenic potential: evidence for inactivation of loci required for basic helix-loop-helix protein activity.
• Mol Cell Biol. 1996; 16: 3901-8
• Display abstract

• Basic helix-loop-helix (bHLH) proteins mediate terminal differentiation in many lineages. By using the bHLH protein MyoD, which can dominantly activate the myogenic differentiation program in numerous cell types, we demonstrated that recessive defects in bHLH protein function are present in human tumor lines. In contrast to prior work with primary cell cultures, MyoD did not activate the myogenic program in six of the eight tumor lines we tested. Cell fusions between the MyoD-defective lines and fibroblasts restored MyoD activity, indicating that the deficiency of a gene or factor prevents bHLH protein function in the tumor lines. Fusions between certain pairings of the MyoD-defective lines also restored MyoD activity, allowing the tumor lines to be assigned to complementation groups on the basis of their ability to execute the myogenic program and indicating that multiple mechanisms exist for abrogation of bHLH protein activity. These groups provide a basis for identifying genes critical for bHLH-mediated differentiation and tumor progression by using genetic complementation.

• Naya FJ, Stellrecht CM, Tsai MJ
• Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor.
• Genes Dev. 1995; 9: 1009-19
• Display abstract

• The insulin gene is one of the best paradigms of tissue-specific gene expression. It is developmentally regulated and is expressed exclusively in the pancreatic beta-cell. This restricted expression is directed by a tissue-specific enhancer, within the promoter, which contains an E-box sequence. The insulin E-box binds an islet-specific protein complex, termed 3a1. E-boxes bind proteins belonging to the basic helix-loop-helix (bHLH) family of transcription factors. The bHLH proteins function as potent transcriptional activators of tissue-specific genes by forming heterodimers between ubiquitous and cell-restricted family members. In addition, the cell-restricted bHLH members play an important role in specifying cell fate. To isolate the tissue-specific bHLH factor controlling insulin gene expression and study its role in islet cell differentiation, a modified yeast two-hybrid system was utilized to clone a novel bHLH factor, BETA2 (beta-cell E-box trans-activator 2), from a hamster insulin tumor (HIT) cell cDNA library. Northern analysis demonstrates that high-level expression of the BETA2 gene is restricted to pancreatic alpha- and beta-cell lines. As expected of tissue-specific bHLH members, BETA2 binds to the insulin E-box sequence with high affinity as a heterodimer with the ubiquitous bHLH factor E47. More importantly, antibody supershift experiments clearly show that BETA2 is a component of the native insulin E-box-binding complex. Transient transfection assays demonstrate that the BETA2/E47 heterodimer synergistically interacts with a neighboring beta-cell-specific complex to activate an insulin enhancer. In contrast, other bHLH factors such as MyoD and E47, which can bind to the insulin E-box with high affinity, fail to do so. Thus, a unique, cooperative interaction is the basis by which the insulin E-box enhancer discriminates between various bHLH factors to achieve tissue-specific activation of the insulin gene.

• Molkentin JD, Black BL, Martin JF, Olson EN
• Cooperative activation of muscle gene expression by MEF2 and myogenic bHLH proteins.
• Cell. 1995; 83: 1125-36
• Display abstract

• Members of the myocyte enhancer factor-2 (MEF2) family of MADS domain transcription factors cannot induce myogenesis in transfected fibroblasts, but when coexpressed with the myogenic basic-helix-loop-helix (bHLH) proteins MyoD or myogenin they dramatically increase the extent of myogenic conversion above that seen with either myogenic bHLH factor alone. This cooperativity required direct interactions between the DNA-binding domains of MEF2 and the myogenic bHLH factors, but only one of the factors needed a transactivation domain, and only one of the factors needed to be bound to DNA. These interactions allow either factor to activate transcription through the other's binding site and reveal a novel mechanism for indirect activation of gene expression via protein-protein interactions between the DNA-binding domains of heterologous classes of transcription factors.

• Cserjesi P et al.
• Scleraxis: a basic helix-loop-helix protein that prefigures skeletal formation during mouse embryogenesis.
• Development. 1995; 121: 1099-110
• Display abstract

• Members of the basic helix-loop-helix (bHLH) family of transcription factors have been shown to regulate growth and differentiation of numerous cell types. Cell-type-specific bHLH proteins typically form heterodimers with ubiquitous bHLH proteins, such as E12, and bind a DNA consensus sequence known as an E-box. We used the yeast two-hybrid system to screen mouse embryo cDNA libraries for cDNAs encoding novel cell-type-specific bHLH proteins that dimerize with E12. One of the cDNAs isolated encoded a novel bHLH protein, called scleraxis. During mouse embryogenesis, scleraxis transcripts were first detected between day 9.5 and 10.5 post coitum (p.c.) in the sclerotome of the somites and in mesenchymal cells in the body wall and limb buds. Subsequently, scleraxis was expressed at high levels within mesenchymal precursors of the axial and appendicular skeleton and in cranial mesenchyme in advance of chondrogenesis; its expression pattern in these cell types foreshadowed the developing skeleton. Prior to formation of the embryonic cartilaginous skeleton, scleraxis expression declined to low levels. As development proceeded, high levels of scleraxis expression became restricted to regions where cartilage and connective tissue formation take place. Scleraxis bound the E-box consensus sequence as a heterodimer with E12 and activated transcription of a reporter gene linked to its DNA-binding site. The expression pattern, DNA-binding properties and transcriptional activity of scleraxis suggest that it is a regulator of gene expression within mesenchymal cell lineages that give rise to cartilage and connective tissue.

• Olson EN, Perry M, Schulz RA
• Regulation of muscle differentiation by the MEF2 family of MADS box transcription factors.
• Dev Biol. 1995; 172: 2-14

• Szymanski P, Levine M
• Multiple modes of dorsal-bHLH transcriptional synergy in the Drosophila embryo.
• EMBO J. 1995; 14: 2229-38
• Display abstract

• Synergistic interactions between the maternal regulatory factor dorsal (dl) and basic helix-loop-helix (bHLH) activators are essential for initiating differentiation of the mesoderm and neuroectoderm in the early Drosophila embryo. Here we present evidence that dl-bHLH interactions mediating gene expression in the neuroectoderm and mesoderm are fundamentally distinct. Close proximity of dl and bHLH binding sites is essential for the synergistic activation of gene expression in the lateral neuroectoderm, where there are diminishing levels of the dl regulatory gradient. In contrast, sharp on/off patterns of gene expression in the presumptive mesoderm do not require linkage of these sites. Analysis of minimal and synthetic promoter elements suggests that dl and bHLH activators, such as twist, might interact with different rate-limiting components of the transcription complex. These results are consistent with two distinct modes of dl-bHLH synergy: cooperative binding to DNA (requiring linkage of sites) and synergistic contact of basal transcription factors (not requiring linkage). Finally, the characterization of a 57 bp synthetic minimal stripe unit (MSU) provides evidence for a third tier of dl-bHLH synergy. Tandem copies of the MSU function as a bona fide enhancer and can mediate neuroectoderm expression in transgenic embryos even when placed 4.5 kb downstream of a test promoter. Multiple copies of the MSU function synergistically only when linked, but not when separated. We propose that this linkage requirement provides the basis for the evolution of modular promoters composed of discrete, non-overlapping enhancers.

• Baudier J, Bergeret E, Bertacchi N, Weintraub H, Gagnon J, Garin J
• Interactions of myogenic bHLH transcription factors with calcium-binding calmodulin and S100a (alpha alpha) proteins.
• Biochemistry. 1995; 34: 7834-46
• Display abstract

• MyoD belongs to a family of myogenic basic helix-loop-helix (bHLH) transcription factors that activate muscle-specific genes. The basic helix I sequence of the bHLH motif contains a consensus sequence for protein kinase C (PKC) substrates. We show here that MyoD is indeed phosphorylated by PKC in vitro on Thr 115 within the basic part of the bHLH motif. By analogy with calmodulin-target peptide models, we also identified within the consensus basic helix I motif of myogenic proteins a conserved putative calmodulin/S100-binding domain. Calcium-dependent interaction between MyoD with calmodulin and the abundant muscle S100a(alpha alpha) proteins was demonstrated by affinity chromatography and cross-linking experiments. The binding of calmodulin and S100a inhibited MyoD phosphorylation by PKC as well as MyoD DNA binding activity. S100a was found to be more efficient than calmodulin in antagonizing DNA binding to MyoD. We next developed a rapid purification method for bacterial recombinant MyoD-bHLH domain by affinity chromatography using a calmodulin-Sepharose column and investigated the phosphorylation of that peptide by PKC and its interactions with calmodulin and S100a. We confirmed the phosphorylation of the threonine residue 115 in the MyoD-bHLH by PKC with a Km of 0.8 microM. Calmodulin and S100a binding inhibited MyoD-bHLH phosphorylation by PKC. A strict calcium-dependent interaction between calcium binding proteins and the MyoD-bHLH was identified by native gel electrophoresis and fluorescence spectroscopy with 5-(dimethylamino)naphthalene-1-sulfonylcalmodulin. The MyoD-bHLH bound to fluorescently labeled 5-(dimethylamino)naphthalene-1-sulfonylcalmodulin with a dissociation constant around 20 nM. S100a inhibited stoichiometrically the binding of the bHLH peptide for labeled calmodulin, suggesting an affinity of S100a for the bHLH peptide at least 1 order of magnitude higher than calmodulin. In favor of an in vivo interaction between S100a and MyoD, we report that S100a- and MyoD-like immunoreactivities colocalize in H9c2 cells, and that a significant amount of MyoD-like immunoreactivity is recovered in the S100a immunoprecipitate from crude H9c2 cell extract in the presence of calcium. We propose that myogenic proteins represent a new family of calmodulin/S100-binding PKC substrates and that calmodulin/S100a could participate in the regulation of the bHLH myogenic protein activities.

• Hollenberg SM, Sternglanz R, Cheng PF, Weintraub H
• Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system.
• Mol Cell Biol. 1995; 15: 3813-22
• Display abstract

• 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.

• Dechesne CA et al.
• E-box- and MEF-2-independent muscle-specific expression, positive autoregulation, and cross-activation of the chicken MyoD (CMD1) promoter reveal an indirect regulatory pathway.
• Mol Cell Biol. 1994; 14: 5474-86
• Display abstract

• Members of the MyoD family of gene-regulatory proteins (MyoD, myogenin, myf5, and MRF4) have all been shown not only to regulate the transcription of numerous muscle-specific genes but also to positively autoregulate and cross activate each other's transcription. In the case of muscle-specific genes, this transcriptional regulation can often be correlated with the presence of a DNA consensus in the regulatory region CANNTG, known as an E box. Little is known about the regulatory interactions of the myogenic factors themselves; however, these interactions are thought to be important for the activation and maintenance of the muscle phenotype. We have identified the minimal region in the chicken MyoD (CMD1) promoter necessary for muscle-specific transcription in primary cultures of embryonic chicken skeletal muscle. The CMD1 promoter is silent in primary chick fibroblast cultures and in muscle cell cultures treated with the thymidine analog bromodeoxyuridine. However, CMD1 and chicken myogenin, as well as, to a lesser degree, chicken Myf5 and MRF4, expressed in trans can activate transcription from the minimal CMD1 promoter in these primary fibroblast cultures. Here we show that the CMD1 promoter contains numerous E-box binding sites for CMD1 and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific expression, autoregulation, or cross activation depends upon the presence of of these E-box or MEF-2 binding sites in the CMD1 promoter. These results demonstrate that the autoregulation and cross activation of the chicken MyoD promoter through the putative direct binding of the myogenic basic helix-loop-helix regulatory factors is mediated through an indirect pathway that involves unidentified regulatory elements and/or ancillary factors.

• Kaushal S, Schneider JW, Nadal-Ginard B, Mahdavi V
• Activation of the myogenic lineage by MEF2A, a factor that induces and cooperates with MyoD.
• Science. 1994; 266: 1236-40
• Display abstract

• Muscle enhancer factor-2A (MEF2A), a member of the MADS family, induced myogenic development when ectopically expressed in clones of nonmuscle cells of human clones, a function previously limited to the muscle basic helix-loop-helix (bHLH) proteins. During myogenesis, MEF2A and bHLH proteins cooperatively activate skeletal muscle genes and physically interact through the MADS domain of MEF2A and the three myogenic amino acids of the muscle bHLH proteins. Thus, skeletal myogenesis is mediated by two distinct families of mutually inducible and interactive muscle transcription factors, either of which can initiate the developmental cascade.

• Anand G, Shapiro DN, Dickman PS, Prochownik EV
• Rhabdomyosarcomas do not contain mutations in the DNA binding domains of myogenic transcription factors.
• J Clin Invest. 1994; 93: 5-9
• Display abstract

• Skeletal myogenesis is regulated by a group of transcription factors (MyoD, myogenin, myf5, and myf6) that are "basic helix-loop-helix" proteins that bind to the promoters of muscle-specific genes and promote their expression. We have previously shown that after a mutation of Leu122 to Arg the DNA binding basic domain of MyoD confers c-myc-like functional characteristics to the protein. In this study we used single-strand conformation polymorphism analysis to determine whether such mutations occur naturally in rhabdomyosarcomas. We have found that the basic domains of all the myogenic factors remain unaltered in rhabdomyosarcomas. Selection against such mutations may be the result of functional redundancy of these myogenic transcription factors.

• Murre C et al.
• Structure and function of helix-loop-helix proteins.
• Biochim Biophys Acta. 1994; 1218: 129-35

• Dias P, Dilling M, Houghton P
• The molecular basis of skeletal muscle differentiation.
• Semin Diagn Pathol. 1994; 11: 3-14
• Display abstract

• In recent years, significant advances have been made in understanding the molecular mechanisms involved in the regulation of skeletal-muscle differentiation. This review focuses on the role of the MyoD family of myogenic transcription factors that includes MyoD, myf-5, myogenin, and MRF4 (herculin or myf-6) in myogenesis. Members of this family share sequence homology for the basic-helix-loop-helix (bHLH) regulatory motif. The basic domain is required for DNA binding, whereas the HLH domain is required for dimerization. The bHLH motif confers both properties of transcriptional activation of muscle specific genes and inhibition of cell growth through collaboration with the E2A gene products (E12 and E47) and the retinoblastoma gene product (pRB). The functions of the MyoD family can be suppressed through inhibition of their expression or activity by various factors. These include peptide growth factors (FGF and TGF-beta), immediate early gene products (Fos, Jun and Myc), other oncogene products (Ras, Src), the Id protein, and innervation. The use of gene-knockout animal models has shown that there is some degree of functional redundancy in that inactivation of either MyoD or myf-5 has no effect on muscle development, whereas inactivation of both genes results in an absolute lack of muscle cells. In contrast, the inactivation of myogenin alone results in mice with gross deficiency of mature muscle.

• Doyle K, Zhang Y, Baer R, Bina M
• Distinguishable patterns of protein-DNA interactions involving complexes of basic helix-loop-helix proteins.
• J Biol Chem. 1994; 269: 12099-105
• Display abstract

• Myogenic factors and TAL1 possess distinguishable DNA binding characteristics when they form a complex with basic helix-loop-helix (bHLH) proteins of class A. These characteristics were evident in electrophoretic mobility shift assays showing that complexes of myogenic factors and HTF4 displayed a relatively high affinity for the enhancer in the muscle creatine kinase gene, whereas TAL1 appeared to greatly attenuate the interaction of HTF4 with this enhancer. In addition, by forming a complex with HTF4 in solution, TAL1 could exert a negative effect on the interactions of HTF4 with elements that include E box motifs of microE2 (CAGCTG) and kappa E2/microE5 (CACCTG) type. Similarly, heterodimers containing TAL1 and the DNA binding domain of E47 exhibited a relatively weak affinity for microE2 and kappa E2/microE5 core motifs. The results of both studies invoked the hypothesis that in vivo TAL1 might act as a negative regulator of microE2 and kappa E2/microE5 sequence motifs by forming a complex with the products of the E2A and HTF4 genes. Support for this hypothesis was obtained by transient expression analyses where TAL1 was found to inhibit the activation effects produced by E2-5 and HTF4a on a reporter gene construct containing repeated microE2 and microE5 motifs, derived from the immunoglobulin gene enhancer.

• Corneliussen B et al.
• Calcium/calmodulin inhibition of basic-helix-loop-helix transcription factor domains.
• Nature. 1994; 368: 760-4
• Display abstract

• The ubiquitous Ca(2+)-binding protein calmodulin (CaM) is a key protein in Ca2+ homeostasis and activation of eukaryotic cells. CaM is the molecular link between free Ca2+ in the cell and the inhibition, or activation, of numerous enzymes. Many nuclear functions are under Ca2+/CaM control, and some transcriptional activators are known to be Ca2+ modulated indirectly through Ca2+/CaM-dependent protein kinases. But Ca2+/CaM has not yet been found to directly modulate any transcription factor or other DNA-binding protein. Transcription factors of the basic-helix-loop-helix (bHLH) group are important regulators in numerous systems. Here we report that binding of Ca(2+)-loaded CaM to the bHLH domains of several bHLH proteins directly inhibits their DNA binding. Other bHLH proteins are either less sensitive or resistant. Ca2+ ionophore selectively inhibits transcriptional activation by Ca2+/CaM-sensitive bHLH proteins in vivo, implying that Ca2+ can directly influence transcription through differential CaM inhibition of bHLH domains.

• Leibham D et al.
• Binding of TFIID and MEF2 to the TATA element activates transcription of the Xenopus MyoDa promoter.
• Mol Cell Biol. 1994; 14: 686-99
• Display abstract

• Members of the MyoD family of helix-loop-helix proteins control expression of the muscle phenotype by regulating the activity of subordinate genes. To investigate processes that control the expression of myogenic factors and regulate the establishment and maintenance of the skeletal muscle phenotype, we have analyzed sequences necessary for transcription of the maternally expressed Xenopus MyoD (XMyoD) gene. A 3.5-kb DNA fragment containing the XMyoDa promoter was expressed in a somite-specific manner in injected frog embryos. The XMyoDa promoter was active in oocytes and cultured muscle cells but not in fibroblasts or nonmuscle cell lines. A 58-bp fragment containing the transcription initiation site, a GC-rich region, and overlapping binding sites for the general transcription factor TFIID and the muscle-specific factor MEF2 was sufficient for muscle-specific transcription. Transcription of the minimal XMyoDa promoter in nonmuscle cells was activated by expression of Xenopus MEF2 (XMEF2) and required binding of both MEF2 and TFIID to the TATA motif. These results demonstrate that the XMyoDa TATA motif is a target for a cell-type-specific regulatory factor and suggests that MEF2 stabilizes and amplifies XMyoDa transcription in mesodermal cells committed to the muscle phenotype.

• Lassar A, Munsterberg A
• Wiring diagrams: regulatory circuits and the control of skeletal myogenesis.
• Curr Opin Cell Biol. 1994; 6: 432-42
• Display abstract

• During the past year, targeted mutagenesis in mice has begun to clarify the roles of individual members of the MyoD family of myogenic regulators in vertebrate development. In this review, we discuss these studies both in the context of tissue interactions necessary to induce skeletal muscle precursor cells during embryogenesis and the molecular circuitry that regulates the terminal differentiation of these cells.

• Olson EN, Klein WH
• bHLH factors in muscle development: dead lines and commitments, what to leave in and what to leave out.
• Genes Dev. 1994; 8: 1-8

• Atchley WR, Fitch WM, Bronner-Fraser M
• Molecular evolution of the MyoD family of transcription factors.
• Proc Natl Acad Sci U S A. 1994; 91: 11522-6
• Display abstract

• Myogenesis in skeletal muscle is a cascade of developmental events whose initiation involves the MyoD family of transcription factors. Evolutionary analyses of amino acid sequences of this family of transcriptional activators suggest that the vertebrate genes MyoD1, myf-5, Myog (myogenin), and myf-6 were derived by gene duplications from a single ancestral gene. A common genetic origin predicts some functional redundancy between MyoD1 and myf-5 and between Myog and myf-6. Experimental studies have suggested that these pairs of genes can substitute for each other during myogenesis. Separate analyses of the conserved basic helix-loop-helix and nonconserved flanking elements yield similar branching sequences but show evolutionary change in the basic helix-loop-helix region has occurred at a much slower rate.

• Olson EN
• Regulation of muscle transcription by the MyoD family. The heart of the matter.
• Circ Res. 1993; 72: 1-6
• Display abstract

• The two striated muscle cell types, skeletal and cardiac muscle, express overlapping sets of muscle-specific genes. Activation of muscle-specific transcription in skeletal muscle is controlled by the MyoD family of regulatory factors, which are expressed exclusively in skeletal muscle. Members of the MyoD family share homology within a basic helix-loop-helix (HLH) motif that mediates DNA binding and dimerization and form heterodimers with widely expressed HLH proteins, referred to as E proteins. Although many of the genes that are regulated by members of the MyoD family are also expressed in cardiac muscle, known members of the MyoD family have never been detected in cardiac muscle, suggesting that cardiac myocytes either express unique cell type-specific HLH proteins or rely on a distinct regulatory strategy for activation of cardiac muscle transcription. This review will summarize current knowledge of the mechanisms through which the MyoD family activates skeletal muscle transcription and will consider potential mechanisms that may regulate gene expression in the heart.

• Taylor DA, Kraus VB, Schwarz JJ, Olson EN, Kraus WE
• E1A-mediated inhibition of myogenesis correlates with a direct physical interaction of E1A12S and basic helix-loop-helix proteins.
• Mol Cell Biol. 1993; 13: 4714-27
• Display abstract

• The observation that adenovirus E1A gene products can inhibit differentiation of skeletal myocytes suggested that E1A may interfere with the activity of myogenic basic helix-loop-helix (bHLH) transcription factors. We have examined the ability of E1A to mediate repression of the muscle-specific creatine kinase (MCK) gene. Both the E1A12S and E1A13S products repressed MCK transcription in a concentration-dependent fashion. In contrast, amino-terminal deletion mutants (d2-36 and d15-35) of E1A12S were defective for repression. E1A12S also repressed expression of a promoter containing a multimer of the MCK high-affinity E box (the consensus site for myogenic bHLH protein binding) that was dependent, in C3H10T1/2 cells, on coexpression of a myogenin bHLH-VP16 fusion protein. A series of coprecipitation experiments with glutathione S-transferase fusion and in vitro-translated proteins demonstrated that E1A12S, but not amino-terminal E1A deletion mutants, could bind to full-length myogenin and E12 and to deletion mutants of myogenin and E12 that spare the bHLH domains. Thus, the bHLH domains of myogenin and E12, and the high-affinity E box, are targets for E1A-mediated repression of the MCK enhancer, and domains of E1A required for repression of muscle-specific gene transcription also mediate binding to bHLH proteins. We conclude that E1A mediates repression of muscle-specific gene transcription through its amino-terminal domain and propose that this may involve a direct physical interaction between E1A and the bHLH region of myogenic determination proteins.

• Wright WE
• Muscle basic helix-loop-helix proteins and the regulation of myogenesis.
• Curr Opin Genet Dev. 1992; 2: 243-8
• Display abstract

• Significant progress has been made in defining the structural motifs that distinguish the muscle-specific from other basic helix-loop-helix proteins. Evidence is accumulating for multiple levels of regulation of the expression and action of the muscle basic helix-loop-helix factors.

• Li L, Zhou J, James G, Heller-Harrison R, Czech MP, Olson EN
• FGF inactivates myogenic helix-loop-helix proteins through phosphorylation of a conserved protein kinase C site in their DNA-binding domains.
• Cell. 1992; 71: 1181-94
• Display abstract

• Myogenin belongs to a family of myogenic helix-loop-helix (HLH) proteins that activate muscle transcription through binding to a conserved DNA sequence associated with numerous muscle-specific genes. Fibroblast growth factor (FGF) inhibits myogenesis by inactivating myogenic HLH proteins. We show that activated protein kinase C (PKC) can substitute for FGF and inhibit transcriptional activity of myogenic HLH proteins. In transfected cells, FGF induces phosphorylation of a conserved site in the DNA-binding domain of myogenin. This site is phosphorylated by PKC in vivo and in vitro and mediates repression of the myogenic program through a loss in DNA binding activity. A myogenin mutant lacking the PKC phosphorylation site is not repressed by FGF, confirming this site as a molecular target for FGF-dependent repression of muscle transcription. These results establish a direct link between the signal transduction pathways that inhibit myogenesis and the transcription factors directly activating muscle-specific genes.

• Schwarz JJ, Chakraborty T, Martin J, Zhou JM, Olson EN
• The basic region of myogenin cooperates with two transcription activation domains to induce muscle-specific transcription.
• Mol Cell Biol. 1992; 12: 266-75
• Display abstract

• Myogenin is a skeletal muscle-specific transcription factor that can activate myogenesis when introduced into a variety of nonmuscle cell types. Activation of the myogenic program by myogenin is dependent on its binding to a DNA sequence known as an E box, which is associated with numerous muscle-specific genes. Myogenin shares homology with MyoD and other myogenic regulatory factors within a basic region and a helix-loop-helix (HLH) motif that mediate DNA binding and dimerization, respectively. Here we show that the basic region-HLH motif of myogenin alone lacks transcriptional activity and is dependent on domains in the amino and carboxyl termini to activate transcription. Analysis of these N- and C-terminal domains through creation of chimeras with the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4 revealed that they act as strong transcriptional activators. These transcription activation domains are dependent for activity on a specific amino acid sequence within the basic region, referred to as the myogenic recognition motif (MRM), when an E box is the target for DNA binding. However, the activation domains function independent of the MRM when DNA binding is mediated through a heterologous DNA-binding domain. The activation domain of the acidic coactivator VP16 can substitute for the myogenin activation domains and restore strong myogenic activity to the basic region-HLH motif. Within a myogenin-VP16 chimera, however, the VP16 activation domain also relies on the MRM for activation of the myogenic program. These findings reveal that DNA binding and transcriptional activation are separable functions, encoded by different domains of myogenin, but that the activity of the transcriptional activation domains is influenced by the DNA-binding domain. Activation of muscle-specific transcription requires collaboration between the DNA-binding and activation domains of myogenin and is dependent on events in addition to DNA binding.

• Sasai Y, Kageyama R, Tagawa Y, Shigemoto R, Nakanishi S
• Two mammalian helix-loop-helix factors structurally related to Drosophila hairy and Enhancer of split.
• Genes Dev. 1992; 6: 2620-34
• Display abstract

• We report the molecular characterization of two novel rat helix-loop-helix (HLH) proteins, designated HES-1 and HES-3, that show structural homology to the Drosophila hairy and Enhancer of split [E(spl)] proteins, both of which are required for normal neurogenesis. HES-1 mRNA, expressed in various tissues of both embryos and adults, is present at a high level in the epithelial cells, including the embryonal neuroepithelial cells, as well as in the mesoderm-derived tissues such as the embryonal muscle. In contrast, HES-3 mRNA is produced exclusively in cerebellar Purkinje cells. HES-1 represses transcription by binding to the N box, which is a recognition sequence of E(spl) proteins. Interestingly, neither HES-1 nor HES-3 alone interacts efficiently with the E box, but each protein decreases the transcription induced by E-box-binding HLH activators such as E47. Furthermore, HES-1 also inhibits the functions of MyoD and MASH1 and effectively diminishes the myogenic conversion of C3H10T1/2 cells induced by MyoD. These results suggest that HES-1 may play an important role in mammalian development by negatively acting on the two different sequences while HES-3 acts as a repressor in a specific type of neurons.

• Funk WD, Wright WE
• Cyclic amplification and selection of targets for multicomponent complexes: myogenin interacts with factors recognizing binding sites for basic helix-loop-helix, nuclear factor 1, myocyte-specific enhancer-binding factor 2, and COMP1 factor.
• Proc Natl Acad Sci U S A. 1992; 89: 9484-8
• Display abstract

• Myogenin is one of four muscle-specific basic helix-loop-helix regulatory factors involved in controlling myogenesis. We here describe various protein complexes that increase the affinity of myogenin for DNA. We mixed an oligonucleotide containing a degenerate center large enough to accommodate multiple binding sites with crude myotube nuclear extracts and used cyclic amplification and selection of targets with an antimyogenin monoclonal antibody to isolate protein-DNA complexes. Since each cycle of selection results in the enrichment for the sequences with the highest affinity, we isolated multicomponent sites in which myogenin binding was increased by its interaction with other DNA binding proteins. Myogenin interacts with members of the nuclear factor 1 family, the muscle-specific factor myocyte-specific enhancer-binding factor 2, and another factor, COMP1 (cooperates with myogenic proteins 1), that binds to the sequence TGATTGAC. Myogenin also exhibits cooperative binding with other proteins that recognize CANNTG motifs, and various constraints on spacing and orientation were observed. The application of this approach to other transcription factors should not only help identify the different functions of myogenin versus other members of the muscle basic helix-loop-helix regulatory family but also help define the general combinatorial mechanisms involved in eukaryotic gene regulation.

• Wright WE, Catala F, Farmer K
• Multimeric structures influence the binding activity of bHLH muscle regulatory factors.
• Symp Soc Exp Biol. 1992; 46: 79-87
• Display abstract

• Sucrose gradients and molecular sieve chromatography were used to determine the native molecular weight of the basic HLH proteins myogenin, MyoD and E12. The muscle bHLH proteins not only formed dimers but also associated in a variety of higher order complexes. Although homodimers bind to DNA sequences such as the MEF-1 site in the creatine kinase enhancer, homotetramers and larger forms do not recognize this DNA sequence. Little evidence for complexes larger than dimers was found for the ubiquitous bHLH protein E12. Most of the myogenin remains in large complexes when myogenin and E12 are mixed. The same result was obtained in nuclear extracts from differentiated myotubes, in which most of the myogenin was found to be present in large complexes that do not bind to the creatine kinase enhancer. A fusion protein that contains only the myogenin HLH region fused to glutathione-S-transferase also forms large homomeric complexes. A model to explain these results is that each helix of the HLH motif can associate with a different subunit to form chains or ring structures. The presence of myogenin in nuclear extracts as both dimers that recognize known DNA sequences as well as higher order complexes that do not raises significant issues concerning the regulation of skeletal muscle bHLH protein activity during myogenesis.

• Olson E
• Activation of muscle-specific transcription by myogenic helix-loop-helix proteins.
• Symp Soc Exp Biol. 1992; 46: 331-41
• Display abstract

• Myogenin is a muscle-specific transcription factor that acts as a molecular switch to induce myogenesis. Myogenin shares homology with MyoD and other myogenic regulatory proteins within a basic region and helix-loop-helix (HLH) motif that mediate binding to a conserved DNA sequence (CANNTG) present in the regulatory regions of numerous muscle-specific genes. Binding of myogenin and other members of the MyoD family to DNA can be augmented upon heterodimerization with the widely expressed HLH protein E12. We have used the muscle creatine kinase (MCK) enhancer as a target to study the mechanism whereby myogenin activates muscle-specific transcription. Full activity of the MCK enhancer requires cooperative interactions between myogenin (or other myogenic HLH proteins that bind the same site) and a complex array of ubiquitous and cell type-specific nuclear factors. To define the domains of myogenin responsible for sequence-specific DNA binding, activation of muscle-specific transcription, and cooperativity with other transcription factors, we have generated an extensive series of mutants by site-directed mutagenesis and domain swapping. These mutants have revealed strong transcriptional activation domains in the N- and C-termini of myogenin that rely on a specific amino acid sequence within the DNA binding domain for activity. Myogenin's ability to induce muscle-specific transcription is subject to negative regulation by growth factor and oncogenic signals. Mechanisms through which growth signals may repress myogenin function are discussed.

• Funk WD, Ouellette M, Wright WE
• Molecular biology of myogenic regulatory factors.
• Mol Biol Med. 1991; 8: 185-95
• Display abstract

• A family of proteins has recently been identified, each member of which has the capacity to initiate muscle differentiation in many non-muscle cell types. These factors, which include MyoD1, myogenin, myf-5 and MRF4, share homologies with each other and belong to a superfamily of Myc-related proteins. Expression of these regulatory proteins results in auto-activation and cross-activation of other members of the family and in the transcriptional activation of the markers of terminal differentiation. Sequence analysis has shown a conserved basic domain in each protein that is required for binding to specific DNA sequences of the E-box type and for myogenic activation. A conserved helix-loop-helix (HLH) domain allows homo- and heterodimerization of these muscle-specific proteins with each other and with ubiquitously expressed proteins such as the E2A gene products (E12/E47). This review describes the discovery and characterization of these muscle regulatory proteins and their actions in the context of proposed models for the determination and differentiation of muscle tissue.

• Brennan TJ, Chakraborty T, Olson EN
• Mutagenesis of the myogenin basic region identifies an ancient protein motif critical for activation of myogenesis.
• Proc Natl Acad Sci U S A. 1991; 88: 5675-9
• Display abstract

• Myogenin is a muscle-specific nuclear factor that acts as a genetic switch to activate myogenesis. Myogenin, MyoD, and a growing number of proteins implicated in transcriptional control share sequence homology within a basic region and an adjacent helix-loop-helix motif. Here we identify by site-directed mutagenesis a 12-amino acid subdomain of the myogenin basic region essential for binding of DNA and activation of myogenesis. The basic region of the widely expressed helix-loop-helix protein E12 is conserved at 8 of these 12 residues and can mediate DNA binding when placed in myogenin, but it cannot activate myogenesis. Replacement of each of the four nonconserved residues of the myogenin basic region with the corresponding residues of E12 reveals two adjacent amino acids (Ala86-Thr) that can impart muscle specificity to the basic region. These residues are specific to, and conserved in, the basic regions of all known myogenic helix-loop-helix proteins from Drosophila to man, suggesting that they constitute part of an ancient protein motif required for activation of the myogenic program.

• Chakraborty T, Brennan T, Olson E
• Differential trans-activation of a muscle-specific enhancer by myogenic helix-loop-helix proteins is separable from DNA binding.
• J Biol Chem. 1991; 266: 2878-82
• Display abstract

• The muscle creatine kinase (MCK) enhancer was used as a target to study the specificity of DNA binding and trans-activation by members of the helix-loop-helix (HLH) family of myogenic regulatory factors, MyoD1, myogenin, myf-5, and MRF4. Whereas all four myogenic factors bound with similar affinities to the MCK enhancer in the presence of the widely expressed HLH protein E12, only MyoD1, myogenin, and myf-5 efficiently trans-activated the enhancer in transiently transfected 10T1/2 and 3T3 cells. That MRF4 binds the MCK enhancer without activating transcription suggests that domains in addition to those required for DNA binding are important for transcriptional activation and supports the notion that the different members of the HLH family of myogenic regulatory factors may selectively regulate unique sets of muscle-specific genes.

• Chakraborty T, Olson EN
• Domains outside of the DNA-binding domain impart target gene specificity to myogenin and MRF4.
• Mol Cell Biol. 1991; 11: 6103-8
• Display abstract

• Myogenin and MRF4 belong to the MyoD family of muscle-specific transcription factors, which can activate myogenesis when introduced into nonmyogenic cells. These proteins share homology within a basic-helix-loop-helix motif that mediates DNA binding and dimerization, but they are divergent in their amino and carboxyl termini. Although myogenin and MRF4 bind the same sequence within the muscle creatine kinase enhancer, only myogenin efficiently transactivates this enhancer. By creating chimeras of myogenin and MRF4, we show that the specificities of these factors for transactivation of the muscle creatine kinase enhancer can be interchanged by swapping their amino and carboxyl termini. Within these chimeras, strong cooperation between the amino and carboxyl termini was observed. These findings suggest that myogenin and MRF4 discriminate between muscle-specific enhancers and that target gene specificity is determined by domains surrounding the basic-helix-loop-helix region.

© 2021 EMBL | Privacy Policy