The E12 inhibitory domain prevents homodimer formation and facilitates selective heterodimerization with the MyoD family of gene regulatory factors.

Although the ubiquitous helix-loop-helix (HLH) protein E12 does not homodimerize efficiently, the myogenic factor MyoD forms an avid DNA-binding heterodimer with E12 through the conserved HLH dimerization domain. However, the mechanism which ensures this selective dimerization is not understood at present. In our functional studies of various amino acid changes in the E12 HLH domain, we found that a single substitution in E12 helix 1 can abolish the effect of the E12 inhibitory domain and results in the efficient DNA binding of the E12 homodimer. Competition experiments revealed that the inhibitory domain, in fact, blocks the dimerization of E12 rather than DNA binding. MyoD contains two glutamic residues in helix 2 that are required for efficient dimerization with E12. More importantly, these residues were not essential for dimerization with E12 mutants in which the dimerization inhibitory domain had been relaxed, or for dimerization with E47 which does not contain the inhibitory domain owing to the use of an alternative exon. The positions of these glutamic residues are conserved among the four myogenic factors. Thus, members of the MyoD family of gene regulatory proteins can overcome the E12 dimerization inhibitory domain through a mechanism involving, in part, the negatively charged amino acid residues in helix 2. This result describes a novel mechanism facilitating the selective formation of the MyoD(MRF)-E12 heterodimer that enhances dimerization specificity and may apply to other members of the E-protein family.     

Regulation of Id1 and its association with basic helix-loop-helix proteins during nerve growth factor-induced differentiation of PC12 cells.

Cell differentiation in the nervous system is dictated by specific patterns of gene expression. We have investigated the role of helix-loop-helix (HLH) proteins during differentiation of PC12 pheochromocytoma cells in response to nerve growth factor. Gel mobility shift assays using PC12 cell nuclear extracts demonstrated that active basic HLH complexes exist throughout differentiation. Addition of exogeneous Id1 protein, a negative regulator of basic HLH proteins, disrupted specific complexes formed by PC12 cell nuclear extracts on a CANNTG consensus oligonucleotide. To identify possible novel basic HLH proteins in these complexes, a glutathione S-transferase-Id1 fusion protein was used to screen a PC12 cell cDNA expression library. A single clone representing the rat E2-2 gene was identified. Sequential immunoprecipitations with antibodies to each HLH protein revealed an association between Id1 and E2-2 that could be detected in both untreated and nerve growth factor-treated PC12 cell lysates. These experiments define a new HLH interaction between Id1 and E2-2 in neuronal cells and suggest that neuronal differentiation may be regulated by HLH proteins in a distinctive manner.     

The loop region of the helix-loop-helix protein Id1 is critical for its dominant negative activity.

Id1, a helix-loop-helix (HLH) protein which lacks a DNA binding domain, has been shown to negatively regulate other members of the HLH family by direct protein-protein interactions, both in vitro and in vivo. In this study, we report the results of site-directed mutagenesis experiments aimed at defining the regions of Id1 which are important for its activity. We have found that the HLH domain of Id1 is necessary and nearly sufficient for its activity. In addition, we show that two amino acid residues at the amino terminus of the Id1 loop are critical for its activity, perhaps by specifying the correct dimerization partners. In this regard, replacing the first four amino acids of the loops of the basic HLH proteins E12 and E47 with the corresponding amino acids of Id1 confers Id1 dimerization specificity. These studies point to the loop region as an important structural and functional element of the Id subfamily of HLH proteins.     

B-cell- and myocyte-specific E2-box-binding factors contain E12/E47-like subunits.

Recent studies have identified a family of DNA-binding proteins that share a common DNA-binding and dimerization domain with the potential to form a helix-loop-helix (HLH) structure. Various HLH proteins can form heterodimers that bind to a common DNA sequence, termed the E2-box. We demonstrate here that E2-box-binding B-cell- and myocyte-specific nuclear factors contain subunits which are identical or closely related to ubiquitously expressed (E12/E47) HLH proteins. These biochemical function for E12/E47-like molecules in mammalian differentiation, similar to the genetically defined function of daughterless in Drosophila development.     

Baculovirus-expressed myogenic determination factors require E12 complex formation for binding to the myosin-light-chain enhancer.

Two recombinant baculoviruses BcV-myf4 and BcV-myf5 have been constructed to synthesize the human myogenic determination factors myogenin (myf4) and myf5 in eucaryotic cells. Both recombinant proteins are localized to the nucleus of virus-infected Spodoroptera frugiperda (sf) insect cells and can be recovered as soluble factors. The virus-produced proteins exhibit high-affinity binding to a muscle-specific DNA sequence in the presence of the ubiquitous helix-loop-helix (HLH) protein E12, but only marginal binding in unsupplemented sf nuclear extracts. Both baculovirus-encoded myogenic factors are able to heterooligomerize with E12 in the absence of DNA-binding sites. We conclude from our results that these muscle-specific HLH proteins produced in eucaryotic cells largely depend on dimerization with E12 or similar HLH proteins to recognize the myosin-light-chain-enhancer-MEF-1-binding site. We have no evidence for intracellular protein modifications exerting major effects on the interaction between these factors and DNA.     

The four human muscle regulatory helix-loop-helix proteins Myf3-Myf6 exhibit similar hetero-dimerization and DNA binding properties.

The muscle regulatory proteins Myf3, Myf4, Myf5, and Myf6 share a highly conserved DNA binding and dimerization domain consisting of a cluster of basic amino acids and a potential helix-loop-helix structure. Here we demonstrate that the four human muscle-specific HLH proteins have similar DNA binding and dimerization properties. The members of this family form protein complexes of comparable stability with the ubiquitously expressed HLH proteins E12, E2-2, and E2-5 and bind to the conserved DNA sequence CANNTG designated as E-box with similar efficiency in vitro. The binding affinities of the various complexes are greatly influenced by the variable internal and flanking nucleotides of the consensus motif. Combinations of Myf proteins with one another and with lyl-1, and HLH protein from human T cells, do not bind to DNA in vitro. Our results suggest that combinatorial associations of the various tissue-specific and more widely expressed HLH factors do not result in differential recognition of DNA sequences by Myf proteins.