Substitution of basic amino acids in the basic region stabilizes DNA binding by E12 homodimers.

The E2A gene encodes two alternatively spliced products, E12 and E47. The two proteins differ in their basic helix-loop-helix motifs (bHLH), responsible for DNA binding and dimerization. Although both E12 and E47 can bind to DNA as heterodimers with tissue-specific bHLH proteins, E12 binds to DNA poorly as homodimers. An inhibitory domain in E12 has previously been found to prevent E12 homodimers from binding to DNA. By measuring the dissociation rates using filter binding and electrophoretic mobility shift assays, we have shown here that the inhibitory domain interferes with DNA binding by destabilizing the DNA-protein complexes. Furthermore, we have demonstrated that substitution of basic amino acids (not other amino acids) in the DNA-binding domain of E12 can increase the intrinsic DNA-binding activity of E12 and stabilize the binding complexes, thus alleviating the repression from the inhibitory domain. This ability of basic amino acids to stabilize DNA-binding complexes may be of biological significance in the case of myogenic bHLH proteins, which all possess two more basic amino acids in their DNA binding domain than E12. To function as heterodimers with E12, the myogenic bHLH proteins may need stronger DNA binding domains.     

Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence

A DNA binding and dimerization motif, with apparent amphipathic helices (the HLH motif), has recently been identified in various proteins, including two that bind to immunoglobulin enhancers (E12 and E47). We show here that various HLH proteins can bind as apparent heterodimers to a single DNA motif and also, albeit usually more weakly, as apparent homodimers. The HLH domain can mediate heterodimer formation between either daughterless, E12, or E47 (Class A) and achaete-scute T3 or MyoD (Class B) to form proteins with high affinity for the kappa E2 site in the immunoglobulin kappa chain enhancer. The achaete-scute T3 and MyoD proteins do not form kappa E2-binding heterodimers together, and no active complex with N-myc was evident. The formation of a heterodimer between the daughterless and achaete-scute T3 products may explain the similar phenotypes of mutants at these two loci and the genetic interactions between them. A role of E12 and E47 in mammalian development, analogous to that of daughterless in Drosophila, is likely.     

Phosphorylation of E47 as a potential determinant of B-cell-specific activity.

The E2A gene encodes two basic helix-loop-helix proteins designated E12 and E47. Although these proteins are widely expressed, they are required only for the B-lymphocyte lineage where DNA binding is mediated distinctively by E47 homodimers. By studying the properties of deltaE47, an N-terminal truncation of E47, we provide evidence that phosphorylation may contribute to B-cell-specific DNA binding by E47. Two serines N terminal to the deltaE47 basic helix-loop-helix domain were found to be phosphorylated in a variety of cell types but were hypophosphorylated in B cells. Phosphorylating these serines in vitro inhibited DNA binding by deltaE47 homodimers but not by deltaE47-containing heterodimers, such as deltaE47:MyoD. These results argue that hypophosphorylation may be a prerequisite for activity of E47 homodimers in B cells, suggesting the use of an inductive (nonstochastic) step in early B-cell development.     

Differential binding of quadruplex structures of muscle-specific genes regulatory sequences by MyoD, MRF4 and myogenin

Four myogenic regulatory factors (MRFs); MyoD, Myf-5, MRF4 and Myogenin direct muscle tissue differentiation. Heterodimers of MRFs with E-proteins activate muscle-specific gene expression by binding to E-box motifs d(CANNTG) in their promoters or enhancers. We showed previously that in contrast to the favored binding of E-box by MyoD-E47 heterodimers, homodimeric MyoD associated preferentially with quadruplex structures of regulatory sequences of muscle-specific genes. To inquire whether other MRFs shared the DNA binding preferences of MyoD, the DNA affinities of hetero- and homo-dimeric MyoD, MRF4 and Myogenin were compared. Similarly to MyoD, heterodimers with E47 of MRF4 or Myogenin bound E-box more tightly than quadruplex DNA. However, unlike homodimeric MyoD or MRF4, Myogenin homodimers associated weakly and nonpreferentially with quadruplex DNA. By reciprocally switching basic regions between MyoD and Myogenin we demonstrated dominance of MyoD in determining the quadruplex DNA-binding affinity. Thus, Myogenin with an implanted MyoD basic region bound quadruplex DNA nearly as tightly as MyoD. However, a grafted Myogenin basic region did not diminish the high affinity of homodimeric MyoD for quadruplex DNA. We speculate that the dissimilar interaction of MyoD and Myogenin with tetrahelical domains in muscle gene promoters may differently regulate their myogenic activities.     

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.     

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.