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.     

An inhibitory domain of E12 transcription factor prevents DNA binding in E12 homodimers but not in E12 heterodimers.

The kappa E2 sequence binding proteins, E12 and E47, are generated by alternative splicing of the E2A gene, giving closely related basic and helix-loop-helix structures crucial for DNA binding and dimerization. Measurements of dimerization constants and binding strengths to the optimal DNA sequence (the kappa E2 site or its near relatives) showed that E47 homodimers and MyoD heterodimers with E12 or E47 dimerized and bound avidly, but E12 homodimerized efficiently and bound to DNA poorly; MyoD homodimerized poorly and bound strongly. An inhibitory domain N-terminal to the basic region of E12 prevents E12 homodimers but not E12/MyoD heterodimers from binding to DNA. Thus, E47 binds to DNA both as a heterodimer with MyoD and as a homodimer, while E12 and MyoD bind to DNA efficiently only as heterodimers.     

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.     

Establishment of distinct MyoD, E2A, and twist DNA binding specificities by different basic region-DNA conformations

Basic helix-loop-helix (bHLH) proteins perform a wide variety of biological functions. Most bHLH proteins recognize the consensus DNA sequence CAN NTG (the E-box consensus sequence is underlined) but acquire further functional specificity by preferring distinct internal and flanking bases. In addition, induction of myogenesis by MyoD-related bHLH proteins depends on myogenic basic region (BR) and BR-HLH junction residues that are not essential for binding to a muscle-specific site, implying that their BRs may be involved in other critical interactions. We have investigated whether the myogenic residues influence DNA sequence recognition and how MyoD, Twist, and their E2A partner proteins prefer distinct CAN NTG sites. In MyoD, the myogenic BR residues establish specificity for particular CAN NTG sites indirectly, by influencing the conformation through which the BR helix binds DNA. An analysis of DNA binding by BR and junction mutants suggests that an appropriate BR-DNA conformation is necessary but not sufficient for myogenesis, supporting the model that additional interactions with this region are important. The sequence specificities of E2A and Twist proteins require the corresponding BR residues. In addition, mechanisms that position the BR allow E2A to prefer distinct half-sites as a heterodimer with MyoD or Twist, indicating that the E2A BR can be directed toward different targets by dimerization with different partners. Our findings indicate that E2A and its partner bHLH proteins bind to CAN NTG sites by adopting particular preferred BR-DNA conformations, from which they derive differences in sequence recognition that can be important for functional specificity.