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.     

Alternative multimeric structures affect myogenin DNA binding activity.

The native molecular weight of the basic helix-loop-helix (bHLH) proteins myogenin, MyoD, and E12 was calculated from their mobilities on sucrose gradients and molecular sieve chromatography. The muscle bHLH proteins associate to form a variety of higher order complexes, most of which are larger than dimers. Homodimers bind to DNA sequences such as the MEF-1 site in the creatine kinase enhancer whereas homotetramers and larger forms do not recognize this DNA sequence. The ubiquitous bHLH protein E12 forms monomers or homodimers with little evidence for higher order complexes. Mixtures of myogenin and E12 show some heterodimeric structures, but most of the myogenin remains in large complexes. This result using purified proteins is also obtained in nuclear extracts from differentiated myotubes, in which most of the myogenin is present in large complexes that do not bind to the creatine kinase enhancer. A fusion protein containing only the myogenin HLH region forms large homomeric complexes. A model is presented in which each helix associates with a different subunit to form chains or ring structures to explain these observations. The partition of myogenin in nuclear extracts into dimers that recognize known DNA sequences and higher order complexes that do not raises important new issues concerning the regulation of skeletal muscle bHLH protein activity during myogenesis.     

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.     

The MyoD DNA binding domain contains a recognition code for muscle-specific gene activation

A 60 amino acid domain of the myogenic determination gene MyoD is necessary and sufficient for sequence-specific DNA binding in vitro and myogenic conversion of transfected C3H10T1/2 cells. We show that a highly basic region, immediately upstream of the helix-loop-helix (HLH) oligomerization motif, is required for MyoD DNA binding in vitro. Replacing helix1, helix2, or the loop of MyoD with the analogous sequence of the Drosophila T4 achaete-scute protein (required for peripheral neurogenesis) has no substantial effect on DNA binding in vitro or muscle-specific gene activation in transfected C3H10T1/2 cells. However, replacing the basic region of MyoD with the analogous sequence of other HLH proteins (the immunoglobulin enhancer binding E12 protein or T4 achaete scute protein) allows DNA binding in vitro, yet abolishes muscle-specific gene activation. These findings suggest that a recognition code that determines muscle-specific gene activation lies within the MyoD basic region and that the capacity for specific DNA binding is insufficient to activate the muscle program.