The basic domain of myogenic basic helix-loop-helix (bHLH) proteins is the novel target for direct inhibition by another bHLH protein, Twist.
In vertebrates, the basic helix-loop-helix (bHLH) protein Twist may be involved in the negative regulation of cellular determination and in the differentiation of several lineages, including myogenesis, osteogenesis, and neurogenesis. Although it has been shown that mouse twist (M-Twist) (i) sequesters E proteins, thus preventing formation of myogenic E protein-MyoD complexes and (ii) inhibits the MEF2 transcription factor, a cofactor of myogenic bHLH proteins, overexpression of E proteins and MEF2 failed to rescue the inhibitory effects of M-Twist on MyoD. We report here that M-Twist physically interacts with the myogenic bHLH proteins in vitro and in vivo and that this interaction is required for the inhibition of MyoD by M-Twist. In contrast to the conventional HLH-HLH domain interaction formed in the MyoD/E12 heterodimer, this novel type of interaction uses the basic domains of the two proteins. While the MyoD HLH domain without the basic domain failed to interact with M-Twist, a MyoD peptide containing only the basic and helix 1 regions was sufficient to interact with M-Twist, suggesting that the basic domain contacts M-Twist. The replacement of three arginine residues by alanines in the M-Twist basic domain was sufficient to abolish both the binding and inhibition of MyoD by M-Twist, while the domain retained other M-Twist functions such as heterodimerization with an E protein and inhibition of MEF2 transactivation. These findings demonstrate that M-Twist interacts with MyoD through the basic domains, thereby inhibiting MyoD.
Hamamori
Y
Y
Institute for Genetic Medicine and Department of Biochemistry and Molecular Biology, University of Southern California School of Medicine, Los Angeles 90033, USA.
Wu
H Y
HY
Sartorelli
V
V
Kedes
L
L
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D002454:Cell Differentiation / Q000235:genetics; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D064326:MEF2 Transcription Factors; D051379:Mice; D008969:Molecular Sequence Data; D017570:MyoD Protein / Q000378:metabolism; D018006:Myogenic Regulatory Factors / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D051879:Twist-Related Protein 1
D001120:Arginine; D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D064326:MEF2 Transcription Factors; D017570:MyoD Protein; D018006:Myogenic Regulatory Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; D051879:Twist-Related Protein 1; C067250:Twist1 protein, mouse