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, osteoge nesis, and neurogenesis. Although it has been shown that mouse twist ( M-Twist) (i) sequesters E proteins, thus preventing formation of myoge nic E protein-MyoD complexes and (ii) inhibits the MEF2 transcription factor, a cofactor of myogenic bHLH proteins, overexpression of E prot eins and MEF2 failed to rescue the inhibitory effects of M-Twist on My oD. We report here that M-Twist physically interacts with the myogenic bHLH proteins in vitro and in vivo and that this interaction is requi red for the inhibition of MyoD by M-Twist. In contrast to the conventi onal HLH-HLH domain interaction formed in the MyoD/E12 heterodimer, th is novel type of interaction uses the basic domains of the two protein s. While the MyoD HLH domain without the basic domain failed to intera ct with M-Twist, a MyoD peptide containing only the basic and helix 1 regions was sufficient to interact with M-Twist, suggesting that the b asic domain contacts M-Twist. The replacement of three arginine residu es by alanines in the M-Twist basic domain was sufficient to abolish b oth the binding and inhibition of MyoD by M-Twist, while the domain re tained other M-Twist functions such as heterodimerization with an E pr otein and inhibition of MEF2 transactivation. These findings demonstra te that M-Tvvist interacts with MyoD through the basic domains, thereb y inhibiting MyoD.