In vivo interactions of MyoD, Id1, and E2A proteins determined by acceptor photobleaching fluorescence resonance energy transfer.
MyoD, a skeletal muscle transcription factor, is rapidly degraded by the ubiquitin-proteasome system. MyoD interacts with ubiquitously expressed E2A or inhibitor of DNA binding (Id) proteins to activate or inhibit transcription, respectively. Furthermore, MyoD has been shown to modulate the ubiquitin-mediated degradation of Id1 and E2A proteins, E12 and E47. The molecular mechanisms governing these events are not clear but are hypothesized to occur via heterodimer formation. Fluorescence resonance energy transfer (FRET) is a technique for evaluation of protein-protein interactions in vivo. Using acceptor photobleaching FRET and chimeric proteins composed of MyoD, Id1, E12, E47, E12(NLS), or MyoD(NLS) and either cyan fluorescent protein or yellow fluorescent protein, we show that each of the wild-type proteins is capable of homodimerization. In addition, heterodimers form between Id1 and E2A proteins, as well as between MyoD and E2A proteins. The Id1:E2A interaction is stronger than the MyoD:E2A interaction, which is consistent with the notion that inhibition of MyoD action occurs by the sequestration of E2A proteins by Id. The stronger interaction of Id1 with E2A may also explain the decrease in the rate of ubiquitin-proteasome degradation of Id1 that is significantly greater than that of MyoD when E2A proteins are abundant. Thus, these studies extend our understanding of the molecular mechanisms of MyoD action.
Lingbeck
Jody M
JM
Edward Mallinckrodt Department of Pediatrics and Molecular Biology and Pharmacology, Washington University School of Medicine and St. Louis Children's Hospital, St. Louis, Missouri 63110, USA.
Trausch-Azar
Julie S
JS
Ciechanover
Aaron
A
Schwartz
Alan L
AL
D041721:3T3-L1 Cells; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D051798:Inhibitor of Differentiation Protein 1 / Q000378:metabolism*; D051379:Mice; D017570:MyoD Protein / Q000378:metabolism*; D038761:Photobleaching; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D051820:TCF Transcription Factors / Q000378:metabolism; D058125:Transcription Factor 7-Like 1 Protein
D051792:Basic Helix-Loop-Helix Proteins; D051798:Inhibitor of Differentiation Protein 1; D017570:MyoD Protein; D011993:Recombinant Fusion Proteins; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; C057363:MyoD1 myogenic differentiation protein; C407909:TCF3 protein, human; C544912:TCF7L1 protein, human; C544913:Tcf7l1 protein, mouse