Inhibition of muscle-specific gene expression by Id3: requirement of the C-terminal region of the protein for stable expression and function.
We have examined the role of an Id-like protein, Id3 (also known as HLH462), in the regulation of muscle-specific gene expression. Id proteins are believed to block expression of muscle-specific genes by preventing the dimerization between ubiquitous bHLH proteins (E proteins) and myogenic bHLH proteins such as MyoD. Consistent with its putative role as an inhibitor of differentiation, Id3 mRNA was detected in proliferating skeletal muscle cells, was further induced by basic fibroblast growth factor (bFGF) and was down-regulated in differentiated muscle cultures. Overexpression of Id3 efficiently inhibited the MyoD-mediated activation of the muscle-specific creatine kinase (MCK) reporter gene. Deletion analysis indicated that the C-terminal 15 amino acids of Id3 are critical for the full inhibitory activity while deleting up to 42 residues from the C-terminus of the related protein, Id2, did not affect its ability to inhibit the MCK reporter gene. Chimeric protein containing the N-terminal region of Id3 and the C-terminus of Id2 was also non-functional in transfected cells. In contrast, wild-type Id3, the C-terminal mutants, and the Id3/Id2 chimera could all interact with the E-protein E47in vitro. Additional studies indicated that truncation of the Id3 C-terminus might have adversely affected the expression level of the mutant proteins but the Id3/Id2 chimera was stably expressed. Taken together, our results revealed a more complex requirement for the expression and proper function of the Id family proteins than was hitherto expected.
Chen
B
B
Department of Pharmacology, School of Medicine, University of Missouri, Columbia, MO 65212, USA.
Han
B H
BH
Sun
X H
XH
Lim
R W
RW
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003402:Creatine Kinase / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D018257:Helix-Loop-Helix Motifs / Q000235:genetics*; D051796:Inhibitor of Differentiation Proteins; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009363:Neoplasm Proteins; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D016415:Sequence Alignment; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors / Q000737:chemistry / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D051796:Inhibitor of Differentiation Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D051820:TCF Transcription Factors; C544912:TCF7L1 protein, human; C544913:Tcf7l1 protein, mouse; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D016222:Fibroblast Growth Factor 2; C075640:ID3 protein, human; D015500:Chloramphenicol O-Acetyltransferase; D003402:Creatine Kinase