Chen B, Han B H, Sun X H, Lim R W
Department of Pharmacology, School of Medicine, University of Missouri, Columbia, MO 65212, USA.
Nucleic Acids Res. 1997 Jan 15;25(2):423-30. doi: 10.1093/nar/25.2.423.
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.
我们研究了一种类Id蛋白Id3（也称为HLH462）在肌肉特异性基因表达调控中的作用。Id蛋白被认为通过阻止普遍存在的bHLH蛋白（E蛋白）与肌源性bHLH蛋白（如MyoD）之间的二聚化来阻断肌肉特异性基因的表达。与它作为分化抑制剂的假定作用一致，在增殖的骨骼肌细胞中检测到Id3 mRNA，碱性成纤维细胞生长因子（bFGF）进一步诱导其表达，而在分化的肌肉培养物中其表达下调。Id3的过表达有效地抑制了MyoD介导的肌肉特异性肌酸激酶（MCK）报告基因的激活。缺失分析表明，Id3的C末端15个氨基酸对于其完全抑制活性至关重要，而从相关蛋白Id2的C末端删除多达42个残基并不影响其抑制MCK报告基因的能力。含有Id3 N末端区域和Id2 C末端的嵌合蛋白在转染细胞中也无功能。相反，野生型Id3、C末端突变体和Id3/Id2嵌合体在体外均能与E蛋白E47相互作用。进一步的研究表明，Id3 C末端的截短可能对突变蛋白的表达水平产生不利影响，但Id3/Id2嵌合体稳定表达。综上所述,我们的结果揭示了Id家族蛋白的表达和正常功能的需求比迄今为止预期的更为复杂。
