Chakraborty T, Brennan T J, Li L, Edmondson D, Olson E N
Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030.
Mol Cell Biol. 1991 Jul;11(7):3633-41. doi: 10.1128/mcb.11.7.3633-3641.1991.
Myogenin is a muscle-specific transcription factor that can activate myogenesis; it belongs to a family of transcription factors that share homology within a basic region and an adjacent helix-loop-helix (HLH) motif. Although myogenin alone binds DNA inefficiently, in the presence of the widely expressed HLH proteins E12 and E47 (encoded by the E2A gene), it forms heterooligomers that bind with high affinity to a DNA sequence known as a kappa E-2 site. In contrast, E47 and to a lesser extent E12 are both able to bind the kappa E-2 site relatively efficiently as homooligomers. To define the relative contributions of the basic regions of myogenin and E12 to DNA binding and muscle-specific gene activation, we created chimeras of the two proteins by swapping their basic regions. We showed that myogenin's weak affinity for the kappa E-2 site is attributable to inefficient homooligomerization and that the myogenin basic domain alone can mediate high-affinity DNA binding when placed in E12. Within a heterooligomeric complex, two basic regions were required to form a high-affinity DNA-binding domain. Basic-domain mutants of myogenin or E2A gene products that cannot bind DNA retained the ability to oligomerize and could abolish DNA binding of the wild-type proteins in vitro. These myogenin and E2A mutants also acted as trans-dominant inhibitors of muscle-specific gene activation in vivo. These findings support the notion that muscle-specific gene activation requires oligomerization between myogenin and E2A gene products and that E2A gene products play an important role in myogenesis by enhancing the DNA-binding activity of myogenin, as well as other myogenic HLH proteins.
肌细胞生成素是一种肌肉特异性转录因子，可激活肌细胞生成；它属于一类转录因子家族，这些转录因子在一个碱性区域和一个相邻的螺旋-环-螺旋（HLH）基序内具有同源性。尽管肌细胞生成素单独与DNA结合的效率较低，但在广泛表达的HLH蛋白E12和E47（由E2A基因编码）存在的情况下，它会形成异源寡聚体，与一种称为κE-2位点的DNA序列以高亲和力结合。相比之下，E47以及程度稍轻的E12都能够作为同源寡聚体相对有效地结合κE-2位点。为了确定肌细胞生成素和E12的碱性区域对DNA结合和肌肉特异性基因激活的相对贡献，我们通过交换它们的碱性区域创建了这两种蛋白质的嵌合体。我们发现，肌细胞生成素对κE-2位点的弱亲和力归因于低效的同源寡聚化，并且当置于E12中时，单独的肌细胞生成素碱性结构域可以介导高亲和力的DNA结合。在异源寡聚体复合物中，需要两个碱性区域来形成高亲和力的DNA结合结构域。无法结合DNA的肌细胞生成素或E2A基因产物的碱性结构域突变体保留了寡聚化的能力，并且在体外可以消除野生型蛋白质的DNA结合。这些肌细胞生成素和E2A突变体在体内也作为肌肉特异性基因激活的反式显性抑制剂起作用。这些发现支持了这样一种观点，即肌肉特异性基因激活需要肌细胞生成素和E2A基因产物之间的寡聚化，并且E2A基因产物通过增强肌细胞生成素以及其他肌源性HLH蛋白的DNA结合活性，在肌细胞生成中发挥重要作用。
