Giese K, Grosschedl R
Howard Hughes Medical Institute, Department of Microbiology, University of California, San Francisco 94143-0414.
EMBO J. 1993 Dec;12(12):4667-76. doi: 10.1002/j.1460-2075.1993.tb06155.x.
Lymphoid enhancer factor 1 (LEF-1) is a member of the high mobility group (HMG) family of proteins and participates in the regulation of the T cell receptor (TCR) alpha enhancer. We have previously shown that DNA binding by the HMG domain of LEF-1 induces a sharp bend in the DNA helix. Together with the dependence of LEF-1 on other factor-binding sites to regulate gene expression, DNA bending induced by the HMG domain suggested an 'architectural' role for LEF-1. In this study, we performed experiments to distinguish between a model in which the HMG domain is the only functional determinant of LEF-1 and a model in which additional domains of LEF-1 are involved in the regulation of gene expression. First, we show that the HMG domain alone is not sufficient to stimulate TCR alpha enhancer function. Second, we replaced the HMG domain of LEF-1 with the DNA-binding domain of the bacterial repressor LexA, which binds a specific nucleotide sequence without inducing a sharp bend in the DNA helix. The chimeric LEF-LexA protein increased the activity of a TCR alpha enhancer in which the LEF-1-binding site had been replaced with a LexA recognition sequence. Transcriptional stimulation by LEF-LexA, however, was less efficient than that observed with endogenous LEF-1. The LEF-LexA-mediated activation of gene expression was dependent upon an amino-terminal region of LEF-1 and a specific context of factor-binding sites in the TCR alpha enhancer. Neither multimerized LexA-binding sites, nor TCR alpha enhancers with altered spatial arrangements of factor-binding sites, were functional for regulation by LEF-LexA. Together, these data suggest that an aminoterminal region in LEF-1 contributes to the context-dependent regulation of the TCR alpha enhancer by LEF-1, presumably by interacting with other enhancer-bound proteins.
淋巴样增强因子1（LEF-1）是高迁移率族（HMG）蛋白家族的成员，参与T细胞受体（TCR）α增强子的调控。我们之前已经表明，LEF-1的HMG结构域与DNA结合会导致DNA螺旋发生急剧弯曲。鉴于LEF-1依赖于其他因子结合位点来调控基因表达，HMG结构域诱导的DNA弯曲表明LEF-1具有“结构”作用。在本研究中，我们进行了实验，以区分HMG结构域是LEF-1唯一功能决定因素的模型和LEF-1的其他结构域参与基因表达调控的模型。首先，我们表明单独的HMG结构域不足以刺激TCRα增强子功能。其次，我们用细菌阻遏物LexA的DNA结合结构域替换了LEF-1的HMG结构域，LexA能结合特定核苷酸序列而不诱导DNA螺旋发生急剧弯曲。嵌合的LEF-LexA蛋白增加了TCRα增强子的活性，其中LEF-1结合位点已被LexA识别序列取代。然而，LEF-LexA介导的转录刺激效率低于内源性LEF-1。LEF-LexA介导的基因表达激活依赖于LEF-1的氨基末端区域和TCRα增强子中因子结合位点的特定背景。多聚化的LexA结合位点，以及因子结合位点空间排列改变后的TCRα增强子，都不能被LEF-LexA调控。总之，这些数据表明LEF-1中的氨基末端区域有助于LEF-1对TCRα增强子进行依赖背景的调控，可能是通过与其他增强子结合蛋白相互作用实现的。
