Haynes T L, Thomas M B, Dusing M R, Valerius M T, Potter S S, Wiginton D A
Department of Pediatrics, University of Cincinnati, OH 45229, USA.
Nucleic Acids Res. 1996 Dec 15;24(24):5034-44. doi: 10.1093/nar/24.24.5034.
Transcriptional activation of eukaryotic genes involves assembly of specific multiprotein complexes on the promoters and enhancers of the genes. Recently, it has been proposed that the role of some of the proteins in the complex may be architectural, involving DNA bending, orchestration of protein-protein interaction and modulation of nucleosome structure. This role has been proposed for the HMG proteins LEF-1 and TCF-1. We examined the role of a LEF-1/TCF-1 binding site in the human adenosine deaminase (ADA) thymic enhancer. Mutational analysis demonstrated that a functional LEF-1/TCF-1 binding site is not required for enhancer-mediated transcriptional activation in transient transfection studies, but is essential for enhancer function in the in vivo chromatin context of transgenic mice. Mutation of the LEF-1/TCF-1 site destroyed the ability of the ADA enhancer/locus control region to specify high level, insertion site-independent transgene expression in thymus. DNase I and DpnII accessibility experiments indicated dramatic changes in the chromatin organization of the ADA enhancer in transgenic mice with a mutated LEF-1/TCF-1 site. This supports the hypothesis that factors binding the LEF-1/TCF-1 site play an architectural role during the in vivo activation of the ADA enhancer, possibly involving chromatin modification.
真核基因的转录激活涉及在基因的启动子和增强子上组装特定的多蛋白复合物。最近，有人提出复合物中某些蛋白质的作用可能是构建性的，包括DNA弯曲、蛋白质-蛋白质相互作用的编排以及核小体结构的调节。HMG蛋白LEF-1和TCF-1就被认为具有这种作用。我们研究了LEF-1/TCF-1结合位点在人腺苷脱氨酶（ADA）胸腺增强子中的作用。突变分析表明，在瞬时转染研究中，增强子介导的转录激活不需要功能性的LEF-1/TCF-1结合位点，但在转基因小鼠体内染色质环境中，该位点对增强子功能至关重要。LEF-1/TCF-1位点的突变破坏了ADA增强子/基因座控制区在胸腺中指定高水平、插入位点无关的转基因表达的能力。DNase I和DpnII可及性实验表明，在具有突变的LEF-1/TCF-1位点的转基因小鼠中，ADA增强子的染色质组织发生了显著变化。这支持了这样一种假设，即结合LEF-1/TCF-1位点的因子在ADA增强子的体内激活过程中发挥构建性作用，可能涉及染色质修饰。
