Dawson P A, Hofmann S L, van der Westhuyzen D R, Südhof T C, Brown M S, Goldstein J L
Department of Molecular Genetics, University of Texas Health Science Center at Dallas 75235.
J Biol Chem. 1988 Mar 5;263(7):3372-9.
A 42-base pair sequence from the 5' flanking region of the low density lipoprotein receptor gene was shown previously to confer sensitivity to sterol-mediated repression when inserted into the herpes simplex virus thymidine kinase promoter. This sequence contains two contiguous 16-base pair repeats, designated repeats 2 and 3, which differ from each other at four positions. In the current study we have analyzed separately the functions of repeats 2 and 3 by altering their sequences, inserting them into the -60 position of the thymidine kinase promoter, and introducing the hybrid promoters into hamster cells by transfection. These studies show that repeat 3 is a constitutive positive transcriptional element that acts in the absence or presence of sterols. Repeat 2 confers strong repression upon repeat 3 when sterols are present. In vitro DNase footprinting and gel retardation assays show that repeat 3, but not repeat 2, binds purified Sp1, a positive transcription factor. Mutants of repeat 3 that abolish transcriptional activity in vivo abolish Sp1 binding in vitro. We suggest that the low density lipoprotein receptor is regulated by a push-pull mechanism in which sterol-regulated binding of a protein to repeat 2 silences the activity of the adjacent Sp1-binding site in repeat 3.
先前的研究表明，低密度脂蛋白受体基因5'侧翼区域的一段42个碱基对的序列，当插入单纯疱疹病毒胸苷激酶启动子时，可赋予对固醇介导的抑制作用的敏感性。该序列包含两个相邻的16个碱基对的重复序列，分别命名为重复序列2和重复序列3，它们在四个位置上彼此不同。在当前的研究中，我们通过改变它们的序列，将它们插入胸苷激酶启动子的-60位置，并通过转染将这些杂交启动子导入仓鼠细胞，分别分析了重复序列2和重复序列3的功能。这些研究表明，重复序列3是一个组成型正转录元件，在有无固醇的情况下均起作用。当存在固醇时，重复序列2对重复序列3具有强烈的抑制作用。体外DNA酶足迹分析和凝胶阻滞分析表明，重复序列3能结合纯化的Sp1（一种正转录因子），而重复序列2则不能。在体内消除转录活性的重复序列3突变体，在体外也消除了Sp1的结合。我们认为，低密度脂蛋白受体受一种推拉机制调控，其中固醇调节的蛋白质与重复序列2的结合使重复序列3中相邻的Sp1结合位点的活性沉默。
