Chang D J, Paik Y K, Leren T P, Walker D W, Howlett G J, Taylor J M
Gladstone Foundation Laboratories for Cardiovascular Disease, Cardiovascular Research Institute, San Francisco, California 94140-0608.
J Biol Chem. 1990 Jun 5;265(16):9496-504.
An enhancer element in the 5' flanking region of the human apolipoprotein E gene, known as upstream regulatory element 1 (URE1), has previously been implicated in the expression of this gene. The URE1 element, which spans nucleotides -193 to -124 of the 5'-flanking region of the human apolipoprotein E gene, contains two sequences that bind to nuclear proteins, as determined by the DNase I footprinting assay. In the present study of URE1, we have characterized these sequences further. Deletion of one of the footprint sequences, at nucleotides -161 to -141, reduced URE1 enhancer activity substantially. A 30-base pair oligonucleotide that included this protein-binding sequence was able, by itself, to act as an enhancer. This sequence, termed the positive element for transcription (PET), was demonstrated by gel retention analysis to bind at least two protein factors, one of which is the transcription factor Sp1. Sp1 appeared to be the only protein required for the enhancer activity of PET to be manifested. In vitro transcription assays showed that the PET sequence was necessary for efficient transcription directed by the apoE promoter and that the PET sequence was the dominant regulatory element in the apoE promoter. Gel filtration chromatography and PET oligonucleotide-affinity chromatography were used to isolate a second PET-binding factor, a Mr = 55,000 protein, from HeLa cell nuclear extracts. It appeared to compete with Sp1 for a common binding site in the PET sequence, but it was not required for enhancer activity. The second footprint sequence in URE1, at nucleotides -184 to -173, also bound Sp1, but it was not required for enhancer activity. A third Sp1-binding region was located at a proximal GC box element (nucleotides -54 to -45). This region had no enhancer activity, but it was required for maximum transcriptional activity of the apoE promoter. Thus, the regulation of apoE gene expression is influenced by different protein-binding sequences, with transcription factor Sp1 playing major roles in both basal promoter activity and enhancer activity.
人类载脂蛋白E基因5'侧翼区域中的一个增强子元件，即上游调控元件1（URE1），此前已被证明与该基因的表达有关。URE1元件跨越人类载脂蛋白E基因5'侧翼区域的核苷酸-193至-124，通过DNA酶I足迹分析确定，它包含两个与核蛋白结合的序列。在目前对URE1的研究中，我们进一步对这些序列进行了表征。删除其中一个足迹序列（核苷酸-161至-141）会大幅降低URE1增强子活性。一个包含该蛋白结合序列的30个碱基对的寡核苷酸本身就能作为增强子发挥作用。这个序列被称为转录正性元件（PET），凝胶滞留分析表明它能结合至少两种蛋白因子，其中之一是转录因子Sp1。Sp1似乎是PET增强子活性表现所需的唯一蛋白。体外转录分析表明，PET序列是载脂蛋白E启动子指导有效转录所必需的，并且PET序列是载脂蛋白E启动子中的主要调控元件。凝胶过滤色谱法和PET寡核苷酸亲和色谱法被用于从HeLa细胞核提取物中分离出另一种PET结合因子，一种分子量为55,000的蛋白。它似乎与Sp1竞争PET序列中的一个共同结合位点，但它不是增强子活性所必需的。URE1中的第二个足迹序列（核苷酸-184至-173）也能结合Sp1，但它不是增强子活性所必需的。第三个Sp1结合区域位于近端GC盒元件（核苷酸-54至-45）处。该区域没有增强子活性，但它是载脂蛋白E启动子最大转录活性所必需的。因此，载脂蛋白E基因表达的调控受到不同蛋白结合序列的影响，转录因子Sp1在基础启动子活性和增强子活性中都发挥着主要作用。
