Walsh M J, Tsao K L, LeLeiko N S
Department of Pediatrics, Mount Sinai School of Medicine, New York, New York 10029.
J Biol Chem. 1992 Apr 5;267(10):7026-35.
We have characterized a DNA-protein interaction within a sequence element distal from the site of transcription initiation within the mouse housekeeping gene (HPRT) promoter region. This interaction occurs within a 35-base pair regulatory element which confers cell type-specific gene transcription, designated as the HPRT cis-acting regulatory element (HCRE). Competition analysis by gel mobility shift electrophoresis indicates that this DNA-protein interaction is novel and not related to many transcription factors previously reported. Cell cycle synchronization experiments and gel mobility shift assays have demonstrated that within the HCRE a specific DNA-protein complex responds to G1 activation of the cell cycle. Experiments to purify specific DNA-binding proteins that interact with the HCRE has resulted in the purification of one sequence-specific DNA-binding protein of approximately 66 kDa. To determine the putative DNA-binding sequence, footprinting analysis has mapped the protection from DNase I hydrolysis which confers a core sequence of GTCTGGGT using both affinity purified protein and crude nuclear extract. This DNA motif represents a novel protein-binding sequence. Interestingly, data base searches have identified the same or homologous sequences of this DNA motif in additional genes, potentially related to cellular growth and proliferation. This consensus was most notable within a region 5' upstream of the ornithine decarboxylase gene. The unique cell type-specific regulation of the HPRT gene in the intestinal mucosa is not completely understood at this time but because of the relationship of ornithine decarboxylase expression to cell proliferation and more specifically, to mucosal cell renewal in the intestine, the function of DNA-protein interactions within the consensus sequence may prove analogous. This may account for the cell type-specific and cell-cycle responsive gene regulation previously demonstrated with HPRT. Identification of one sequence-specific DNA-binding protein within the HCRE suggest that this protein contributes to the trans-activation of specific genes during the immediate-early response of the cell cycle.
我们已经对小鼠管家基因（HPRT）启动子区域内转录起始位点远端的一个序列元件中的DNA-蛋白质相互作用进行了表征。这种相互作用发生在一个35碱基对的调控元件内，该元件赋予细胞类型特异性基因转录，被命名为HPRT顺式作用调控元件（HCRE）。凝胶迁移率变动分析的竞争分析表明，这种DNA-蛋白质相互作用是新颖的，与先前报道的许多转录因子无关。细胞周期同步实验和凝胶迁移率变动分析表明，在HCRE内，一种特定的DNA-蛋白质复合物对细胞周期的G1期激活有反应。纯化与HCRE相互作用的特定DNA结合蛋白的实验已导致纯化出一种约66 kDa的序列特异性DNA结合蛋白。为了确定假定的DNA结合序列，足迹分析已绘制出对DNase I水解的保护图谱，使用亲和纯化蛋白和粗核提取物确定其核心序列为GTCTGGGT。这个DNA基序代表一个新颖的蛋白质结合序列。有趣的是，数据库搜索在其他基因中鉴定出了这个DNA基序的相同或同源序列，这些基因可能与细胞生长和增殖有关。这种共有序列在鸟氨酸脱羧酶基因5'上游区域最为显著。目前，肠道黏膜中HPRT基因独特的细胞类型特异性调控尚未完全明确，但由于鸟氨酸脱羧酶表达与细胞增殖，更具体地说，与肠道黏膜细胞更新之间的关系，共有序列内DNA-蛋白质相互作用的功能可能被证明是类似的。这可能解释了先前用HPRT证明的细胞类型特异性和细胞周期响应性基因调控。在HCRE内鉴定出一种序列特异性DNA结合蛋白表明，该蛋白在细胞周期的早期反应中有助于特定基因的反式激活。
