Ma J, Saito H, Oka T, Vijay I K
Department of Animal Sciences, University of Maryland, College Park 20742, USA.
J Biol Chem. 1996 May 10;271(19):11197-203. doi: 10.1074/jbc.271.19.11197.
The gene encoding UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT), the enzyme that initiates the pathway for the biosynthesis of asparagine-linked glycoproteins, is ubiquitously expressed in eukaryotic cells. However, its expression in the mammary gland is developmentally and hormonally regulated; transcription of the mouse mammary GPT gene is stimulated by the lactogenic hormones, insulin, glucocorticoid, and prolactin. The involvement of cisacting elements in regulating the expression of the mouse GPT (mGPT) gene was investigated by transient transfections of various GPT promoter/luciferase (Luc) constructs into primary mouse mammary epithelial cells. A series of 5'-deletions of the GPT promoter identified a distal negative regulatory region (base pairs -1057 to -968) and deletion of this region results in enhanced hormonal induction (approximately 7-fold) with no effect on basal promoter activity. Electrophoretic mobility shift assays (EMSA) performed with nuclear extracts from different developmental stages of mouse mammary gland demonstrated that the binding activity of the nuclear proteins to the distal negative regulatory region was predominant in virgin stage as compared with pregnant and lactating stages. EMSA performed with nuclear extracts from virgin explants showed that the binding activity was markedly decreased after cultivation with the combination of the three lactogenic hormones. DNase I footprinting analysis identified two pentamer direct repeat motifs, AGGAA and GAAAC, within the negative regulatory region. EMSA competition experiments showed that mutations within the direct repeats failed to compete for binding of the nuclear proteins to labeled wild type oligonucleotide. Transcription from the promoter containing the mutated direct repeats was increased greatly, consistent with the conclusion that these motifs functions in vivo to repress GPT gene expression. These data suggest the importance of the negative regulatory region in hormonal control of mGPT gene expression in mammary gland.
编码UDP-葡萄糖胺：磷酸多萜醇N-乙酰葡糖胺-1-磷酸转移酶（GPT）的基因启动了天冬酰胺连接糖蛋白的生物合成途径，该酶在真核细胞中普遍表达。然而，其在乳腺中的表达受到发育和激素的调控；小鼠乳腺GPT基因的转录受泌乳激素、胰岛素、糖皮质激素和催乳素的刺激。通过将各种GPT启动子/荧光素酶（Luc）构建体瞬时转染到原代小鼠乳腺上皮细胞中，研究了顺式作用元件在调节小鼠GPT（mGPT）基因表达中的作用。一系列GPT启动子的5'端缺失鉴定出一个远端负调控区域（碱基对-1057至-968），缺失该区域会导致激素诱导增强（约7倍），而对基础启动子活性无影响。对来自小鼠乳腺不同发育阶段的核提取物进行的电泳迁移率变动分析（EMSA）表明，与怀孕和泌乳阶段相比，处女阶段核蛋白与远端负调控区域的结合活性占主导。对来自处女外植体的核提取物进行的EMSA表明，在用三种泌乳激素组合培养后，结合活性明显降低。DNase I足迹分析在负调控区域内鉴定出两个五聚体直接重复基序AGGAA和GAAAC。EMSA竞争实验表明，直接重复序列内的突变未能竞争核蛋白与标记的野生型寡核苷酸的结合。含有突变直接重复序列的启动子的转录大大增加，这与这些基序在体内抑制GPT基因表达的结论一致。这些数据表明负调控区域在乳腺中mGPT基因表达的激素控制中的重要性。
