Pit-1/GHF-1 binds to TRH-sensitive regions of the rat thyrotropin beta gene.
Pit-1/GHF-1 binds to TRH-sensitive regions of the rat thyrotropin beta gene.
复制标题
Pit-1/GHF-1 与大鼠促甲状腺素 β 基因的 TRH 敏感区域结合。
DOI：
10.1021/bi00085a026
复制
发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Shupnik,MA
中科院分区：
文献类型：
--
作者：
Mason,ME;Friend,KE;Copper,J;Shupnik,MA
1993年6月16日接收的修订版Mandarin pt摘要：TSH/3基因5 '侧翼区域内的三个区域具有富含AT的序列，其与垂体特异性POU结构域转录因子Pit-1/GHF-1的结合位点具有序列相似性。这三个区域被称为TSH A（-274至-258 bp）、TSH B（-336至-326 bp）和TSH C（-402至-384 bp）。在GH 3垂体细胞中的瞬时表达测定中，TSH A和TSH C能够赋予异源病毒胸苷激酶（tk）启动子2-6倍的TRH刺激; TSH C也可以赋予基础增强子活性3-10倍的增加。基于凝胶迁移率变动分析，TSH A、B和C DNA均与来自GH 3细胞核提取物的Pit-1结合，其中抗Pit-1抗体阻止特异性DNA-GH 3核蛋白复合物的形成。TSH A和TSH C还各自形成了几种额外的DNA-核蛋白复合物，而TSH B未观察到。这些复合物中的一些可能含有Pit-1，因为它们的形成被Pit-1抗体抑制;然而，其他复合物不被抗体处理改变。所有三种富含AT的元件结合体外翻译的Pit-1，计算的亲和力分别为360（A）、125（B）和38（C）nM。为了测试Pit-1反式激活TSH/3基因的能力，将含有同源rTSH/3基因启动子和S ′-侧翼区或与异源HSV胸苷激酶（tk）启动子融合的合成TSH基因元件的荧光素酶报告基因构建体转染到缺乏Pit-1的293细胞中。将Pit-1表达载体与TSH/3-荧光素酶构建体共转染到293细胞中，同源TSH/8启动子活性增加3-10倍，表明Pit-1可以反式激活该基因。嵌合tk-TSH A-荧光素酶构建体的表达在Pit-1共转染实验中没有被刺激;然而，tk-TSH C-荧光素酶活性被刺激高达10倍。这些数据表明Pit-1可能在大鼠TSH/3基因的基础和TRH刺激的表达中起作用。下丘脑肽TRH对促甲状腺激素（TSH）1的分泌和合成很重要（Hershman & Pekar，1985; Shupnik et al.，1989），以及TSH/β-和α-亚基mRNA（Franklyn等人，1986; Taylor埃塔尔，1990; Murakami埃塔尔，1991年）。用TRH处理大鼠垂体细胞培养物导致两个亚基基因的转录速率增加2-6倍，观察到TSH/3亚基的作用更显著（Shupnik et al.，1986年）。将大鼠TSH/3基因的5 ′-侧翼区与报告基因融合并转染入GH 3或正常垂体细胞的基因转移实验已经证明该基因的上游部分赋予刺激性TRH应答（卡尔等人，1989年）。突变缺失实验表明，该基因的TRH敏感区至少部分位于主要转录起始位点上游的-520和-204 bp之间（Shupnik等人，1990年）。在这个TRH反应区域，
Revised Manuscript Received June 16, 1993 abstract: Three regions within the 5'-flanking region of the TSH/3 gene have AT-rich sequences which have sequence similarity to binding sites for the pituitary-specific POU domain transcription factor Pit-l/GHF-1. These three regions have been termed TSH A (-274 to-258 bp), TSH B (-336 to-326 bp), and TSH C (-402 to-384 bp). TSH A and TSH C are able to confer 2-6-fold TRH stimulation to the heterologous viral thymidine kinase (tk) promoter in transient expression assays in GH3 pituitary cells; TSH C can confer a 3-10-fold increase in basal enhancer activity as well. TSH A, B, andC DNAs all bound Pit-1 from GH3 cell nuclear extracts, based on gel mobility shift analysis in which antibody against Pit-1 prevented the formation of specific DNA-GH3 nuclear protein complexes. TSH A and TSH C also each formed several additional DNA-nuclear protein complexes which were not observed with TSH B. Some of these complexes may contain Pit-1 as their formation was inhibited by the addition of Pit-1 antibody; other complexes, however, were not altered by antibody treatment. All three AT-rich elements bound in vitro translated Pit-1, with calculated affinities of 360 (A), 125 (B), and 38 (C) nM, respectively. In order to test the ability of Pit-1 to transactivate the TSH/3 gene, luciferase reporter constructs containing either the homologous rTSH/3 gene promoter and S'-flanking region or synthetic TSH gene elements fused to the heterologous HSV thymidine kinase (tk) promoter were transfected into 293 cells which lack Pit-1. Cotransfection of a Pit-1 expression vector with TSH/3-luciferase constructs into 293 cells increased homologous TSH/8 promoter activity 3-10-fold, indicating that Pit-1 could transactivate the gene. Chimeric tk-TSH A-luciferase construct expression was not stimulated in Pit-1 cotransfection experiments; however, tk-TSH C-luciferase activity was stimulated up to 10-fold. These data suggest that Pit-1 may play a role in thebasal and TRH-stimulated expression of the rat TSH/3 gene.The hypothalamic peptide TRH is important for thyrotropin (TSH) 1 secretion and synthesis (Hershman & Pekar, 1985; Shupnik et al., 1989), and the continuous expression of the TSH/?-and a-subunit mRNAs (Franklyn et al., 1986; Taylor etal., 1990; Murakami etal., 1991). Treatmentof rat pituitary cell cultures with TRH resulted in 2-6-fold stimulation of the transcription rate of both subunit genes, with more dramatic effects observed with the TSH/3 subunit (Shupnik et al., 1986). Gene-transfer experiments in which the 5'-flanking region of the rat TSH/3 gene was fused toreporter genes and transfected into either GH3 or normal pituitary cells have demonstrated that the upstreamportion of the gene conferred a stimulatory TRH response (Carr et al., 1989). Mutation deletion experiments indicated that the TRH-sensitive region of the gene lies at least partially between-520 and-204 bp upstream of the major transcriptional start site (Shupnik et al., 1990). Within this TRH-responsive area of the geneare three DNA