Ohmori M, Shimura H, Shimura Y, Kohn L D
Laboratory of Biochemistry and Metabolism, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Mol Endocrinol. 1996 Jan;10(1):76-89. doi: 10.1210/mend.10.1.8838147.
The decanucleotides in a tandem repeat, -162 to -140 bp, are suppressor elements that decrease TSH receptor (TSHR) gene expression by different mechanisms. A factor(s) interacting with the 3'-decanucleotide compete for proteins that bind the cAMP response element, -139 to -132 bp, a constitutive enhancer necessary for efficient TSHR expression. The 5'-decanucleotide is in a CT-rich, S1 nuclease-sensitive region of the promoter; its suppressor activity has been related to its ability to bind a nonthyroid-specific protein to its coding strand. In this report we clone a complementary DNA encoding a single strand DNA-binding protein that forms a specific protein-DNA complex with the coding strand of the 5'- but not the 3'-decanucleotide and not with the 5'-decanucleotide noncoding or double strand. We show, by cotransfection with TSHR promoter-chloramphenicol acetyltransferase chimeras, that the protein is a suppressor that regulates the function of the 5'- but not the 3'-decanucleotide. The protein is a Y-box protein that was previously cloned as an enhancer factor from the rat liver; it is, however, 95% identical to human YB-1, which suppresses major histocompatibility class II gene expression, and to human nuclease-sensitive element protein-1, a Y-box protein identified by its ability to bind single strand, CT-rich, nuclease-sensitive elements of genes that, like the TSHR, have GC-rich promoters. Unexpectedly, the Y-box protein binds two other sites in the minimal TSHR promoter in a single strand-specific fashion and acts a suppressor at each of these sites. One is associated with the insulin response element of the minimal TSHR promoter and is not in an overtly CT-rich region. The other is located 3' to the cAMP response element in a region termed the S-box, -120 to -113 bp, because of its homology to the S-box of the major histocompatibility class II promoter; this site is in a CT-rich area and, as in the class II promoter, is linked to cAMP-induced gene suppression. A conserved CCTC sequence in each site is important for the binding and suppressor function of the Y-box protein.
串联重复序列中 -162 至 -140 bp 的十聚核苷酸是抑制元件，可通过不同机制降低促甲状腺激素受体（TSHR）基因的表达。与 3' 端十聚核苷酸相互作用的一个或多个因子会竞争与 cAMP 反应元件（-139 至 -132 bp）结合的蛋白质，该元件是 TSHR 高效表达所必需的组成型增强子。5' 端十聚核苷酸位于启动子富含 CT 且对 S1 核酸酶敏感的区域；其抑制活性与其在编码链上结合一种非甲状腺特异性蛋白质的能力有关。在本报告中，我们克隆了一个互补 DNA，其编码一种单链 DNA 结合蛋白，该蛋白与 5' 端十聚核苷酸的编码链形成特异性蛋白质 - DNA 复合物，但不与 3' 端十聚核苷酸以及 5' 端十聚核苷酸的非编码链或双链形成复合物。通过与 TSHR 启动子 - 氯霉素乙酰转移酶嵌合体共转染，我们表明该蛋白是一种抑制因子，可调节 5' 端而非 3' 端十聚核苷酸的功能。该蛋白是一种 Y 盒蛋白，此前作为增强子因子从大鼠肝脏中克隆得到；然而，它与人类 YB - 1 有 95% 的同源性，后者可抑制主要组织相容性复合体 II 类基因的表达，并且与人类核酸酶敏感元件蛋白 - 1 同源，该 Y 盒蛋白因其能够结合基因的单链、富含 CT 且对核酸酶敏感的元件而被鉴定，这些基因与 TSHR 一样具有富含 GC 的启动子。出乎意料的是，Y 盒蛋白以单链特异性方式结合最小 TSHR 启动子中的另外两个位点，并在每个位点发挥抑制作用。其中一个位点与最小 TSHR 启动子的胰岛素反应元件相关，但不在明显富含 CT 的区域。另一个位点位于 cAMP 反应元件的 3' 端，在一个称为 S 盒（-120 至 -113 bp）的区域，因其与主要组织相容性复合体 II 类启动子的 S 盒具有同源性；该位点位于富含 CT 的区域，并且与 II 类启动子一样，与 cAMP 诱导的基因抑制有关。每个位点中保守的 CCTC 序列对于 Y 盒蛋白的结合和抑制功能很重要。
