Schoderbek W E, Kim K E, Ridgway E C, Mellon P L, Maurer R A
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242.
Mol Endocrinol. 1992 Jun;6(6):893-903. doi: 10.1210/mend.6.6.1379672.
Transient transfection studies have been used to determine the DNA sequences of the glycoprotein hormone alpha-subunit gene that are required for tissue-specific expression. In the initial phase of these studies, a variant mouse alpha gene was identified which contains a fully palindromic cAMP response element (CRE). The corresponding region of a previously cloned and sequenced mouse alpha gene contains a single point mutation that disrupts the symmetrical nature of this element. DNase footprint studies demonstrate that the fully palindromic CRE binds the CRE-binding protein with much higher affinity than the imperfect palindrome. Transfection experiments using both mouse alpha gene variants demonstrate differences in basal and cAMP-induced expression. Studies of the cAMP response of the human alpha gene indicated that this gene contains sequences other than the known CRE that are sufficient to permit a transcriptional response to cAMP in both placental and pituitary cells. Expression of human and mouse alpha-subunit genes has been examined in cells of the gonadotrope, thyrotrope, and trophoblast lineages to identify DNA sequences that mediate selective transcription of the alpha gene in these cells. The results demonstrate that sequences between about -500 and -200 are important for expression in the pituitary, but not the placenta. Clustered point mutations were used to further characterize sequences required for expression in the pituitary. Two regions, one at positions -445 to -438 and one at positions -337 to -330, were required for expression in cells of the gonadotrope lineage. One of these regions, at -337 to -330, is also important for expression in thyrotropes. When linked to a minimal promoter, multiple copies of the -344 to -300 region had transcriptional enhancer activity in gonadotropes and thyrotropes, but not in several other cell types. These results are consistent with a model involving different combinations of regulatory elements that determine cell-specific alpha expression in gonadotropes and thyrotropes.
瞬时转染研究已被用于确定糖蛋白激素α亚基基因中组织特异性表达所需的DNA序列。在这些研究的初始阶段，鉴定出了一个变异的小鼠α基因，它包含一个完全回文的cAMP反应元件（CRE）。先前克隆和测序的小鼠α基因的相应区域包含一个单点突变，该突变破坏了该元件的对称性质。DNA酶足迹研究表明，完全回文的CRE与CRE结合蛋白的结合亲和力比不完全回文的要高得多。使用两种小鼠α基因变体的转染实验表明了基础表达和cAMP诱导表达的差异。对人α基因的cAMP反应研究表明，该基因除了已知的CRE外，还包含其他序列，这些序列足以使胎盘细胞和垂体细胞对cAMP产生转录反应。已在促性腺激素细胞、促甲状腺激素细胞和滋养层细胞系的细胞中检测了人和小鼠α亚基基因的表达，以鉴定介导这些细胞中α基因选择性转录的DNA序列。结果表明，约-500至-200之间的序列对于在垂体而非胎盘中的表达很重要。使用成簇点突变进一步表征垂体表达所需的序列。促性腺激素细胞系细胞表达需要两个区域，一个在-445至-438位，另一个在-337至-330位。这些区域之一，即-337至-330位，对促甲状腺激素细胞的表达也很重要。当与最小启动子相连时，-344至-300区域的多个拷贝在促性腺激素细胞和促甲状腺激素细胞中具有转录增强活性，但在其他几种细胞类型中则没有。这些结果与一个涉及调控元件不同组合的模型一致，该模型决定了促性腺激素细胞和促甲状腺激素细胞中细胞特异性α的表达。
