Flug F, Copp R P, Casanova J, Horowitz Z D, Janocko L, Plotnick M, Samuels H H
J Biol Chem. 1987 May 5;262(13):6373-82.
In GC cells, a growth hormone-producing rat pituitary cell line, 3,5,3'-triiodo-L-thyronine (L-T3) rapidly stimulates the transcription rate of the growth hormone gene which parallels the level of chromatin-associated L-T3-receptor complexes (Yaffe, B. M., and Samuels, H. H. (1984) J. Biol. Chem. 259, 6284-6291). In this study we have functionally mapped the elements of the gene which are involved in mediating basal and hormone-regulated expression. Stable transformation studies indicate that transcriptional regulation of the gene by L-T3 is mediated by sequences in the 5'-flanking region. Transient expression studies were performed using a series of chimeric plasmids in which 5'-flanking DNA was ligated to the chloramphenicol acetyltransferase gene. Transient expression occurred only in cells which expressed the endogenous growth hormone gene. Sequences between -104 and +7 were found to be essential for basal expression. One of the most highly conserved regions (-105 to -145) contains elements which further enhance the level of basal expression but are not necessary for regulated expression by L-T3. DNA between -210 and -181 was found to be essential for stimulation by L-T3 and was shown to function most efficiently with the homologous rat growth hormone promoter (-104 to +7). Sequences from -206 to -198 show about 80% homology with a sequence in the 5'-flanking region of two other rat genes which are regulated by thyroid hormone. Glucocorticoid hormones, which also transcriptionally stimulate the rat growth hormone gene, elicited only minimal effects in both stable and transient expression studies. This suggests that the elements which mediate glucocorticoid regulation of the endogenous gene are found either upstream of the cloned 5'-flanking region (1800 base pairs) or 3' of the cap site.
在生长激素分泌型大鼠垂体细胞系GC细胞中，3,5,3'-三碘-L-甲状腺原氨酸（L-T3）能迅速刺激生长激素基因的转录速率，这与染色质相关的L-T3受体复合物水平平行（亚菲，B.M.，和塞缪尔斯，H.H.（1984年）《生物化学杂志》259，6284 - 6291）。在本研究中，我们对该基因中参与介导基础表达和激素调节表达的元件进行了功能定位。稳定转化研究表明，L-T3对该基因的转录调控是由5'侧翼区的序列介导的。使用一系列嵌合质粒进行瞬时表达研究，其中5'侧翼DNA与氯霉素乙酰转移酶基因连接。瞬时表达仅发生在内源生长激素基因表达的细胞中。发现-104至+7之间的序列对于基础表达至关重要。其中一个高度保守区域（-105至-145）包含能进一步提高基础表达水平但对L-T3调节表达并非必需的元件。发现-210至-181之间的DNA对于L-T3的刺激至关重要，并且与同源大鼠生长激素启动子（-104至+7）一起作用时效率最高。-206至-198的序列与另外两个受甲状腺激素调节的大鼠基因5'侧翼区的一个序列具有约80%的同源性。糖皮质激素也能转录刺激大鼠生长激素基因，但在稳定和瞬时表达研究中仅产生最小影响。这表明介导内源性基因糖皮质激素调节的元件要么位于克隆到的5'侧翼区（1800个碱基对）上游，要么位于帽位点的3'端。
