Wood W M, Kao M Y, Gordon D F, Ridgway E C
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
J Biol Chem. 1989 Sep 5;264(25):14840-7.
In TtT 97 cells, a thyrotropin-producing mouse pituitary tumor, thyroid hormone rapidly inhibits the transcription rate of both the thyrotropin alpha- and beta-subunit (TSH beta) genes, and this closely parallels the increase in nuclear thyroid hormone receptor occupancy. In this study, we have identified regions of the mouse TSH beta gene which are involved in mediating tissue-specific and thyroid hormone-regulated expression. Transient expression studies were performed using a series of chimeric plasmids in which 5'-flanking DNA was ligated to the firefly luciferase gene. Following transfection by electroporation, efficient expression of TSH beta 5'-flanking luciferase constructs occurred only in cells derived from TtT 97 tumors which express the endogenous TSH beta gene. Deletion analysis demonstrated that the region of the 5'-flanking DNA between positions -271 and -80 relative to the major transcriptional start site is important for TSH beta promoter activity in thyrotropes. No expression was measurable in mouse L cells, a fibroblast line, whereas a low level of expression was seen in MGH 101A cells derived from a thyrotropic tumor which no longer expresses the TSH beta gene. Reduced expression of TSH beta constructs was also found in GH3 and GH4 pituitary tumor lines. Addition of thyroid hormone effectively inhibited the level of transient TSH beta promoter activity in TtT 97 cells in a dose-dependent manner. The inhibitory effect was more pronounced and more accurately reflected the transcription rate data when transfected cells were derived from tumors treated with thyroid hormone for 5 days prior to transfection. Deletion of all but 46 base pairs of TSH beta gene 5'-flanking DNA and 3 base pairs of the first exon had no effect on thyroid hormone inhibition. This indicates that signals sufficient for transcriptional regulation of the TSH beta gene by thyroid hormone reside in the vicinity of the proximal promoter and may act by interfering with basal transcriptional factors.
在促甲状腺素生成的小鼠垂体瘤TtT 97细胞中，甲状腺激素能迅速抑制促甲状腺素α亚基和β亚基（TSHβ）基因的转录速率，且这与核甲状腺激素受体占有率的增加密切相关。在本研究中，我们鉴定出了小鼠TSHβ基因中参与介导组织特异性和甲状腺激素调节表达的区域。使用一系列嵌合质粒进行瞬时表达研究，其中5'侧翼DNA与萤火虫荧光素酶基因相连。通过电穿孔转染后，TSHβ 5'侧翼荧光素酶构建体仅在源自表达内源性TSHβ基因的TtT 97肿瘤的细胞中高效表达。缺失分析表明，相对于主要转录起始位点，位于-271至-80位之间的5'侧翼DNA区域对于促甲状腺细胞中TSHβ启动子活性很重要。在小鼠成纤维细胞系L细胞中未检测到表达，而在源自不再表达TSHβ基因的促甲状腺肿瘤的MGH 101A细胞中观察到低水平表达。在GH3和GH4垂体瘤细胞系中也发现TSHβ构建体的表达降低。添加甲状腺激素以剂量依赖的方式有效抑制了TtT 97细胞中瞬时TSHβ启动子活性水平。当转染细胞源自转染前用甲状腺激素处理5天的肿瘤时，抑制作用更明显，并且更准确地反映了转录速率数据。缺失除TSHβ基因5'侧翼DNA的46个碱基对和第一个外显子的3个碱基对外的所有序列，对甲状腺激素抑制没有影响。这表明甲状腺激素对TSHβ基因转录调控的足够信号位于近端启动子附近，并且可能通过干扰基础转录因子起作用。
