Clayton R N, Lalloz M R, Salton S R, Roberts J L
Endocrinology Research Group, Clinical Research Centre, Harrow, Middlesex, U.K.
Mol Cell Endocrinol. 1991 Sep;80(1-3):193-202. doi: 10.1016/0303-7207(91)90156-m.
In this study we determined the activity of the rat luteinising hormone-beta gene promoter in a heterologous rat pituitary cell line (GH3 cells). 1.7 kb of LH-beta 5' flanking sequence and the first 5 bp of the 5' untranslated region were ligated to the chloramphenicol acetyltransferase (CAT) receptor gene (LH-beta-CAT) and transiently transfected by calcium phosphate precipitation into subconfluent cultures of GH3 cells. Basal low-level CAT activity was only detected in GH3 cells, being absent in two non-pituitary cell lines (BeWo and HeLa) RNase analysis revealed that mRNA from transfected GH3 cells protected a fragment of labelled antisense probe of correct size for transcription initiation from the LH-beta CAP site, confirming that promoter activity reflected correctly initiated LH-beta-CAT fusion gene transcripts. CAT activity was consistently induced by an average of 3-5-fold from the full-length 1.7 kb promoter, in a dose- and time-dependent manner, by forskolin, dibutyryl cAMP, and 8-bromo cAMP implying presence of a cAMP-responsive cis-acting domain in the LH-beta promoter region. Transfection of deletion mutants delta-615-CAT, delta-385-CAT and delta-250-CAT each reduced forskolin inducibility to 1.7-fold but did not abolish induction completely suggesting a domain between -1.7 and -0.6 kb contained a cAMP-responsive element(s) (CRE). Further deletion of LH-beta 5' flanking sequences to delta-85-CAT restored forskolin induction to wild-type levels (3-5-fold), suggesting the presence of a weak inhibitory element between -600 and -85 kb, and a cAMP-responsive domain in the proximal promoter region. The LH-beta promoter does not contain perfect tandem repeat palindromic CRE DNA sequences, though there are several octanucleotide sequences differing by only 1 bp from AP-2 binding sites, the consensus CRE, and the vasointestinal peptide gene CRE. Although these data suggest that the LH-beta gene is cAMP responsive this is likely mediated by several and complex protein interactions with multiple DNA sequences in the proximal and distal LH-beta promoter enhancer.
在本研究中，我们测定了大鼠促黄体生成素β基因启动子在异源大鼠垂体细胞系（GH3细胞）中的活性。将1.7 kb的LH-β 5'侧翼序列和5'非翻译区的前5个碱基与氯霉素乙酰转移酶（CAT）报告基因（LH-β-CAT）连接，并通过磷酸钙沉淀法将其瞬时转染至亚汇合的GH3细胞培养物中。基础低水平的CAT活性仅在GH3细胞中检测到，在两种非垂体细胞系（BeWo和HeLa）中未检测到。核糖核酸酶分析表明，转染的GH3细胞中的mRNA保护了一段正确大小的标记反义探针片段，用于从LH-β CAP位点起始转录，证实启动子活性反映了正确起始的LH-β-CAT融合基因转录本。福司可林、二丁酰环磷腺苷和8-溴环磷腺苷以剂量和时间依赖性方式持续诱导全长1.7 kb启动子的CAT活性平均增加3至5倍，这意味着LH-β启动子区域存在一个cAMP反应性顺式作用结构域。缺失突变体delta-615-CAT、delta-385-CAT和delta-250-CAT的转染分别将福司可林诱导性降低至1.7倍，但并未完全消除诱导作用，这表明在-1.7至-0.6 kb之间的一个区域含有一个cAMP反应元件（CRE）。将LH-β 5'侧翼序列进一步缺失至delta-85-CAT可将福司可林诱导恢复至野生型水平（3至5倍），这表明在-600至-85 kb之间存在一个弱抑制元件，并且在近端启动子区域存在一个cAMP反应结构域。LH-β启动子不包含完美的串联重复回文CRE DNA序列，尽管有几个八核苷酸序列与AP-2结合位点、共有CRE和血管活性肠肽基因CRE仅相差1个碱基。虽然这些数据表明LH-β基因对cAMP有反应，但这可能是由近端和远端LH-β启动子增强子中多个DNA序列与多种复杂蛋白质相互作用介导的。
