Sp3/Sp1 in the parathyroid gland: Identification of an Sp1 deoxyribonucleic acid element in the parathyroid hormone promoter
Sp3/Sp1 in the parathyroid gland: Identification of an Sp1 deoxyribonucleic acid element in the parathyroid hormone promoter
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DOI：
10.1210/en.2003-0119
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发表时间：
2003-07-01
期刊：
影响因子：
4.8
通讯作者：
Koszewski, NJ
中科院分区：
文献类型：
--
作者：
Alimov, AP;Langub, MC;Koszewski, NJ
利用BLAST (basic local alignment search tool) 2序列比对，在PTH启动子中发现了一个高度保守的区域。使用牛甲状旁腺（bPTG）核提取物，用DNA探针观察到含有许多计算机导出的EMSA保守序列的强特异性复合物。乙基化干扰足迹表明，主要复合物与Sp1结合位点的序列惊人地相似。Sp3在主要的dna结合复合体中是明显的，而Sp1的贡献要弱得多。其他未识别的bPTG核因子的特异性结合也很明显。免疫细胞化学和Western blotting分析证实Sp1和Sp3阳性定位于bPTG主细胞和预期分子量的细胞核中，其中Sp3的表达尤其强烈。使用牛PTH Sp1元件的亲和DNA结合实验表明，尽管这两种蛋白的很大一部分无法与亲和标记的DNA相互作用，但特异性恢复了完整的Sp3和Sp1蛋白。然而，用磷酸酶处理bPTG核提取物可显著提高Sp1/Sp3复合物的dna结合能力。瞬时转染分析表明，牛sp1样元件是外源基因表达的增强子。本研究在PTH基因的启动子中发现了Sp1元件，该基因代表一个复杂的dna结合位点，主要与Sp1/Sp3蛋白相互作用。因此，这些数据强调了Sp家族通过与激素启动子中的Sp1 DNA元件相互作用来调节PTH基因表达的可能性。
A highly conserved region in the PTH promoter was identified using the basic local alignment search tool ( BLAST) 2 Sequences comparison. Strong specific complexes were observed with a DNA probe that contained much of the computer-derived conserved sequence in the EMSA using bovine parathyroid gland ( bPTG) nuclear extracts. Ethylation interference footprinting indicated that the major complex made contacts to a sequence strikingly similar to an Sp1 binding site. Sp3 was evident in the major DNA-binding complexes, whereas the contribution by Sp1 was substantially weaker. Specific binding by additional unidentified bPTG nuclear factors was also evident. Immunocytochemical and Western blotting analyses established that Sp1 and Sp3 were positively localized in the nuclei of chief cells of the bPTG and of the expected molecular weights, with particularly robust expression of Sp3. Affinity DNA-binding experiments using the bovine PTH Sp1 element demonstrated specific recovery of intact Sp3 and Sp1 proteins, although a significant portion of both proteins failed to interact with the affinity-tagged DNA. Treatment of the bPTG nuclear extracts with phosphatase, however, significantly increased the DNA-binding capacity of the Sp1/Sp3 complexes. Finally, transient transfection analysis indicated that the bovine Sp1-like element acted as an enhancer of heterologous gene expression. The present study identified an Sp1 element in the promoter of the PTH gene that represents a complex DNA-binding site involving interactions primarily with Sp1/Sp3 proteins. The data, therefore, highlight the likely involvement of the Sp family in regulating PTH gene expression through interactions with an Sp1 DNA element in the hormone's promoter.