GATA-4 and GATA-6 modulate tissue-specific transcription of the human gene for P450c17 by direct interaction with Sp1
GATA-4 and GATA-6 modulate tissue-specific transcription of the human gene for P450c17 by direct interaction with Sp1
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DOI：
10.1210/me.2003-0342
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发表时间：
2004-05-01
影响因子：
--
通讯作者：
Miller, WL
中科院分区：
文献类型：
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作者：
Flück, CE;Miller, WL
细胞色素 P450c17 催化类固醇生成 17α-羟化酶和 17,20 裂解酶活性。 P450c17 基因的表达是 cAMP 依赖性的、组织特异性的、发育程序性的，并且在物种之间存在差异。 Sp1、Sp3 和 NF1-C（核因子 1-C）与 5' 侧翼 DNA (- 227/LUC) 的前 227 bp 的结合对于人 NCI-H295A 肾上腺细胞中的基础转录至关重要。人胎盘 JEG-3 细胞含有 Sp1、Sp3 和 NF1，但不表达 - 227/LUC，即使用表达类固醇生成因子 1 (SF-1) 的载体转染也是如此。因此，其他因素对于 P450c17 的基础表达至关重要。脱氧核糖核酸酶 I 足迹和 EMSAs 在 - 64/ - 58 处识别出 GATA 共有位点，在 - 58/ - 50 处识别出 SF-1 位点。RT-PCR 在 NCI-H295A 细胞中识别出 GATA-4、GATA-6 和 SF-1，在 JEG-3 细胞中识别出 GATA-2 和 GATA-3，但未识别出 GATA-4、GATA-6 或 SF-1。在JEG-3细胞中，没有SF-1的GATA-4或GATA-6的共转染激活了- 227/LUC，但是有或没有SF-1的GATA-2或GATA-3的共转染则没有。令人惊讶的是，-227/LUC 中 GATA 结合位点的突变增加了 GATA-4 或 GATA-6 诱导的活性，而 Sp1/Sp3 位点的突变则降低了它。此外，包括GATA位点但不包括-196/-188位点的Sp1/Sp3位点的启动子构建体不被GATA-4或GATA-6激活，表明Sp1/Sp3与GATA-4或GATA-6之间存在相互作用。谷胱甘肽-S-转移酶下拉实验和免疫共沉淀证明了 GATA-4 或 GATA-6 与 Sp1 之间的相互作用，但与 Sp3 之间没有相互作用。染色质免疫沉淀测定证实，GATA-4/6 与 Sp1 的相互作用发生在 NCI-H295A 细胞中 P450c17 启动子的 Sp 位点。用 5-aza-2-deoxycytidine 去甲基化允许 JEG-3 细胞表达内源性 P450c17、SF-1、GATA-4、GATA-6，并转染 - 227/LUC。因此，GATA-4或GATA-6和Sp1共同调节肾上腺NCI-H295A细胞中P450c17的表达，并且P450c17、GATA-4和GATA-6的甲基化沉默胎盘JEG-3细胞中P450c17的表达。
Cytochrome P450c17 catalyzes steroidogenic 17alpha-hydroxylase and 17,20 lyase activities. Expression of the gene for P450c17 is cAMP dependent, tissue specific, developmentally programmed, and varies among species. Binding of Sp1, Sp3, and NF1-C ( nuclear factor 1-C) to the first 227 bp of 5' flanking DNA ( - 227/LUC) is crucial for basal transcription in human NCI-H295A adrenal cells. Human placental JEG-3 cells contain Sp1, Sp3, and NF1, but do not express - 227/LUC, even when transfected with a vector expressing steroidogenic factor 1 (SF-1). Therefore, other factors are essential for basal expression of P450c17. Deoxyribonuclease I footprinting and EMSAs identified a GATA consensus site at - 64/ - 58 and an SF-1 site at - 58/ - 50. RT-PCR identified GATA-4, GATA-6, and SF-1 in NCI-H295A cells and GATA-2 and GATA-3, but not GATA-4, GATA-6, or SF-1 in JEG-3 cells. Cotransfection of either GATA-4 or GATA-6 without SF-1 activated - 227/LUC in JEG-3 cells, but cotransfection of GATA-2 or GATA-3 with or without SF-1 did not. Surprisingly, mutation of the GATA binding site in - 227/LUC increased GATA-4 or GATA-6 induced activity, whereas mutation of the Sp1/Sp3 site decreased it. Furthermore, promoter constructs including the GATA site, but excluding the Sp1/Sp3 site at - 196/ - 188, were not activated by GATA-4 or GATA-6, suggesting an interaction between Sp1/Sp3 and GATA-4 or GATA-6. Glutathione-S-transferase pull-down experiments and coimmunoprecipitation demonstrated interaction between GATA-4 or GATA-6 and Sp1, but not Sp3. Chromatin immunoprecipitation assays confirmed that this GATA-4/6 interaction with Sp1 occurred at the Sp site in the P450c17 promoter in NCI-H295A cells. Demethylation with 5-aza-2-deoxycytidine permitted JEG-3 cells to express endogenous P450c17, SF-1, GATA-4, GATA-6, and transfected - 227/LUC. Thus, GATA-4 or GATA-6 and Sp1 together regulate expression of P450c17 in adrenal NCI-H295A cells and methylation of P450c17, GATA-4 and GATA-6 silence the expression of P450c17 in placental JEG-3 cells.