Sp1 trans-activates the murine H(+)-K(+)-ATPase alpha(2)-subunit gene.
Sp1 trans-activates the murine H(+)-K(+)-ATPase alpha(2)-subunit gene.
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Sp1 反式激活鼠 H( )-K( )-ATPase α(2)-亚基基因。
DOI：
10.1152/ajprenal.00039.2009
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Kone,BruceC
中科院分区：
文献类型：
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作者：
Yu,Zhiyuan;Li,Mei;Zhang,Dongyu;Xu,William;Kone,BruceC
肾集合管和远端结肠的H+-K+-ATP酶α2（HKα2）基因在钾和酸碱平衡中起着核心作用，但其转录调控的特征仍不清楚。我们以前证明，其5′-侧翼区的近端177 bp赋予小鼠内髓集合管（mIMCD 3）细胞的基础转录活性，NF-κB和CREB-1结合该区域以改变转录。在本研究中，我们试图确定-144/-135 Sp元件是否影响这些细胞中基础HKα2基因的转录。使用−154/−127探针的电泳迁移率变化和超迁移分析显示，在mIMCD 3细胞的核提取物中含有Sp1的DNA-蛋白质复合物。染色质免疫沉淀（ChIP）分析表明，Sp1，而不是Sp3，在体内与mIMCD 3细胞中HKα2基因的启动子区结合。在瞬时转染试验中，在-144/-135 Sp元件中具有点突变的HKα2最小启动子-荧光素酶构建体表现出比野生型启动子低得多的活性。在缺少Sp因子的mIMCD 3细胞和SL 2昆虫细胞中，Sp1的过表达反式激活了HKα2近端启动子-荧光素酶结构，而Sp3没有。相反，小干扰RNA敲低Sp1抑制内源性HKα2 mRNA表达，并抑制Sp1与近端HKα2启动子相关染色质的结合，而不改变NF-κB p65或CREB-1对近端HK α2启动子的结合或调节作用。结论：Sp1在体内外调控mIMCD 3细胞HKα2基因基础表达中起重要作用。
The H+-K+-ATPase α2(HKα2) gene of the renal collecting duct and distal colon plays a central role in potassium and acid-base homeostasis, yet its transcriptional control remains poorly characterized. We previously demonstrated that the proximal 177 bp of its 5′-flanking region confers basal transcriptional activity in murine inner medullary collecting duct (mIMCD3) cells and that NF-κB and CREB-1 bind this region to alter transcription. In the present study, we sought to determine whether the −144/−135 Sp element influences basal HKα2 gene transcription in these cells. Electrophoretic mobility shift and supershift assays using probes for −154/−127 revealed Sp1-containing DNA-protein complexes in nuclear extracts of mIMCD3 cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that Sp1, but not Sp3, binds to this promoter region of the HKα2 gene in mIMCD3 cells in vivo. HKα2 minimal promoter-luciferase constructs with point mutations in the −144/−135 Sp element exhibited much lower activity than the wild-type promoter in transient transfection assays. Overexpression of Sp1, but not Sp3,trans-activated an HKα2 proximal promoter-luciferase construct in mIMCD3 cells as well as in SL2 insect cells, which lack Sp factors. Conversely, small interfering RNA knockdown of Sp1 inhibited endogenous HKα2 mRNA expression, and binding of Sp1 to chromatin associated with the proximal HKα2 promoter without altering the binding or regulatory influence of NF-κB p65 or CREB-1 on the proximal HKα2 promoter. We conclude that Sp1 plays an important and positive role in controlling basal HKα2 gene expression in mIMCD3 cells in vivo and in vitro.