Specific GATA-binding elements in the GnRH promoter are required for gene expression pulse activity: role of GATA-4 and GATA-5 in this intermittent process.
Specific GATA-binding elements in the GnRH promoter are required for gene expression pulse activity: role of GATA-4 and GATA-5 in this intermittent process.
复制标题
GnRH 启动子中的特定 GATA 结合元件是基因表达脉冲活性所必需的：GATA-4 和 GATA-5 在这一间歇过程中的作用。
DOI：
10.1159/000115952
复制
发表时间：
2008
影响因子：
4.1
通讯作者：
Boockfor,FredricR
中科院分区：
文献类型：
--
作者：
Leclerc,GillesM;Bose,SudeepK;Boockfor,FredricR
最近的证据表明，几个加塔因子作为多功能的转录调节神经内分泌基因的表达。大鼠促性腺激素释放激素启动子以一种偶发的方式表达，需要一部分称为神经元特异性增强子（NSE）的启动子才能发挥活性。在这项研究中，我们检查是否某些加塔调节元件的NSE是必要的，这种间歇性的活动。当注射到单个活GT 1 -7细胞中时，含有GATA-A-或GATA-B-结合位点突变的荧光素酶报告基因构建体导致基因表达脉冲频率显著降低，而两个位点的突变几乎消除了脉冲。在随后的研究中，RT-PCR和western blot分析首次揭示了加塔-5和加塔-6在GT 1 -7细胞中表达，但电泳迁移率变动分析进一步证明了加塔-5结合到这些加塔位点之一：GATA-A。染色质免疫沉淀分析显示，所有三个因素，加塔-4，加塔-5，和加塔-6，在体内与GnRH启动子。然而有趣的是，免疫中和加塔-5或加塔-4（据报道结合GATA-B）消除了基因表达脉冲，但注射加塔-6抗体没有，表明这些因子中只有加塔-5和加塔-4对间歇活性是关键的。最后，凝胶移位竞争实验揭示了在GATA-A位点结合的蛋白质和与相邻的OCT 1位点相关联的蛋白质之间的相互作用，先前显示出脉冲形成所必需的。这些发现表明，间歇性GnRH基因表达脉冲由加塔-5和加塔-4介导，可能通过GnRH NSE区域的GATA结合位点起作用。此外，我们的观察，与加塔网站相关的因素也可能与OCT 1网站，这两个都是至关重要的脉冲活动提出了有趣的可能性，GnRH脉冲制作是一个高度复杂的过程，可能需要协调的相互作用的几个NSE结合元件的GnRH启动子。
Recent evidence reveals that several GATA factors act as versatile transcriptional modulators in neuroendocrine gene expression. The rat GnRH promoter is expressed in an episodic fashion that requires a portion of the promoter termed the neuron-specific enhancer (NSE) for activity. In this study, we examined whether certain GATA regulatory elements in the NSE are necessary for this intermittent activity. When injected into individual living GT1-7 cells, luciferase reporter constructs containing mutations of either GATA-A-or GATA-B-binding sites resulted in a marked reduction in gene expression pulse frequency, while mutations of both sites virtually abolished pulses. In subsequent studies, RT-PCR and western blot analysis revealed for the first time that GATA-5 and GATA-6 were expressed in GT1-7 cells, but electrophoretic mobility shift assays demonstrated further that GATA-5 bound to one of these GATA sites: GATA-A. Chromatin immunoprecipitation analysis revealed that all three factors, GATA-4, GATA-5, and GATA-6, were associated with the GnRH promoter in vivo. Interestingly though, immunoneutralization of GATA-5 or GATA-4 (reported to bind GATA-B) abolished gene expression pulses, but injection of GATA-6 antibody did not, indicating that of these factors just GATA-5 and GATA-4 are critical for intermittent activity. Finally, gel shift competition experiments revealed an interaction between proteins binding at the GATA-A site and those associating with an adjacent OCT1 site, previously shown to be necessary for pulse formation. These findings indicate that episodic GnRH gene expression pulses are mediated by GATA-5 and GATA-4, likely acting through the GATA-binding sites in the GnRH NSE region. Moreover, our observations that factors associated with GATA sites may also interact with OCT1 sites and that both are critical for pulse activity raise the intriguing possibility that GnRH pulse elaboration is a highly complex process that may require the coordinated interaction of several NSE-binding elements of the GnRH promoter.