Leclerc Gilles M, Bose Sudeep K, Boockfor Fredric R
Laboratory of Molecular Dynamics, Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC, USA.
Neuroendocrinology. 2008;88(1):1-16. doi: 10.1159/000115952. Epub 2008 Feb 6.
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.
最近的证据表明，几种GATA因子在神经内分泌基因表达中充当多功能转录调节因子。大鼠促性腺激素释放激素（GnRH）启动子以间歇性方式表达，其活性需要启动子的一部分，即神经元特异性增强子（NSE）。在本研究中，我们检测了NSE中某些GATA调控元件对于这种间歇性活性是否必要。当将含有GATA-A或GATA-B结合位点突变的荧光素酶报告基因构建体注射到单个活的GT1-7细胞中时，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，逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析首次显示GATA-5和GATA-6在GT1-7细胞中表达，但电泳迁移率变动分析进一步表明GATA-5与这些GATA位点之一：GATA-A结合。染色质免疫沉淀分析表明，GATA-4、GATA-5和GATA-6这三种因子在体内均与GnRH启动子相关。然而，有趣的是，GATA-5或GATA-4（据报道与GATA-B结合）的免疫中和消除了基因表达脉冲，但注射GATA-6抗体则没有，这表明在这些因子中，只有GATA-5和GATA-4对间歇性活性至关重要。最后，凝胶迁移竞争实验揭示了在GATA-A位点结合的蛋白质与与相邻OCT1位点结合的蛋白质之间存在相互作用，先前已证明该OCT1位点是脉冲形成所必需的。这些发现表明，间歇性GnRH基因表达脉冲由GATA-5和GATA-4介导，可能通过GnRH NSE区域中的GATA结合位点起作用。此外，我们观察到与GATA位点相关的因子也可能与OCT1位点相互作用，并且两者对脉冲活性都至关重要，这引发了一个有趣的可能性，即GnRH脉冲的形成是一个高度复杂的过程，可能需要GnRH启动子的几个NSE结合元件的协同相互作用。
