Analysis of the GABA-A receptor alpha-4 promoter

描述(申请人提供)：本研究项目的目的是 GABAA受体A4亚单位表达调控的研究 (GABAA-R)，它显示了在 GABAA-R亚基家族，在 各种高兴奋性综合征，如慢性戒断 间歇性乙醇(CIE)暴露。导致这种疾病的遗传因素 将研究对小鼠GABAA-R A4亚单位表达的控制。这个 本提议的具体目标是：1)克隆编码 小鼠GABAA-R A4亚单位(GABRA4)，包括推测的5‘-调节域， 并鉴定基因结构和外显子/内含子边界。2)至 研究编码该基因的信使核糖核酸的转录起始位置 GABAA-R A4亚基。3)调查和表征假定的消音器， 小鼠体内的增强子、启动子和/或位置调节元件/S A4亚基基因。A4亚基的表达仅限于特定的 在非神经元细胞中不存在--这可能是由于 存在一个或多个神经元限制性消音器元件 5‘-该基因的调节域。4)培养细胞系或原代神经元 研究GABAA-RA4亚基表达调控的培养体系， 这将概括在体内诱导该基因的关键特征 过度兴奋的模型。反复饮酒，要么是为了 原代培养的神经元，或细胞系模型，会导致诱导 A4亚单位的表达，这种增加的表达可以是 受抑制相关的特定信号通路的调节 转录调控。这些目标的实现将增加我们的 与行为相关的GABAA-R亚基的知识 极度兴奋，并表现出极大的可塑性。这一知识将是 在研究涉及诱导的转录机制方面很有用 GABAA-R亚单位的表达，并可能在设计旨在 阻断与戒断过度兴奋相关的基因组机制。

DESCRIPTION (provided by applicant): The objective of this research project is to study the regulation of expression of the a4 subunit of the GABAA receptor (GABAA-R), which shows a degree of plasticity that is remarkable among the family of GABAA-R subunits, with a striking increase in expression during a variety of hyper-excitability syndromes, such as withdrawal from chronic intermittent ethanol (CIE) exposure. The genetic elements responsible for the control of expression of the mouse GABAA-R a4 subunit will be studied. The specific aims of the present proposal are: 1) To clone the gene encoding the mouse GABAA-R a4 subunit (GABRA4), including the putative 5'-regulatory domain, and to characterize the gene structure and exon/intron boundaries. 2) To investigate the transcriptional start sites for mRNA species encoding the GABAA-R a4 subunit. 3) To investigate and characterize putative silencer, enhancer, initiator and/or positional regulator element/s in the mouse a4-subunit gene. Expression of the a4 subunit is limited to specific populations of neurons and is absent in non-neuronal cells - this may be due to the existence of one or more neuron-restrictive silencer elements in the 5'-regulatory domain of the gene. 4) To develop a cell line or primary neuronal culture system to study the regulation of expression of the GABAA-R a4 subunit, that will recapitulate key features of the induction of this gene in vivo in models of hyperexcitability. Repeated administration of alcohol, either to neurons in primary culture, or to a cell line model, results in induction of expression of the a4 subunit, and that this increased expression can be regulated by the inhibition of specific signaling pathways associated with transcriptional regulation. The completion of these Aims will increase our knowledge of a GABAA-R subunit that is associated with behavioral hyperexcitability and shows substantial plasticity. This knowledge will be useful in the study of transcriptional mechanisms involved in the induction of GABAA-R subunit expression, and potentially in designing interventions aimed at interrupting genomic mechanisms associated with withdrawal hyperexcitability.