Transcriptional Mechanisms Regulating Activity Dependent

表观遗传和遗传因素有助于神经系统和骨骼肌在发育过程中的重塑。突触后靶点的特性在很大程度上由转录决定，受到不同模式的神经去极化和释放的神经因子的作用的影响。细胞谱系和发育环境有助于这些表观遗传因素引起的反应。分子神经生物学部分感兴趣的是阐明有助于神经元和骨骼肌活动依赖的发育可塑性的分子途径。为此，我们正在研究中枢神经元中NMDA受体和骨骼肌中肌钙蛋白I的发育调节。神经递质谷氨酸激活NMDA受体是许多兴奋性突触的活性可塑性所必需的。我们已经研究了该受体的NR2A和NR2C亚基的调节。克隆了NR2A型基本启动子及其邻近序列，并对其功能进行了检测。含有9kb NR2A上游序列的转基因小鼠在神经元中特异性地表达荧光素酶报告基因。对导入大脑皮层神经元的构建体进行缺失分析，确定了对发育和神经元特异转录重要的序列。目前正在进行研究，以确定调控NR2A发育表达的因素。在其他研究中，我们发现NR2C基因在小脑颗粒细胞中的表达被神经活动和神经调节蛋白(NRG)大大增强，神经调节蛋白(NRG)是一种与表皮生长因子密切相关的因子。最近，其他研究小组发现，NRG还调节乙酰胆碱和GABA的神经递质受体的表达，以及神经胶质细胞的存活。由于Nrgs在中枢神经系统发育中的核心作用，我们正在研究这些因素对突触可塑性的调节和贡献。我们发现这3个NRG基因(NRG1-3)及其4个受体(ErbB1-4)具有不同的区域和发育表达模式。有趣的是，NMDA和ErbB受体在突触后密度(PSD)处共表达，在那里它们与含有PDZ蛋白相互作用结构域的相同蛋白质相互作用。PDZ结构域蛋白很重要，因为它们将受体和通道搭建到信号分子上，从而将突触活动耦合到突触后神经元的信号级联反应中。我们目前正在研究这些互动的重要性。为了了解选择性调节不同肌肉基因的信号通路，我们鉴定并鉴定了肌钙蛋白I慢(TnI)和快(TnIf)基因的增强子。我们的结果表明，转录因子的复合体与这些增强子相互作用，调节转录。使用酵母-1-杂交系统，我们分离出了赋予肌肉纤维类型特异性的假定因子。其中一种被称为GTF-3的因子编码一种具有5-6个内部重复的蛋白质，该蛋白质可能与一系列转录因子相互作用。有趣的是，在患有威廉姆斯-博伦综合征(WBS)的患者中，GTF-3基因以及该基因家族的另一个成员都被缺失了。威廉姆斯-博伦综合征是由染色体7q11.23上的微缺失引起的。据报道，WBS患者有不同程度的智力低下、主动脉瓣上狭窄和肌病。研究正在进行中，以了解GTF-3在与WBS相关的缺陷中的作用。

Epigenetic and genetic factors contribute to the remodeling of the nervous system and skeletal muscles during development. The properties of postsynaptic targets, largely determined by transcription, are modified by distinct patterns neural depolarization and the action of released neuronal factors. Cell lineage and developmental context contribute to the response elicited by these epigenetic factors. The Section on Molecular Neurobiology is interested in elucidating the molecular pathways that contribute to the activity-dependent developmental plasticity of neurons and skeletal muscles. To this end, we are studying the developmental regulation of NMDA receptors in central neurons and Troponin I in skeletal muscle. Activation of NMDA receptors (NRs) by the neurotransmitter glutamate is necessary for activity-dependent plasticity at numerous excitatory synapses. We have studied the regulation of the NR2A and NR2C subunits of the receptor. The NR2A basal promoter and adjacent sequences were cloned, tested for function. Transgenic mice harboring 9 kilobases of NR2A upstream sequence express the luciferase reporter specifically in neurons. Deletion analysis of constructs transfected into cortical neurons identified sequences important for developmental- and neuronal-specific transcription. Studies are in progress to identify factors regulating the developmental expression of NR2A. In other studies we found that expression of the NR2C gene in cerebellar granule cells is greatly enhanced by neural activity and Neuregulin (Nrg), a factor distantly related to the epidermal growth factor. Recently, other groups found that Nrg also regulates expression of neurotransmitter receptors for acetylcholine and GABA, as well as survival of glial cells. Because of the central role of Nrgs in CNS development, we are studying the regulation and contribution of these factors to synaptic plasticity. We found that the 3 Nrg genes (Nrg 1-3), as well as their four receptors (ErbB 1-4), have distinct regional and developmental expression patterns. Interestingly, NMDA and ErbB receptors are co-expressed at postsynaptic densities (PSD) where they interact with the same proteins harboring PDZ protein-protein interaction domains. The PDZ-domain proteins are important because they scaffold receptors and channels to signaling molecules, thus coupling synaptic activity to signaling cascades in the postsynaptic neurons. We are presently studying the importance of these interactions. To understand the signaling pathways that selectively regulate distinct muscle genes, we have identified and characterized the enhancers of the troponin I slow (TnIs) and fast (TnIf) genes. Our results suggest that a complex of transcription factors interact with these enhancers to regulate transcription. Using a yeast-1-hybrid system, we have isolated putative factors that confer muscle fiber-type-specificity. One of these factors, known as GTF-3, encodes a protein with 5-6 internal repeats that may interact with a series of transcription factors. Interestingly, the GTF-3 gene, as well another member of this gene family, is deleted in persons afflicted with the Williams-Beuren Syndrome (WBS) which results from a micro-deletion on chromosome 7q11.23. Persons with WBS are reported to have variable degrees of mental retardation, supra valvular aortic stenosis, and myopathies. Studies are in progress to understand the role of GTF-3 in the deficiencies associated with WBS.