Functional Genomic Studies of Neuronal Differentiation

描述(申请人提供)：脊椎动物神经系统内神经元的精确分化是由转录因子相互作用的复杂遗传程序指定的，以及环境提示对这些相互作用的调节。虽然许多特定的转录因子和信号通路在神经元分化中的作用已经被表征，但我们对其中涉及的详细机制的了解还远远不完整。这一建议利用了几种哺乳动物基因组的基因组序列信息，脊椎动物神经系统发育在整个进化过程中高度保守的基本观察结果，以及功能基因组学的最新进展，提出了一种高通量的神经元分化功能基因组分析。这项提议中要检验的假设是，在神经元分化过程中诱导的基因周围进化保守的序列对神经元诱导的编排很重要。在第一个特定目标中，将诱导P19胚胎癌细胞经历神经元分化，并将进行微阵列杂交研究，以确定转录调控基因。在第二个特定目标中，最近被提出代表顺式调控序列簇的进化保守的小鼠基因组非基因序列(CNGs)将使用高通量微阵列转染方法在P19细胞中分析转录调控。在第三个特定目标中，将描述蛋白激酶和转录因子在调节这些顺式作用序列中的作用。这些研究的最终结果将是详细了解神经诱导基因的重要顺式调控序列，以及特定信号转导途径在其转录激活调控中的作用。这些研究的结果将对未来治疗包括阿尔茨海默氏症和帕金森氏病在内的神经退行性疾病的治疗方法具有重要意义。

DESCRIPTION (provided by applicant): The precise differentiation of neurons within the vertebrate nervous system is specified by a complex genetic program of transcription factor interactions, as well as modulation of these interactions by environmental cues. Although the roles of many specific transcription factors and signaling pathways have been characterized in neuronal differentiation, our understanding of the detailed mechanisms involved is still far from complete. This proposal employs genomic sequence information of several mammalian genomes, the fundamental observation that vertebrate nervous system development is highly conserved throughout evolution, and recent developments in functional genomics to propose a high-throughput functional genomic analysis of neuronal differentiation. The hypothesis to be tested in this proposal is that evolutionary conserved sequences surrounding genes induced during neuronal differentiation are important to the orchestration of neuronal induction. In the first specific aim, P19 embryonic carcinoma cells will be induced to undergo neuronal differentiation and microarray hybridization studies will be carried out to identify genes that are transcriptionally regulated. In the second specific aim, evolutionarily conserved non-genic sequences (CNGs) of the mouse genome, that have recently been proposed to represent clusters of cis- regulatory sequences, will be analyzed for transcriptional regulation using a high-throughput microarray transfection method in P19 cells. In the third specific aim, the role of protein kinases and transcription factors in the regulation of these cis-acting sequences will be characterized. The end result of these studies will be a detailed understanding of important cis-regulatory sequences of neuronally induced genes and the role of specific signal transduction pathways in the modulation of their transcriptional activation. The results of these studies will be important for future therapeutic approaches to treatment of neurodegenerative diseases including Alzheimer's and Parkinson's diseases.