Genome Wide Identification of PTF1-J Targets in Dorsal Neural Tube

描述（由申请人提供）：Ptf1a是脊髓背侧、小脑和视网膜中gaba能抑制神经元所必需的。在缺乏Ptf1a的情况下，神经祖细胞不能产生抑制性神经元，并异常地呈现兴奋性神经元表型。Ptf1a是II类bHLH因子，可与I类bHLH因子（e蛋白）异源二聚，在体外结合E-box DNA共识序列。然而，Ptf1a在体内的功能是在一个独特的转录因子复合体中，称为PTF1-J，它包含Ptf1a和一个e蛋白，加上第三个因子Rbpj。这个复合体结合了包含E-box和TC- box的二部分DNA序列。Rbpj作为Notch信号的转录效应因子具有独特的功能。Notch-Rbpj信号抑制神经元分化，而Ptf1a-Rbpj信号在神经元亚型规范中起作用。该建议是使用体内染色质免疫沉淀结合大规模平行测序平台（ChIP-seq），通过全基因组鉴定其转录靶点来描绘Ptf1a的功能。ChIP-seq鉴定的体内Ptf1a靶基因将通过结合背神经管中Ptf1a细胞的表达谱结果，评估Ptf1a突变体的表达变化，以及在携带和不携带外源Ptf1a的鸡神经管中检测靶调控区域的报告基因试验来验证和表征。确定Ptf1a的靶点将为控制抑制性和兴奋性神经元平衡的发育过程提供分子洞察力。

DESCRIPTION (provided by applicant): Ptf1a is required for GABAergic inhibitory neurons in the dorsal spinal cord, cerebellum, and retina. In the absence of Ptf1a, neural progenitor cells fail to generate inhibitory neurons and aberrantly assume an excitatory neuronal phenotype. Ptf1a is a class II bHLH factor that can heterodimerize with class I bHLH factors (E-proteins) to bind E-box DNA consensus sequences in vitro. However, the in vivo function of Ptf1a is in a unique transcription factor complex, called PTF1-J, that contains Ptf1a and an E-protein, plus a third factor Rbpj. This complex binds a bipartite DNA sequence containing the E-box plus a TC- box. Rbpj has a distinct function as the transcriptional effector of Notch signaling. Notch-Rbpj signaling inhibits neuronal differentiation, while Ptf1a-Rbpj functions in neuronal subtype specification. The proposal is to use in vivo chromatin immunoprecipitation combined with massive parallel sequencing platforms (ChIP-seq) to delineate the function of Ptf1a by genome wide identification of its transcription targets. ChIP-seq identified target genes of Ptf1a in vivo will be validated and characterized by combining results from expression profiling of Ptf1a cells in the dorsal neural tube, evaluating changes in expression in Ptf1a mutants, and by testing target regulatory regions in reporter assays in chick neural tube with and without exogenous Ptf1a. Identifying targets of Ptf1a will provide molecular insight into developmental processes controlling the balance of inhibitory and excitatory neurons. PUBLIC HEALTH RELEVANCE: Normal nervous system function requires the correct balance of inhibitory and excitatory neurons. Alterations in this balance are thought to underlie diverse neurological disorders from epilepsy to autism to hyperalgesia. Successful completion of these aims will substantially advance efforts to define transcriptional networks regulating the balance of inhibitory and excitatory neuronal specification. This is a unique opportunity to combine multiple technical advances to begin to tackle the challenging goal of defining the molecular rationale controlling the generation of neuronal diversity.