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

描述(申请人提供)：Ptf1a是背侧脊髓、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，神经前体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是一种II类bHLH因子，在体外可以与I类bHLH因子(E蛋白)异源二聚体结合E-box DNA共同序列。然而，Ptf1a在体内的功能是在一个名为PTF1-J的独特的转录因子复合体中完成的，该复合体包含Ptf1a和一个E蛋白，以及第三个因子Rbpj。该复合体与含有E-box和TC-box的两部分DNA序列结合。Rbpj作为Notch信号的转录效应因子具有独特的功能。Noch-Rbpj信号抑制神经元分化，而Ptf1a-Rbpj信号在神经元亚型规范中发挥作用。建议使用体内染色质免疫沉淀结合大规模并行测序平台(CHIP-SEQ)来描述Ptf1a的功能，方法是在全基因组范围内鉴定其转录靶标。ChIP-SEQ确定的体内Ptf1a靶基因将通过结合背侧神经管Ptf1a细胞表达谱的结果、评估突变体中Ptf1a表达的变化以及在有和没有外源Ptf1a的鸡神经管中检测目标调节区来验证和表征。识别Ptf1a的靶点将提供对控制抑制性和兴奋性神经元平衡的发育过程的分子洞察。 公共卫生相关性：正常的神经系统功能需要抑制性和兴奋性神经元的正确平衡。这种平衡的改变被认为是从癫痫到自闭症再到痛觉过敏的各种神经疾病的基础。这些目标的成功完成将极大地推动定义转录网络的努力，以调节抑制性和兴奋性神经元规范的平衡。这是一个独特的机会，可以结合多种技术进步，开始解决确定控制神经元多样性产生的分子原理这一具有挑战性的目标。

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.