Motor neuron selector genes and mechanism of their action

描述（由申请人提供）：细胞身份的转录编程在基础发育和临床水平上越来越重要。虽然通过转录因子的强制表达的细胞编程和重编程的现象学被很好地描述，但编程因子的作用机制或导致细胞采用新身份的调控事件的序列在很大程度上是未知的。我们建议联合收割机干细胞生物学的优势与基因组和计算方法来表征运动神经元（MN）身份的转录编程过程。我们开发了有效的方法，通过表达定义的转录因子诱导分化胚胎干细胞（ESCs）的MN身份。利用该系统，我们将结合联合收割机生化、基因组和计算分析来解决以下问题：i）编程因子是否直接调节终末运动神经元效应基因或启动级联中间转录程序; ii）编程因子对DNA的募集是否是合作的，哪些因子决定DNA结合的特异性; iv）鉴定的Onecut和Ebf转录因子的二级结合基序是否有助于MN特异性基因表达的生产性调节; V）额外的二级基序是否将辅助转录因子募集到NIL结合的增强子，所述增强子有助于MN特异性基因表达的生产性调节。总之，这些研究将提供基本的洞察发展过程的基础规范的定义细胞身份， 为疾病建模、研究和药物发现提供新的和有效的运动神经元来源。

DESCRIPTION (provided by applicant): Transcriptional programming of cell identity is gaining importance at both basic developmental and clinical level. While the phenomenology of cell programming and reprogramming by forced expression of transcription factors is well described, the mechanism of action of programming factors or the sequence of regulatory events resulting in a cell adopting a new identity are largely unknown. We propose to combine the strengths of stem cell biology with genomic and computational approaches to characterize the process of transcriptional programming of motor neuron (MN) identity. We developed efficient methods for the induction of MN identity in differentiating embryonic stem cells (ESCs) by the expression of defined transcription factors. Using the system we will combine biochemical, genomic and computational analysis to address following questions: i) whether programming factors directly regulate terminal motor neuron effector genes or initiate a cascade of intermediate transcription programs; ii) whether recruitment of programming factors to DNA is cooperative and which factors determine the specificity of DNA binding; iii) whether identified MN enhancers are inaccessible for programming factor binding in cell types refractory to MN programming; iv) whether identified secondary binding motifs for Onecut and Ebf transcription factors contribute to productive regulation of MN specific gene expression; v) whether additional secondary motifs recruit ancillary transcription factors to NIL bound enhancers that contribute to productive regulation of MN specific gene expression. Together these studies will provide fundamental insight into the developmental processes underlying specification of defined cell identity and will provide novel and efficient source of motor neurons for disease modeling, study and drug discovery.