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都越来越重要。虽然通过强制表达转录因子来对细胞编程和重新编程的现象学已经被很好地描述，但编程因子的作用机制或导致细胞采用新身份的调节事件序列在很大程度上是未知的。我们建议将干细胞生物学的优势与基因组和计算方法结合起来，以表征运动神经元(MN)身份的转录编程过程。我们开发了通过表达已定义的转录因子来诱导分化胚胎干细胞(ESCs)中MN身份的有效方法。利用该系统，我们将结合生化、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控末端运动神经元效应器基因或启动一系列中间转录程序；ii)编程因子与DNA的招募是否具有协同性以及哪些因素决定了DNA结合的特异性；iii)已确定的MN增强子是否无法与MN编程难于编程的细胞类型的因子结合；iv)已确定的ONECUT和EBF转录因子的二级结合基序是否有助于对MN特异基因表达的生产性调控；v)额外的二级基序是否向NIL结合增强子招募辅助转录因子，以促进对MN特异性基因表达的生产性调控。总之，这些研究将提供对已定义细胞身份和意志的潜在发展过程的基本洞察 为疾病建模、研究和药物开发提供新颖高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.