MOLECULAR MECHANISMS OF FATE CHOICE IN NEURAL STEM CELLS

bHLH转录因子Olig 1和Olig 2调节CNS祖细胞形成神经元或少突胶质细胞的命运选择决定。尽管Olig 1和Olig 2编码具有重叠表达模式的结构相关蛋白质，但它们具有不同的生物学功能。该项目的假设是Olig 1和Olig 2的非重叠生物学功能反映了与不同转录辅助调节蛋白的相互作用。该项目的主要目标是通过鉴定和表征Olig 1和Olig 2共调节子来验证这一假设。该研究计划建立在初步工作的基础上，其中我们发现Olig 2蛋白与Nkx2.2蛋白形成物理复合物（37）。在发育中的神经管中观察到该复合物的功能性泛音，其中各种研究表明Olig 2和Nkx2.2在少突胶质细胞祖细胞的特化和分化中合作。我们开始与Olig 2/Nkx2.2复合物的结构和功能的重点关注，然后进展到额外的辅助调节，可能与Olig 1和Olig 2的差异相互作用，以控制基础的转录机制和修改染色质的结构。我们有五个具体目标： 目的一是确定Olig 2：Nkx2.2相互作用是直接的还是间接的。目标二是 确定Olig 2和Nkx2.2之间的复合物是否在体内形成并与DNA相互作用。目的三是检验Olig 1和Olig 2与Nkx2.2差异相互作用的假设。目的四是利用酵母双杂交捕获技术鉴定、克隆和鉴定新的、细胞类型特异性的Olig 1和Olig 2共调节蛋白。两种Olig蛋白的氨基和羧基末端结构域（它们彼此相当不同）将用作“诱饵”。目的五是利用免疫亲和纯化结合飞行时间质谱技术对Olig 1和Olig 2相关蛋白进行鉴定、比较和对比。这种生物化学方法将补充酵母遗传筛选，其可能错过i）在酵母中错误折叠或错误定位，ii）与寡核苷酸bHLH结构域相互作用或iii）仅在DNA或染色质存在下与寡核苷酸相互作用的共调节子。该研究计划与其他项目互动，并依赖于基因表达和生物信息学的核心设施。Olig伴侣蛋白的鉴定可能会转化为多发性硬化症，脊髓损伤和中枢神经系统原发性癌症的新疗法。

The bHLH transcription factors Olig1 and Olig2 regulate the fate choice decision of CNS progenitor cells to form neurons or oligodendrocytes. Although they are encode structurally related proteins with overlapping expression patterns, Olig1 and Olig2 have distinct biological functions. The hypothesis of this project is that non-overlapping biological functions of Olig1 and Olig2 reflect interactions with distinct transcription co-regulator proteins. The broad goal of this project is to test the hypothesis through identification and characterization of Olig1 and Olig2 co-regulators. The study plan builds upon preliminary work wherein we have found that Olig2 protein forms a physical complex with Nkx2.2 protein (37). Functional overtones of this complex are seen in developing neural tube where a variety of studies indicate that Olig2 and Nkx2.2 cooperate in the specification and differentiation of oligodendrocyte progenitors. We begin with a focused look at structure and fimction of the Olig2/Nkx2.2 complex and then progress to additional co-regulators that may interact differentially with Olig1 and Olig2 to control the basal transcriptional machinery and modify the structure of chromatin. We have five specific aims: Aim one is to determine whether the Olig2:Nkx2.2 interaction is direct or indirect. Aim two is to determine whether the complex between Olig2 and Nkx2.2 forms in vivo and interacts with DNA. Aim three is to test the hypothesis that Olig1 and Olig2 interact differentially with Nkx2.2. Aim four is to use yeast two-hybrid trapping technology to identify, clone and characterize novel, cell type-specific co-regulator proteins for Olig1 and Olig2. The amino and carboxyl terminal domains of the two Olig proteins (which are quite divergent from each other) will be used as "bait". Aim five is use immunoaffinity purification coupled with time-of-flight mass spectroscopy to identify, compare and contrast proteins that associate with Olig1 and Olig2. This biochemical approach will complement the yeast genetic screen, which could miss co-regulators that are i) misfolded or mislocalized in yeast, ii) interact with the Olig bHLH domain or iii) interact with Oligs only in the presence of DNA or chromatin. The study plan is interactive with the other projects and dependent upon core facilities for gene expression and bioinformatics. Identification of Olig partner proteins might translate into new therapies for multiple sclerosis, spinal cord injuries and primary cancers of the central nervous system.