MOLECULAR MECHANISMS OF FATE CHOICE IN NEURAL STEM CELLS

BHLH转录因子Orig1和Opol2调控中枢神经系统前体细胞形成神经元或少突胶质细胞的命运选择。尽管它们编码的是具有重叠表达模式的结构相关的蛋白质，但它们具有不同的生物学功能。该项目的假设是，寡核苷酸1和寡核苷酸2的不重叠的生物学功能反映了与不同的转录辅助调节蛋白的相互作用。这个项目的主要目标是通过识别和表征Otl1和Otl2共同调节因子来检验这一假说。该研究计划建立在前期工作的基础上，其中我们发现Orig2蛋白与Nkx2.2蛋白(37)形成物理复合体。在发育中的神经管中可以看到这种复合体的功能含义，各种研究表明，Orig2和Nkx2.2在少突胶质前体细胞的指定和分化中起着协同作用。我们首先重点研究Otl2/Nkx2.2复合体的结构和功能，然后进一步研究其他可能与Otl1和Olob2相互作用的协同调控因子，以控制基本的转录机制和改变染色质的结构。我们有五个具体目标： 目的一是确定Orig2：Nkx2.2的相互作用是直接的还是间接的。目标二是 确定寡核苷酸和Nkx2.2之间的复合体是否在体内形成并与DNA相互作用。目的三是验证寡核苷酸1和寡核苷酸2与Nkx2.2存在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、细胞类型特异的寡核苷酸1和寡核苷酸2的协同调节蛋白。这两个Olig蛋白的氨基和羧基末端结构域(彼此非常不同)将被用作“诱饵”。目的五是利用免疫亲和纯化与飞行时间质谱仪相结合的方法，鉴定、比较和对比与寡核苷酸1和寡核苷酸2相关的蛋白质。这种生化方法将补充酵母遗传筛选，它可能会错过共同调控因子：i)在酵母中错误折叠或错误定位；ii)与Olig bHLH域相互作用；或iii)仅在DNA或染色质存在的情况下与寡核苷酸相互作用。该研究计划与其他项目相互作用，并依赖于基因表达和生物信息学的核心设施。Olig伙伴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.