MOLECULAR MECHANISMS OF FATE CHOICE IN NEURAL STEM CELLS

点击翻译按钮获取中文摘要

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.