Oligodendrocyte Lineage Gene Function in the CNS

描述(申请人提供)：本研究的主要目标是确定调节bHLH转录因子Orig2对神经前体细胞增殖和分化的相反功能的分子机制。在初步研究中，我们已经鉴定了Oli2氨基末端的三个丝氨酸残基(“三重S基序”)，它们在循环神经前体细胞中被磷酸化，但在分化的后代中不被磷酸化。突变分析表明，寡核苷酸的三重S基序的磷酸化是神经干细胞自我更新所必需的，但对于依赖寡核苷酸的未成熟少突胶质细胞的规格则不是必需的。这里提出的工作建立在这些初步观察的基础上。我们有五个特定的目标：第一个目标是利用小分子激酶抑制剂和一个动态组范围的ShRNAi文库鉴定能够磷酸化寡聚糖2的蛋白激酶(S)。目的二是利用“TAP-TAG”野生型、无磷蛋白和仿磷蛋白，确定受磷酸化调控的寡聚蛋白-蛋白相互作用。目的三是确定寡核苷酸磷酸化的转录功能，以决定神经前体细胞自我更新或退出细胞周期和分化的决定。我们将使用CHIP/SEQ协议来确定磷酸化的寡核苷酸的直接遗传靶点。目的四是建立一种新型的双功能寡核苷酸-TVA-cre转基因小鼠神经管外植体系统，以确定在脊髓胚胎发育和运动神经元发育过程中对寡核苷酸三重S磷酸化的可能需求。目的五是确定寡突胶质细胞成熟和体内中枢神经系统肿瘤发生过程中对寡聚糖2磷酸化的可能需求。原位移植研究将确定寡核苷酸磷酸化是否是少突胶质细胞髓鞘形成的关键，或者是恶性胶质瘤小鼠模型的肿瘤形成。

DESCRIPTION (provided by applicant): The broad objective of the research proposed here is to define the molecular mechanisms that regulate oppositional functions of the bHLH transcription factor Olig2 on proliferation and differentiation of neural progenitor cells. In preliminary studies, we have identified a cluster of three serine residues ("Triple-S motif") in the amino terminus of Olig2 that are phosphorylated in cycling neural progenitors, but not in differentiated progeny. Mutational analysis indicates that phosphorylation of the Olig2 Triple-S motif is required for self-renewal of neural stem cells but is not required for Olig2-dependent specification of immature oligodendrocytes. The work proposed here builds upon these preliminary observations. We have five Specific Aims: Aim One is to identify the protein kinase(s) that phosphorylate Olig2 using small molecule kinase inhibitors and a kinome-wide ShRNAi library. Aim Two is to define Olig2 protein-protein interactions that are regulated by phosphorylation using "TAP- Tagged" wild type, phospho-null and phosphomimetic Olig2 proteins. Aim Three is to define the transcriptional functions of Olig2 phosphorylation to dictate the decision of neural progenitors to self renew or exit the cell cycle and differentiate. We will use ChIP/Seq protocols to identify direct genetic targets of phosphorylated Olig2. Aim Four is to determine a possible requirement for Olig2 Triple-S phosphorylation in embryonic patterning of spinal cord and in motor neuron development using a novel, bifunctional Olig2-tva-cre transgenic mouse neural tube explant system. Aim Five is to determine a possible requirement for Olig2 phosphorylation during oligodendrocyte maturation and CNS tumorgenesis in vivo. Orthotopic grafting studies will determine if Olig2 phosphorylation is essential for oligodendrocyte myelination, or alternatively, tumorgenesis in a mouse model of malignant glioma.