Oligodendrocyte Lineage Gene Function in the CNS

描述（由申请人提供）：这里提出的研究的主要目的是确定bHLH转录因子Olig2对神经祖细胞增殖和分化的调控拮抗功能的分子机制。在初步研究中，我们在Olig2的氨基端发现了一个由三个丝氨酸残基组成的簇（“Triple-S基序”），在循环神经祖细胞中被磷酸化，但在分化的后代中没有磷酸化。突变分析表明，Olig2 Triple-S基序的磷酸化对于神经干细胞的自我更新是必需的，但对于未成熟少突胶质细胞的Olig2依赖性规范不是必需的。这里提出的工作建立在这些初步观察的基础上。我们有五个具体目标：目标一是利用小分子激酶抑制剂和全激酶ShRNAi文库鉴定磷酸化Olig2的蛋白激酶。目的二是定义由“TAP标记”野生型、无磷酸化和拟磷型Olig2蛋白磷酸化调控的Olig2蛋白-蛋白相互作用。目的三是明确Olig2磷酸化的转录功能，以决定神经祖细胞是自我更新还是退出细胞周期并分化。我们将使用ChIP/Seq协议来确定磷酸化Olig2的直接遗传靶点。目的四是利用一种新型的双功能Olig2-tva-cre转基因小鼠神经管移植系统，确定在脊髓胚胎模式和运动神经元发育中Olig2 Triple-S磷酸化的可能要求。目的五是确定体内少突胶质细胞成熟和中枢神经系统肿瘤发生过程中Olig2磷酸化的可能需求。原位移植研究将确定Olig2磷酸化是否对少突胶质细胞髓鞘形成或恶性胶质瘤小鼠模型中的肿瘤发生至关重要。

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.