Chromatin Changes During CNS Migration and Circuit Formation

这项拟议的研究将研究染色质如何在中枢神经系统中调节基因表达。 以小脑颗粒细胞为模型进行迁移和环路形成。这项工作建立在我们最近的基础上 发现在小脑发育过程中染色质重塑基因水平发生显著变化 发育，包括组蛋白修饰基因和Tet基因的变化，Tet基因氧化5-甲基-2-甲基-3-酮. 将胞嘧啶(5mC)转化为5-羟甲基胞嘧啶(5hmC)，这是一种大脑特异的DNA修饰。我们的研究 结果表明，5hmC水平的增加与离子通道基因和轴突的水平的增加有关 迁移后神经元中的引导(树突)基因(朱等人，2016)。此外，RNAi介导的 体外组织切片中迁移的小脑颗粒细胞中Tet基因的敲除阻止了这一转变 从双极细胞，迁移到多极细胞，延伸树突并形成突触接触 具有嵌入性传入纤维和苔藓纤维(朱等人，2016)。这些发现提供了第一个证据 染色质变化的想法是小脑回路形成的基础。 在拟议的研究中，我们将与我们的同事David Allis博士和Erica Korb博士合作， 他们是染色质生物学方面的权威，以表征四种基本组蛋白甲基化的修饰 调节转录激活和抑制的标记，组蛋白3赖氨酸残基4(H3K4)，H3K9， H3K27和H3K36，在一个特征良好的中枢神经系统神经元，小脑颗粒细胞(GC)中，在 就在胶质引导的迁移之后，随着小脑回路的形成。然后我们将确定基因的变化 与这些组蛋白甲基化相关的表达变化。最后，我们将使用RNAi方法来 研究组蛋白甲基转移酶和去甲基酶在这些组蛋白生成中的作用 体外小脑片上的迁移、树突伸展和突触形成的标记。这些研究 将提供有关中枢神经系统迁移和回路形成的染色质变化的关键信息 小脑。

The proposed research will examine how chromatin regulates gene expression during CNS migration and circuit formation, using the cerebellar granule cell as a model. The work builds on our recent discovery that dramatic changes occur in the levels of chromatin remodeling genes during cerebellar development, including changes in histone modifying genes and in Tet genes, which oxidize 5-methyl- cytosine (5mC) into 5-hydroxymethyl-cytosine (5hmC), a brain-specific DNA modification. Our studies showed that increased levels of 5hmC correlated with increased levels of ion channel genes and of axon guidance (dendritic) genes in post-migratory neurons (Zhu et al, 2016). Moreover, RNAi-mediated knockdown of Tet genes in migrating cerebellar granule cells in ex vivo tissue slices blocked the transition from a bipolar, migrating cell into a multipolar cell that is extending dendrites and forming synaptic contacts with ingrowing afferent, mossy fibers (Zhu et al, 2016). These findings provided the first evidence for the idea that chromatin changes underlie the formation of the cerebellar circuitry. In the proposed research, we will collaborate with our colleagues, Dr. David Allis and Dr. Erica Korb, who are authorities on chromatin biology, to characterize modifications in four basic histone methylation marks that regulate transcriptional activation and repression, histone 3 lysine residue 4 (H3K4), H3K9, H3K27 and H3K36, in a well-characterized CNS neuron, the cerebellar granule cell (GC), before, during and just after glial-guided migration, as the cerebellar circuitry forms. We will then identify changes in gene expression associated with these histone methylation changes. Finally, we will use RNAi methodology to study the function of histone methyltransferases and demethylases involved in generating these histone marks in migration, dendrite extension and synapse formation on ex vivo cerebellar slices. These studies will provide critical information on chromatin changes underlying CNS migration and circuit formation in the cerebellum.