Gene regulatory networks in spinal cord development

摘要 过去对外源性信号和内源性转录因子（TF）的研究极大地增强了我们对转录因子的认识。 了解CNS中不同神经元细胞类型的命运特化和分化是如何 基因编码染色质调控标记及其产生酶的研究进展 这些标记使我们能够将染色质调节的潜在重要作用与转录因子的活性结合起来， CNS发展。然而，这一努力面临着一些挑战。1)发展中的中枢神经系统包括 在不同的发育状态下，细胞具有深刻的异质性，这使得研究染色质如何 每一种细胞类型都在协调变化。2)虽然细胞类型特异性调控元件（本文称为 作为顺式元件）被预测在整个过程中经历最重要的功能性染色质变化 发展，这样的顺式元件是全球定义不清。3)虽然多种染色质调节因子 在每种细胞类型的发育过程中，将被动员到细胞类型特异性顺式元件， 确定特定的染色质因子在起作用。基于我们先前解决这些限制的努力，目前 研究脊髓运动神经元（MN）发育的基因调控网络是一项开创性的研究， 解决CNS发育中染色质调节的关键问题。 我们在过去的资助周期中的发现确定了转录密码和基因调控元件， 基于MN命运的承诺和规范，建立了一个理想的细胞模型来研究染色质 CNS发育的调控。在发育中的脊髓中，Olig 2，一种基本的螺旋-环-螺旋（bHLH）TF， 在MN祖细胞（pMNs）中表达，在建立pMN结构域和保持 pMN细胞过早分化为MN。随着pMN细胞开始分化为MN，LIM 同源结构域（HD）TF Isl 1和Lhx 3与bHLH TF Ngn 2一起沿着上调。Isl 1和Lhx 3形成a 复合物（Isl 1-Lhx 3），其通过MN基因与Ngn 2的协同反式激活来指导MN命运。在这 建议整合这些遗传程序与染色质调控，我们假设，在MN 细胞类型特异性TF（Olig 2、Ngn 2和Isl 1-Lhx 3）和细胞类型特异性TF（Olig 2、Ngn 2和Isl 1-Lhx 3）的协调作用。 染色质修饰物（Ezh 2，Jmjd 3和CBP/p300）协调了MN基因的染色质变化， 转录平衡/抑制到活性状态，使得能够及时获得MN命运和细胞 分化基于我们的初步数据，我们特别假设，在pMN细胞中，Olig 2募集Ezh 2 对于MN基因，建立转录抑制染色质标记，三甲基化组蛋白H3-赖氨酸27 （H3K27me3）。随着Olig 2表达在分化的pMN细胞中下降，Isl 1-Lhx 3和Ngn 2抓住MN特异性 增强子和募集Jmjd 3，其去除H3 K27 me 3并允许CBP/p300建立乙酰化的H3 K27 （H3 K27 ac），转录活性染色质标记。我们将用一组细胞， 生物化学、遗传学和全基因组方法。

ABSTRACT Past studies on extrinsic signals and intrinsic transcription factors (TFs) have greatly enhanced our understanding of how the fate specification and differentiation of diverse neuronal cell types in CNS are genetically coded. The recent identification of diverse chromatin regulatory marks and the enzymes producing those marks allows us to integrate the potentially vital action of chromatin regulation with the activity of TFs in CNS development. However, this effort is riddled with a few challenges. 1) The developing CNS consists of profoundly heterogeneous cells at different developmental states, making it difficult to study how chromatin changes are orchestrated in each cell type. 2) While cell type-specific regulatory elements (herein referred to as cis-elements) are predicted to undergo the most functionally critical chromatin changes throughout development, such cis-elements are globally ill-defined. 3) Although multiple chromatin regulatory factors would be mobilized to cell type-specific cis-elements throughout development of each cell type, it is tough to identify the specific chromatin factors in action. Building on our prior effort to solve these limitations, the current study of the gene regulatory networks for spinal motor neuron (MN) development is a pioneering study in addressing the critical issue of chromatin regulation in CNS development. Our findings in the past funding cycles identified transcription codes and gene regulatory elements that underlie commitment and specification of MN fate, establishing an ideal cellular model to investigate chromatin regulation in CNS development. In the developing spinal cord, Olig2, a basic helix-loop-helix (bHLH) TF expressed in progenitors for MNs (pMNs), plays essential roles in establishing the pMN domain and keeping pMN cells from prematurely differentiating to MNs. As pMN cells begin to differentiate to MNs, the LIM homeodomain (HD) TFs Isl1 and Lhx3 are upregulated, along with the bHLH TF Ngn2. Isl1 and Lhx3 form a complex (Isl1-Lhx3), which directs MN fate via synergistic transactivation of MN genes with Ngn2. In this proposal to integrate these genetic programs with chromatin regulation, we hypothesize that during MN development, coordinated actions of the cell type-specific TFs (Olig2, Ngn2 and Isl1-Lhx3) and the chromatin modifiers (Ezh2, Jmjd3 and CBP/p300) orchestrate the chromatin changes in MN genes from transcriptionally poised/repressive to active state, enabling the timely acquisition of MN fate and cell differentiation. Based on our preliminary data, we specifically postulate that, in pMN cells, Olig2 recruits Ezh2 to MN genes, instituting the transcriptionally repressive chromatin mark, trimethylated histone H3-lysine 27 (H3K27me3). As Olig2 expression declines in differentiating pMN cells, Isl1-Lhx3 and Ngn2 seize MN-specific enhancers and recruit Jmjd3, which removes H3K27me3 and allows CBP/p300 to set up acetylated H3K27 (H3K27ac), a transcriptionally active chromatin mark. We will test our hypothesis using an ensemble of cellular, biochemical, genetic and genome-wide approaches.