Gene regulatory networks in spinal cord development

摘要 过去对外在信号和内在转录因子(TF)的研究大大增强了我们的 了解中枢神经系统中不同神经细胞类型的命运指定和分化是如何 基因编码的。不同染色质调控标记及其产酶的最新鉴定 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在一起 中枢神经系统的发展。然而，这一努力充满了一些挑战。1)正在开发的CNS包括 处于不同发育状态的细胞严重不同，这使得研究染色质是如何 在每种单元格类型中都编排了更改。2)虽然细胞类型特定的调节元件(这里指的是 作为顺式元件)被预测在整个过程中经历最关键的染色质变化 在发展方面，这类独联体要素在全球范围内定义不清。3)尽管多种染色质调节因子 将被动员到细胞类型特定的顺式元件，在每种细胞类型的发育过程中，很难 确定起作用的特定染色质因子。在我们解决这些限制的先前努力的基础上，目前 脊髓运动神经元(MN)发育的基因调控网络研究是 解决中枢神经系统发育中染色质调节的关键问题。 我们在过去的资金周期中的发现确定了转录密码和基因调控元件 为MN命运的承诺和规范奠定基础，建立研究染色质的理想细胞模型 中枢神经系统发育的调控。在发育中的脊髓中，寡核苷酸是一种基本的螺旋-环-螺旋(BHLH)转铁蛋白 在MN祖细胞(PMN)中表达，在PMN结构域的建立和保存中起着至关重要的作用 PMN细胞从早期分化为MNS。随着PMN细胞开始分化为MN，LIM 同源域(HD)转录因子Isl1和Lhx3以及bHLHTF Ngn2上调。Isl1和Lhx3形成一个 复合体(Isl1-Lhx3)，通过与Ngn2协同反式激活MN基因来指导MN的命运。在这 将这些遗传程序与染色质调节相结合的建议，我们假设在MN期间 细胞类型特异的转录因子(Orig2、Ngn2和Isl1-Lhx3)的发育、协调作用和 染色质修饰物(Ezh2、Jmjd3和CBP/p300)协调来自 转录稳定/抑制到活跃状态，使MN命运和细胞能够及时获得 差异化。根据我们的初步数据，我们特别假设，在PMN细胞中，寡核苷酸2招募了Ezh2 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)。随着中性粒细胞分化过程中寡核苷酸表达的下降，Isl1-Lhx3和Ngn2夺取MN特异性 增强剂和招募Jmjd3，它去除H3K27me3并允许CBP/p300建立乙酰化的H3K27 (H3K27ac)，一个转录活性的染色质标记。我们将使用一个细胞集合来验证我们的假设， 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.