TRANSCRIPTIONAL REGULATION BY GEMININ

描述(申请人提供)：未分化的神经前体细胞可以不限数量地从胚胎干细胞或重新编程的体细胞(IPS)细胞衍生而来，具有广泛的分化潜力，可以通过基因工程进行修饰或针对特定特性进行克隆选择，对于iPS细胞，可以与患者匹配以避免免疫不相容。因此，这些细胞对于人类神经系统疾病的体外建模和开发基于移植的治疗方法以替代或刺激受损组织的修复具有重要意义。考虑到这些细胞的广泛用途，我们对此知之甚少，令人惊讶 细胞固有的调控网络，指导神经命运的最初获得，并维持神经前体处于未分化状态。这大大限制了我们对神经发育的理解，并阻碍了对来自不同起始细胞来源或方案的神经前体细胞进行比较和操作的努力。在脊椎动物胚胎发育过程中，Ginein和Zic家族的锌指转录因子控制着细胞对神经诱导的最早的内在反应，并在初始神经规范和维持未分化状态中发挥重要作用。对这些转录因子的突变分析支持它们在发育和人类疾病中的核心作用：完全丧失Ginin会导致小鼠早期胚胎死亡，而人类的Zic1-4突变会导致一系列胚胎和神经系统畸形以及神经疾病。然而，这些转录因子发挥作用的调控网络和机制在很大程度上仍不清楚。在这里，我们将阐明转录程序和他们在全基因组水平上控制的直接靶标，并将使用这些数据来构建神经命运获取的基础调控网络。我们将通过与不同的表观遗传调控复合体的相互作用，检验双子座是神经性与非神经性命运选择的中心调节因子的假设。这项工作将定义表观遗传调控机制，这些机制指导胚胎细胞命运获得的最早方面。我们将致力于以下具体目标：1.确定Ginin激活神经基因表达以促进神经细胞命运的机制；2.定义并比较Zic转录因子和Ginin控制神经规范的转录调控网络；3.检验在胚胎细胞早期Fate获取过程中，Ginin与Polycomb协同作用抑制非神经、中内胚层基因表达的假设。总之，这项工作将定义控制初始神经命运获得的调控网络，将识别新的神经调控活动和调控模式，并将确定几个表观遗传调控因子在早期命运获得过程中集中控制细胞状态和发育潜力的机制。这些数据将填补我们对早期细胞命运获得的理解的一个根本空白。它们将告诉我们对出生缺陷的理解，并为操纵这些调控网络在许多生物环境中控制神经细胞规格提供必要的基础。

DESCRIPTION (provided by applicant): Undifferentiated neural precursor cells can be derived from embryonic stem cells or reprogrammed somatic (iPS) cells in unlimited quantities, have broad differentiation potential, can be modified by genetic engineering or clonally selected for particular properties and, for iPS cells, can be patient-matched to avoid immunological incompatibilities. Therefore, these cells are of high significance for in vitro modeling of human neurological diseases and for developing transplantation-based therapies to replace or stimulate repair of damaged tissue. Given the broad utility of these cells, we know surprisingly little about cell intrinsic regulatory networks that direct the initial acquisition of a neural fate and that maintain neural precursors in an undifferentiated state. This significantly limits our understanding of neural development and hampers efforts to compare and manipulate neural precursor cells derived from different starting cell sources or protocols. During vertebrate embryogenesis, Geminin and the Zic family of zinc finger transcription factors control the earliest cell intrinsic responses to neural induction and have essential roles in initial neural specification and maintenance of the undifferentiated state. Mutational analyses of these transcription factors supports their central roles in development and contribution to human disease: complete loss of Geminin results in early embryonic lethality in mice, while Zic1-4 mutations in humans cause a range of embryonic and nervous system malformations and neurological disorders. However, regulatory networks and mechanisms through which these transcription factors act remain largely unknown. Here, we will elucidate transcriptional programs and direct targets that they control at a genome-wide level and will use these data to construct regulatory networks underlying neural fate acquisition. We will test the hypothesis that Geminin is a central regulator of neural versus non-neural fate choice, through interactions with distinct epigenetic regulatory complexes. This work will define epigenetic regulatory mechanisms that direct the earliest aspects of embryonic cell fate acquisition. We will pursue the following Specific Aims: 1. Determine mechanisms by which Geminin activates neural gene expression to promote neural cell fate, 2. Define and compare the transcriptional regulatory networks through which the Zic transcription factors and Geminin control neural specification, and 3. Test the hypothesis that Geminin acts cooperatively with Polycomb to repress non-neural, mesendodermal gene expression during early fate acquisition of embryonic cells. Together, this work will define the regulatory networks controlling initial neural fate acquisition will identify new neural regulatory activities and modes of regulation, and will determine mechanisms through which several epigenetic regulators centrally control cell state and developmental potential during early fate acquisition. These data will fill a fundamental gap in our understanding of early cell fate acquisition. They will inform our understanding of birth defects and provide an essential foundation for manipulating these regulatory networks to control neural cell specification in many biological contexts.