Molecular Analysis of Hindbrain Development

描述(申请人提供)：将胚胎脊椎动物的后脑分割成菱形脑确保后脑衍生品的正确定位，对于正常的神经活动是必不可少的。菱形核的形成由HOX基因调控，其表达受到仔细的协调，以确保转录本的正确时间和空间分布。HOX的表达受维甲酸(RA)介导的信号转导和HOX基因相互调控的共同调控。我们的初步数据显示，在HOX启动子上形成了一个核小体耗尽区(NDR)，以响应RA。我们还发现，Prep和PBX辅助因子在转录开始之前就占据了Hox启动子，这些辅助因子的存在与组蛋白的活性修饰和RNA聚合酶II的招募有关。我们发现这一系列因子不足以驱动转录-相反，Hox蛋白似乎是启动转录所必需的。根据这些数据，我们假设HOX表达的激活是通过：1)RA诱导HOX启动子的NDR，2)Prep和PBX辅助因子结合在NDR上，3)PBX和Prep募集组蛋白修饰酶和RNA聚合酶，4)结合Hox蛋白激活转录。我们提出了两个目的来验证这一假说：1)确定辅因子和Hox蛋白在启动Hox基因转录中的顺序作用。为此，我们将确定HOX基因表达所需的每个因子的作用顺序和特定角色，2)确定核小体缺失区域在激活后脑HOX基因表达中的作用。在这一目标中，我们将确定NDR是如何形成的，以及它们在控制HOX基因表达方面发挥了什么作用。我们的工作将通过探索胚胎神经系统是如何形成的来影响人类健康。人类HOX基因突变会导致脑干发育障碍，并导致自闭症等认知缺陷，这一事实突显了这一重要性。由于我们的工作将提高我们对神经基因在未分化细胞中的表达方式的理解，我们的结果也将为旨在推动前体细胞(如ES和iPS细胞)分化为神经命运的研究提供信息，用于基础和临床应用。

DESCRIPTION (provided by applicant): Segmentation of the embryonic vertebrate hindbrain into rhombomeres ensures proper positioning of hindbrain derivatives and is essential for normal neural activity. Rhombomere formation is regulated by hox genes whose expression is carefully coordinated to ensure correct timing and spatial distribution of transcripts. hox expression is regulated by a combination of retinoic acid (RA) mediated signaling and by hox genes regulating each other's expression. Our preliminary data reveal a nucleosome-depleted region (NDR) forming at hox promoters in response to RA. We also find that Prep and Pbx cofactors occupy hox promoters well before onset of transcription and that the presence of these cofactors correlates with 'active' histone modifications and recruitment of RNA Polymerase II. We find this constellation of factors to be insufficient to drive transcription - instead, Hox proteins appear required for initiation of transcription. Based on these data we hypothesize that activation of hox expression is achieved by: 1) RA inducing an NDR at hox promoters, 2) Prep and Pbx cofactors binding at the NDR, 3) recruitment of histone-modifying enzymes and RNA Polymerase by Pbx and Prep, 4) binding of Hox proteins to activate transcription. We have developed two aims to test this hypothesis: 1) Define sequential action of cofactors and Hox proteins in initiation of hox gene transcription. In this aim we will establish te order of action and specific roles for each factor required for hox gene expression, 2) Define the role of nucleosome-depleted regions in the activation of hindbrain hox gene expression. In this aim we will determine how NDRs are formed and what role they play in control of hox gene expression. Our work will impact human health by exploring how the embryonic nervous system forms. The importance is underscored by the fact that human hox mutations produce developmental disruptions of the brainstem and cause cognitive defects such as autism. Since our work will improve our understanding of how neural gene expression is initiated in undifferentiated cells in general, our results will also inform studies aimed at driving the differentiation of precursor cells (such as ES and iPS cells) towards a neural fate for basic and clinical applications.