Molecular Analysis of Hindbrain Development

描述（由申请人提供）：将脊椎动物胚胎后脑分割成菱脑，确保后脑衍生物的正确定位，对正常的神经活动至关重要。扁状体的形成是由hox基因调控的，hox基因的表达是精心协调的，以确保转录本的正确时间和空间分布。hox的表达受视黄酸（RA）介导的信号和hox基因相互调节表达的组合调控。我们的初步数据显示，在响应RA的hox启动子处形成核小体耗尽区（NDR）。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“活性”组蛋白修饰和RNA聚合酶II的募集相关。我们发现这些因素不足以驱动转录-相反，Hox蛋白似乎是转录起始所必需的。基于这些数据，我们假设hox的表达激活是通过以下途径实现的：1)RA在hox启动子处诱导NDR， 2) Prep和Pbx辅因子在NDR处结合，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。为了验证这一假设，我们制定了两个目标：1)确定在Hox基因转录起始过程中辅因子和Hox蛋白的顺序作用。在这个目标中，我们将建立hox基因表达所需的每个因子的作用顺序和具体作用。2)定义核小体缺失区域在后脑hox基因表达激活中的作用。在这个目标中，我们将确定ndr是如何形成的，以及它们在控制hox基因表达中起什么作用。我们的工作将通过探索胚胎神经系统如何形成来影响人类健康。人类hox突变导致脑干发育中断，并导致自闭症等认知缺陷，这一事实强调了其重要性。由于我们的工作将提高我们对神经基因表达如何在未分化细胞中启动的理解，我们的结果也将为旨在推动前体细胞（如ES和iPS细胞）向基础和临床应用的神经命运分化的研究提供信息。

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.