Transcriptional Regulation of the Intestinal Epithelium

描述(由申请人提供)： 候选人和导师的选择：这份K01申请的目的是继续我在胃肠道发育和疾病转录机制方面的培训，长期目标是追求并有望治愈胃肠道紊乱和疾病的分子病因。我对转录机制的兴趣始于大学，在加州大学旧金山分校布莱恩·布莱克博士的研究生培训期间成为我的重点，在那里我研究了MEF2C的转录调控，这是一种在心血管发育中具有重要功能的基因。这些研究对个体基因是如何调控的产生了重要的见解。对于我的博士后工作，我感兴趣的是扩大这些研究，不仅要了解一个基因是如何受到调控的，而且要利用新技术来揭示整个基因调控计划。我寻找了一位研究胃肠功能分子机制的导师，他在培训年轻科学家方面有着出色的记录，拥有转录调控方面的专业知识。Ramesh Shivdasani和他的实验室将提供一个出色的培训环境，以探索控制肠道上皮功能的转录调控机制。 项目：肠道经历持续的细胞周转，因为细胞从干细胞和祖细胞分化为分化的肠道细胞，这些细胞迅速执行它们的功能，然后被排入肠腔。祖细胞和分化细胞有非常不同的基因表达模式，但控制这些表达模式的因素尚未完全描述。同源结构域转录因子CDX2在非肠道组织中主要诱导肠型上皮细胞，具有肠道主要调节因子的特性。由于CDX2在祖细胞和分化细胞中都有表达并具有已知的靶基因，因此它被认为在分化过程中发挥了调节基因表达变化的作用。然而，尽管CDX2具有如此深刻的功能，但CDX2作用的分子细节仍未完全确定--特别是在分化过程中。 我的建议应用最新的技术进步(全基因组染色质免疫沉淀)来全面描述肠道祖细胞及其分化的后代中CDX2功能的分子细节。首先，在祖细胞和分化细胞的基因组中定位CDX2结合位点，以确定结合位点是否随着细胞状态的变化而变化。这项工作将使我们能够定义CDX2如何在分化过程中介导基因表达的变化。其次，我们有初步证据表明CDX2分别与祖细胞和分化细胞中的特定转录因子GATA6和HNF4()协同作用。CDX2是否与这些转录因子合作，以及这些多因子复合体是如何在DNA上组装的，将被确定。最后，将产生一个适当的遗传模型来确定成人肠道对CDX2的需求。将分析CDX2的可诱导基因敲除，以检验该因子在体内平衡的成人肠道中是肠道分化所必需的假说。总之，这些实验将为一种重要的转录因子如何调节肠道分化提供分子见解。这项工作有望为作为一名独立科学家的未来研究开辟道路，我希望在那里定义容易受到侮辱的基因程序，这些侮辱会导致肠道疾病和癌症。 培训环境。达纳·法伯癌症研究所和哈佛医学院附属医院共同提供了大量资源来执行这一培训计划。已经建立了几个指导计划，帮助后期博士后研究员转变为独立科学家。除了完全使用所有必要的最先进的研究设备和设施外，哈佛医学界将成为一项资产，为我提供接触优秀教员的机会，我已经从那里组建了一个由胃肠生物学和转录机制专家组成的指导委员会，该委员会将监督我的培训进度，并指导我追求独立研究计划。再加上强大的导师、有根有据的研究计划和培训计划，我希望这项提议将为研究胃肠道疾病的分子基础的职业生涯提供一个良好的开端。 公共卫生相关性： 肠上皮是一层单细胞厚的层，允许营养物质，同时抵抗肠腔内的毒素和病原体。这一关键的上皮层容易感染炎症性疾病和癌症，因此必须了解构建正常功能的分子框架。拟议的培训拨款将专注于发展职业生涯，致力于研究肠上皮细胞维持的潜在转录机制。

DESCRIPTION (provided by applicant): Candidate and choice of Mentor: The purpose of this K01 application is to continue my training in transcriptional mechanisms of gastrointestinal development and disease with the long-term goal of a career pursuing and hopefully curing the molecular etiologies of GI disorders and diseases. My interest in transcriptional mechanisms began in college and became my focus during graduate training with Dr. Brian Black at UCSF where I studied the transcriptional regulation of MEF2C, a gene with important functions in cardiovascular development. These studies yielded important insights on how an individual gene is regulated. For my postdoctoral work I was interested in expanding these studies to understand not how only one gene was regulated, but to employ new technologies to uncover entire gene regulatory programs. I sought a mentor pursuing the molecular mechanisms of gastrointestinal function with a fantastic track-record in training young scientists with expertise in transcriptional regulation. Ramesh Shivdasani and his laboratory will provide an outstanding training environment to pursue the transcriptional regulatory mechanisms controlling intestinal epithelial functions. Project: The intestine undergoes continual cell turnover as cells differentiate from stem and progenitor cells to differentiated intestinal cells that quickly carry out their functions and then are shed into the lumen. Progenitor and differentiated cells have very different gene expression patterns but the factors controlling these expression patterns are incompletely described. The homeodomain transcription factor CDX2 has the properties of a master regulator of the intestine in that it can dominantly induce intestine-type epithelium in non-intestinal tissues. Because CDX2 is expressed and has known target genes in both progenitor and differentiated cells, it is believed to play a role in mediating gene expression changes that occur during differentiation. Yet despite such profound functions, the molecular details of CDX2 actions are incompletely defined - particularly during the course of differentiation. My proposal applies recent advances in technology (whole genome chromatin immunoprecipitation) to comprehensively describe the molecular details of CDX2 function in both intestinal progenitor cells and their differentiated descendants. Firstly, CDX2 binding sites will be mapped in the genomes of progenitor cells and differentiated cells to determine whether the binding sites change concurrent with cellular state. This work will allow us to define how CDX2 can mediate gene expression changes during differentiation. Secondly, we have preliminary evidence to suggest CDX2 differentially cooperates with specific transcription factors in progenitor and differentiated cells, GATA6 and HNF4(, respectively. Whether CDX2 partners with these transcription factors and how these multi-factor complexes are assembled on the DNA will be determined. Finally, a proper genetic model to determine the requirement for CDX2 in the adult intestine will be generated. An inducible knockout of CDX2 will be analyzed to test the hypothesis that this factor is required for intestinal differentiation in the homeostatic adult intestine. Together, these experiments will provide molecular insights into how an important transcription factor mediates intestinal differentiation. This work will hopefully open avenues for future studies as an independent scientist where I hope to define gene programs susceptible to insults that result in intestinal disorders and cancers. Training Environment. The Dana Farber Cancer Institute and the affiliated hospitals of Harvard Medical School together provide tremendous resources to execute this training plan. Several instructional programs have been established to help transition late-stage postdoctoral fellows into independent scientists. In addition to full access to all necessary state-of-the-art research equipment and facilities, the Harvard Medical Community will be an asset by providing access to outstanding faculty from which I have assembled a mentoring committee comprised of experts in gastrointestinal biology and transcriptional mechanisms that will oversee my training progress and council me in my pursuit of an independent research program. Together with a strong mentor, well-founded research program and training plan, I hope this proposal will provide a strong beginning to a career investigating the molecular underpinnings of GI disease. PUBLIC HEALTH RELEVANCE: The intestinal epithelium is a one-cell-thick layer that is permissive to nutrients while resistant to toxins and pathogens within the intestinal lumen. This critical epithelial layer is susceptible to inflammatory diseases and cancer making it imperative to understand the molecular framework upon which normal functions are built. The proposed training grant will focus on developing a career pursuing the underlying transcriptional mechanisms of intestinal epithelial cell maintenance.