Transcriptional Regulation of the Intestinal Epithelium

描述（由申请人提供）： 候选人和导师的选择：本次 K01 申请的目的是继续我在胃肠道发育和疾病的转录机制方面的培训，长期目标是追求并希望治愈胃肠道疾病的分子病因学。我对转录机制的兴趣始于大学时期，并成为我在加州大学旧金山分校跟随 Brian Black 博士进行研究生培训期间的重点，在那里我研究了 MEF2C 的转录调控，MEF2C 是一种在心血管发育中具有重要功能的基因。这些研究对单个基因如何调控产生了重要的见解。在我的博士后工作中，我有兴趣扩大这些研究，不仅了解一个基因是如何受到调控的，而且采用新技术来揭示整个基因调控程序。我寻找一位研究胃肠功能分子机制的导师，他在培训具有转录调控专业知识的年轻科学家方面有着出色的记录。 Ramesh Shivdasani 和他的实验室将为研究控制肠上皮功能的转录调控机制提供出色的培训环境。 项目：肠道经历持续的细胞更替，细胞从干细胞和祖细胞分化为分化的肠细胞，这些细胞快速执行其功能，然后脱落到肠腔中。祖细胞和分化细胞具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 具有肠道主调节因子的特性，因为它可以在非肠组织中显着诱导肠型上皮。由于 CDX2 在祖细胞和分化细胞中表达并具有已知的靶基因，因此人们相信它在介导分化过程中发生的基因表达变化中发挥作用。然而，尽管具有如此深远的功能，CDX2 作用的分子细节尚未完全确定，特别是在分化过程中。 我的建议应用最新的技术进展（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后代中 CDX2 功能的分子细节。首先，将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与细胞状态同时变化。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有差异地合作。CDX2是否与这些转录因子合作以及这些多因子复合物如何在DNA上组装将被确定。最后，将生成一个适当的遗传模型来确定成体肠道中对CDX2的需求。将分析CDX2的诱导型敲除，以检验该因子是成体肠道分化所必需的假设。总之，这些实验将为重要转录因子如何介导肠道分化提供分子见解，作为一名独立科学家，我希望能够定义易受损伤的基因程序，从而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。已经建立了一些教学计划来帮助后期博士后研究员转变为独立科学家。除了能够充分使用所有必要的最先进的研究设备和设施外，哈佛医学界还将成为一项资产，因为它可以提供优秀的教师，我从这些教师中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，并为我追求独立研究计划提供建议。加上强大的导师、基础良好的研究计划和培训计划，我希望该提案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受到炎症性疾病和癌症的影响，因此必须了解构建正常功能的分子框架。拟议的培训补助金将重点发展探索肠上皮细胞维持的基本转录机制的职业。

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.