Mechanisms of gastrointestinal cell responses to Notch Signaling

描述（由申请人提供）：我有兴趣了解正常发育和疾病机制之间的关系。因此，作为一名研究生，我在分子生物学，生物化学和发育遗传学方面打下了坚实的基础。作为一名博士后研究员，我使用消化道作为模型系统来研究胚胎和成年小鼠干细胞更新和分化的基本发育问题。Notch信号在这些过程中起着关键作用。使用强大的小鼠遗传工具，我已经证明了Notch信号转导及其关键下游丝氨酸限制性转录因子Atoh 1在肠道干细胞和祖细胞增殖，去分化和癌症中的作用，导致了第一作者的出版物。为了进一步了解胃肠道细胞对上皮细胞中Notch信号的反应机制，我现在的目标是研究Notch信号下游的转录因子。为了系统地识别在肠细胞分化中发挥重要作用的新型转录因子，并研究潜在的转录机制，我将学习和应用染色质免疫沉淀结合高通量DNA测序（ChIP-Seq）的新的全基因组方法。我将分析分泌转录因子Atoh 1和Tcf 4的全基因组结合，并测试特定的机制假设，它们在细胞复制和肠分泌细胞系的分化中的相互作用。在胃中，不表达关键的下游因子Atoh 1，我将测试新的假设，胃限制同系物，Ascl 1，行为下游的Notch信号调节上皮细胞增殖和肠内分泌分化。因此，我提出的研究将为胃肠道上皮分化的中枢机制提供新的和重要的见解，并有助于阐明胃肠道上皮分化的分子机制。 从先天性疾病和炎症到癌症的疾病基础。我的目标是成为胃肠道生物学领域的独立首席研究员，拥有谱系分化方面的专业知识。K99/R 00奖将有助于我通过连续的指导和独立阶段过渡到完全独立。在短期内，这个奖项将使我能够学习新技术，包括ChIP-Seq和生物信息学分析，从而扩大我的专业知识，包括系统生物学和基因组学的知识，在Ramesh Shivdasani的指导下。他的实验室已经成功完成了应用这些方法的项目并发表了重要论文，提供了完美的培训环境。此外，丹娜-法伯癌症研究所和哈佛医学院的特殊机构环境将通过提供机构计划，设施，合作机会和广泛的指导，进一步使我能够向独立过渡。我的指导委员会包括世界领先的哈佛教师在胃肠道生物学，转录和基因组学领域，为发展我自己的研究计划提供指导和支持。此外，哈佛干细胞研究所和丹娜-法伯癌症研究所的功能性癌症表观遗传学中心（CFCE）不仅为我提供了完善我的方法的绝佳机会，而且还使我能够与干细胞生物学和表观基因组学领域的杰出科学家进行互动和合作。因此，K99/R 00奖将在我职业生涯的关键阶段提供关键支持，并使我能够长期致力于研究胃肠道疾病背后的独特生物学。作为一名成功的独立研究者，我将致力于了解胃肠道疾病的基本机制，领导与临床和计算生物学家的合作，并教育下一代基础和临床科学家。 公共卫生相关性：胃肠道表面通过分化成专门细胞类型的胃和肠干细胞的活动不断补充。虽然这些细胞具有发展癌症和炎症等疾病的风险，但对其分子调控的理解并不完整。K99/R 00奖的这一提议侧重于重要的器官特异性转录因子，即，这些蛋白质控制着数百个其他基因，我打算研究这些蛋白质如何在一个著名的发育信号通路Notch中发挥作用。这些研究将有助于阐明胃肠道细胞分化的分子机制，并为消化道疾病的治疗找到新的靶点。

DESCRIPTION (provided by applicant): I am interested in understanding the relationship between normal development and mechanisms of disease. I therefore built a strong foundation in molecular biology, biochemistry and developmental genetics as a graduate student. As a postdoctoral fellow, I have used the digestive tract as a model system to study fundamental developmental questions of stem cell renewal and differentiation in fetal and adult mice. Notch signaling plays critical roles in these processes. Using powerful mouse genetic tools, I have demonstrated roles for Notch signaling and its key downstream intestine-restricted transcription factor Atoh1 in gut stem and progenitor cell proliferation, dedifferentiation and cancer, leading t 4 first-author publications. To further understand mechanisms of gastrointestinal cell response to Notch signaling in epithelial cells, I now aim to investigate transcription factors that act downstream of Notch signaling. To systemically identify novel transcription factors that play important roles in intestinal cell differentiation and study the underlying transcriptional mechanisms, I will learn and apply the new genome-wide approach of chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-Seq). I will analyze genome-wide binding of the secretory transcription factors Atoh1 and Tcf4, and test specific mechanistic hypotheses regarding their interactions in cell replication and in differentiation of te intestinal secretory cell lineage. In the stomach, which does not express the crucial downstream factor Atoh1, I will test the novel hypothesis that a stomach-restricted homologue, Ascl1, acts downstream of Notch signaling to regulate epithelial cell proliferation and enteroendocrine differentiation. The studies I propose will hence provide novel and significant insights into central mechanisms of gastrointestinal epithelial differentiation and help illuminate the molecular basis of diseases ranging from congenital disorders and inflammation to cancer. I aim to become an independent principal investigator in the area of gastrointestinal biology, with expertise in lineage differentiation. A K99/R00 award will facilitate my transition to full independence through sequential mentored and independent phases. In the short-term, this award will allow me to learn new technologies including ChIP-Seq and bioinformatics analysis, hence broadening my expertise to include knowledge of systems biology and genomics, under the mentorship of Ramesh Shivdasani. His lab has successfully completed projects and published important papers that apply such approaches, providing a perfect training environment. Furthermore, the exceptional institutional environment at the Dana-Farber Cancer Institute and Harvard Medical School will further enable my transition to independence by providing institutional programs, facilities, opportunities for collaboration, and broad mentorship My mentorship committee includes world-leading Harvard faculty in the fields of gastrointestinal biology, transcription and genomics to provide guidance and support towards developing my own research program. Furthermore, the Harvard Stem Cell Institute and the Center for Functional Cancer Epigenetics (CFCE) at Dana-Farber Cancer institute will not only give me outstanding opportunities to refine my methods but will also allow me to interact and collaborate with eminent scientists in stem cell biology and epigenomics. Thus, a K99/R00 award will provide critical support at a crucial stage in my career and permit me to make a long-term commitment toward studying the unique biology that underlies gastrointestinal diseases. In the long-term future as a successful independent investigator, I will devote myself to understanding basic mechanisms of gastrointestinal diseases, leading collaborative efforts with clinical and computational biologists, and educating the next generation of basic and clinical scientists. PUBLIC HEALTH RELEVANCE: The gastrointestinal surface is continually replenished through the activity of stomach and intestinal stem cells that differentiate into specialized cell types. Although these cells harbor the risk of developing diseases such as cancer and inflammation, understanding of their molecular regulation is incomplete. This proposal for a K99/R00 award focuses on important organ-specific transcription factors, i.e., proteins that control hundreds of other genes, and I propose to study how these proteins function within a prominent developmental signaling pathway known as Notch. These studies will help define molecular mechanisms of gastrointestinal cell differentiation and identify new targets for therapy of digestive tract diseases.