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.
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.
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