Role of TGF-? Signaling in Embryonic Stem Cell Maintenance and Endodermal Differe

描述（由申请人提供）：TGF-2信号传导指导多种细胞反应，包括分化、增殖和细胞周期阻滞，并通过这些反应在人类发育和疾病中发挥核心作用。TGF-2受体的激活导致转录因子Smad2和Smad3的磷酸化（Smad2/3）。一旦被磷酸化，Smad2/3就会聚集在细胞核中，在那里它们必须与其他转录因子相互作用，才能结合DNA并调节转录。Smad2/3的激活如何指导这些不同反应的机制尚不清楚。据报道，特定的转录因子与Smad2/3相互作用，但这些转录因子存在于许多不同的细胞类型中，仅在少数基因上与Smad2/3相互作用。我们的初步数据为理解TGF-2信号的不同作用提供了线索。我们发现谱系特异性转录因子可能决定了Smad2/3调控哪些基因。TGF-2信号是维持人类胚胎干细胞所必需的，也是指导胚胎干细胞向内胚层分化所必需的，内胚层将产生肝脏、肠道和胰腺细胞。胚胎干细胞可以分化为肝细胞，但产量低和污染细胞是培养用于人类治疗的肝细胞需要克服的主要障碍。该项目的目标是了解TGF-2信号如何指导内胚层分化，并利用这一知识提高我们培养用于再生医学的肝细胞的能力。为了实现这些目标，该项目将1)确定人类胚胎干细胞中与Smad2/3共占据DNA位点的关键转录因子，2)确定内胚层分化过程中与Smad2/3共占据DNA位点的关键转录因子，以及内胚层分化过程中需要的关键转录因子，3)确定哪些因子直接与Smad2/3相互作用，募集其在胚胎干细胞和内胚层分化过程中结合DNA。鉴定确定发育细胞对TGF-2信号如何反应的特定蛋白-蛋白相互作用，将为调节发育过程中的TGF-2信号提供潜在靶点，并提高产生用于治疗的肝细胞和胰腺细胞的能力。这项研究将由Mullen博士在Whitehead研究所Richard Young博士的实验室进行，该研究所是干细胞生物学、转录和基因组学领域的领导者。他获得了免疫学博士学位和胃肠病学临床培训。他基于分子遗传学和细胞生物学的背景以及对肝脏疾病的临床兴趣，提出了一项应用基因组学和系统生物学来理解TGF-2信号在胚胎干细胞维持和内胚层分化中的作用的建议。怀特黑德研究所是进行这项研究的理想环境，因为它有合作的氛围、设施、教育机会和培养成功医生科学家的经验。Mullen博士还将与他在麻省总医院的共同导师Ramnik Xavier博士密切合作，Ramnik Xavier博士在基因组学和系统生物学在信号通路中的应用方面具有专业知识。此外，他还成立了一个咨询委员会，以支持他的研究和他的发展成为一个独立的调查员。委员会成员是干细胞生物学、发育和信号转导方面的专家，并具有指导内科科学家的丰富经验。！

DESCRIPTION (provided by applicant): TGF-2 signaling directs diverse cellular responses including differentiation, proliferation, and cell cycle arrest and through these responses plays a central role human development and disease. Activation of the TGF-2 receptor leads to phosphorylation of the transcription factors, Smad2 and Smad3 (Smad2/3). Once phosphorylated, Smad2/3 accumulates in the nucleus where they must interact with other transcription factors in order to bind DNA and regulate transcription. The mechanism by which activation of Smad2/3 directs such diverse responses remains unclear. Specific transcription factors have been reported to interact with Smad2/3 but these transcription factors are present in many different cell types and interact with Smad2/3 at only a few genes. Our preliminary data provides a clue to understand the diverse effects of TGF-2 signaling. We found that lineage-specific transcription factors may determine which genes Smad2/3 regulate. TGF-2 signaling is required to maintain human embryonic stem cells and is also required to direct embryonic stem cells to differentiate into endoderm, which will give rise to the cells of the liver, gut and pancreas. ES cells can be differentiated into hepatocytes, but low yields and contaminating cells are major hurdles to overcome in growing hepatocytes for human therapy. The goal of this project is to understand how TGF-2 signaling can direct endodermal differentiation and use this knowledge to improve our ability to grow hepatocytes for use in regenerative medicine. To achieve these goals the project will 1) identify the key transcription factors that co- occupy DNA sites with Smad2/3 in human ES cells, 2) identify key transcription factors that co-occupy DNA sites with Smad2/3 during endodermal differentiation and are required for endodermal differentiation, and 3) determine which factors directly interact with Smad2/3 to recruit them to bind DNA in embryonic stem cells and during endodermal differentiation. Identification of specific protein-protein interactions that determine how developing cells respond to TGF-2 signaling would provide potential targets to modulate TGF-2 signaling in during development and improve the ability to produce hepatocytes and pancreatic cells for therapy. This research will be performed by Dr. Mullen in the laboratory of Dr. Richard Young at the Whitehead Institute, a leader in the fields of stem cell biology, transcription and genomics. Dr. Mullen received his Ph.D. training in immunology and clinical training in gastroenterology. He is building on a background in molecular genetics and cell biology and a clinical interest in liver disease to develop a proposal to apply genomics and systems biology to understand the role of TGF-2 signaling in embryonic stem cells maintenance and endodermal differentiation. The Whitehead Institute is the ideal environment in which to perform this research given the collaborative atmosphere, facilities, educational opportunities and experience training successful physician scientist. Dr. Mullen will also work closely with his co-mentor at Massachusetts General Hospital, Dr. Ramnik Xavier who has expertise in genomics and application of systems biology to signaling pathways. In addition, Dr. Mullen has formed an advisory committee to support his research and his development into an independent investigator. The committee members are experts in stem cell biology, development and signal transduction and have extensive experience mentoring physician scientists. ! PUBLIC HEALTH RELEVANCE: TGF-2 signaling plays an essential role in human development and in many human diseases (1-4). The goal of this project is to learn how the TGF-2 signaling pathway can be required to regulate pluripotency in human embryonic stem (ES) cells yet also direct human ES cells to differentiate into endoderm, the precursor of cells of the liver and gut. Understanding which factors control this change in activity has direct application in regenerative medicine and the production of hepatocytes for therapy. !