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

描述（由申请人提供）：TGF-2信号传导指导不同的细胞应答，包括分化、增殖和细胞周期停滞，并通过这些应答在人类发育和疾病中发挥核心作用。TGF-2受体的活化导致转录因子Smad 2和Smad 3（Smad 2/3）的磷酸化。一旦磷酸化，Smad 2/3在细胞核中积累，它们必须与其他转录因子相互作用以结合DNA并调节转录。Smad 2/3的激活指导这种不同反应的机制仍不清楚。已经报道了特定的转录因子与Smad 2/3相互作用，但是这些转录因子存在于许多不同的细胞类型中，并且仅在少数基因上与Smad 2/3相互作用。我们的初步数据为了解TGF-2信号转导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哪些基因Smad 2/3调节。TGF-2信号传导是维持人类胚胎干细胞所必需的，也是指导胚胎干细胞分化成内胚层所必需的，内胚层将产生肝、肠和胰腺的细胞。ES细胞可以分化为肝细胞，但低产量和污染细胞是人类治疗肝细胞生长中要克服的主要障碍。该项目的目标是了解TGF-2信号传导如何指导内胚层分化，并利用这些知识来提高我们培养肝细胞用于再生医学的能力。为了实现这些目标，该项目将1）鉴定在人ES细胞中与Smad 2/3共占据DNA位点的关键转录因子，2）鉴定在内胚层分化期间与Smad 2/3共占据DNA位点并且是内胚层分化所需的关键转录因子，和3）确定哪些因子直接与Smad 2/3相互作用以募集它们在胚胎干细胞中和在内胚层分化期间结合DNA。确定发育细胞如何响应TGF-2信号传导的特定蛋白质-蛋白质相互作用的鉴定将提供在发育期间调节TGF-2信号传导的潜在靶点，并提高产生肝细胞和胰腺细胞用于治疗的能力。这项研究将由马伦博士在怀特黑德研究所理查德杨博士的实验室进行，怀特黑德研究所是干细胞生物学、转录和基因组学领域的领导者。马伦博士获得了博士学位。免疫学培训和胃肠病学临床培训。他正在建立在分子遗传学和细胞生物学的背景和对肝病的临床兴趣的基础上，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。Whitehead研究所是进行这项研究的理想环境，提供合作氛围，设施，教育机会和培训成功的医生科学家的经验。马伦博士还将与他在马萨诸塞州总医院的共同导师Ramnik Xavier博士密切合作，他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长。此外，马伦博士还成立了一个咨询委员会，以支持他的研究和他成为一名独立调查员的发展。委员会成员是干细胞生物学、发育和信号转导方面的专家，并具有指导医生科学家的丰富经验。!

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. !