Mechanisms governing context-dependent Wnt activity in C. elegans embryogenesis

秀丽隐杆线虫胚胎发生中背景依赖性 Wnt 活性的控制机制

• 批准号: 8766240
• 负责人: Amanda Lucille Zacharias
• 金额: $ 7.32万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8766240
• 关键词: Adopted; Affect; Award; Beryllium; Binding; Binding Sites; Bioinformatics; Biological Assay; Biological Models; Caenorhabditis elegans; Cells; ChIP-seq; Code; DNA; Data; Defect; Development; Developmental Gene; Disease; Embryo; Embryonic Development; Enhancers; Environment; Exhibits; Faculty; Functional RNA; Future; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Genome engineering; Grant; Homeostasis; Human; Human Development; Image; Knowledge; Learning; Life; Logic; Malignant Neoplasms; Measures; Memory; Mentors; Mentorship; Mutation; Outcomes Research; Parents; Pathway interactions; Pattern; Pennsylvania; Phase; Play; Positioning Attribute; Postdoctoral Fellow; Process; Property; Regulation; Regulator Genes; Regulatory Element; Reporter; Reporter Genes; Research; Research Personnel; Resolution; Role; Signal Pathway; Signal Transduction; Signaling Pathway Gene; Site; Stem cells; System; TCF Transcription Factor; Techniques; Testing; Thinking; Time; Training; Transgenes; Transgenic Organisms; Universities; Vertebrates; Work; Writing; Yeasts; career; career development; combinatorial; design; developmental genetics; extracellular; human disease; improved; in vivo; medical schools; member; novel; phase 1 study; programs; public health relevance; response; transcription factor

DESCRIPTION (provided by applicant): A small number of extracellular signaling pathways control a diverse array of processes during development and homeostasis by altering transcription in target cells. An important question in the regulation of genes by signaling pathways is how the same signal can activate different targets in different cells. The Wnt signaling pathway and its role in development are conserved among all metazoans and disruption of the pathway causes developmental defects and disease in humans, including cancer. I propose to uncover the fundamental rules that determine which genes are regulated by the Wnt pathway in different embryonic contexts by analyzing the enhancers of Wnt target genes and investigating the expression of synthetic enhancers. I will leverage synthetic enhancer strategies pioneered in yeast to systematically study enhancer function for the targets of the Wnt pathway over the course of developmental time in C. elegans embryos. To do this, I will use a powerful automated lineage tracing system that measures dynamic expression patterns quantitatively in a live developing embryo; because C. elegans has an invariant lineage, I can directly compare expression between reporters on a cellular level and identify cell fates without the need for separate markers. In preliminary work, I identified sixteen transcription factors that are targets of the Wnt pathway and found that contrary to previous thinking, some targets required the Wnt effector transcription factor, TCF, only to repress expression in unsignaled cells, while others required TCF only to activate expression in signaled cells. Furthermore, I found that the response to Wnt signaling is not uniform throughout the embryo; instead, cells with parents who received a Wnt signal exhibit a stronger response to a Wnt signal than cells with unsignaled parents. This indicates the cells possess transmitotic memory of Wnt signaling, a novel finding. In the mentored phase of this study, I will characterize enhancers that are direct targets of Wnt signaling and determine how TCF and context transcription factors contribute to the regulation of Wnt target genes. In the first aim, I will identify sites bound by TCF during embryogenesis and evaluate the ability of TCF binding sites to drive expression in synthetic enhancers. In the second aim, I will characterize the enhancers of Wnt target genes, identify the context factors that co-regulate them, and use synthetic enhancers to determine how context factors sites combine with TCF binding sites to regulate expression. The data I gather and techniques I learn in the mentored phase will help me be successful in undertaking the third aim in the independent phase. For this third aim, I will use synthetic enhancers to identify the properties that are essential for Wnt targets, including the numbers, strength, orientation, and spacing of TCF and context factor binding sites. I will then predict and test the effect of targeted mutations on endogenous target enhancers and generate synthetic enhancers with novel expression patterns to demonstrate understanding of the underlying rules. This research will significantly advance our ability to identify Wnt target genes and predict their expression by sequence alone. Due to evolutionary conservation, some of the rules that govern Wnt regulation in C. elegans will likely be universal principles that also govern regulation of this and other important pathways in vertebrates. During the initial phase of this award, I will receive mentoring from my primary mentor, Dr. John Murray, my co-mentor, Dr. Klaus Kaestner, and two additional faculty members with relevant expertise. This mentorship committee will assist me with learning new techniques, including genome engineering, ChIP-seq, and bioinformatic enhancer identification that will be essential in my future career as an independent investigator in developmental genetics. My training will also include coursework, grant writing, and a mentored faculty position search to allow me to transition to an independent career. The proposed training will take place at the Perelman School of Medicine at the University of Pennsylvania, which possesses outstanding facilities, an excellent collaborative environment with many opportunities for postdoctoral fellows to present their research, and an office of Biomedical Postdoctoral Programs that organizes training in career development and responsible conduct in research.

描述（由申请人提供）：少数细胞外信号传导途径通过改变靶细胞中的转录来控制发育和稳态期间的各种过程。通过信号通路调节基因的一个重要问题是，相同的信号如何激活不同细胞中的不同靶点。Wnt信号通路及其在发育中的作用在所有后生动物中是保守的，并且该通路的破坏会导致人类的发育缺陷和疾病，包括癌症。我建议通过分析Wnt靶基因的增强子和研究合成增强子的表达来揭示决定哪些基因在不同胚胎环境中受Wnt通路调控的基本规则。我将利用酵母中开创的合成增强子策略，系统地研究在C.线虫胚胎为了做到这一点，我将使用一个强大的自动谱系追踪系统，该系统定量地测量活的发育胚胎中的动态表达模式;由于线虫具有不变的谱系，我可以直接在细胞水平上比较报告基因之间的表达，并确定细胞命运，而不需要单独的标记物。 在前期工作中，我鉴定了16种Wnt通路的靶点转录因子，并发现与以前的想法相反，一些靶点只需要Wnt效应转录因子TCF来抑制无信号细胞中的表达，而另一些靶点只需要TCF来激活有信号细胞中的表达。此外，我发现对Wnt信号的反应在整个胚胎中并不一致;相反，父母接受Wnt信号的细胞对Wnt信号的反应比父母没有信号的细胞更强。这表明细胞具有Wnt信号传导记忆，这是一个新的发现。 在本研究的指导阶段，我将描述增强子是Wnt信号的直接靶点，并确定TCF和上下文转录因子如何有助于Wnt靶基因的调节。在第一个目标中，我 将鉴定胚胎发生过程中TCF结合的位点，并评估TCF结合位点驱动合成增强子表达的能力。在第二个目标中，我将描述Wnt靶基因的增强子，鉴定协同调节它们的背景因子，并使用合成增强子来确定背景因子位点如何与TCF结合位点联合收割机结合来调节表达。我在辅导阶段收集的数据和学到的技术将帮助我在独立阶段成功地实现第三个目标。对于第三个目标，我将使用合成增强子来识别Wnt靶标所必需的特性，包括TCF和上下文因子结合位点的数量、强度、方向和间距。然后，我将预测和测试靶向突变对内源性靶增强子的影响，并产生具有新表达模式的合成增强子，以证明对潜在规则的理解。 这项研究将显著提高我们识别Wnt靶基因并仅通过序列预测其表达的能力。由于进化上的保守性，C.优雅的原则很可能是普遍的原则， 控制着脊椎动物中这一和其他重要途径的调节。 在这个奖项的初始阶段，我将接受我的主要导师，约翰·默里博士，我的共同导师，克劳斯·凯斯特纳博士，和另外两名具有相关专业知识的教师的指导。这个指导委员会将帮助我学习新技术，包括基因组工程，ChIP-seq和生物信息学增强子识别，这对我未来作为发育遗传学独立研究者的职业生涯至关重要。我的培训还将包括课程作业，赠款写作和指导教师职位搜索，让我过渡到一个独立的职业生涯。拟议的培训将在宾夕法尼亚大学佩雷尔曼医学院进行，该学院拥有出色的设施，良好的合作环境，为博士后研究员提供许多机会来展示他们的研究，以及组织职业发展培训和负责任的研究行为的生物医学博士后项目办公室。