Dynamic mechanisms of transcriptional coactivator function in Notch signaling

项目摘要 转录因子是控制靶基因表达的蛋白质。关于转录的关键问题 因子的功能包括：它们的DNA结合如何激活转录？激活域如何相互作用 有特定的辅助因子吗活动需要哪些序列特征？作为解决这些问题的模型系统， 为了解决这些基本问题，我将使用Notch信号通路的转录输出。Notch途径 是多种组织发育所必需的，其异常活性与疾病有关， 癌在信号激活后，Notch在DNA上组装转录复合物（NTC），其由DNA 结合蛋白RBPJ、Notch胞内结构域（NICD）和MAML家族共激活剂。MAML蛋白 被认为是激活转录所需的辅助因子组装的平台，但彻底的 这些因子的鉴定、它们如何调节转录以及MAML如何与它们相互作用尚不清楚。 这项研究将联合收割机的各种实验方法，使时间分辨率的转录 响应信号传导激活的诱导和转录复合物组装。这项工作将确定共同- 使用基于APEX 2的邻近性与人类中的主要MAML蛋白MAML 1相互作用的激活剂 标记，并将询问使用新生转录组学的转录反应的动力学（目的1）。 在这个目标的独立阶段，将确定这些共激活子如何与NTC一起工作，以诱导基因表达。 表情这项研究将直接可视化MAML 1核动力学响应Notch激活， 评估Notch依赖性转录中高阶蛋白组装的作用（Aim 2）。文件还将提出 赋予转录激活活性的MAML 1的序列特征（Aim 3）。成功完成 这些目标将阐明Notch信号如何激活转录，并更广泛地提供关键的 对转录激活结构域功能的机械洞察。这一多学科的建议提供了一个 为PI提供了一个极好的培训机会，使她能够扩展自己的技术库，以研究转录 在他们的细胞环境中的因素，并加强她在基因调控的专业知识。联合辅导， 博士Stephen Blacklow，Notch信号结构机制专家，Karen Adelman博士， 转录调节机制，将为PI完成这项工作提供一个特殊的环境 并获得过渡到独立所需的技能。广泛的培训计划将使PI能够 实现了她的目标，运行一个独立的实验室，研究转录因子 激活特定的基因表达模式。参与MOSAIC UE 5计划将扩大PI的 建立网络并提供指导和领导技能，使PI能够促进学术科学的多样性。

Project Summary Transcription factors are proteins that control the expression of target genes. Key questions about transcription factor function include: how does their DNA binding activate transcription? How do activation domains interact with specific co-factors? What sequence features are required for activity? As a model system to address these fundamental questions, I will use the transcriptional output of the Notch signaling pathway. The Notch pathway is essential for development of multiple tissues and its aberrant activity is associated with disease including cancer. Upon signal activation, Notch assembles a transcription complex (NTC) on DNA, consisting of the DNA binding protein RBPJ, the Notch intracellular domains (NICD), and a MAML family co-activator. MAML proteins are thought to act as a platform for assembly of co-factors required to activate transcription, but a thorough identification of these factors, how they regulate transcription, and how MAMLs interacts with them are unknown. This study will combine a variety of experimental approaches that allow temporal resolution of transcriptional induction and transcriptional complex assembly in response to signaling activation. This work will identify co- activators that interact with the primary MAML protein in humans, MAML1, using APEX2-based proximity labeling, and will interrogate the dynamics of the transcriptional response using nascent transcriptomics (Aim 1). In the independent phase of this aim, it will determine how these co-activators work with the NTC to induce gene expression. This study will directly visualize MAML1 nuclear dynamics in response to Notch activation, and assess the role of higher order protein assemblies in Notch dependent transcription (Aim 2). It will also identify the sequence features of MAML1 that confer transcriptional activation activity (Aim 3). Successful completion of these aims will both elucidate how Notch signaling activates transcription, and more broadly provide key mechanistic insight into transcriptional activation domain function. This multidisciplinary proposal provides an excellent training opportunity for the PI, enabling her to expand her repertoire of techniques to study transcription factors in their cellular context, and strengthen her expertise in gene regulation. The combined mentorship from Dr. Stephen Blacklow, expert in structural mechanisms of Notch signaling, and Dr. Karen Adelman, leader in mechanisms of transcriptional regulation, will provide an exceptional environment for the PI to complete this work and gain the skills necessary to transition to independence. The extensive training plan will equip the PI to achieve her goal of running an independent laboratory studying mechanisms by which transcription factors activate specific gene expression patterns. Participation in the MOSAIC UE5 programs will expand the PI’s network and provide mentoring and leadership skills to empower the PI to promote diversity in academic science.