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。关于抄写的关键问题 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 有特定的辅助因素吗？活动需要哪些序列特征？作为一个模型系统来解决这些问题 基本问题，我将使用Notch信号通路的转录输出。Notch小路 对多种组织的发育是必不可少的，其异常活性与疾病有关，包括 癌症。在信号激活时，Notch在DNA上组装一个转录复合体(NTC)，由DNA组成 结合蛋白RBPJ、Notch胞内域(NICD)和MAML家族共激活因子。MAML蛋白 被认为是组装激活转录所需的辅助因子的平台，但 这些因子的鉴定，它们如何调控转录，以及MAML如何与它们相互作用尚不清楚。 这项研究将结合各种实验方法，允许转录的时间分辨 响应信号激活的诱导和转录复合体组装。这项工作将确定共同的- 与人类主要MAML蛋白MAML1相互作用的激活子，使用基于APEX2的邻近作用 标记，并将使用新生转录组学来询问转录反应的动态(目标1)。 在这个目标的独立阶段，它将确定这些共激活子如何与NTC一起诱导基因 表情。这项研究将直接可视化MAML1对Notch激活的反应，以及 评估高阶蛋白组件在Notch依赖转录中的作用(目标2)。它还将确定 赋予转录激活活性的MAML1的序列特征(目标3)。成功完成 这些目的既将阐明Notch信号如何激活转录，更广泛地提供关键 对转录激活结构域功能的机械性洞察。这项多学科计划提供了 为PI提供了极好的培训机会，使她能够扩大自己学习转录的技巧 在他们的细胞环境中的因素，并加强她在基因调控方面的专业知识。联合导师来自 Notch信号结构机制专家Stephen Blacklow博士和 转录调控机制将为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具备以下能力 实现她的目标，经营一个独立的实验室，研究转录因子 激活特定的基因表达模式。参与MASIC UE5计划将扩大PI 建立网络，并提供指导和领导技能，以增强私营部门的能力，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.