Dynamic mechanisms of transcriptional coactivator function in Notch signaling

项目概要 转录因子是控制靶基因表达的蛋白质。有关转录的关键问题 因子功能包括：它们的DNA结合如何激活转录？激活域如何相互作用 与特定的辅因子？活动需要哪些序列特征？作为解决这些问题的模型系统 对于基本问题，我将使用 Notch 信号通路的转录输出。 Notch通路 对于多种组织的发育至关重要，其异常活性与疾病相关，包括 癌症。信号激活后，Notch 在 DNA 上组装转录复合物 (NTC)，该复合物由 DNA 组成 结合蛋白 RBPJ、Notch 胞内结构域 (NICD) 和 MAML 家族共激活剂。 MAML蛋白 被认为是作为激活转录所需的辅因子组装的平台，但彻底的 这些因子的识别、它们如何调节转录以及 MAML 如何与它们相互作用尚不清楚。 这项研究将结合多种实验方法，以实现转录的时间分辨率 响应信号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 APEX2 的邻近性与人类主要 MAML 蛋白 MAML1 相互作用的激活剂 标记，并将使用新生的转录组学来询问转录反应的动态（目标 1）。 在该目标的独立阶段，将确定这些共激活剂如何与NTC一起诱导基因 表达。这项研究将直接可视化响应 Notch 激活的 MAML1 核动力学，以及 评估更高阶蛋白质组装体在 Notch 依赖性转录中的作用（目标 2）。它还将识别 赋予转录激活活性的 MAML1 序列特征（目标 3）。顺利完成 这些目标将阐明 Notch 信号传导如何激活转录，并更广泛地提供关键 对转录激活域功能的机制洞察。这项多学科提案提供了 为 PI 提供了极好的培训机会，使她能够扩展研究转录的技术 因素的细胞环境，并加强她在基因调控方面的专业知识。联合指导来自 斯蒂芬·布莱克洛 (Stephen Blacklow) 博士是 Notch 信号传导结构机制方面的专家，凯伦·阿德尔曼 (Karen Adelman) 博士是 转录调控机制，将为PI提供一个特殊的环境来完成这项工作 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，即运行一个独立的实验室，研究转录因子的机制 激活特定的基因表达模式。参与 MOSAIC UE5 项目将扩大 PI 的范围 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.