Dynamic mechanisms of transcriptional coactivator function in Notch signaling

About This Grant

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.