Characterizing the interpretation and generation of Erk signaling dynamics using high-throughput genetics - PROJECT SUMMARY Cells reuse the same signaling pathways to initiate differing responses and distinct cell fates. This plasticity in cellular decision-making is crucial in biological contexts ranging from development to cancer biology, where identical cells may acquire distinct properties such as differentiated cell fates or drug resistance phenotypes. The Erk pathway is responsible for heterogeneous fate decisions in many biological contexts. Recent studies have uncovered two potential sources of heterogeneity in Erk signaling. First, different cells may have different Erk signaling dynamics, ranging from constant high or low signaling states to repetitive pulses of Erk kinase activity. Second, even a uniform field of Erk-active cells do not all transcribe canonical downstream target genes. Connecting single-cell behavior to a multitude of distinct cellular responses and cell fates requires an understanding of the molecular mechanisms generating pathway dynamics and interpreting pathway activity into a transcriptional response. Here, we propose to identify molecular drivers for heterogeneity in Erk-induced responses using genomic methods, and a novel Erk biosensor: the Recorder of Erk Activity Dynamics, or READer. READer selectively transcribes GFP only in response to pulses of Erk activation, allowing for rapid and high-throughput identification of cells that have both undergone an Erk pulse and initiated immediate-early gene transcription. In this proposed work, the READer system will be paired with live-cell imaging, RNA sequencing and scalable CRISPR screening technologies to 1) investigate cell states mediating Erk response and 2) dissect the molecular mechanisms responsible for Erk pulse generation and regulation. Together, these data will provide key insights into how differences in single-cells can arise, and how this heterogeneity effects signaling outcomes. Understanding the origins and consequences of heterogeneity is crucial to our understanding of development and cancer biology, as well as for the development of novel cell- based therapeutics, which require precise control of engineered cell fates for clinical applications. The proposed work will take place at Princeton University in the Department of Molecular Biology under the direct supervision of sponsor Dr. Jared Toettcher and co-sponsor Dr. Britt Adamson. The sponsors’ laboratories, the departmental community and Princeton’s overall graduate program provide an excellent supportive environment for the proposed project to succeed. Princeton University has stellar core facilities and resources available and accessible to the fellow that will enable efficient, high-quality and thorough research-training and data collection. The training plan will enable the fellow to develop skills in next-generation sequencing analysis, live-cell imaging, and synthetic biology, as well as supporting her growth and development in mentorship and scientific communication.
