Determinants of cell state reprogramming PROJECT SUMMARY Forced expression of transcription factors (TFs) can reprogram cell state. The discovery of direct reprogramming has been a catalyst for our understanding of the molecular and genetic drivers of cell state. However, despite its successes, only a handful of successful reprogramming cocktails have been identified, the conversion process is often inefficient, and the mechanistic reasons for reprogramming failure are often unclear. Thus, a key obstacle to further progress is our incomplete understanding of the determinants and mechanisms of cell state conversion. Our long term goal is to understand the genetic and molecular basis of cell state, which has important implications for synthetic control of cell state for regenerative medicine. Towards this goal, the objective of this proposal is to elucidate the molecular and cellular determinants of cell state reprogramming. Specifically, we seek to address several fundamental gaps in knowledge on cell state reprogramming with three central research directions: 1) Epigenetic determinants of cellular reprogramming, 2) Genetic and temporal determinants of cellular differentiation, and 3) Initial cell state determinants on reprogramming. This scope is made possible by our innovative platform that couples multifactorial, pooled perturbation with multidimensional readouts for each cell spanning perturbations (induced TFs), mechanisms (epigenome), and phenotypes (transcriptome). By tracing thousands of individual cells on their path to reprogramming across a multitude of distinct perturbations, our high throughput functional experiments will reveal insights and suggest mechanisms for the determinants of reprogramming. Our rationale is that learning these fundamental rules of reprogramming will improve our understanding of the molecular basis of cell state and enable improved approaches to manipulate it.