Illuminating the Molecular Mechanisms of Replication and Transcription Coordination

Project Summary/Abstract The goal of this proposal is to understand how replication and transcription are spatially and temporally coordinated at molecular scales during embryonic development. A long-standing assertion is that RNA Pol II (Pol II) is completely evicted from DNA during replication to avoid steric conflict between transcription and replication machinery. This eviction hypothesis rests largely on in vitro or population and time-averaged genomics or biochemical experiments. These either may not recapitulate endogenous contexts or are unable to capture the interactions kinetics of Pol II’s association with chromatin with adequate temporal resolution (of seconds to minutes). In fact, recent in vivo experiments suggest that Pol II is not completely evicted from chromatin but remains in proximity (~40nm) to promoters and gene bodies during replication. This relationship is facilitated by Pol II interacting with PCNA, a sliding clamp protein associated with actively replicating domains in the nucleus that grow and disappear as replication completes. These contradictory findings call into question the molecular mechanisms that govern the coordination of transcription and replication machinery. I propose to use advanced live microscopy and single molecule tracking to directly measure the interaction kinetics of transcription and replication proteins in real time in developing Drosophila embryos. This work will provide molecular scale insights on the coordination of replication and transcription in an in vivo context. Our lab has established high resolution light-sheet microscopy which enables tracking of single protein molecules within the nuclei of live Drosophila embryos. Single molecule tracking reveals how individual proteins move within the nucleus and the kinetics of protein-protein and protein-chromatin interactions. Using these approaches along with perturbations to replication and transcription, I will investigate the chromatin binding kinetics of Pol II and PCNA as transcription is activated post-replication. To further understand how transcription is reactivated post-replication I will similarly investigate the distributions and kinetics of the pioneer transcription factor Zelda that is a ubiquitous activator in Drosophila embryos. Preliminary data from our lab shows that Zelda is excluded from domains of active replication marked by PCNA yet is detected near these sites. I hypothesize that i) Pol II is retained within actively replicating domains to avoid complete eviction from chromatin to enable rapid re-engagement and that ii) Zelda swiftly transitions from its excluded state post-replication to engage at these sites to facilitate transcriptional activation.