Transcription elongation can be sufficient, but is not necessary, to advance replication timing

Abstract DNA replication timing (RT) often correlates with transcription during cell fate transitions, yet notable exceptions indicate a complex relationship. Using a reductionist system in mouse embryonic stem cells, we manipulate transcriptional length and strength at a single locus upstream of the silent, late-replicating Pleiotrophin (Ptn) gene. Small reporter genes driven by two of four promoters advance RT, whereas all promoters advance RT when driving the 96-kb endogenous Ptn gene. Inducible transcription of Ptn, but not the reporter, triggers a rapid and reversible RT advance, providing a system to manipulate RT independent of differentiation. Strikingly, deletion of the Ptn promoter and enhancers abolishes transcription yet does not prevent the developmental RT switch to early replication during neural differentiation. These findings, supported by parallel genome-wide analyses during differentiation, demonstrate that transcriptional elongation can causally advance RT in a rate-dependent and context-specific manner, but that transcription is neither necessary nor sufficient for RT advancement. Our results provide a solid empirical base with which to re-evaluate decades of seemingly contradictory literature.