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

Abstract

DNA replication timing (RT) is correlated with transcription during cell fate changes but there are many exceptions and our understanding of this relationship suffers from a paucity of reductionist approaches. Here, we manipulated length and strength of transcription in hybrid-genome mouse embryonic stem cells (mESCs) at a single locus upstream of the silent, late replicating, Pleiotrophin (Ptn) gene, directly comparing RT to nascent transcription rates at engineered vs. wild-type alleles. First, we inserted four reporter genes that differ only in their promoter. Two promoters transcribed the reporter gene at high rates and advanced RT. The other two transcribed at lower rates and did not advance RT. Since these promoters may prove useful in applications where effects on RT are undesirable, we confirmed the inability of one of them to advance RT at numerous ectopic sites. We next juxtaposed these same four promoters upstream of the Ptn transcription start site where they all transcribed the 96kb Ptn gene and advanced RT to different extents correlated with transcription rates. Indeed, a doxycycline-responsive promoter, which could not advance RT when induced as a small reporter gene, elicited a rapid and reversible RT advance proportional to the rate of transcription, providing direct evidence that transcription itself can advance RT. However, deletion of the Ptn promoter and enhancer, followed by directed differentiation to neural precursors, eliminated induction of transcription throughout the entire Ptn replication domain, without preventing the switch to early replication. Our results provide a solid empirical base with which to re-evaluate many decades of literature, demonstrating that length and strength of transcription is sufficient but not necessary to advance RT. Our results also provide a robust system in which to rapidly effect an RT change, permitting mechanistic studies of the role of transcription in RT and the consequences of RT changes to epigenomic remodeling.