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

DNA replication timing (RT) is correlated with transcription during cell fate changes but there are many exceptions, underscoring a need for reductionist approaches. Here, we manipulated length and strength of transcription at a single site upstream of the silent, late-replicating, Pleiotrophin (Ptn) gene in mouse embryonic stem cells (mESCs). Reporter genes driven by two of four promoters elicited an RT advance while all four promoters advanced RT when driving the endogenous Ptn gene. Inducible transcription of the Ptn gene, but not the reporter gene, elicited a rapid and reversible RT advance. Deletion of the Ptn promoter and enhancer, followed by differentiation to neural precursors, eliminated transcription without attenuating the natural Ptn domain RT advance. Together with new genome-wide analyses, our results provide a solid empirical base with which to re-evaluate many decades of seemingly contradictory literature. We also identify vectors that do not disturb RT and provide a robust system to induce RT changes, permitting mechanistic studies of transcription's role in RT and the consequences of RT changes to epigenomic remodeling. Overall design: To investigate the relationship between RT regulation and transcription, we focused on mouse Pleiotrophin (Ptn) gene locus the replication timing and transcription of which are developmentally regulatged; switching from late RT and transcription "OFF" in embryonic stem cell to early RT and transcription "ON" in NPC. We inserted various promoters in one allele on the same locus in F121-9 hybrid embryonic stem cell expressing the tetracycline transactivator from the Rosa26 locus to see how transcription induction and replication timing differ between the WT allele and manipulated allele. In addition, to investigate position effect, we inserted hPGK promoter-eGFP casette to multiple loci using PiggyBac as described in PMC7073462. We also deleted Ptn gene transcription start site in another mESC line 46C and induced the differentiation to NPC to observed how the depletion of Ptn gene transcription affects RT in NPC.