GSE160262
The Asymmetric Distribution of RNAPII and Nucleosomes on Replicated Daughter Genomes is caused by Differences in Replication Timing between the Lagging and the Leading Strand [ChIP-seq]
Chromatin features are thought to have a role in the epigenetic transmission of transcription states from one cell generation to the next. It is unclear how chromatin structure survives disruptions caused by genomic replication or if chromatin features are instructive of the transcription state of the underlying gene. We developed a method to monitor budding yeast replication, transcription and chromatin maturation dynamics on each daughter genome in parallel, with which we identified clusters of secondary origins surrounding known origins. We find a difference in the timing of lagging and leading strand replication on the order of minutes at most yeast genes. We propose a model in which the majority of old histones and RNAPII bind to the gene copy that replicated first, while newly synthesized nucleosomes are assembled on the copy that replicated second. RNAPII enrichment then shifts to the sister copy that replicated second. The order of replication is largely determined by genic orientation: if transcription and replication are co-directional the leading strand replicates first; if they are counter-directional the lagging strand replicates first. A mutation in the Mcm2 subunit of the replicative helicase Mcm2-7 which impairs Mcm2 interactions with histone H3 slows down replication forks but does not qualitatively change the asymmetry in nucleosome distribution observed in the WT. Active transcription states are inherited simultaneously and independently of their underlying chromatin states through the recycling of the transcription machinery and old histones, respectively. Transcription thus actively contributes to the reestablishment of the active chromatin state. Overall design: We have measured the genome-wide dynamics of H3K4me3, H3, H3K36me3, H3K56ac and RNAPol2 (Rpb3) distribution on both replicated daughter genomes in a timecourse during budding yeast S-phase in wt and mutant backgrounds. We  used EdU (ethylene deoxy uridine) to mark nascent (replicated) DNA strands. We have enriched for DNA fragments bound by the above proteins by immunoprecipitation at different times in budding yeast S-phase. EdU incorporated into nascent ChIPed (the ChIP-NChAP (Nascent Chromatin Avidin Pulldown) fraction) and nascent bulk DNA ( the NChAP fraction) strands was biotinylated using Click chemistry and nascent DNA strand fragments were subsequently isolated by pulldown on Streptavidin coated magnetic beads for strand specific NGS library preparation.