SummaryChromatin is thought to carry epigenetic information from one generation to the next, although it is unclear how such information survives the disruptions of nucleosomal architecture occurring during genomic replication. Here, we measure a key aspect of chromatin structure dynamics during replication—how rapidly nucleosome positions are established on the newly replicated daughter genomes. By isolating newly synthesized DNA marked with 5-ethynyl-2′-deoxyuridine (EdU), we characterize nucleosome positions on both daughter genomes of S. cerevisiae during chromatin maturation. We find that nucleosomes rapidly adopt their mid-log positions at highly transcribed genes, which is consistent with a role for transcription in positioning nucleosomes in vivo. Additionally, experiments in hir1Δ mutants reveal a role for HIR in nucleosome spacing. We also characterized nucleosome positions on the leading and lagging strands, uncovering differences in chromatin maturation dynamics at hundreds of genes. Our data define the maturation dynamics of newly replicated chromatin and support a role for transcription in sculpting the chromatin template.
Dynamic disruption and reassembly of promoter-proximal nucleosomes is a conserved hallmark of transcriptionally active chromatin. Histone H3-K56 acetylation (H3K56Ac) enhances these turnover events and promotes nucleosome assembly during S phase. Here we sequence nascent transcripts to investigate the impact of H3K56Ac on transcription throughout the yeast cell cycle. We find that H3K56Ac is a genome-wide activator of transcription. While H3K56Ac has a major impact on transcription initiation, it also appears to promote elongation and/or termination. In contrast, H3K56Ac represses promiscuous transcription that occurs immediately following replication fork passage, in this case by promoting efficient nucleosome assembly. We also detect a stepwise increase in transcription as cells transit S phase and enter G2, but this response to increased gene dosage does not require H3K56Ac. Thus, a single histone mark can exert both positive and negative impacts on transcription that are coupled to different cell cycle events.
Most protein half-lives are equal to cell-cycle length Net synthesis rates of 18 test proteins are 2-fold higher than their decay rates Most tested proteins segregate stochastically between mothers and daughters ''Old'' Rxt3p, Tup1p, and Fpr4p and low abundance proteins are retained in the mother
The budding yeast SIR complex (Silent Information Regulator) is the principal actor in heterochromatin formation, which causes epigenetically regulated gene silencing phenotypes.The maternal chromatin bound SIR complex is disassembled during replication.Consequently, if heterochromatin is to be restored on both daughter strands, the SIR complex has to be reformed on both strands to pre-replication levels. The dynamics of SIR complex maintenance and re-formation during the cell-cycle and in different growth conditions are however not clear. Understanding exchange rates of SIR subunits during the cell cycle and their distribution pattern to daughter chromatids after replication has important implications for how heterochromatic states may be inherited and therefore how epigenetic states are maintained from one cellular generation to the next. We used the tag switch RITE system to measure genome wide turnover rates of the SIR subunit Sir3 before and after exit from stationary phase and show that maternal Sir3 subunits are completely replaced with newly synthesized Sir3 at subtelomeric regions during the first cell cycle after release from stationary phase. The SIR complex is therefore not "inherited" and the silenced state has to be established de novo upon exit from stationary phase. Additionally, our analysis of genomewide transcription dynamics shows that precise Sir3 dosage is needed for the optimal upregulation of "growth" genes during the first cell-cycle after release from stationary phase.
Asymmetric division is a prerequisite for cellular differentiation. Phenotypic transformation during differentiation is a poorly understood epigenetic phenomenon, in which chromatin theoretically plays a role. The assumption that chromatin components segregate asymmetrically in asymmetric divisions has however not been systematically tested. We have developed a live cell imaging method to measure how 18 chromatin proteins are inherited in asymmetric divisions of budding yeast. We show that abundant and moderately abundant maternal proteins segregate stochastically and symmetrically between the two cells with the exception of Rxt3, Fpr4 and Tup1, which are retained in the mother. Mother retention seems to be the norm for low abundance proteins with the exception of Sir2 and the linker histone H1. Our in vivo analysis of chromatin protein behavior in single cells highlights general trends in protein biology during the cell cycle such as coupled protein synthesis and decay, and a correlation between half-lives and cell cycle duration.
Asymmetric division is a prerequisite for cellular differentiation. Phenotypic transformation during differentiation is a poorly understood epigenetic phenomenon, in which chromatin theoretically plays a role. The assumption that chromatin components segregate asymmetrically in asymmetric divisions has however not been systematically tested. We have developed a live cell imaging method to measure how 18 chromatin proteins are inherited in asymmetric divisions of budding yeast. We show that abundant and moderately abundant maternal proteins segregate stochastically and symmetrically between the two cells with the exception of Rxt3, Fpr4 and Tup1, which are retained in the mother. Mother retention seems to be the norm for low abundance proteins with the exception of Sir2 and the linker histone H1. Our in vivo analysis of chromatin protein behavior in single cells highlights general trends in protein biology during the cell cycle such as coupled protein synthesis and decay, and a correlation between half-lives and cell cycle duration.
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