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Replication-coupled nucleosome assembly and positioning by ATP-dependent chromatin remodeling enzymes

ABSTRACT: We have previously shown that Okazaki fragments in Saccharomyces cerevisiae are sized according to the chromatin repeat. Here we demonstrate that nucleosome positioning is rapidly established on newly synthesized DNA. Using deep sequencing, we demonstrate that ATP-dependent chromatin remodeling enzymes, Isw1 and Chd1, collaborate with histone chaperones, such as CAF-1 and Rtt106, to remodel nucleosomes, as they are loaded. Importantly, we find that nucleosome positioning is ultimately specified by select sequence-specific DNA-binding factors, which serve as physical cues for chromatin remodeling. Our results provide a mechanistic understanding of how chromatin structures are replicated in vivo, and show that chromatin structure at gene promoters is rapidly established after DNA replication. Altogether, our data provide evidence for coordinated “loading and remodeling” of nucleosomes behind the replication fork, in collaboration with packing of nucleosomes against a barrier.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE63583 | GEO | 2016/04/14

SECONDARY ACCESSION(S): PRJNA268316

REPOSITORIES: GEO

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Replication-coupled nucleosome assembly and positioning by ATP-dependent chromatin remodeling enzymes

Project description:We have previously shown that Okazaki fragments in Saccharomyces cerevisiae are sized according to the chromatin repeat. Here we demonstrate that nucleosome positioning is rapidly established on newly synthesized DNA. Using deep sequencing, we demonstrate that ATP-dependent chromatin remodeling enzymes, Isw1 and Chd1, collaborate with histone chaperones, such as CAF-1 and Rtt106, to remodel nucleosomes, as they are loaded. Importantly, we find that nucleosome positioning is ultimately specified by select sequence-specific DNA-binding factors, which serve as physical cues for chromatin remodeling. Our results provide a mechanistic understanding of how chromatin structures are replicated in vivo, and show that chromatin structure at gene promoters is rapidly established after DNA replication. Altogether, our data provide evidence for coordinated âloading and remodelingâ of nucleosomes behind the replication fork, in collaboration with packing of nucleosomes against a barrier. 7 samples, including a wild type, are included. All are single-end sequenced via Ion Torrent PGM methodology.

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Project description:The positioning of nucleosomes within the coding regions of eukaryotic genes is aligned with respect to transcriptional start sites. This organization is likely to influence many genetic processes, requiring access to the underlying DNA. Here we show that the combined action of Isw1 and Chd1 nucleosome spacing enzymes is required to maintain this organization. In the absence of these enzymes regular positioning of the majority of nucleosomes is lost. Exceptions include the region upstream of the promoter, the +1 nucleosome and a subset of locations distributed throughout coding regions where other factors are likely to be involved. These observations indicated that ATP-dependent remodeling enzymes are responsible for directing the positioning of the majority of nucleosomes within the Saccharomyces cerevisiae genome. Agilent two-color experiment,Organism: Saccharomyces cerevisiae ,Slides: Agilent Gene Expression S. cerevisiae 8x15k array AMADID: 016333, Labeling kit: Agilent’s Quick-Amp labeling Kit (p/n5190-0444)Method: T7 promoter based-linear amplification to generate labeled complementary RNA.

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Project description:Replication of the eukaryotic genome occurs in the context of chromatin, a nucleoprotein packaging state consisting of repeating nucleosomes. Chromatin is commonly thought to carry epigenetic information from one generation to the next, although it is unclear how such information survives the disruptions of nucleosomal architecture that occur during genomic replication. Here, we sought to directly 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 the nucleotide analogue EdU, we characterize nucleosome positions on both daughter genomes of budding yeast during a time course of chromatin maturation. We find that nucleosomes rapidly adopt their mid log positions at highly-transcribed genes, and that this process was impaired upon treatment with the transcription inhibitor thiolutin, consistent with a role for transcription in positioning nucleosomes in vivo. Additionally, experiments in the Hir1Δ background reveal a role for HIR in nucleosome spacing. Using strand-specific EdU libraries, we characterize nucleosome positions on the leading and lagging strand daughter genomes, uncovering differences in chromatin maturation dynamics between the two daughter genomes 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.

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