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MNase-seq analysis of Col-0 and chr11-1 chr17-1

ABSTRACT: Chromatin has a highly organized structure with the repeating nucleosome subunits. The position of nucleosomes on the chromatin is dynamically regulated by ATP-dependent chromatin remodeling factors (remodelers), therefore providing specific epigenetic information. However, the in vivo nucleosome distribution pattern in plants and how plant remodelers control the pattern formation are not yet clear. Here we use the Micrococcal Nuclease digestion followed by deep sequencing (MNase-seq) assay to show the genome-wide nucleosome pattern in plants and the role of the Arabidopsis thaliana Imitation Switch (AtISWI) subfamily remodelers in the pattern formation. Our data revealed three patterns in wild-type plants: 1) Promoters have a relative low nucleosome density compared with gene bodies; 2) Nucleosome density is negatively associated with gene expression levels; 3) The evenly and periodically spaced nucleosomes in gene bodies are positively associated with gene expression levels. Double mutations in the AtISWI genes CHROMATIN REMODELING 11 (CHR11) and CHR17 resulted in loss of the evenly-spaced-nucleosome pattern in gene bodies, but did not affect nucleosome density, suggesting that the primary role of AtISWI is to slide nucleosomes in gene bodies for promotion of transcription.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE50242 | GEO | 2014/03/30

SECONDARY ACCESSION(S): PRJNA217328

REPOSITORIES: GEO

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MNase-seq analysis of Col-0 and chr11-1 chr17-1

Project description:Chromatin has a highly organized structure with the repeating nucleosome subunits. The position of nucleosomes on the chromatin is dynamically regulated by ATP-dependent chromatin remodeling factors (remodelers), therefore providing specific epigenetic information. However, the in vivo nucleosome distribution pattern in plants and how plant remodelers control the pattern formation are not yet clear. Here we use the Micrococcal Nuclease digestion followed by deep sequencing (MNase-seq) assay to show the genome-wide nucleosome pattern in plants and the role of the Arabidopsis thaliana Imitation Switch (AtISWI) subfamily remodelers in the pattern formation. Our data revealed three patterns in wild-type plants: 1) Promoters have a relative low nucleosome density compared with gene bodies; 2) Nucleosome density is negatively associated with gene expression levels; 3) The evenly and periodically spaced nucleosomes in gene bodies are positively associated with gene expression levels. Double mutations in the AtISWI genes CHROMATIN REMODELING 11 (CHR11) and CHR17 resulted in loss of the evenly-spaced-nucleosome pattern in gene bodies, but did not affect nucleosome density, suggesting that the primary role of AtISWI is to slide nucleosomes in gene bodies for promotion of transcription. The overall design of the experiment: (1) Nuclear extraction from Col-0 and chr11-1 chr17-1. (2) MNase treatment. (3) DNA purification. (4) Library construction. (5) Deep sequencing. (6) Data analysis.

2014-03-30 | E-GEOD-50242 | biostudies-arrayexpress

Nucleosome recognition and DNA distortion by the Chd1 remodeler revealed at 2.6 Å resolution

Project description:Chromatin remodelers are ATP-dependent enzymes that reorganize nucleosomes within all eukaryotic genomes. The Chd1 remodeler specializes in shifting nucleosomes into evenly spaced arrays, a defining characteristic of chromatin in gene bodies that blocks spurious transcription initiation. Linked to some forms of autism and commonly mutated in prostate cancer, Chd1 is essential for maintaining pluripotency in stem cells. Here we report a complex of yeast Chd1 bound to a nucleosome in a nucleotide-free state, determined by cryo-electron microscopy (cryo-EM) to 2.6 Å resolution. The structure shows a bulge of the DNA tracking strand where the ATPase motor engages the nucleosome, consistent with an initial stage in DNA translocation. Unlike other remodeler-nucleosome complexes, nucleosomal DNA compensates for the remodeler-induced bulge with a bulge of the complementary DNA strand one helical turn downstream from the ATPase motor. Unexpectedly, the structure also reveals an N-terminal binding motif, called ChEx, which binds on the exit-side acidic patch of the nucleosome. The ChEx motif can displace a LANA-based peptide from the acidic patch, which suggests a means by which Chd1 remodelers may block competing chromatin remodelers from acting on the opposite side of the nucleosome.

2022-02-23 | PXD025287 | Pride

A single fiber view of the nucleosome organization of eukaryotic chromatin

Project description:Nucleosomes arrange into extended arrays, much like beads on a string. They are often phased at genomic landmarks and are thought to be evenly spaced. Here we tested to what extent this stereotypic organization describes the nucleosome landscape in Saccharomyces cerevisiae using a long-read nucleosome-sequencing technique called Fiber-Seq. Fiber-Seq maps the nucleosome pattern on individual chromatin fibers. As such, it is ideally suited to measure the density of nucleosomes per read and quantitate the nucleosome occupancy throughout the genome. We document substantial deviations from the stereotypical nucleosome organization, with unexpectedly long linker DNAs between individual nucleosomes, genomic regions lacking entire nucleosomes, heterogeneous phasing of arrays, truly irregular spacing of arrays and read-to-read variation in nucleosome densities. We exploited the technology to test mechanistic models for the biogenesis of nucleosome arrays. We can rule out transcription elongation playing a decisive role in array formation and detect signatures for a clamping activity of remodelers of the ISWI and CHD1 families after acute nucleosome depletion in vivo. Given that nucleosomes are cis-regulatory elements, the cell-to-cell heterogeneity that Fiber-Seq uncovers provides much needed information to understand chromatin structure and function.

2024-01-17 | GSE224713 | GEO

Ruler elements in chromatin remodelers set nucleosome array spacing and phasing

Project description:Arrays of regularly spaced nucleosomes dominate chromatin and are often phased, i.e., aligned at reference sites like active promoters. How distances between nucleosomes and distances between phasing sites and nucleosomes are determined remained unclear, specifically, the role of ATP dependent chromatin remodelers in it. Here, we used a genome-wide reconstitution system to probe how yeast remodelers generate phased nucleosome arrays. We find that remodelers bear a structural element named the ‘ruler’ that sets nucleosome spacing, in the order Chd1 < ISW1a < ISW2 < INO80. Structure-based mutagenesis confirmed the functional significance of the ruler element in INO80. Differences in the ruler elements of different remodelers explain the observed nucleosome array features. More generally, we propose that remodelers use their rulers to regulate the direction of nucleosome sliding in response to nucleosome density and environment, leading to nucleosome positioning relative to other nucleosomes, DNA bound factors or DNA sequence elements.

2020-02-10 | GSE140614 | GEO

Mechanism of nucleosome positioning, spacing and regularity in Saccharomyces cerevisiae

Project description:Numerous nucleosome remodeling enzymes tightly regulate nucleosome positions in eukaryotic cells. Transcription and statistical positioning of nucleosomes may also contribute to proper nucleosome organization. Individual contributions remain controversial due to strong redundancy of processes acting on the nucleosome landscape. By incisive yeast genome engineering we radically decreased their redundancy. We find the transcriptional machinery to be disruptive of evenly spaced nucleosomes, and proper nucleosome density critical for their biogenesis. INO80 spaces nucleosomes in vivo and positions the first nucleosome covering genes. It requires its Arp8 and Ies2 subunits, but unexpectedly not the Nhp10 module, for spacing. Whereas H2A.Z stimulates INO80 in vitro, its presence is dispensable for INO80 +1 positioning function in vivo. DNA damage, recombination and transposon integration assays suggest that evenly spaced nucleosomes protect cells against genotoxic stress. We derive a unifying model of the biogenesis of the nucleosome landscape and suggest that it evolved not only to regulate but also to protect the genome.

2021-10-20 | GSE141007 | GEO

Insights into promoter chromatin remodeling from the structure of ISW1a complex bound to the dinucleosome

Project description:Nucleosomes, the basic repeating units of chromatin, form evenly spaced arrays inside the cell. Chromatin remodelers alter the positions of nucleosomes, and are vital in regulating chromatin organization and gene expression. Here we report the cryoEM structure of the chromatin remodeler ISW1a complex bound to the dinucleosome. Both subunits of the complex recognize one nucleosome. The motor subunit binds at the canonical position of the nucleosome, with two arginine anchors recognizing the acidic patch. The DNA-binding module interacts with the entry linker DNA at the nucleosome edge, consistent with the protein ruler model in nucleosome spacing. The Ioc3 subunit recognizes the disk face of the adjacent nucleosome, being important for the spacing activity in vitro, and for chromatin organization in vivo. Together, our findings illustrate a new mode of chromatin remodeling in the context of higher-order chromatin

2023-05-15 | GSE203073 | GEO

Hrp3-TAP_Purification from Schizosaccharomyces pombe

Project description:Hrp3_Purification from Schizosaccharomyces pombe 972h- Eukaryotic genome is composed of repeating units of nucleosomes to form chromatin arrays. A canonical gene is marked by nucleosome free region (NFR) at its 5’ end followed by uniformly spaced arrays of nucleosomes. In fission yeast we show both biochemically and in vivo that both Hrp1 and Hrp3 are key determinants of uniform spacing of genic arrays.

2014-05-16 | PXD000041 | Pride

Dynamics of yeast ISWI and CHD chromatin remodeler genomic association (part 1)

Project description:Chromatin remodelers influence genetic processes by altering nucleosome occupancy, positioning, and composition. In vitro, yeast ISWI and CHD remodelers require > 20 bp of extranucleosomal DNA for remodeling, but linker DNA in S. cerevisiae averages < 20 bp. To resolve this paradox, we have mapped the genomic distributions of the yeast Isw1, Isw2, and Chd1 remodelers at base-pair resolution. Surprisingly, remodelers are highly enriched at promoter nucleosome depleted regions (5' NDRs), where they bind to regions of extended linker DNA. Remodelers are also enriched in the bodies of genes displaying high nucleosome turnover. We hypothesize that remodelers bind but do not act at 5' NDRs, remaining in physical proximity to gene bodies, where they act on regions of transient nucleosome depletion following transcriptional elongation. We have analyzed the dynamics of yeast ISWI and CHD chromatin remodeler genomic association at base-pair resolution using native chromatin immunoprecipitation followed by sequencing (N-ChIP-seq).

2013-03-01 | E-GEOD-39430 | biostudies-arrayexpress

Dynamics of yeast ISWI and CHD chromatin remodeler genomic association (part 2)

2013-03-01 | E-GEOD-39431 | biostudies-arrayexpress

Asymmetric nucleosomes flank promoters in the budding yeast genome

Project description:Nucleosomes in active chromatin are dynamic, but whether they have distinct structural conformations is unknown. To identify nucleosomes with alternative structures genome-wide, we used H4S47C-anchored cleavage mapping, which revealed that nucleosomes at 5% of budding yeast nucleosome positions have asymmetric histone-DNA interactions. These asymmetric interactions are enriched at nucleosome positions that flank promoters. Micrococcal nuclease (MNase) sequence-based profiles of asymmetric nucleosome positions revealed a corresponding asymmetry in MNase protection near the dyad axis, suggesting that the loss of DNA contacts around H4S47 is accompanied by protection of the DNA from MNase. Chromatin immunoprecipitation mapping of selected nucleosome remodelers indicated that asymmetric nucleosomes are bound by the RSC chromatin remodeling complex, which is required for maintaining nucleosomes at asymmetric positions. These results imply that the asymmetric nucleosome-RSC complex is a metastable intermediate representing partial unwrapping and protection of nucleosomal DNA on one side of the dyad axis during chromatin remodeling. We have analyzed the chromatin landscape of the yeast genome using paired-end MNase-seq and the chromatin binding of yeast remodelers Swr1, Ino80 and RSC at base-pair resolution using native chromatin immunoprecipitation followed by sequencing (N-ChIP-seq).

2014-12-04 | E-GEOD-59523 | biostudies-arrayexpress

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