Mechanism of nucleosome positioning, spacing and regularity in Saccharomyces cerevisiae
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE141007 | GEO | 2021/10/20
REPOSITORIES: GEO
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Project description:Chromatin remodelers regulate genes by organizing nucleosomes around promoters, but their individual contributions are obfuscated by the complex in vivo milieu of factor redundancy and indirect effects. Genome-wide reconstitution of promoter nucleosome organization with purified proteins resolves this problem and is therefore a critical goal. Here we reconstitute four stages of nucleosome architecture using purified components: Yeast genomic DNA, histones, sequence-specific Abf1/Reb1, and remodelers RSC, ISW2, INO80, and ISW1a. We identify direct, specific and sufficient contributions that in vivo observations validate. First, RSC clears promoters by translating poly(dA:dT) into directional nucleosome removal. Second, partial redundancy is recapitulated where INO80 alone, or ISW2 at Abf1/Reb1sites, positions +1 nucleosomes. Third, INO80 and ISW2 each align downstream nucleosomal arrays. Fourth, ISW1a tightens the spacing to canonical repeat lengths. Such a minimal set of rules and proteins establishes core mechanisms by which promoter chromatin architecture arises through a blend of redundancy and specialization.
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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.
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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
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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.
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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
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
Project description:Nucleosomes are important for gene regulation because their arrangement on the genome can control which proteins bind to DNA. Currently, few human nucleosomes are thought to be consistently positioned across cells; however, this has been difficult to assess due to the limited resolution of existing data. We performed paired-end sequencing of micrococcal nuclease-digested chromatin (MNase-seq) from seven lymphoblastoid cell lines and mapped over 3.6 billion MNase-seq fragments to the human genome to create the highest-resolution map of nucleosome occupancy to date in a human cell type. In contrast to previous results, we find that most nucleosomes have more consistent positioning than expected by chance and a substantial fraction (8.7%) of nucleosomes have moderate to strong positioning. In aggregate, nucleosome sequences have 10 bp periodic patterns in dinucleotide frequency and DNase I sensitivity; and, across cells, nucleosomes frequently have translational offsets that are multiples of 10 bp. We estimate that almost half of the genome contains regularly spaced arrays of nucleosomes, which are enriched in active chromatin domains. Single nucleotide polymorphisms that reduce DNase I sensitivity can disrupt the phasing of nucleosome arrays, which indicates that they often result from positioning against a barrier formed by other proteins. However, nucleosome arrays can also be created by DNA sequence alone. The most striking example is an array of over 400 nucleosomes on chromosome 12 that is created by tandem repetition of sequences with strong positioning properties. In summary, a large fraction of nucleosomes are consistently positioned-in some regions because they adopt favored sequence positions, and in other regions because they are forced into specific arrangements by chromatin remodeling or DNA binding proteins. MNase-seq of 7 human lymphoblastoid cell lines from the HapMap project
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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.
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Project description:We have used micrococcal nuclease (MNase) digestion followed by deep sequencing in order to obtain a higher-resolution map than previously available of nucleosome positions in the fission yeast, Schizosaccharomyces pombe. Our data confirm an unusually short average nucleosome repeat length, ~152 bp, in fission yeast and that transcriptional start sites (TSSs) are associated with nucleosome-depleted regions (NDRs), ordered nucleosome arrays downstream and less regularly spaced upstream nucleosomes. In addition, we found enrichments for associated function in four of eight groups of genes clustered according to chromatin configurations near TSSs. At replication origins, our data revealed asymmetric localization of Pre-Replication Complex (pre-RC) proteins within large NDRsM-bM-^@M-^Ta feature that is conserved in fission and budding yeast and is therefore likely to be conserved in other eukaryotic organisms. Examination of nucleosome positioning in Schizosaccharomyces pombe
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Project description:Nucleosomes are important for gene regulation because their arrangement on the genome can control which proteins bind to DNA. Currently, few human nucleosomes are thought to be consistently positioned across cells; however, this has been difficult to assess due to the limited resolution of existing data. We performed paired-end sequencing of micrococcal nuclease-digested chromatin (MNase-seq) from seven lymphoblastoid cell lines and mapped over 3.6 billion MNase-seq fragments to the human genome to create the highest-resolution map of nucleosome occupancy to date in a human cell type. In contrast to previous results, we find that most nucleosomes have more consistent positioning than expected by chance and a substantial fraction (8.7%) of nucleosomes have moderate to strong positioning. In aggregate, nucleosome sequences have 10 bp periodic patterns in dinucleotide frequency and DNase I sensitivity; and, across cells, nucleosomes frequently have translational offsets that are multiples of 10 bp. We estimate that almost half of the genome contains regularly spaced arrays of nucleosomes, which are enriched in active chromatin domains. Single nucleotide polymorphisms that reduce DNase I sensitivity can disrupt the phasing of nucleosome arrays, which indicates that they often result from positioning against a barrier formed by other proteins. However, nucleosome arrays can also be created by DNA sequence alone. The most striking example is an array of over 400 nucleosomes on chromosome 12 that is created by tandem repetition of sequences with strong positioning properties. In summary, a large fraction of nucleosomes are consistently positioned-in some regions because they adopt favored sequence positions, and in other regions because they are forced into specific arrangements by chromatin remodeling or DNA binding proteins.
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