Chemical Mapping Reveals Novel Features of The Nucleosome Landscape in Mouse Embryonic Stem Cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE82127 | GEO | 2016/11/23
SECONDARY ACCESSION(S): PRJNA324099
REPOSITORIES: GEO
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Project description:Nucleosome remodeling factors regulate the occupancy and positioning of nucleosomes genome-wide through ATP-driven DNA translocation. While many nucleosomes are well- and consistently positioned, some nucleosomes and nucleosome-like structures are more sensitive to nuclease digestion or transitory in nature. To better understand the role of nucleosome remodeling factors in generating and clearing these alternative nucleosome structures, we depleted murine embryonic stem cells of the remodeler ATPases BRG1 and SNF2H then performed MNase-seq in murine embryonic stem cells. We performed MNase-seq under high- and low-MNase conditions to assess the effects of nucleosome remodeling factors on nuclease-sensitive or “fragile” nucleosome occupancy. In parallel, we gel-extracted MNase-digested fragments to enrich for another alternative nucleosome structure, the overlapping dinucleosome. Overlapping dinucleosomes are composed of two canonical nucleosomes, asymmetrically lacking one H2A:H2B dimer and wrapped by ~250 bp of DNA. In vitro studies of nucleosome remodeling have suggested that the collision of adjacent nucleosomes by nucleosome sliding can stimulate formation of overlapping dinucleosomes. Using these methods, we were able to identify fragile nucleosomes and overlapping dinucleosomes near transcription start sites and gene-distal DNaseI hypersensitive sites in mouse embryonic stem cells, among other loci. We find that BRG1 consistently stimulates occupancy of fragile nucleosomes but represses occupancy of overlapping dinucleosomes through its nucleosome remodeling function, while SNF2H expression slightly increases fragile nucleosome occupancy.
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Project description:The position of nucleosomes influences DNA accessibility to DNA-binding proteins. Genome-wide nucleosome profiles often report the observation of a canonical nucleosome organization at gene promoters where arrays of well-positioned nucleosomes emanate from nucleosome-depleted regions. It is unclear how this canonical promoter nucleosome organization forms and how it is related to transcription activation and the establishment of histone marks during development. Here we report the genome-wide organization of nucleosomes during zebrafish embryogenesis and show that well-positioned nucleosome arrays appear in thousands of promoters during the activation of the zygotic genome. The formation of canonical promoter nucleosome organization cannot be explained by DNA sequence preference, and is independent of transcription and the presence of RNA polymerase II, but strongly correlates with the presence of Histone H3 Lysine 4 trimethylation (H3K4me3). Our study further suggests that promoter nucleosome structure primes genes to future transcription activation. To determine whether the occlusions are consistent in mammalian pluripotent cells, we performed the same analyses in mouse embryonic stem cells and found similar relationships. MNase-seq to generate nucleosome organization in mouse embryonic stem cell (J1)
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Project description:Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and their normal localization is correlated with proper insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) are also required for recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy binding site DNA, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization.
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Project description:The structural complexity of nucleosomes underlies their functional versatility. Here we report a new type of complexity – nucleosome fragility, manifested as high sensitivity to micrococcal nuclease, in contrast to the common presumption that nucleosomes are similar in resistance to MNase digestion. Using differential MNase digestion of chromatin and high-throughput sequencing, we have identified a special group of nucleosomes termed fragile nucleosomes throughout the yeast genome, nearly one thousand of which are at previously determined “nucleosome free” loci. Nucleosome fragility is broadly implicated in multiple chromatin processes, including transcription, translocation and replication, in correspondence to specific physiological states of cells. In the environmental-stress-response genes, the presence of fragile nucleosomes prior to the occurrence of environmental changes suggests that nucleosome fragility poises genes for swift up-regulation in response to the environmental changes. We propose that nucleosome fragility underscores distinct functional statuses of the chromatin and provides a new dimension for portraying the landscape of genome organization. Comparing nucleosome occupancy under different MNase digestion levels and growth conditions.
2011-01-24 | E-GEOD-26412 | biostudies-arrayexpress
Project description:Nucleosome organization is critical for gene regulation. In living cells, this organization is determined by multiple factors, including the action of chromatin remodelers, competition with site-specific DNA-binding proteins, and the DNA sequence preferences of the nucleosomes themselves. However, it has been difficult to estimate the relative importance of each of these mechanisms in vivo, because in vivo nucleosome maps reflect the combined action of all influencing factors. Here, we determine the importance of DNA sequence preferences experimentally by measuring the genome-wide occupancy of nucleosomes assembled on purified yeast genomic DNA. The resulting map, in which nucleosome occupancy is governed only by the intrinsic sequence preferences of nucleosomes, is remarkably similar to in vivo nucleosome maps generated in three different growth conditions. In vitro, nucleosome depletion is evident at many transcription factor binding sites and around gene start and end sites, suggesting that nucleosome depletion at these sites in vivo is partially encoded in the genome. We confirm these results with a micrococcal nuclease-independent experiment that measures the relative affinity of nucleosomes for ~40,000 double-stranded 150bp oligonucleotides. Using our in vitro data, we devise a computational model of nucleosome sequence preferences that is significantly correlated with in vivo nucleosome occupancy in C. elegans. Our results indicate that the intrinsic DNA sequence preferences of nucleosomes play a central role in determining the organization of nucleosomes in vivo.
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Project description:Micrococcal nuclease (MNase) is commonly used to map nucleosomes genome-wide, but nucleosome maps are affected by the degree of digestion. It has been proposed that many yeast promoters are not nucleosome-free but occupied by easily digested, unstable, “fragile” nucleosomes. We analyzed the histone content of all MNase-sensitive complexes by MNase-ChIP-seq and Sonication-ChIP-seq. We find that yeast promoters are predominantly bound by non-histone protein complexes, with little evidence for fragile nucleosomes. We do detect MNase-sensitive nucleosomes elsewhere in the genome, including transcription termination sites. However, they have high A/T-content, suggesting that MNase sensitivity does not indicate instability, but the preference of MNase for A/T-rich DNA, such that A/T-rich nucleosomes are digested faster than G/C-rich nucleosomes. We confirm our observations by analyzing ChIP-exo, chemical mapping and ATAC-seq data from other laboratories. Thus, histone ChIP-seq experiments are essential to distinguish nucleosomes from other DNA-binding proteins that protect against MNase.
2016-12-15 | GSE83123 | GEO
Project description:The structural complexity of nucleosomes underlies their functional versatility. Here we report a new type of complexity – nucleosome fragility, manifested as high sensitivity to micrococcal nuclease, in contrast to the common presumption that nucleosomes are similar in resistance to MNase digestion. Using differential MNase digestion of chromatin and high-throughput sequencing, we have identified a special group of nucleosomes termed fragile nucleosomes throughout the yeast genome, nearly one thousand of which are at previously determined “nucleosome free” loci. Nucleosome fragility is broadly implicated in multiple chromatin processes, including transcription, translocation and replication, in correspondence to specific physiological states of cells. In the environmental-stress-response genes, the presence of fragile nucleosomes prior to the occurrence of environmental changes suggests that nucleosome fragility poises genes for swift up-regulation in response to the environmental changes. We propose that nucleosome fragility underscores distinct functional statuses of the chromatin and provides a new dimension for portraying the landscape of genome organization.
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Project description:The position of nucleosomes influences DNA accessibility to DNA-binding proteins. Genome-wide nucleosome profiles often report the observation of a canonical nucleosome organization at gene promoters where arrays of well-positioned nucleosomes emanate from nucleosome-depleted regions. It is unclear how this canonical promoter nucleosome organization forms and how it is related to transcription activation and the establishment of histone marks during development. Here we report the genome-wide organization of nucleosomes during zebrafish embryogenesis and show that well-positioned nucleosome arrays appear in thousands of promoters during the activation of the zygotic genome. The formation of canonical promoter nucleosome organization cannot be explained by DNA sequence preference, and is independent of transcription and the presence of RNA polymerase II, but strongly correlates with the presence of Histone H3 Lysine 4 trimethylation (H3K4me3). Our study further suggests that promoter nucleosome structure primes genes to future transcription activation. Together, this study reveals that genome activation but not transcription underlies the organization of nucleosome arrays during early embryogenesis. MNase-seq to generate nucleosome organization in two stages of zebrafish development; two biological replicates for each stage. 7 ChIP-seq experiments in three stages.
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Project description:Open chromatin plays key roles in regulating gene expression in eukaryotic genomes. However, functional implications of fragile nucleosome overlapping open chromatin are still largely unknown, especially in plants. Here, through simultaneous measurement of MHSs and nucleosomes, we detected two distinct types of open chromatin, one with nucleosome-free (type-I MHSs) and the other with fragile nucleosomes (type-II MHSs) in maize and Arabidopsis. Both types of MHSs can function as cis-regulatory elements and have similar TF binding motifs. Furthermore, fragile nucleosomes with MHSs have unique DNA sequence features with less AT and more GC dinucleotides, whereas other fragile nucleosomes without overlapping MHSs have more AT and less GC dinucleotides. In particular, we observed distinct fragile nucleosome positioning patterns occurred around the summit of type-I and type-II MHSs, phased fragile nucleosome arrays and only two well-phased fragile nucleosomes surrounding the summit of type-I and type-II MHSs, respectively. We finally found that type-II MHS-related genes with BCTSS or SCTSS express more than type-I-related genes with the corresponding CTSS, which is mediated by combined actions of MHSs with TFs, fragile nucleosomes, Pol II and epigenetic marks.
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