Novel nucleosomal particles containing core histones and linker DNA but no histone H1
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus Synthetic plasmid Saccharomyces cerevisiae
PROVIDER: GSE65889 | GEO | 2015/09/23
SECONDARY ACCESSION(S): PRJNA275314
REPOSITORIES: GEO
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Project description:The nucleosome plays a central role in genome regulation. Traditional methods for mapping nucleosomes depend on the resistance of the nucleosome core to micrococcal nuclease (MNase). However, the lengths of the protected DNA fragments are heterogeneous, limiting the accuracy of nucleosome position information. To resolve this problem, we removed residual linker DNA by simultaneous digestion of yeast chromatin with MNase and exonuclease III (ExoIII). Paired-end sequencing of mono-nucleosomes revealed not only core particles (145-147 bp), but also intermediate particles in which ~8 bp project from one side (154 bp) or both sides (161 bp) of the nucleosome core. We term these particles "pseudo-chromatosomes" because they are present in yeast lacking linker histone. They are also observed after MNase-ExoIII digestion of chromatin reconstituted using recombinant core histones. We propose that the pseudo-chromatosome provides a DNA framework to facilitate H1 binding. Comparison of budding yeast nucleosome sequences obtained using micrococcal nuclease (MNase-seq) and MNase + exonuclease III (ExoIII) (MNase-ExoIII-seq): wild type cells and hho1-null cells. Nucleosome sequences from native chromatin and H1-depleted chromatin from mouse liver. Nucleosome sequences from a plasmid reconstituted into nucleosomes using recombinant yeast histones or native chicken erythrocyte histones.
2015-09-23 | E-GEOD-65889 | biostudies-arrayexpress
Project description:Histone H1 variants, known as linker histones, are essential chromatin components in higher eukaryotes, yet compared to the core histones relatively little is known about their in vivo functions. The filamentous fungus Neurospora crassa encodes a single H1 protein that is not essential for viability. To investigate the role of N. crassa H1, we constructed a functional FLAG-tagged H1 fusion protein and performed genomic and molecular analyses. Cell fractionation experiments showed that H1-FLAG is a chromatin binding protein. Chromatin-immunoprecipitation combined with sequencing (ChIP-seq) revealed that H1-3XFLAG is globally enriched throughout the genome with a subtle preference for promoters of expressed genes. In mammals, the stochiometery of H1 impacts nucleosome repeat length. To determine if H1 impacts nucleosome occupancy or nucleosome positioning in N. crassa, we performed micrococcal nuclease digestion in wildtype and the ∆hH1 strain followed by sequencing (MNase-Seq). Deletion of hH1 did not significantly impact nucleosome positioning or nucleosome occupancy. Analysis of DNA methylation using methylC-sequencing (mC-Seq) revealed a modest but global increase in DNA methylation in the ∆hH1 mutant. Together, these data suggest that H1 acts as a non-specific chromatin binding protein that can limit accessibility of the DNA methylation machinery in N. crassa.
2016-04-20 | GSE78157 | GEO
Project description:Eukaryotic chromosomes are composed of chromatin, in which regularly spaced nucleosomes containing ~147 bp of DNA are separated by linker DNA. Most eukaryotic cells have a characteristic average nucleosome spacing of ~190 bp, corresponding to a ~45 bp linker. However, cortical neurons have a shorter average spacing of ~165 bp. The significance of this atypical global chromatin organization is unclear. We have compared the chromatin structures of purified mouse dorsal root ganglia (DRG) neurons, cortical oligodendrocyte precursor cells (OPCs) and cortical astrocytes. DRG neurons have short average spacing (~165 bp), whereas OPCs (~182 bp) and astrocytes (~183 bp) have longer spacing. We measured nucleosome positions by MNase-seq and gene expression by RNA-seq. Most genes in all three cell types have a promoter chromatin organization typical of active genes: a nucleosome-depleted region at the promoter flanked by regularly spaced nucleosomes phased relative to the transcription start site. In DRG neurons, the spacing of phased nucleosomes downstream of promoters (~178 bp) is longer than expected from the genomic average, whereas phased nucleosome spacing in OPCs and astrocytes is similar to the global average (~183 bp). Thus, the atypical nucleosome spacing of neuronal chromatin does not extend to promoter-proximal regions.
2019-10-07 | GSE133745 | GEO
Project description:Eukaryotic chromosomes are composed of chromatin, in which regularly spaced nucleosomes containing ~147 bp of DNA are separated by linker DNA. Most eukaryotic cells have a characteristic average nucleosome spacing of ~190 bp, corresponding to a ~45 bp linker. However, cortical neurons have a shorter average spacing of ~165 bp. The significance of this atypical global chromatin organization is unclear. We have compared the chromatin structures of purified mouse dorsal root ganglia (DRG) neurons, cortical oligodendrocyte precursor cells (OPCs) and cortical astrocytes. DRG neurons have short average spacing (~165 bp), whereas OPCs (~182 bp) and astrocytes (~183 bp) have longer spacing. We measured nucleosome positions by MNase-seq and gene expression by RNA-seq. Most genes in all three cell types have a promoter chromatin organization typical of active genes: a nucleosome-depleted region at the promoter flanked by regularly spaced nucleosomes phased relative to the transcription start site. In DRG neurons, the spacing of phased nucleosomes downstream of promoters (~178 bp) is longer than expected from the genomic average, whereas phased nucleosome spacing in OPCs and astrocytes is similar to the global average (~183 bp). Thus, the atypical nucleosome spacing of neuronal chromatin does not extend to promoter-proximal regions.
2019-10-07 | GSE133966 | GEO
Project description:Linker histone H1 is a core chromatin component that binds to nucleosome core particles and the linker DNA between nucleosomes. It has been implicated in chromatin compaction and gene regulation and is anticipated to play a role in higher-order genome structure. We find that depletion of histone H1 changes the epigenetic signature of thousands of potential regulatory sites across the genome. Many of them show cooperative loss or gain of multiple chromatin marks. Epigenetic alterations cluster to gene-dense topologically associated domains (TADs) that already showed a high density of corresponding chromatin features. Genome organization at the three-dimensional level is largely intact, but we find changes in the structural segmentation of chromosomes specifically for the epigenetically most modified TADs.
2015-11-30 | GSE75426 | GEO
Project description:Nucleosomes are part of the first level of chromatin packaging. They each consist of a histone heterooctamer around which DNA wraps 1.6 times. The histone heterooctomamer is made up of two copies of histones 2A, 2B, 3 and 5. The segment of DNA wrapped around the histones, the so-called "core" fragment, is 147 base pairs long. Neighboring nucleosomes are separated from one another by a stretch of DNA called the "linker," whose size varies depending on organism, cell type, and even chromatin activity. Certain chromatin remodeling factors govern accessibility of DNA to regulatory proteins by repositioning nucleosomes to reveal regulatory sites that would otherwise be occluded by a nucleosome. In contrast to histone modifications such as methylation or acetylation, which are investigated by ChIP-seq, nucleosome positioning data are generated without immunoprecipitation (see Methods below). Instead, micrococcal nuclease is used to digest chromatin to apparent completion, the (well-defined and clearly visible) mononucleosomal core fragment fraction is isolated by gel purification, and one end is then sequenced. Mapping the sequence tag back to the genome reveals the precise position of one end of the core fragment that was protected by the nucleosome; the position of the other end can then simply be inferred by extending the read to a virtual length of 147 bases. Statistical analyses such as occupancy and positioning stringency can then be employed to analyze the local nucleosome landscape anywhere in the (mappable) genome or to infer global parameters of nucleosome organization. In the context of the ENCODE project, nucleosome positioning data are particularly valuable for analysis of the relationship between transcription factor binding, histone modifications, and gene activity. For a general primer on these types of data and analyses, refer to Valouev et al. (2008). For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf To isolate mononucleosome core DNA fragments from the GM12878 and K562 ENCODE cell lines we followed the micrococcal nuclease (MNase) digestion and isolation protocol as described in Johnson et al. (2006), Valouev et al. (2008), and Valouev et al. (2011) with the following modifications. The precise concentrations of the two flash-frozen cell samples received from the Snyder Lab were not known so, per our standard procedure, we performed a series of digestions titrating the amount of MNase to determine the concentration of MNase for optimal digestion of each sample. Final concentrations of 25 U/µL and 50 U/µL of MNase were used to digest the GM12878 cells and K562 cells respectively at 20°C for 12 min. All other steps in the digestion and isolation protocol were as described. Cells were grown according to the approved ENCODE cell culture protocols (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/cell). K562 and GM12878 were each grown to ~2.5×108 cells. The cells were harvested, frozen and the nucleosome core isolation followed (Valouev et al. 2008). The SOLiD reads were mapped in color-space with the probabilistic mapper, DNAnexus (https://dnanexus.com/). The DNAnexus mapper measures and propagates mapping uncertainty by including both quality values and mismatches in the alignment score calculation. The scores are then scaled across all possible mappings of the read to estimate the posterior probability for alignment to each genomic location. Reads corresponding to posterior probability of correct mapping > 0.9 were reported. Nucleosome density signal maps (bedgraph and bigwig files) were generated by first shifting reads by 74 bp in the 5´ to 3´ direction and counting the total number of reads starting at each genomic coordinate on both strands. These counts are then smoothed using un-normalized kernel density smoothing with a triweight kernel. A bandwidth of 30 bp is used which is equivalent to a smoothing window of 60 bp. The smoothed counts at each position are then divided by the expected number of reads from an equivalent uniform distribution of reads in a ± 30 bp window around that position. If less than 25% of the positions in a ± 30 bp window around a genomic location are uniquely mappable or if the location is part of an assembly gap, the signal value at that position is considered unreliable and not recorded in the signal files. Hence, genomic coordinates that do not have any associated signal value should be considered missing or unreliable data. Genomic coordinates associated with a signal value of 0 are reliably mapable but do not have any signal in the dataset.
2012-04-19 | E-GEOD-35586 | biostudies-arrayexpress
Project description:Linker histones are involved in the formation of higher-order chromatin structure. Although linker histones have been implicated in the regulation of specific genes, it still remains unclear what their principal binding determinants are and how their repressive function in vitro can be reconciled with presumed broad binding in vivo. We generated a full genome, high resolution binding map of linker histone H1 in Drosophila Kc cells, using DamID. H1 binds at similar levels across much of the genome, both in classical euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites of active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes. Rather, all regions with low binding of H1 show enrichment of the histone variant H3.3 which itself has been linked to high nucleosome turnover. Upon knockdown of H3.3, we find that H1 levels increase at sites previously not covered with H1 with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1 at genomic sites with high rates of histone turnover. This antagonism provides a mechanism to keep diverse genomic sites in an open chromatin conformation. For this study, we generated DamID profiles of histone H1 and RpII18 in Drosophila Kc167 cells. Additionally, we generated H1 profiles in cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Nucleosome positioning profiles were generated in untreated cells and cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Profiles of expression changes were generated for H3.3B RNAi and H3.3A and H3.3B RNAi. DamID experiments for H1 and RpII18 were performed in Drosophila cell cultures. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing. Formaldehyde-assisted isolation of regulatotry elements (FAIRE) was performed in Drosophila Kc167 cells. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing over non-crosslinked genomic DNA. Nucleosome positioning profiles were made by hybridizing mononucleosomal DNA over MNase digested purified genomic DNA on 380k NimbleGen arrays with 10 bp probe spacing. Expression profiles were made as H3.3 RNAi over white RNAi cohybridizations on spotted INDAC long oligo arrays. Every experiment was done in duplicate in the reverse dye orientation.
2010-06-21 | E-GEOD-16884 | biostudies-arrayexpress
Project description:Linker histones are involved in the formation of higher-order chromatin structure. Although linker histones have been implicated in the regulation of specific genes, it still remains unclear what their principal binding determinants are and how their repressive function in vitro can be reconciled with presumed broad binding in vivo. We generated a full genome, high resolution binding map of linker histone H1 in Drosophila Kc cells, using DamID. H1 binds at similar levels across much of the genome, both in classical euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites of active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes. Rather, all regions with low binding of H1 show enrichment of the histone variant H3.3 which itself has been linked to high nucleosome turnover. Upon knockdown of H3.3, we find that H1 levels increase at sites previously not covered with H1 with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1 at genomic sites with high rates of histone turnover. This antagonism provides a mechanism to keep diverse genomic sites in an open chromatin conformation. For this study, we generated DamID profiles of histone H1 and RpII18 in Drosophila Kc167 cells. Additionally, we generated H1 profiles in cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Nucleosome occupancy profiles were generated in untreated cells and cells treated with RNAi against white or H3.3A and H3.3B. Profiles of expression changes were generated for H3.3B RNAi and H3.3A and H3.3B RNAi. DamID experiments for H1 and RpII18 were performed in Drosophila cell cultures. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing. Formaldehyde-assisted isolation of regulatotry elements (FAIRE) was performed in Drosophila Kc167 cells. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing over non-crosslinked genomic DNA. Nucleosome positioning profiles were made by hybridizing mononucleosomal DNA over MNase digested purified genomic DNA on 380k NimbleGen arrays with 10 bp probe spacing. Expression profiles were made as H3.3 RNAi over white RNAi cohybridizations on spotted INDAC long oligo arrays. Every experiment was done in duplicate in the reverse dye orientation.
2010-06-23 | E-GEOD-16882 | biostudies-arrayexpress
Project description:Linker histones are involved in the formation of higher-order chromatin structure. Although linker histones have been implicated in the regulation of specific genes, it still remains unclear what their principal binding determinants are and how their repressive function in vitro can be reconciled with presumed broad binding in vivo. We generated a full genome, high resolution binding map of linker histone H1 in Drosophila Kc cells, using DamID. H1 binds at similar levels across much of the genome, both in classical euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites of active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes. Rather, all regions with low binding of H1 show enrichment of the histone variant H3.3 which itself has been linked to high nucleosome turnover. Upon knockdown of H3.3, we find that H1 levels increase at sites previously not covered with H1 with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1 at genomic sites with high rates of histone turnover. This antagonism provides a mechanism to keep diverse genomic sites in an open chromatin conformation. For this study, we generated DamID profiles of histone H1 and RpII18 in Drosophila Kc167 cells. Additionally, we generated H1 profiles in cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Nucleosome positioning profiles were generated in untreated cells and cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Profiles of expression changes were generated for H3.3B RNAi and H3.3A and H3.3B RNAi. DamID experiments for H1 and RpII18 were performed in Drosophila cell cultures. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing. Formaldehyde-assisted isolation of regulatotry elements (FAIRE) was performed in Drosophila Kc167 cells. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing over non-crosslinked genomic DNA. Nucleosome positioning profiles were made by hybridizing mononucleosomal DNA over MNase digested purified genomic DNA on 380k NimbleGen arrays with 10 bp probe spacing. Expression profiles were made as H3.3 RNAi over white RNAi cohybridizations on spotted INDAC long oligo arrays. Every experiment was done in duplicate in the reverse dye orientation.
2010-06-21 | E-GEOD-16883 | biostudies-arrayexpress
Project description:Linker histones are involved in the formation of higher-order chromatin structure. Although linker histones have been implicated in the regulation of specific genes, it still remains unclear what their principal binding determinants are and how their repressive function in vitro can be reconciled with presumed broad binding in vivo. We generated a full genome, high resolution binding map of linker histone H1 in Drosophila Kc cells, using DamID. H1 binds at similar levels across much of the genome, both in classical euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites of active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes. Rather, all regions with low binding of H1 show enrichment of the histone variant H3.3 which itself has been linked to high nucleosome turnover. Upon knockdown of H3.3, we find that H1 levels increase at sites previously not covered with H1 with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1 at genomic sites with high rates of histone turnover. This antagonism provides a mechanism to keep diverse genomic sites in an open chromatin conformation. For this study, we generated DamID profiles of histone H1 and RpII18 and a FAIRE profile in Drosophila Kc167 cells. Additionally, we generated H1 profiles in cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Nucleosome positioning profiles were generated in untreated cells and cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Profiles of expression changes were generated for H3.3B RNAi and H3.3A and H3.3B RNAi. DamID experiments for H1 and RpII18 were performed in Drosophila cell cultures. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing. Formaldehyde-assisted isolation of regulatotry elements (FAIRE) was performed in Drosophila Kc167 cells. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing over non-crosslinked genomic DNA. Nucleosome positioning profiles were made by hybridizing mononucleosomal DNA over MNase digested purified genomic DNA on 380k NimbleGen arrays with 10 bp probe spacing. Expression profiles were made as H3.3 RNAi over white RNAi cohybridizations on spotted INDAC long oligo arrays. Every experiment was done in duplicate in the reverse dye orientation.
2010-05-16 | E-GEOD-19764 | biostudies-arrayexpress