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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Proteogenomics analysis reveals specific genomic orientations at distal regulatory regions composed by non-canonical histone variants

ABSTRACT: Histone variants (H3.1, H3.3, H2A, and macroH2A) were studied about their proteomic and genomic distribution in Hela cells HeLa S3 cells stably expressing either FLAG-tagged H3.1, and H3.3 were grown in suspension in Joklik media containing 10% newborn calf serum (Hyclone), 1% GlutaMAX (Invitrogen), and 1% penicillin-streptomycin, and cells were harvested at log phase. Nuclei were isolated, mononucleosomes were subsequently obtained from these cell lines. Cells were lysed in hypotonic TMSD buffer to isolate nuclei, which were then digested with micrococcal nuclease. The resulting mononucleosomes were immunoprecipitated with anti-FLAG M2-agarose beads (Sigma) and eluted with FLAG peptide (Sigma)

ORGANISM(S): Homo sapiens

SUBMITTER: Kyoung Jae Won

PROVIDER: E-GEOD-64652 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Proteogenomics analysis reveals specific genomic orientations of distal regulatory regions composed by non-canonical histone variants.

Won Kyoung-Jae KJ Choi Inchan I LeRoy Gary G Zee Barry M BM Sidoli Simone S Gonzales-Cope Michelle M Garcia Benjamin A BA

Epigenetics & chromatin 20150410

<h4>Background</h4>Histone variants play further important roles in DNA packaging and controlling gene expression. However, our understanding about their composition and their functions is limited.<h4>Results</h4>Integrating proteomic and genomic approaches, we performed a comprehensive analysis of the epigenetic landscapes containing the four histone variants H3.1, H3.3, H2A.Z, and macroH2A. These histones were FLAG-tagged in HeLa cells and purified using chromatin immunoprecipitation (ChIP). B ...[more]

PMID: 25878728

Similar Datasets

Proteogenomics analysis reveals specific genomic orientations at distal regulatory regions composed by non-canonical histone variants

Project description:Histone variants (H3.1, H3.3, H2A, and macroH2A) were studied about their proteomic and genomic distribution in Hela cells

2015-12-22 | GSE64652 | GEO

Proteo-Genomic Characterization and Mapping of Nucleosomes Decoded by Brd and HP1 Proteins (Chip-Seq data)

Project description:Background: Histone post-translational modifications (PTMs) constitute a branch of epigenetic mechanisms that can control the expression of eukaryotic genes in a heritable manner. Recent studies have identified several PTM-binding proteins containing diverse specialized domains whose recognition of specific PTM sites leads to gene activation or repression. Here, we present a high-throughput proteogenomic platform designed to characterize the nucleosomal make-up of chromatin enriched with a set of histone PTM-binding proteins known as histone PTM readers. We support our findings with gene expression data correlating to PTM distribution. Results: We isolated human mononucleosomes bound by the bromodomain-containing proteins Brd2, Brd3 and Brd4, and by the chromodomain-containing heterochromatin proteins HP1alpha and HP1beta. Histone PTMs were quantified by mass spectrometry (ChIP-qMS), and their associated DNAs were mapped using deep sequencing. Our results reveal that Brd- and HP1-bound nucleosomes are enriched in histone PTMs consistent with actively transcribed euchromatin and silent heterochromatin, respectively. Data collected using RNA-Seq (GSM301568) show that Brd-bound sites correlate with highly expressed genes. In particular, Brd3 and Brd4 are most enriched on nucleosomes located within HOX gene clusters, whose expression is reduced upon Brd4 depletion by shRNA. Conclusions: Proteogenomic mapping of histone PTM readers, alongside the characterization of their local chromatin environments and transcriptional information, should prove useful for determining how histone PTMs are bound by these readers and how they contribute to distinct transcriptional states. Examination of Brd and HP1 proteins-binding sites in HEK293 cells.

2012-07-31 | E-GEOD-39579 | biostudies-arrayexpress

De novo deposition of the histone H3 variants H3.1 and H3.3 during DNA replication

Project description:We developed a new sequencing assay to track the de novo deposition of the histone H3 variants H3.1 and H3.3 during S phase. We use cells stably expressing H3.1-SNAP or H3.3-SNAP, and synchronize them in G1/S by double-thymidine block. The SNAP-tag enables to discriminate newly synthesized histones from preexisting ones, via a quench-chase-capture strategy. We applied this strategy to isolate new H3.1 and H3.3 after releasing cells into S phase, and probed their distribution by MNase digestion and sequencing. We could thus characterize H3.1 and H3.3 dynamics from early to mid S phase at genome-wide resolution. We further applied our method to investigate the consequences of perturbations upon deletion of the H3.3 chaperone HIRA. We used HIRA knockout and control cells, and compared H3.1 and H3.3 distribution to early replication patterns by EdU labeling and sequencing of nascent DNA.

2022-03-31 | E-MTAB-10619 | biostudies-arrayexpress

Dynamic Deposition of the Histone H3.3 Variant Accompanies Developmental Remodeling of Arabidopsis Transcriptome (mRNA-Seq)

Project description:In animals, replication coupled histone H3.1 can be distinguished from replication independent histone H3.3. H3.3 variants are enriched at active genes and their promoters. Furthermore, H3.3 is specifically incorporated upon gene activation. Histone H3 variants evolved independently in plants and animals and it is unclear whether different replication independent H3.3 variants developed similar properties in both phyla. We studied Arabidopsis H3 variants in order to find core properties of this class of histones. Here we present genome-wide maps of H3.3 and H3.1 enrichment and the dynamic changes of their profiles upon cell division arrest. We find H3.3 enrichment to positively correlate with gene expression and to be biased towards the transcription termination site. While heterochromatic regions are mostly depleted of H3.3, H3.1 shows a more even distribution along the genome. We report that in planta, dynamic changes in H3.3 profiles are associated with the extensive remodeling of the transcriptome that occurs during cell differentiation. We propose that H3.3 dynamics are linked to transcription and are involved in resetting covalent histone marks at a genomic scale during plant development. Similarities between plant and animal H3.3 deposition profiles indicate that H3 variants likely result from functionally convergent evolution. Analysis of 2 different histone H3 variants and transcriptome in 2 conditions.

2012-05-15 | E-GEOD-36626 | biostudies-arrayexpress

Dynamic Deposition of the Histone H3.3 Variant Accompanies Developmental Remodeling of Arabidopsis Transcriptome (ChIP-Seq)

2012-05-15 | E-GEOD-36629 | biostudies-arrayexpress

Genome-wide analysis of histone H3.1 and H3.3 variants in Arabidopsis thaliana

Project description:Nucleosomes package eukaryotic DNA and are composed of four different histone proteins, H3, H4, H2A and H2B. Histone H3 has two main variants, H3.1 and H3.3, which show different genomic localization patterns in animals. We profiled H3.1 and H3.3 variants in the genome of the plant Arabidopsis thaliana and show that the localization of these variants shows broad similarity in plants and animals, in addition to some unique features. H3.1 was enriched in silent areas of the genome including regions containing the repressive chromatin modifications H3 lysine 27 methylation, H3 lysine 9 methylation, and DNA methylation. In contrast, H3.3 was enriched in actively transcribed genes, especially peaking at the 3’ end of genes, and correlated with histone modifications associated with gene activation such as histone H3 lysine 4 methylation, and H2B ubiquitylation, as well as by RNA Pol II occupancy. Surprisingly, both H3.1 and H3.3 were enriched on defined origins of replication, as was overall nucleosome density, suggesting a novel characteristic of plant origins. Our results are broadly consistent with the hypothesis that H3.1 acts as the canonical histone that is incorporated during DNA replication, whereas H3.3 acts as the replacement histone that can be incorporated outside of S-phase during chromatin disrupting processes like transcription. ChIP-seq - 4 samples: 2 experiment and 2 controls RNA-seq - 1 sample

2012-03-05 | E-GEOD-34840 | biostudies-arrayexpress

Genome-wide analysis of H3.3 dissociation in mouse embryonic stem cells

Project description:Using Tet-inhibited expression of epitope-tagged H3.3 combined with ChIP-Seq we undertook genome-wide measurements of H3.3 dissociation rates across the ESC genome and examined the relationship between H3.3-nucleosome turnover and ESC-specific transcription factors, chromatin modifiers and epigenetic marks. To measure dissociation rates of H3.3, we utilized a TET-repressible ESC line, ES[MC1R(20)], with the expression cassette integrated at the ROSA26 locus. We transfected MC1R ESCs with HA/FLAG-tagged H3.3 controlled by tetracycline response elements. For ‘TET-OFF’ experiments, ESCs were cultured over several passages (weeks) on feeder cells in the absence of DOX and were subsequently passaged onto feeder-free plates prior to the inhibition of HA-H3.3 expression. To repress HA/FLAG-H3.3 expression we treated cells with 2 ?g/ml doxycycline hyclate before crosslinking with formaldehyde at various time points. Measurement of H3.3-HA enrichment over time-course was performed using two replicates of ChIP-seq experiments. As a control, input DNA sequencing was performed for every time point

2015-05-14 | E-GEOD-63641 | biostudies-arrayexpress

Quantitative middle-down MS analysis of histone H3 proteoforms throughout lifespan in mice

Project description:Replication-independent deposition of histone variant H3.3 into chromatin is essential for many biological processes, including development, oogenesis and nuclear reprogramming. Unlike replication-dependent H3.1/2 isoforms, H3.3 is expressed throughout the cell cycle and becomes enriched in postmitotic cells with age. However, lifelong dynamics of H3 variant replacement and the impact of this process on chromatin organization remain largely undefined. To address this, we investigated genome-wide changes in histone H3 variants composition and H3 modification abundances throughout the lifespan in mice using quantitative mass spectrometry (MS) – based middle-down proteomics strategy. Using middle-down MS we demonstrate that H3.3 accumulates in the chromatin of various somatic mouse tissues throughout life, resulting in near complete replacement of H3.1/2 isoforms by the late adulthood. Accumulation of H3.3 is associated with profound changes in the global level of H3 methylation. H3.3-containing chromatin exhibits distinct stable levels of H3R17me2 and H3K36me2, different from those on H3.1/H3.2-containing chromatin, indicating a direct link between H3 variant exchange and histone methylation dynamics with age. In summary, our study provides the first time comprehensive characterization of dynamic changes in the H3 modification landscape during mouse lifespan and links these changes to the age-dependent accumulation of histone variant H3.3.

2017-07-28 | PXD005300 | Pride

HDAC mediated suppression of histone turnover promotes epigenetic stability of heterochromatin

Project description:Analysis of histone H3 turnover in wild-type and mutant fission yeast cells by using MNase-ChIP-chip Exponentially growing cultures of fission yeast cells expressing an ectopic copy of FLAG tagged histone H3 under the control of inv1 promoter were synchronized and expression of H3-FLAG was induced by changing the carbon source of the medium to Sucrose. Cells were crosslinked with 1% Formaldehyde and chromatin was fragmented into mononucleosomes by using micrococcal nuclease (MNase). Chromatin immunoprecipitated DNA recovered with anti-FLAG antibody from wild-type and mutant fission yeast cells and whole cell extracts DNA were amplified by random priming and respectively labeled with Cy5 and Cy3. Labeled DNA samples were mixed and hybridized onto 44k pombe Agilent oligo arrays. Data were processed using Agilent scanner and Feature Extraction software.

2013-04-21 | E-GEOD-45378 | biostudies-arrayexpress

Factors influencing in vivo deposition of histone variants H3.1 and H3.3

Project description:Establishment of a proper chromatin landscape is central to genome function. Here, we explain H3 variant distribution by specific targeting and dynamics of deposition involving the CAF-1 and HIRA histone chaperones. Impairing replicative H3.1 incorporation via CAF-1 enables an alternative H3.3 deposition at replication sites via HIRA. Conversely, the H3.3 incorporation throughout the cell cycle via HIRA cannot be replaced by H3.1. ChIP-seq analyses reveal correlation between HIRA-dependent H3.3 accumulation and RNA pol II at transcription sites and specific regulatory elements, further supported by their biochemical association. Remarkably, the HIRA complex shows unique DNA binding properties and depleting HIRA increases DNA sensitivity to nucleases. We propose that protective gap-filling of naked DNA by HIRA leads to a broad distribution of H3.3, and HIRA association with Pol II ensures local H3.3 enrichment at specific sites. Examination of genome-wide localization of two histone H3 variants.

2011-12-16 | E-GEOD-31794 | biostudies-arrayexpress

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