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CATCH-IT method for measuring nucleosome turnover

ABSTRACT: Direct measurement of nucleosome turnover dynamics by using co-translational incorporation of the methionine (Met) surrogate azidohomoalaine (Aha) into proteins and subsequent ligation of biotin to Aha-containing proteins through the [3+2] cycloaddition reaction between the azide group of Aha and an alkyne linked to biotin. To measure turnover rates, we treat cells briefly with Aha, couple biotin to nucleosomes containing newly incorporated histones, affinity purify with strepavidin, wash stringently to remove non-histone proteins and H2A/H2B dimers, and analyze the affinity-purified DNA using tiling microarrays. We call this strategy 'CATCH-IT' for Covalent Attachment of Tags to Capture Histones and Identify Turnover. Keywords: Chromatin affinity-purification on microarray All experiments were done using strepavidin pulldown DNA cohybridized with total input DNA to the same array. Two channels per array, Cy5 and Cy3, were used in each experiment.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Jorja Henikoff

PROVIDER: E-GEOD-19788 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Genome-wide kinetics of nucleosome turnover determined by metabolic labeling of histones.

Deal Roger B RB Henikoff Jorja G JG Henikoff Steven S

Science (New York, N.Y.) 20100501 5982

Nucleosome disruption and replacement are crucial activities that maintain epigenomes, but these highly dynamic processes have been difficult to study. Here, we describe a direct method for measuring nucleosome turnover dynamics genome-wide. We found that nucleosome turnover is most rapid over active gene bodies, epigenetic regulatory elements, and replication origins in Drosophila cells. Nucleosomes turn over faster at sites for trithorax-group than polycomb-group protein binding, suggesting th ...[more]

PMID: 20508129

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CATCH-IT method for measuring nucleosome turnover

Project description:Direct measurement of nucleosome turnover dynamics by using co-translational incorporation of the methionine (Met) surrogate azidohomoalaine (Aha) into proteins and subsequent ligation of biotin to Aha-containing proteins through the [3+2] cycloaddition reaction between the azide group of Aha and an alkyne linked to biotin. To measure turnover rates, we treat cells briefly with Aha, couple biotin to nucleosomes containing newly incorporated histones, affinity purify with strepavidin, wash stringently to remove non-histone proteins and H2A/H2B dimers, and analyze the affinity-purified DNA using tiling microarrays. We call this strategy 'CATCH-IT' for Covalent Attachment of Tags to Capture Histones and Identify Turnover. Keywords: Chromatin affinity-purification on microarray

2010-05-27 | GSE19788 | GEO

Baculovirus-encoded protein expression for epigenomic profiling in Drosophila cells

Project description:Baculovirus-encoded proteins can be efficiently produced following infection of different Drosophila cell lines. Epitope-tagged proteins produced in baculovirus-infected cells provide genome-wide profiles of the histone variant H2Av that are comparable to those produced by stably transformed cell lines. Keywords: Chromatin affinity-purification on microarray All experiments were done using strepavidin or FLAG pulldown DNA cohybridized with total input DNA to the same array. Two channels per array, Cy5 and Cy3, were used in each experiment.

2010-07-12 | E-GEOD-21416 | biostudies-arrayexpress

INTACT method for gene expression and chromatin profiling of individual cell types within a tissue

Project description:We used in vivo biotin labeling of a nuclear envelope protein in individual cell types followed by affinity isolation of labeled nuclei to measure gene expression and chromatin features of the hair and non-hair cell types of the Arabidopsis root epidermis. Keywords: Chromatin affinity-purification on microarray All experiments were done using two channels per chip. Two types of experiments were performed. Expression profiling was performed by comparing cDNAs synthesized from nuclear RNA isolated from INTACT-prepared Arabidopsis nuclei to total sonicated genomic DNA isolated from Arabidopsis plants. Chromatin profiling was performed using INTACT-prepared Arabidopsis nuclei. DNA isolated by Chromatin immunoprecipitation (ChIP) using antibodies to modified histones was compared to histone H3 ChIP DNA from the same sample.

2010-06-13 | E-GEOD-19654 | biostudies-arrayexpress

Doxorubicin enhances nucleosome turnover around promoters

Project description:We use a metabolic labeling strategy for directly measuring nucleosome turnover to examine the effect of doxorubicin on chromatin dynamics in squamous cell carcinoma cell lines derived from genetically defined mice. We find that doxorubicin enhances nucleosome turnover around gene promoters, and turnover correlates with gene expression level. Keywords: Chromatin affinity-purification on microarray 26 CATCH-IT arrays and 8 expression arrays.

2013-04-19 | E-GEOD-43753 | biostudies-arrayexpress

The nucleosomal barrier to promoter escape by RNA Polymerase II is overcome by the chromatin remodeler Chd1 [NimbleGen]

Project description:We use a metabolic labeling strategy for directly measuring nucleosome turnover to determine the role of chromatin remodeler Chd1 on chromatin dynamics in mouse embryonic fibroblasts expressing with wildtype-Chd1 and dominant negative K510R-Chd1. We find that expression of K510R-Chd1 decreases nucleosome turnover at the promoter and increases turnover within the gene body. 10 Illumina sequencing samples and 4 Nimblegen array samples

2014-04-15 | E-GEOD-52347 | biostudies-arrayexpress

Chem-seq data from 5'-biotin-DRB-labelled human U2OS cells

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Time-ChIP in ESCs and NSCs

Project description:Histone H3.3 turnover in ESCs and NSCs was determined using time-ChIP. This was performed by biotin-labeling H3.3_SNAP and following the loss of labeled histones over time. H3.1 time-ChIP was also performed although the resolution of these data is limited due to the depth of sequencing required.

2016-06-20 | GSE78876 | GEO

Histone H2A.Z and DNA methylation are mutually antagonistic chromatin marks

Project description:Eukaryotic chromatin is separated into functional domains differentiated by posttranslational histone modifications, histone variants, and DNA methylation. Methylation is associated with repression of transcriptional initiation in plants and animals, and is frequently found in transposable elements. Proper methylation patterns are critical for eukaryotic development, and aberrant methylation-induced silencing of tumor suppressor genes is a common feature of human cancer. In contrast to methylation, the histone variant H2A.Z is preferentially deposited by the Swr1 ATPase complex near 5' ends of genes where it promotes transcriptional competence. How DNA methylation and H2A.Z influence transcription remains largely unknown. Here we show that in the plant Arabidopsis thaliana, regions of DNA methylation are quantitatively deficient in H2A.Z. Exclusion of H2A.Z is seen at sites of DNA methylation in the bodies of actively transcribed genes and in methylated transposons. Mutation of the MET1 DNA methyltransferase, which causes both losses and gains of DNA methylation, engenders opposite changes in H2A.Z deposition, while mutation of the PIE1 subunit of the Swr1 complex that deposits H2A.Z17 leads to genome-wide hypermethylation. Our findings indicate that DNA methylation can influence chromatin structure and effect gene silencing by excluding H2A.Z, and that H2A.Z protects genes from DNA methylation. Keywords: Affinity-purification on microarray All experiments were done using two channels per chip. DNA methylation experiments compared immunoprecipitated, methylated DNA to control genomic DNA. H2A.Z experiments compared whole micrococcal nuclease-treated affinity-purified chromatin to input chromatin used for affinity purification. Affinity purification was performed using either biotin-tagged H2A.Z, pulled down using streptavidin, or endogenous H2A.Z pulled down using an anti-H2A.Z antibody.

2008-10-02 | E-GEOD-12212 | biostudies-arrayexpress

Tissue-specific nuclei purification from animal models for genome-wide expression and chromatin profiling.

Project description:Here we describe a method for purifying nuclei from specific tissues of animal models that allows simultaneous analysis of both expression and chromatin profiles. The method is based on in vivo labeling of the nuclear envelope and subsequent affinity purification. We describe the use of the method to isolate nuclei from muscle. We determined genes preferentially expressed in muscle cells of adult C. elegans by analyzing RNA from total and affinity-puridied muscle nuclei using microarrays. We also determined the nucleosome occupancy in these nuclei by micrococcal nuclease mapping and paired-end sequencing.

2011-12-05 | E-GEOD-31602 | biostudies-arrayexpress

Genome-wide profiling of salt fractions maps physical properties of chromatin

Project description:We applied genome-wide profiling to successive salt-extracted fractions of micrococcal nuclease-treated Drosophila chromatin. Chromatin fractions extracted with 80mM or 150mM NaCl after digestion contain predominantly mononucleosomes and represent calssical 'active' chromatin. Profiles of these low-salt-soluble fractions display phased nucleosomes over transcriptionally active genes that are locally depleted of histone H3.3 and correspond closely to profiles of RNA polymerase II. Nearly quantitative recovery of chromatin is obtained with 600mM NaCl, however, the remaining insoluble chromatin is enriched in actively transcribed regions. Salt-insoluble chromatin likely represents oligonucleosomes that are attached to large protein complexes. Both low-salt extracted and insoluble chromatin are rich in sequences that correspond to epigenetic regulatory elements genome-wide. The presence of active chromatin at both extremes of salt solubility suggests that these salt fractions capture bound and unbound intermediates in active processes, thus providing a simple, powerful strategy for mapping epigenome dynamics. Keywords: Chromatin affinity-purification on microarray For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf All experiments were done using two channels per chip, comparing DNAs extracted from either salt-extracted or insoluble chromatin to whole nuclear chromatin, whole nuclear chromatin to randomly fragmented genomic DNA, streptavidin-bound biotin-tagged histone-variant-containing chromatin to salt-extracted chromatin, gel-purified mononucleosomes to whole EDTA-extracted soluble chromatin, streptavidin-bound biotin-tagged histone-variant-containing chromatin to whole EDTA-extracted soluble chromatin, or oligo(dT)-primed cDNA to randomly fragmented genomic DNA from S2 cell nuclei.

2008-12-11 | E-GEOD-13217 | biostudies-arrayexpress

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