共查询到20条相似文献,搜索用时 31 毫秒
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Genomewide histone acetylation microarrays 总被引:2,自引:0,他引:2
Histone acetylation and methylation are important regulators of gene activity. Chromatin immunoprecipitation (ChIP or ChrIP) has made it possible to examine not only the state of histone acetylation at a gene but also that of histone methylation and may soon be extended to other histone modifications such as phosphorylation and ubiquitination. In principle such studies are possible as long as an antibody is available to the particular histone modification. Once a target gene is identified it is instructive to see the effect of mutating putative enzymes responsible for the modification to determine how a particular enzyme is responsible for altering chromatin of that gene. Although specific target genes have been studied that contain such modifications recent technical advances have made it possible to study histone modifications genomewide. This not only allows for alternate views of particular paradigms to be investigated, but also uncovers chromosomal patterns of histone modification that would be missed in analyzing individual genes. We describe here an approach to rapidly study histone modifications genomewide by combining chromatin immunoprecipitation and DNA microarrays. 相似文献
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Shingo Nishikori Takamitsu Hattori Stephen M. Fuchs Norihisa Yasui John Wojcik Akiko Koide Brian D. Strahl Shohei Koide 《Journal of molecular biology》2012,424(5):391-399
Antibodies directed against histone posttranslational modifications (PTMs) are critical tools in epigenetics research, particularly in the widely used chromatin immunoprecipitation (ChIP) experiments. However, a lack of quantitative methods for characterizing such antibodies has been a major bottleneck in accurate and reproducible analysis of histone modifications. Here, we report a simple and sensitive method for quantitatively characterizing polyclonal and monoclonal antibodies for histone PTMs in a ChIP-like format. Importantly, it determines the apparent dissociation constants for the interactions of an antibody with peptides harboring cognate or off-target PTMs. Analyses of commercial antibodies revealed large ranges of affinity, specificity and binding capacity as well as substantial lot-to-lot variations, suggesting the importance of quantitatively characterizing each antibody intended to be used in ChIP experiments and optimizing experimental conditions accordingly. Furthermore, using this method, we identified additional factors potentially affecting the interpretation of ChIP experiments. 相似文献
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Kayla M. Harmeyer Paul F. South Brett Bishop Joe Ogas Scott D. Briggs 《Nucleic acids research》2015,43(6):e38
Genome-wide chromatin immunoprecipitation (ChIP) studies have brought significant insight into the genomic localization of chromatin-associated proteins and histone modifications. The large amount of data generated by these analyses, however, require approaches that enable rapid validation and analysis of biological relevance. Furthermore, there are still protein and modification targets that are difficult to detect using standard ChIP methods. To address these issues, we developed an immediate chromatin immunoprecipitation procedure which we call ZipChip. ZipChip significantly reduces the time and increases sensitivity allowing for rapid screening of multiple loci. Here we describe how ZipChIP enables detection of histone modifications (H3K4 mono- and trimethylation) and two yeast histone demethylases, Jhd2 and Rph1, which were previously difficult to detect using standard methods. Furthermore, we demonstrate the versatility of ZipChIP by analyzing the enrichment of the histone deacetylase Sir2 at heterochromatin in yeast and enrichment of the chromatin remodeler, PICKLE, at euchromatin in Arabidopsis thaliana. 相似文献
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The Current State of Chromatin Immunoprecipitation 总被引:2,自引:0,他引:2
Philippe Collas 《Molecular biotechnology》2010,45(1):87-100
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Peach SE Rudomin EL Udeshi ND Carr SA Jaffe JD 《Molecular & cellular proteomics : MCP》2012,11(5):128-137
The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code." We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living cells. 相似文献
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Chromatin Immuno Precipitation (ChIP) profiling detects in vivo protein-DNA binding, and has revealed a large combinatorial complexity in the binding of chromatin associated proteins and their post-translational modifications. To fully explore the spatial and combinatorial patterns in ChIP-profiling data and detect potentially meaningful patterns, the areas of enrichment must be aligned and clustered, which is an algorithmically and computationally challenging task. We have developed CATCHprofiles, a novel tool for exhaustive pattern detection in ChIP profiling data. CATCHprofiles is built upon a computationally efficient implementation for the exhaustive alignment and hierarchical clustering of ChIP profiling data. The tool features a graphical interface for examination and browsing of the clustering results. CATCHprofiles requires no prior knowledge about functional sites, detects known binding patterns "ab initio", and enables the detection of new patterns from ChIP data at a high resolution, exemplified by the detection of asymmetric histone and histone modification patterns around H2A.Z-enriched sites. CATCHprofiles' capability for exhaustive analysis combined with its ease-of-use makes it an invaluable tool for explorative research based on ChIP profiling data. CATCHprofiles and the CATCH algorithm run on all platforms and is available for free through the CATCH website: http://catch.cmbi.ru.nl/. User support is available by subscribing to the mailing list catch-users@bioinformatics.org. 相似文献
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Post-translational histone modifications and histone variants generate complexity in chromatin to enable the many functions of the chromosome. Recent studies have mapped histone modifications across the Saccharomyces cerevisiae genome. These experiments describe how combinations of modified and unmodified states relate to each other and particularly to chromosomal landmarks that include heterochromatin, subtelomeric chromatin, centromeres, origins of replication, promoters and coding regions. Such patterns might be important for the regulation of heterochromatin-mediated silencing, chromosome segregation, DNA replication and gene expression. 相似文献
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Chromatin immunoprecipitation (ChIP) is routinely used to examine epigenetic modification of histones at specific genomic locations. However, covalent modifications of histone tails can serve as docking sites for chromatin regulatory factors. As such, association of these regulatory factors with chromatin could cause steric hindrance for antibody recognition, resulting in an underestimation of the relative enrichment of a given histone modification at specific loci. To overcome this problem, we have developed a native ChIP protocol to study covalent modification of histones that takes advantage of hydroxyapatite (HAP) chromatography to wash away chromatin-associated proteins before the immunoprecipitation of nucleosomes. This fast and simple procedure consists of five steps: nuclei isolation from cultured cells; fragmentation of chromatin using MNase; purification of nucleosomes using HAP; immunoprecipitation of modified nucleosomes; and qPCR analysis of DNA associated with modified histones. Nucleosomes prepared in this manner are free of contaminating proteins and permit an accurate evaluation of relative abundance of different covalent histone modifications at specific genomic loci. Completion of this protocol requires approximately 1.5 d. 相似文献
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Genome-scale ChIP-chip analysis using 10,000 human cells 总被引:2,自引:0,他引:2