The Experts below are selected from a list of 54552 Experts worldwide ranked by ideXlab platform
Hodaka Fujii - One of the best experts on this subject based on the ideXlab platform.
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identification of telomere associated molecules by engineered dna binding molecule mediated Chromatin Immunoprecipitation enchip
Scientific Reports, 2013Co-Authors: Toshitsugu Fujita, Yoshinori Asano, Junko Ohtsuka, Yoko Takada, Kazunobu Saito, Rieko Ohki, Hodaka FujiiAbstract:Biochemical analysis of molecular interactions in specific genomic regions requires their isolation while retaining molecular interactions in vivo. Here, we report isolation of telomeres by engineered DNA-binding molecule-mediated Chromatin Immunoprecipitation (enChIP) using a transcription activator-like (TAL) protein recognizing telomere repeats. Telomeres recognized by the tagged TAL protein were immunoprecipitated with an antibody against the tag and subjected to identification of telomere-binding molecules. enChIP-mass spectrometry (enChIP-MS) targeting telomeres identified known and novel telomere-binding proteins. The data have been deposited to the ProteomeXchange with identifier PXD000461. In addition, we showed that RNA associated with telomeres could be isolated by enChIP. Identified telomere-binding molecules may play important roles in telomere biology. enChIP using TAL proteins would be a useful tool for biochemical analysis of specific genomic regions of interest.
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efficient isolation of specific genomic regions and identification of associated proteins by engineered dna binding molecule mediated Chromatin Immunoprecipitation enchip using crispr
Biochemical and Biophysical Research Communications, 2013Co-Authors: Toshitsugu Fujita, Hodaka FujiiAbstract:Isolation of specific genomic regions retaining molecular interactions is necessary for their biochemical analysis. Here, we established a novel method, engineered DNA-binding molecule-mediated Chromatin Immunoprecipitation (enChIP), for purification of specific genomic regions retaining molecular interactions. We showed that enChIP using the CRISPR system efficiently isolates specific genomic regions. In this form of enChIP, specific genomic regions are immunoprecipitated with antibody against a tag(s), which is fused to a catalytically inactive form of Cas9 (dCas9), which is co-expressed with a guide RNA (gRNA) and recognizes endogenous DNA sequence in the genomic regions of interest. enChIP-mass spectrometry (enChIP-MS) targeting endogenous loci identified associated proteins. enChIP using the CRISPR system would be a convenient and useful tool for dissecting Chromatin structure of genomic regions of interest.
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direct identification of insulator components by insertional Chromatin Immunoprecipitation
PLOS ONE, 2011Co-Authors: Toshitsugu Fujita, Hodaka FujiiAbstract:Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. However, non-biased methods to identify molecules bound to specific genomic loci in vivo are limited. Here, we applied insertional Chromatin Immunoprecipitation (iChIP) to direct identification of components of insulator complexes, which function as boundaries of Chromatin domain. We found that the chicken β-globin HS4 (cHS4) insulator complex contains an RNA helicase protein, p68/DDX5; an RNA species, steroid receptor RNA activator 1; and a nuclear matrix protein, Matrin-3, in vivo. Binding of p68 and Matrin-3 to the cHS4 insulator core sequence was mediated by CCCTC-binding factor (CTCF). Thus, our results showed that it is feasible to directly identify proteins and RNA bound to a specific genomic region in vivo by using iChIP.
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insertional Chromatin Immunoprecipitation a method for isolating specific genomic regions
Journal of Bioscience and Bioengineering, 2009Co-Authors: Akemi Hoshino, Hodaka FujiiAbstract:We established a novel method, insertional Chromatin Immunoprecipitation (iChIP), for isolation of specific genomic regions. In iChIP, specific genomic domains are immunoprecipitated with antibody against a tag, which is fused to the DNA-binding domain of an exogenous DNA-binding protein, whose recognition sequence is inserted into the genomic domains of interest. The iChIP method will be a useful tool for dissecting Chromatin structure of genomic region of interest.
Kevin Struhl - One of the best experts on this subject based on the ideXlab platform.
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chipper discovering transcription factor targets from Chromatin Immunoprecipitation microarrays using variance stabilization
Genome Biology, 2005Co-Authors: Francis D Gibbons, Kevin Struhl, Markus Proft, Frederick P RothAbstract:Chromatin Immunoprecipitation combined with microarray technology (Chip2) allows genome-wide determination of protein-DNA binding sites. The current standard method for analyzing Chip2 data requires additional control experiments that are subject to systematic error. We developed methods to assess significance using variance stabilization, learning error-model parameters without external control experiments. The method was validated experimentally, shows greater sensitivity than the current standard method, and incorporates false-discovery rate analysis. The corresponding software ('Chipper') is freely available. The method described here should help reveal an organism's transcription-regulatory 'wiring diagram'.
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genomic studies with escherichia coli melr protein applications of Chromatin Immunoprecipitation and microarrays
Journal of Bacteriology, 2004Co-Authors: David C Grainger, Kevin Struhl, Tim W Overton, Nikos Reppas, Joseph T Wade, Eiji Tamai, Jon L Hobman, Chrystala Constantinidou, George M Church, Stephen J W BusbyAbstract:Escherichia coli MelR protein is a transcription activator that is essential for melibiose-dependent expression of the melAB genes. We have used Chromatin Immunoprecipitation to study the binding of MelR and RNA polymerase to the melAB promoter in vivo. Our results show that MelR is associated with promoter DNA, both in the absence and presence of the inducer melibiose. In contrast, RNA polymerase is recruited to the melAB promoter only in the presence of inducer. The MelR DK261 positive control mutant binds to the melAB promoter but cannot recruit RNA polymerase. Further analysis of immunoprecipitated DNA, by using an Affymetrix GeneChip array, showed that the melAB promoter is the major, if not the sole, target in E. coli for MelR. This was confirmed by a transcriptomics experiment to analyze RNA in cells either with or without melR.
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Chromatin Immunoprecipitation for determining the association of proteins with specific genomic sequences in vivo
Current protocols in pharmacology, 2004Co-Authors: Oscar M Aparicio, Joseph V. Geisberg, Kevin StruhlAbstract:Chromatin Immunoprecipitation (ChIP) is a powerful and widely applied technique for detecting the association of individual proteins with specific genomic regions in vivo. Live cells are treated with formaldehyde to generate protein-protein and protein- DNA cross-links between molecules in close proximity on the Chromatin template in vivo. DNA sequences that cross-link with a given protein are selectively enriched and reversal of the formaldehyde cross-link permits recovery and quantitative analysis of the immunoprecipitated DNA. As formaldehyde inactivates cellular enzymes essentially immediately upon addition to cells, ChIP provides snapshots of protein-protein and protein- DNA interactions at a particular time point, and hence is useful for kinetic analysis of events occurring on chromosomal sequences in vivo. In addition, ChIP can be combined with microarray technology to identify the location of specific proteins on a genome-wide basis. This unit describes the ChIP protocol for Saccharomyces cerevisiae; however, it is also applicable to other organisms.
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Chromatin Immunoprecipitation for determining the association of proteins with specific genomic sequences in vivo
Current protocols in molecular biology, 2004Co-Authors: Oscar M Aparicio, Joseph V. Geisberg, Edward A Sekinger, Annie Yang, Zarmik Moqtaderi, Kevin StruhlAbstract:Chromatin Immunoprecipitation (ChIP) is a powerful and widely applied technique for detecting the association of individual proteins with specific genomic regions in vivo. Live cells are treated with formaldehyde to generate protein-protein and protein-DNA cross-links between molecules that are in close proximity on the Chromatin template in vivo. DNA sequences that cross-link with a given protein are selectively enriched, and reversal of the formaldehyde cross-linking permits recovery and quantitative analysis of the immunoprecipitated DNA. As formaldehyde inactivates cellular enzymes essentially immediately upon addition to cells, ChIP provides snapshots of protein-protein and protein-DNA interactions at a particular time point, and hence is useful for kinetic analysis of events occurring on chromosomal sequences in vivo. In addition, ChIP can be combined with microarray technology to identify the location of specific proteins on a genome-wide basis. Basic Protocol 1 in this unit describes the ChIP procedure for Saccharomyces cerevisiae; Basic Protocol 2 describes the corresponding steps for mammalian cells.
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quantitative sequential Chromatin Immunoprecipitation a method for analyzing co occupancy of proteins at genomic regions in vivo
Nucleic Acids Research, 2004Co-Authors: Joseph V. Geisberg, Kevin StruhlAbstract:Sequential Chromatin Immunoprecipitation (SeqChIP) is a procedure in which formaldehyde-crosslinked, protein–DNA complexes from living cells are subjected to two sequential Immunoprecipitations with antibodies of different specificity. SeqChIP has been used to address, in a qualitative manner, whether two proteins can simultaneously co-occupy a stretch of DNA in vivo. Here, we expand on our earlier work and describe theoretical and practical considerations for performing and interpreting SeqChIP experiments in a quantitative manner. We provide a detailed experimental procedure for designing and performing SeqChIP experiments as well as experimental examples of the three possible outcomes: full co-occupancy, no co-occupancy and partial co-occupancy. In some cases of partial co-occupancy, the order of Immunoprecipitations in SeqChIP can strongly influence the outcome. We experimentally confirm a quantitative parameter that provides a measure of co-occupancy of two proteins on a given region of DNA and provide information on how to interpret the results of SeqChIP experiments. Our quantitative treatment of SeqChIP data substantially expands the usefulness of the technique for elucidating molecular mechanisms in vivo.
Toshitsugu Fujita - One of the best experts on this subject based on the ideXlab platform.
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identification of telomere associated molecules by engineered dna binding molecule mediated Chromatin Immunoprecipitation enchip
Scientific Reports, 2013Co-Authors: Toshitsugu Fujita, Yoshinori Asano, Junko Ohtsuka, Yoko Takada, Kazunobu Saito, Rieko Ohki, Hodaka FujiiAbstract:Biochemical analysis of molecular interactions in specific genomic regions requires their isolation while retaining molecular interactions in vivo. Here, we report isolation of telomeres by engineered DNA-binding molecule-mediated Chromatin Immunoprecipitation (enChIP) using a transcription activator-like (TAL) protein recognizing telomere repeats. Telomeres recognized by the tagged TAL protein were immunoprecipitated with an antibody against the tag and subjected to identification of telomere-binding molecules. enChIP-mass spectrometry (enChIP-MS) targeting telomeres identified known and novel telomere-binding proteins. The data have been deposited to the ProteomeXchange with identifier PXD000461. In addition, we showed that RNA associated with telomeres could be isolated by enChIP. Identified telomere-binding molecules may play important roles in telomere biology. enChIP using TAL proteins would be a useful tool for biochemical analysis of specific genomic regions of interest.
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efficient isolation of specific genomic regions and identification of associated proteins by engineered dna binding molecule mediated Chromatin Immunoprecipitation enchip using crispr
Biochemical and Biophysical Research Communications, 2013Co-Authors: Toshitsugu Fujita, Hodaka FujiiAbstract:Isolation of specific genomic regions retaining molecular interactions is necessary for their biochemical analysis. Here, we established a novel method, engineered DNA-binding molecule-mediated Chromatin Immunoprecipitation (enChIP), for purification of specific genomic regions retaining molecular interactions. We showed that enChIP using the CRISPR system efficiently isolates specific genomic regions. In this form of enChIP, specific genomic regions are immunoprecipitated with antibody against a tag(s), which is fused to a catalytically inactive form of Cas9 (dCas9), which is co-expressed with a guide RNA (gRNA) and recognizes endogenous DNA sequence in the genomic regions of interest. enChIP-mass spectrometry (enChIP-MS) targeting endogenous loci identified associated proteins. enChIP using the CRISPR system would be a convenient and useful tool for dissecting Chromatin structure of genomic regions of interest.
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direct identification of insulator components by insertional Chromatin Immunoprecipitation
PLOS ONE, 2011Co-Authors: Toshitsugu Fujita, Hodaka FujiiAbstract:Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. However, non-biased methods to identify molecules bound to specific genomic loci in vivo are limited. Here, we applied insertional Chromatin Immunoprecipitation (iChIP) to direct identification of components of insulator complexes, which function as boundaries of Chromatin domain. We found that the chicken β-globin HS4 (cHS4) insulator complex contains an RNA helicase protein, p68/DDX5; an RNA species, steroid receptor RNA activator 1; and a nuclear matrix protein, Matrin-3, in vivo. Binding of p68 and Matrin-3 to the cHS4 insulator core sequence was mediated by CCCTC-binding factor (CTCF). Thus, our results showed that it is feasible to directly identify proteins and RNA bound to a specific genomic region in vivo by using iChIP.
Philippe Collas - One of the best experts on this subject based on the ideXlab platform.
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The Current State of Chromatin Immunoprecipitation
Molecular Biotechnology, 2010Co-Authors: Philippe CollasAbstract:The biological significance of interactions of nuclear proteins with DNA in the context of gene expression, cell differentiation, or disease has immensely been enhanced by the advent of Chromatin Immunoprecipitation (ChIP). ChIP is a technique whereby a protein of interest is selectively immunoprecipitated from a Chromatin preparation to determine the DNA sequences associated with it. ChIP has been widely used to map the localization of post-translationally modified histones, histone variants, transcription factors, or Chromatin modifying enzymes on the genome or on a given locus. In spite of its power, ChIP has for a long time remained a cumbersome procedure requiring large numbers of cells. These limitations have sparked the development of modifications to shorten the procedure, simplify sample handling and make ChIP amenable to small numbers of cells. In addition, the combination of ChIP with DNA microarray and high-throughput sequencing technologies has in recent years enabled the profiling of histone modification, histone variants, and transcription factor occupancy on a genome-wide scale. This review highlights the variations on the theme of the ChIP assay, the various detection methods applied downstream of ChIP, and examples of their application.
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fish n chips Chromatin Immunoprecipitation in the zebrafish embryo
Methods of Molecular Biology, 2009Co-Authors: Leif C Lindeman, Linn T Vogtkielland, Peter Alestrom, Philippe CollasAbstract:Chromatin Immunoprecipitation (ChIP) is arguably the assay of choice to determine the genomic localization of DNA- or Chromatin-binding proteins, including post-translationally modified histones, in cells. The increasing importance of the zebrafish, Danio rerio, as a model organism in functional genomics has recently sparked investigations of ChIP-based genome-scale mapping of modified histones on promoters, and studies on the role of specific transcription factors in developmental processes. ChIP assays used in these studies are cumbersome and conventionally require relatively large number of embryos. To simplify the procedure and to be able to apply the ChIP assay to reduced number of embryos, we re-evaluated the protocol for preparation of embryonic Chromatin destined to ChIP. We found that manual homogenization of embryos rather than protease treatment to remove the chorion enhances ChIP efficiency and quickens the assay. We also incorporated key steps from a recently published ChIP assay for small cell numbers. We report here a protocol for Immunoprecipitation of modified histones from mid-term blastula zebrafish embryos.
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μchip a rapid micro Chromatin Immunoprecipitation assay for small cell samples and biopsies
Nucleic Acids Research, 2008Co-Authors: John Arne Dahl, Philippe CollasAbstract:Chromatin Immunoprecipitation (ChIP) is a powerful technique for studying protein–DNA interactions. Drawbacks of current ChIP assays however are a requirement for large cell numbers, which limits applicability of ChIP to rare cell samples, and/or lengthy procedures with limited applications. There are to date no protocols for fast and parallel ChIPs of post-translationally modified histones from small cell numbers or biopsies, and importantly, no protocol allowing for investigations of transcription factor binding in small cell numbers. We report here the development of a micro (μ) ChIP assay suitable for up to nine parallel quantitative ChIPs of modified histones or RNA polymerase II from a single batch of 1000 cells. μChIP can also be downscaled to monitor the association of one protein with multiple genomic sites in as few as 100 cells. μChIP is applicable to small fresh tissue biopsies, and a cross-link-while-thawing procedure makes the assay suitable for frozen biopsies. Using μChIP, we characterize transcriptionally permissive and repressive histone H3 modifications on developmentally regulated promoters in human embryonal carcinoma cells and in osteosarcoma biopsies. μChIP creates possibilities for multiple parallel and rapid transcription factor binding and epigenetic analyses of rare cell and tissue samples.
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a rapid micro Chromatin Immunoprecipitation assay microchip
Nature Protocols, 2008Co-Authors: John Arne Dahl, Philippe CollasAbstract:Interactions of proteins with DNA mediate many critical nuclear functions. Chromatin Immunoprecipitation (ChIP) is a robust technique for studying protein-DNA interactions. Current ChIP assays, however, either require large cell numbers, which prevent their application to rare cell samples or small-tissue biopsies, or involve lengthy procedures. We describe here a 1-day micro ChIP (microChIP) protocol suitable for up to eight parallel histone and/or transcription factor Immunoprecipitations from a single batch of 1,000 cells. MicroChIP technique is also suitable for monitoring the association of one protein with multiple genomic sites in 100 cells. Alterations in cross-linking and Chromatin preparation steps also make microChIP applicable to approximately 1-mm(3) fresh- or frozen-tissue biopsies. From cell fixation to PCR-ready DNA, the procedure takes approximately 8 h for 16 ChIPs.
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chop it chip it check it the current status of Chromatin Immunoprecipitation
Frontiers in Bioscience, 2008Co-Authors: Philippe Collas, John Arne DahlAbstract:Abstract Our understanding of the significance of interactions of proteins with DNA in the context of gene expression, cell differentiation or to some extent disease has immensely been enhanced by the advent of Chromatin Immunoprecipitation (ChIP). ChIP has been widely used to map the localization of post-translationally modified histones or histone variants on the genome or on a specific gene locus, or to map the association of transcription factors or Chromatin modifying enzymes to the genome. In spite of its power, ChIP is a cumbersome procedure and typically requires large numbers of cells. This review outlines variations elaborated on the ChIP assay to shorten the procedure, make it suitable for small cell numbers and unravel the multiplicity of histone modifications on a single locus. In addition, the combination of ChIP assays with DNA microarray and high-throughput sequencing technologies has in recent years enabled the profiling of histone modifications and transcription factor occupancy sites throughout the genome and in a high-resolution manner throughout a genomic region of interest. We also review applications of ChIP to the mapping of histone modifications or transcription factor binding at the genome-wide level. Finally, we speculate on future perspectives opened by the combination of emerging ChIP-related technologies.
Ido Amit - One of the best experts on this subject based on the ideXlab platform.
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high throughput Chromatin Immunoprecipitation for genome wide mapping of in vivo protein dna interactions and epigenomic states
Nature Protocols, 2013Co-Authors: Ronnie Blechergonen, Zohar Barnettitzhaki, Diego Jaitin, Daniela Amannzalcenstein, David Laraastiaso, Ido AmitAbstract:Dynamic protein binding to DNA elements regulates genome function and cell fate. Although methods for mapping in vivo protein-DNA interactions are becoming crucial for every aspect of genomic research, they are laborious and costly, thereby limiting progress. Here we present a protocol for mapping in vivo protein-DNA interactions using a high-throughput Chromatin Immunoprecipitation (HT-ChIP) approach. By using paramagnetic beads, we streamline the entire ChIP and indexed library construction process: sample transfer and loss is minimized and the need for manually labor-intensive procedures such as washes, gel extraction and DNA precipitation is eliminated. All of this allows for fully automated, cost effective and highly sensitive 96-well ChIP sequencing (ChIP-seq). Sample preparation takes 3 d from cultured cells to pooled libraries. Compared with previous methods, HT-ChIP is more suitable for large-scale in vivo studies, specifically those measuring the dynamics of a large number of different Chromatin modifications/transcription factors or multiple perturbations.