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At least 19 recordsLinked to original sources
PubMed2004

Native chromatin immunoprecipitation.

Chromatin immunoprecipitation (ChIP) is a technique widely used for determining the genomic location of modified histones and other chromatin-associated factors. Here we describe the methodology we have used in our laboratory for the immunoprecipitation of chromatin isolated from cells in the absence of crosslinking. Chromatin released from nuclei by micrococcal nuclease digestion is centrifuged through sucrose gradients to allow selection of mono- or dinucleosomes. This allows a protein or modification at a particular gene or locus to be mapped at higher resolution than in a crosslinked ChIP experiment. Two methods for the immunoprecipitation of chromatin are described: a large-scale fractionation by which it is possible to visualize the proteins of the immunoprecipitate by polyacrylamide gel electrophoresis, PAGE and a small-scale method that is more appropriate when the quantity of chromatin is limited. The sequence content of DNA extracted from the immunoprecipitated chromatin is analyzed by hybridization of Southern or slot blots, or by quantitative polymerase chain reaction. Enrichment of particular sequences in the immunoprecipitated fraction reveals the presence and extent of the modification at this location.

Animals↗
PubMed2004

Characterization of protein-DNA association in vivo by chromatin immunoprecipitation.

Chromatin immunoprecipitation (ChIP) is one of the most powerful methods to identify and characterize the association of proteins with specific genomic regions in the context of intact cells. In this method, cells are first treated with formaldehyde to crosslink protein-protein and protein-DNA complexes in situ. Next, the crosslinked chromatin is sheared by sonication to generate small chromatin fragments, and the fragments associated with the protein of interest are immunoprecipitated using antibodies to the protein. Finally, protein-DNA crosslinks are reversed and the DNA is examined for the presence of particular sequences by quantitative polymerase chain reaction (PCR). Enrichment of specific sequences in the precipitate indicates that the sequences are associated with the protein of interest in vivo. The ChIP method described here is intended for studying protein-DNA association in the budding yeast Saccharomyces cerevisiae, but it can be easily implemented in other cell types, including fly, mammalian, and plant cells.

Animals↗
PubMed2006

Assessment of multiple different estrogen receptor-beta antibodies for their ability to immunoprecipitate under chromatin immunoprecipitation conditions.

Several different antibodies to total estrogen receptor (ER)beta, ERbeta1 and ERbeta2/cx have been tested and compared for their ability to immunoprecipitate ERbeta specific isoforms under chromatin immunoprecipitation conditions (ChIP). The rabbit polyclonal antibodies AP-ERbeta1 and AP-ERbeta2/cx, specific for ERbeta1 and ERbeta2/cx isoforms, respectively, were the most efficient for ChIP. The monoclonal antibody MCA1974/PPG5/10 was also able to ChIP ERbeta1, but less efficiently than AP-ERbeta1. All other antibodies tested were not suitable for ChIP analyses although most antibodies tested immunoprecipitated the appropriate ERbeta isoforms under standard conditions. To identify antibodies that can also be used to verify in-vivo expression profiles, a comparison of the antibodies to detect ERbeta isoforms by western blotting and immunohistochemistry was also undertaken. Under the tissue processing and autostaining conditions used at the Manitoba Breast Tumor Bank 385P/GC17, MCA1974/PPG5/10, Ab288/14C8 and MCA2279S/57/3 were found to be the best for IHC of ERbeta isoforms in human breast tissue biopsy sections, while Ab14021, AP-ERbeta1 and AP-ERbeta2/cx were best for western blot detection of ERbeta isoforms.

Animals↗
PubMed2006

Fast chromatin immunoprecipitation assay.

Chromatin immunoprecipitation (ChIP) is a widely used method to explore in vivo interactions between proteins and DNA. The ChIP assay takes several days to complete, involves several tube transfers and uses either phenol-chlorophorm or spin columns to purify DNA. The traditional ChIP method becomes a challenge when handling multiple samples. We have developed an efficient and rapid Chelex resin-based ChIP procedure that dramatically reduces time of the assay and uses only a single tube to isolate PCR-ready DNA. This method greatly facilitates the probing of chromatin changes over many time points with several antibodies in one experiment.

Animals↗
PubMed2006

Chip-on-beads: flow-cytometric evaluation of chromatin immunoprecipitation.

BACKGROUND: Chromatin immunoprecipitation (ChIP) is a widely used technique for the detection of in vivo DNA-protein interactions underlying epigenetic regulation. The standard readout of ChIP is based on semi-quantitative or quantitative PCR measurements; however, the development of alternative platforms with high throughput potentialities is expected to facilitate the introduction of this method into routine diagnostics. METHODS: We have established a flow-cytometry-based alternative for the evaluation of ChIP data. The method is based on the capture of the products of a conventional PCR run to low cycle numbers, on microbeads. RESULTS: In vivo histone H4 acetylation and H3 lysine 4 methylation was detected at the promoter of the tissue transglutaminase type 2 gene. These results were confirmed by QPCR measurements. The levels of modifications decreased significantly upon apoptosis and were accompanied by the down-regulation of TGM2 mRNA expression. CONCLUSIONS: This method that we named ChIP-on-beads, a combination of flow cytometry and conventional PCR, is a reliable and efficient alternative in the quantitative analysis of ChIP results, especially promising when high throughput monitoring of epigenetic markers of diagnostic importance is required. The method is simple enough to be easily implemented in a routine flow-cytometric laboratory.

Acetylation↗
PubMed2005

Two-step cross-linking method for identification of NF-kappaB gene network by chromatin immunoprecipitation.

The chromatin immunoprecipitation (ChIP) assay has recently been exploited as a powerful and versatile technique for probing protein-DNA interactions within the chromatin environment. In this method, intact cells are fixed with a reversible DNA-protein cross-linking agent (formaldehyde), and associated DNA is enriched by immunoprecipitating a target DNA binding protein. The bound DNA in the immune complexes is then used to identify that specific DNA binding protein's endogenous genomic targets. Nuclear factor kappaB (NF-kappaB) is a highly inducible transcription factor that controls genetic networks important for pathogen- or cytokine-induced inflammation, immune response, and cellular survival. In our studies of the genetic network under control of the inducible NF-kappaB transcription factor, we found that the conventional ChIP technique using a single formaldehyde cross-linking step did not reproducibly cross-link it to DNA. As a result, we have developed a novel ChIP assay using a two-step cross-linking procedure, incorporating N-hydroxysuccinimide (NHS)-ester-mediated protein-protein cross-linking prior to conventional DNA-protein cross-linking. We demonstrate that this technique is highly efficient, cross-linking virtually all NF-kappaB/Rel A into covalent complexes, resulting in quantitative and robust identification of inducible NF-kappaB family binding to a variety of validated NF-kappaB-dependent genomic targets. To demonstrate the general utility of this two-step cross-linking procedure, we performed enhanced capture of cytokine-inducible signal transducer and activator of transcription-3 (STAT3) binding to one of its known target genes. Our method represents a significant improvement in the efficiency of ChIP analysis in the study of endogenous targets for rare transcription factors.

Binding Sites↗
PubMed2004

Chromatin immunoprecipitation assays.

Chromatin immunoprecipitation (ChIP) is a powerful tool to study protein-DNA interaction and is widely used in many fields to study proteins associated with chromatin, such as histone and its isoforms and transcription factors, across a defined DNA domain. Here, we show the step-by-step methods currently used in our lab to immunoprecipitate the formaldehyde crosslinked chromatin and further analyze the immuprecipitated DNA by semiquantitative PCR.

Chromatin↗
PubMed2004

ChIP-chip: considerations for the design, analysis, and application of genome-wide chromatin immunoprecipitation experiments.

Chromatin immunoprecipitation (ChIP) is a well-established procedure to investigate interactions between proteins and DNA. Coupled with whole-genome DNA microarrays, ChIPS allow one to determine the entire spectrum of in vivo DNA binding sites for any given protein. The design and analysis of ChIP-microarray (also called ChIP-chip) experiments differ significantly from the conventions used for locus ChIP approaches and ChIP-chip experiments, and these differences require new methods of analysis. In this light, we review the design of DNA microarrays, the selection of controls, the level of repetition required, and other critical parameters for success in the design and analysis of ChIP-chip experiments, especially those conducted in the context of mammalian or other relatively large genomes.

Animals↗
PubMed2002

Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation.

The standard chromatin immunoprecipitation (ChIP) assay is used to examine the specific association of transcription factors with DNA in the context of living cells. Here we review two modifications to this protocol which are designed to identify novel target genes of transcription factors in mammalian cells. The main advantage to both of these approaches is that only DNA sequences directly bound by a factor within the context of a living cell will be identified. Therefore, artifacts associated with overexpression and/or alterations in signaling pathways are avoided. The first modification we describe, a ChIP cloning strategy, can be used to isolate any genomic fragment specifically associated with a particular factor. It requires no special equipment or reagents other than a high-affinity antibody to be used for immunoprecipitation of the factor of interest. However, it is most useful for the isolation of a small number of genomic targets. In contrast, the second modification, which combines ChIP with specialized CpG microarrays, is ideal for a more global analysis of target genes. Advantages, common problems, and detailed protocols for these two ChIP techniques are discussed.

Animals↗
PubMed2002

An algorithm for finding protein-DNA binding sites with applications to chromatin-immunoprecipitation microarray experiments.

Chromatin immunoprecipitation followed by cDNA microarray hybridization (ChIP-array) has become a popular procedure for studying genome-wide protein-DNA interactions and transcription regulation. However, it can only map the probable protein-DNA interaction loci within 1-2 kilobases resolution. To pinpoint interaction sites down to the base-pair level, we introduce a computational method, Motif Discovery scan (MDscan), that examines the ChIP-array-selected sequences and searches for DNA sequence motifs representing the protein-DNA interaction sites. MDscan combines the advantages of two widely adopted motif search strategies, word enumeration and position-specific weight matrix updating, and incorporates the ChIP-array ranking information to accelerate searches and enhance their success rates. MDscan correctly identified all the experimentally verified motifs from published ChIP-array experiments in yeast (STE12, GAL4, RAP1, SCB, MCB, MCM1, SFF, and SWI5), and predicted two motif patterns for the differential binding of Rap1 protein in telomere regions. In our studies, the method was faster and more accurate than several established motif-finding algorithms. MDscan can be used to find DNA motifs not only in ChIP-array experiments but also in other experiments in which a subgroup of the sequences can be inferred to contain relatively abundant motif sites. The MDscan web server can be accessed at http://BioProspector.stanford.edu/MDscan/.

Algorithms↗
PubMed2007

Faster and easier chromatin immunoprecipitation assay with high sensitivity.

Chromatin immunoprecipitation (ChIP) assays are widely used to investigate where chromatin-binding proteins bind to the genome. The standard assay is very time consuming. We have developed a rapid ChIP assay in which the immunoprecipitates serve directly as PCR templates. This assay eliminates the step to reverse the crosslinking, shortening the assay by 1 day. It also requires a less immunoprecipitating antibody, permits many samples to be tested simultaneously, and is more sensitive than the standard ChIP assay.

Chromatin Immunoprecipitation↗
PubMed2005

Chipper: discovering transcription-factor targets from chromatin immunoprecipitation microarrays using variance stabilization.

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'.

Analysis of Variance↗
PubMed2004

Using chromatin immunoprecipitation to monitor 1alpha,25-dihydroxyvitamin D3-dependent chromatin activity on the human CYP24 promoter.

We applied the chromatin immunoprecipitation (ChIP) method for the analysis of 1alpha,25-dihydroxyvitamin D(3) (1,25-D(3))-dependent chromatin activity on the human 24-hydroxylase (CYP24) promoter in MCF-7 human breast cancer cells. In this pilot study we concentrated on the proximal promoter (+22 to -424) of the CYP24 gene, which includes the known 1,25-D(3) response element (VDRE) cluster. A constitutively active region of the human histone 4a gene (-40 to +285) served for normalization. Chromatin activity snapshots were taken 0, 30, 60, 120, 180, 240 and 300 min after the onset of stimulation with 1,25-D(3) and anti-acetylated histone 4 antibodies were used for ChIP. Our results suggest that ChIP is suitable for monitoring 1,25-D(3)-dependent changes of chromatin organization and can be used to reveal information about chromatin activity in living cells.

Base Sequence↗
PubMed2006

Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations.

Chromatin immunoprecipitation (ChIP) defines the genomic distribution of proteins and their modifications but is limited by the cell numbers required (ideally >10(7)). Here we describe a protocol that uses carrier chromatin and PCR, 'carrier' ChIP (CChIP), to permit analysis of as few as 100 cells. We assayed histone modifications at key regulator genes (such as Nanog, Pou5f1 (also known as Oct4) and Cdx2) by CChIP in mouse embryonic stem (ES) cells and in inner cell mass (ICM) and trophectoderm of cultured blastocysts. Activating and silencing modifications (H4 acetylation and H3K9 methylation) mark active and silent promoters as predicted, and we find close correlation between values derived from CChIP (1,000 ES cells) and conventional ChIP (5 x 10(7) ES cells). Studies on genes silenced in both ICM and ES cells (Cdx2, Cfc1, Hhex and Nkx2-2, also known as Nkx) show that the intensity of silencing marks is relatively diminished in ES cells, indicating a possible relaxation of some components of silencing on adaptation to culture.

Animals↗
PubMed2005

A hypothesis-based approach for identifying the binding specificity of regulatory proteins from chromatin immunoprecipitation data.

MOTIVATION: Genome-wide chromatin-immunoprecipitation (ChIP-chip) detects binding of transcriptional regulators to DNA in vivo at low resolution. Motif discovery algorithms can be used to discover sequence patterns in the bound regions that may be recognized by the immunoprecipitated protein. However, the discovered motifs often do not agree with the binding specificity of the protein, when it is known. RESULTS: We present a powerful approach to analyzing ChIP-chip data, called THEME, that tests hypotheses concerning the sequence specificity of a protein. Hypotheses are refined using constrained local optimization. Cross-validation provides a principled standard for selecting the optimal weighting of the hypothesis and the ChIP-chip data and for choosing the best refined hypothesis. We demonstrate how to derive hypotheses for proteins from 36 domain families. Using THEME together with these hypotheses, we analyze ChIP-chip datasets for 14 human and mouse proteins. In all the cases the identified motifs are consistent with the published data with regard to the binding specificity of the proteins.

Algorithms↗
PubMed2001

Use of chromatin immunoprecipitation to clone novel E2F target promoters.

We have taken a new approach to the identification of E2F-regulated promoters. After modification of a chromatin immunoprecipitation assay, we cloned nine chromatin fragments which represent both strong and weak in vivo E2F binding sites. Further characterization of three of the cloned fragments revealed that they are bound in vivo not only by E2Fs but also by members of the retinoblastoma tumor suppressor protein family and by RNA polymerase II, suggesting that these fragments represent promoters regulated by E2F transcription complexes. In fact, database analysis indicates that all three fragments correspond to genomic DNA located just upstream of start sites for previously identified mRNAs. One clone, ChET 4, corresponds to the promoter region for beclin 1, a candidate tumor suppressor protein. We demonstrate that another of the clones, ChET 8, is strongly bound by E2F family members in vivo but does not contain a consensus E2F binding site. However, this fragment functions as a promoter whose activity can be repressed by E2F1. Finally, we demonstrate that the ChET 9 promoter contains a consensus E2F binding site, can be activated by E2F1, and drives expression of an mRNA that is upregulated in colon and liver tumors. Interestingly, the characterized ChET promoters do not display regulation patterns typical of known E2F target genes in a U937 cell differentiation system. In summary, we have provided evidence that chromatin immunoprecipitation can be used to identify E2F-regulated promoters which contain both consensus and nonconsensus binding sites and have shown that not all E2F-regulated promoters show identical expression profiles.

Apoptosis Regulatory Proteins↗
PubMed2025

Chromatin Immunoprecipitation for Standard, Rare, or Weakly Binding Proteins.

Various proteins interact with specific genome regions, playing crucial roles in gene regulation. Chromatin Immunoprecipitation (ChIP) is the most commonly used method to study protein-DNA interactions in vivo. By combining ChIP with high-throughput sequencing, ChIP-seq allows for studying the genome-wide localization of proteins. Although several ChIP protocols are available for plant tissues, they are primarily designed for histone modifications and abundant proteins with high DNA-binding affinity, which are considered as the "standard targets." Here we describe a ChIP protocol for plant tissues not only optimized for the standard targets but also adapted for proteins with low abundances or weak DNA-binding ability. Successful execution of the protocol enables reliable generation of DNA templates for quantitative PCR or libraries for next-generation sequencing, which makes it an effective tool for analyzing genomic interactions of a wide range of proteins.

Chromatin Immunoprecipitation↗