Chromatin Immunoprecipitation Assays
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Abstract:
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.Keywords:
Chromatin immunoprecipitation
ChIP-on-chip
ChIP-sequencing
Immunoprecipitation
ChIA-PET
In the post-genomic era, identifying and characterzing various DNA-protein interactions are a major challenge in the research of gene transcriptional regulation. Now, chromatin immunoprecipitation assay (ChIP) is ideally suited for investigating DNA-protein interactions in vivo. The conditions of chromatin immunoprecipitation assay was optimized and utilized to screen unknown target genes of transcription factor AP-2 alpha. The results yielded a more complete understanding of AP-2α-regulated signaling pathways and further elucidated the complex transcriptional networks.
Chromatin immunoprecipitation
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Chromatin immunoprecipitation
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Proper gene expression involves communication between the regulatory elements and promoters of genes. Because regulatory elements can be located over a large range of genomic distances (from as close as a few hundred bp to as much as several Mb away), contact and communication between regulators and the core transcriptional machinery at promoters are mediated through DNA looping. Today, chromosome conformation capture (3C)-based methods efficiently probe chromosome folding in the nucleus and thus provide a molecular description of physical proximity between enhancer(s) and their target promoter(s). One such method, chromatin interaction analysis using paired-end-tag (ChIA-PET) sequencing, is a leading high-throughput method for detection of genome wide chromatin interactions. Briefly, the method involves cross-linkage of chromatin (-DNA) fibers in cells in situ, fragmentation of the fixed chromatin-DNA complexes by sonication, followed by enrichment of the chromatin complexes with a dedicated antibody through the process of immunoprecipitation (IP). Next, application of the ChIA-PET protocol followed by deep sequencing and mapping of reads to the reference genome reveals both binding sites and remote chromatin interactions mediated by the protein factors of interest. The method detailed here focuses on ChIP sample preparation and can be completed in ∼5 d. The ChIA-PET method is detailed in an associated protocol. Because not all chromatin immunoprecipitation protocols are suitable for ChIA-PET, it is important to strictly follow this procedure before performing the ChIA-PET protocol.
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ChIP-on-chip
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Chromatin immunoprecipitation
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Expression of eukaryotic genes during development requires complex spatial-temporal regulation. This complex regulation is often achieved through the coordinated interaction of transcription regulatory elements in the promoters of the target genes. The identification and mapping of regulatory elements in genome scale is crucial to understand how gene expression is regulated. Chromatin immunoprecipitation is a standard method for assessing the occupancy of DNA binding proteins in vivo in their native chromatin context using antibodies. However, standard chromatin immunoprecipitation procedure is time consuming, labor intensive and not suited for analyzing many samples simultaneously.
Recently, we have developed a simple ChIP protocol that requires fewer steps and less hands-on time. This protocol is compatible with both 96-well plate and single tube formats, and enables higher sensitivity and more reliable performance, as compared to conventional approaches.
We have successfully used this protocol to map various clinically relevant chromatin marks and controls across several cell types to quantitatively measure chromatin states. This analysis included a variety of marks corresponding to repressed, poised and active promoters, strong and weak enhancers, putative insulators, transcribed regions, as well as large-scale repressed and inactive domains. This study demonstrates the utility of this approach for the characterization of model cellular systems in perturbation studies with chemical probes.
Chromatin immunoprecipitation
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ChIP-on-chip
Tiling array
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Chromatin immunoprecipitation
ChIP-on-chip
ChIP-sequencing
ChIA-PET
Immunoprecipitation
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Chromatin immunoprecipitation
ChIP-sequencing
ChIA-PET
Immunoprecipitation
ChIP-on-chip
Transcription
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Abstract Expression of eukaryotic genes during development requires complex spatial-temporal regulation. This complex regulation is often achieved through the coordinated interaction of transcription regulatory elements in the promoters of the target genes. The identification and mapping of regulatory elements in genome scale is crucial to understand how gene expression is regulated. Chromatin immunoprecipitation is a standard method for assessing the occupancy of DNA binding proteins in vivo in their native chromatin context using antibodies. However, standard chromatin immunoprecipitation procedure is time consuming, labor intensive and not suited for analyzing many samples simultaneously. Recently, we have developed a simple high throughput chromatin immunoprecipitation protocol that utilizes high capacity protein A/G coated magnetic beads and 96 well plates. This protocol requires fewer steps and less hands-on time, has higher sensitivity and more reliable performance as compared to conventional approaches. The method is also compatible with multi-channel pipetting and liquid handling systems. We have successfully used this protocol to map various clinically relevant chromatin marks and controls across several cell types to quantitatively measure chromatin states. This analysis included a variety of marks corresponding to repressed, poised and active promoters, strong and weak enhancers, putative insulators, transcribed regions, as well as large-scale repressed and inactive domains. This study demonstrates the utility of this approach for the characterization of model cellular systems in perturbation studies with chemical probes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2209. doi:1538-7445.AM2012-2209
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Chromatin structure plays a critical role in eukaryotic gene expression. Chromatin immunoprecipitation assay (ChIP) provides a powerful tool to analyze the interaction of trans-acting factors with specific chromatin regions in vivo, as well as the role of histone modifications in gene regulation. A simple ChIP protocol is established and used to study the H3 acetylation pattern of the β-globin locus in MEL cells. DMSO induction results in a dramatic increase in H3 acetylation at hypersensitive site HS2 and active gene (βmaj) promoter, whereas the inactive gene (Ey) promoter maintains low acetylation. This result indicates that the ChIP method is feasible.
Chromatin immunoprecipitation
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