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Two-step cross-linking for analysis of protein-chromatin interactions
Bing Tian1, Jun Yang, Allan R Brasier
1Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, USA.
Methods in Molecular Biology (Clifton, N.J.)
|November 25, 2011
Summary
A novel two-step crosslinking ChIP (XChIP) method enhances the capture of dynamic protein-DNA interactions, improving the study of gene regulation and chromatin accessibility.
Area of Science:
- Molecular Biology
- Genetics
- Epigenetics
Background:
- Eukaryotic gene regulation involves transcription factors binding to DNA.
- Analyzing these interactions in native chromatin is crucial for understanding gene timing and chromatin modifier effects.
- Conventional Chromatin Immunoprecipitation (ChIP) has limitations in capturing dynamic or transient protein-DNA interactions.
Purpose of the Study:
- To develop and validate a versatile and efficient "two-step" crosslinking ChIP (XChIP) method.
- To improve the analysis of transcription factor and coactivator interactions with chromatin.
- To enhance the study of gene regulation and promoter accessibility.
Main Methods:
- A "two-step" XChIP protocol involving sequential protein-protein and protein-DNA fixation.
- Formaldehyde-mediated crosslinking optimized for capturing dynamic interactions.
- Modifications in DNA extraction and sonication for downstream analysis.
Main Results:
- The two-step XChIP method successfully captured transcription factors (NF-κB, STAT3), RNA Polymerase II, coactivators (CBP/p300, CDK9), and modified histones.
- Demonstrated improved preservation of protein-DNA interactions compared to single-step crosslinking.
- Enabled downstream target identification using quantitative genomic PCR and next-generation sequencing.
Conclusions:
- The two-step XChIP method is a versatile and efficient technique for analyzing protein-chromatin interactions.
- This improved method facilitates systematic studies of gene regulation timing and chromatin accessibility.
- XChIP provides a more comprehensive analysis of transcription factor and coactivator binding dynamics.
Related Concept Videos
02:36Chromatin Immunoprecipitation- ChIP
Chromatin immunoprecipitation, or ChIP, is an antibody-based technique used to identify sites on DNA that bind to transcription factors of interest or histone proteins. It also helps determine the type of histone modifications such as acetylation, phosphorylation, or methylation.
Types of ChIP
ChIP can be divided into two types - X-ChIP and N-ChIP. X-ChIP involves in vivo cross-linking of histones and regulatory proteins to DNA, fragmenting the DNA by sonication, and isolating the protein-DNA...
Types of ChIP
ChIP can be divided into two types - X-ChIP and N-ChIP. X-ChIP involves in vivo cross-linking of histones and regulatory proteins to DNA, fragmenting the DNA by sonication, and isolating the protein-DNA...
01:20Immunoprecipitation
Immunoprecipitation, or IP, is a widely used technique that employs protein-antibody interactions to isolate proteins or protein complexes in their native state for studying protein-protein interactions, quaternary structures, or supramolecular complexes. Various modifications of the technique, including chromatin IP, cross-linking IP, and fluorescence IP, are commonly used.
Chromatin Immunoprecipitation
Chromatin immunoprecipitation, also known as ChIP, is used to study protein-DNA or...
Chromatin Immunoprecipitation
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