This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.
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Method Article
Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells
In This Article
Summary
Abstract
In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3.
It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.
Protocol
PLANNING
The day before a ChIP experiment is planned, cells should be cultured so that a sufficient number is available. Assume that each ChIP assay will require ~1-1.5 x 107 cells. Always plan to do both the negative (IgG) and positive (pan histone antibody) controls and duplicate experiments.
Tip - For 2FTGH cells, each ChIP assay requires about one 15 cm tissue culture dish at 80% confluency.
PART 1: PREPARE CHROMATIN AND PERFORM ChIP
- Remove culture media and induce cells for the times required with 20 mL of 10% cosmic calf serum (CCS)/DMEM containing IFN-gamma....
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Discussion
The ChIP protocol outlined has been used successfully in the lab to assay more than twenty different histone modifications. In addition, we have characterized changes in the occupancy of transcription factors, histone-modifying enzymes, proteins that recognize histone modifications and the RNA Polymerase II transcription machinery, at several IFN-γ induced genes. We have also used the procedure to characterize changes in histone modifications during the differentiation of a cancer stem cell10.
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Disclosures
No conflicts of interest declared.
Acknowledgements
This work is supported by the University of Virginia, the University of Virginia Cancer Center and The Thomas F. and Kate Miller Jeffress Memorial Trust. We thank the James E. Darnell Lab (Rockefeller University) the Robert Ross Lab (Fordham University) for cell lines.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 37% Formaldehyde | Fisher Scientific | BP531-500 | |
| Chloroform/Isoamyl alcohol | Acros Organics | AC32715-5000 | |
| Complete Mini Protease Inhibitors | Roche Group | 1836153 | |
| Cosmic Calf Serum | Hyclone | SH30087.04N | |
| DMEM | Hyclone | SH30243 | |
| DTT | Sigma-Aldrich | D0632 | |
| EDTA | Fisher Scientific | BP118-500 | |
| Ethanol | Pharmco Products, Inc. | zp1110EP | |
| Glycine | Roche Group | 100149 | |
| Glycogen | Roche Group | 901393 | |
| HEPES | Sigma-Aldrich | H3784 | |
| IFN-gamma | R&D Systems | 285-IF-100 | |
| IgG | Jackson ImmunoResearch | 109-005-003 | |
| KCl | MP Biomedicals | ||
| LiCl | Sigma-Aldrich | L4408 | |
| MgCl2 | Sigma-Aldrich | M8266 | |
| Sodium Acetate | Fisher Scientific | BP333-500 | |
| Deoxycholic Acid Sodium Salt | Fisher Scientific | BP349-100 | |
| NaCl | Fisher Scientific | 7647-14-5 | |
| NP40 | US Biological | N3500 | |
| Anti-Histone H3, CT, pan | EMD Millipore | 07-690 | |
| Phenol/chloroform/isoamyl | Fisher Scientific | 108-95-2 | |
| Phosphate Buffered Saline | Fisher Scientific | 7647-14-5 | |
| PMSF | EMD Millipore | 50-230-0316 | |
| Proteinase K | 5 PRIME | 2500140 | |
| RNAse A | 5 PRIME | 2500130 | |
| Salmon Sperm DNA/Protein A Agarose | EMD Millipore | 16-157 | |
| Salmon Sperm DNA/Protein G Agaorse | EMD Millipore | 16-201 | |
| SDS | Fisher Scientific | BP166-500 | |
| Tris-Cl | Sigma-Aldrich | T5941 | |
| Triton X-100 | Acros Organics | 327372500 | |
| Anti-K36me3 | Abcam | ab9050 | |
| Anti-STAT1 | Santa Cruz Biotechnology, Inc. | sc-345X | |
| Anti-RNA Polymerase II | Santa Cruz Biotechnology, Inc. | sc-899 |
References
- Levy, D., Darnell, J. E. Signalling: Stats: transcriptional control and biological impact. Nature Reviews Molecular Cell Biology. 3, 651-662 (2002).
- Bromberg, J., Darnell, J. E. Jr The role of STATs in transcriptional control and their impact on ....
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