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.
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.
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
PART 2: COLLECT IMMUNOCOMPLEXES
PART 3: PURIFY ChIP’d AND INPUT DNA
PART 4: PURIFY ChIP’d AND INPUT DNA CONTINUED
Part 5: REPRESENTATIVE RESULTS:
ChIP and Input DNA samples are quantified with Real-Time PCR 7,8 (Applied Biosystems 7500 Fast Real-Time PCR System) using primers specific to the gene locus of interest. Primers that target genomic sequence known to be enriched in or lacking in the histone modifications being assayed can be used as positive and negative controls as well. Data is presented as Percent of Input. The ChIP procedure should be wholly repeated with three biological replicates to ensure reported changes in histone modifications are statistically significant.
There are several important issues to consider when using real-time PCR to quantify DNA, including primer design, PCR efficiency, and the appropriate way to calculate the percent of input and normalization procedures. When profiling histone modification occupancy across a gene, the PCR efficiency must be consistent among all primer pairs. The real-time PCR system manufacturer can provide the necessary information and training.
Figure 1 shows representative results of chromatin immunoprecipitated during STAT1 activation of the IRF1 gene9, triggered by IFN-γ.
Figure 1. Occupancy of STAT1, RNA Polymerase II and H3K36me3 is dynamic during STAT1 activation of the IRF1 gene. (A, B, C) ChIP with α-H3K36me3, α-STAT1 and α-Pol II of chromatin collected from 2FTGH cells induced with IFN-γ for 30 minutes (red squares), 1.5 hours (blue circles), 5 hours (green triangles) or uninduced (black diamonds). IgG is the negative control (grey crosses). (D) Pan H3 is the positive control and shows the histone occupancy across the locus in the induced (blue circles) and uninduced conditions (black diamonds). The dip around -5 bp is due to the nucleosome depletion that is found at transcription start sites.
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.
The quality of ChIP data ultimately depends upon the effectiveness of the antibody used and there is considerable variability among antibodies. The failure to ChIP a modification, therefore, cannot be interpreted to mean that said modification is not present. Furthermore, changes in a particular modification observed in a ChIP experiment may simply reflect a change in the recognition of the epitope by the antibody caused by additional histone modifications at nearby residues. Therefore, ChIP results must be validated with approaches that will relate the observed changes in a histone modification to a biological outcome. When this is done, ChIP is a powerful way to study chromatin-based mechanisms that contribute to the regulation of gene expression.
The authors have nothing to disclose.
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.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
37% Formaldehyde | Fisher | BP531-500 | ||
Chloroform/Isoamyl alcohol | Acros | AC32715-5000 | ||
Complete Mini Protease Inhibitors | Roche | 1836153 | ||
Cosmic Calf Serum | Hyclone | SH30087.04N | ||
DMEM | Hyclone | SH30243 | ||
DTT | Sigma | D0632 | ||
EDTA | Fisher | BP118-500 | ||
Ethanol | Pharmco | zp1110EP | ||
Glycine | Roche | 100149 | ||
Glycogen | Roche | 901393 | ||
HEPES | Sigma | H3784 | ||
IFN-gamma | R&D Systems | 285-IF-100 | ||
IgG | Jackson Immunoresearch |
109-005-003 |
||
KCl | MP Biologicals | |||
LiCl | Sigma | L4408 | ||
MgCl2 | Sigma | M8266 | ||
Sodium Acetate | Fisher | BP333-500 | ||
Deoxycholic Acid Sodium Salt | Fisher | BP349-100 | ||
NaCl | Fisher | 7647-14-5 | ||
NP40 | US Biological | N3500 | ||
Anti-Histone H3, CT, pan | Millipore | 07-690 | ||
Phenol/chloroform/isoamyl | Fisher | 108-95-2 | ||
Phosphate Buffered Saline | Fisher | 7647-14-5 | ||
PMSF | EMD | 50-230-0316 | ||
Proteinase K | 5-Prime | 2500140 | ||
RNAse A | 5-Prime | 2500130 | ||
Salmon Sperm DNA/Protein A Agarose | Millipore | 16-157 | ||
Salmon Sperm DNA/Protein G Agaorse | Millipore | 16-201 | ||
SDS | Fisher | BP166-500 | ||
Tris-Cl | Sigma | T5941 | ||
Triton X-100 | Acros | 327372500 | ||
Anti-K36me3 | Abcam | ab9050 | ||
Anti-STAT1 | Santa Cruz | sc-345X | ||
Anti-RNA Polymerase II | Santa Cruz | sc-899 |