Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.
To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.
Gene expression in mammalian cells is tightly and dynamically regulated, and transcription is one of the key steps. Gene transcription is mainly regulated by transcription factors and histones. A transcription factor is a protein that binds to specific DNA sequences and controls gene transcription. These factors either promote or inhibit the recruitment of RNA polymerase II (PolII), which initiates mRNA synthesis from genomic DNA as a template1. Histone modifications such as acetylation and methylation of histone tail residues positively and negatively affect gene transcription by changing the chromatin structure2. Since alterations in gene expression affect the cellular context, it is essential to examine the molecular mechanisms by which transcription is regulated.
To date, several methods for investigating the regulation of gene transcription are available. Electrophoretic mobility shift assay (EMSA), also called a gel shift assay, is used for analyzing a protein-DNA interaction3. A nuclear extract from cells of interest is incubated with a radioactive isotope (for example, 32P)-labeled DNA probe and electrophoresed on a polyacrylamide gel. Its autoradiogram shows that the DNA-protein complex migrates slower than the probe in a gel. In the presence of an antibody against the protein, the DNA-protein-antibody complex migrates in a gel more slowly than the DNA-protein complex. This supershifted band reveals specific binding between the DNA and protein. However, EMSA only determines a specific DNA-protein interaction in a cell-free system, and therefore it remains unknown whether the interaction controls transcription in living cells. The transient reporter assay, commonly called luciferase reporter assay, was developed to address gene expression regulation in cells. Typically, an upstream genomic region of a gene of interest is inserted into a reporter plasmid, transiently transfected into cells, and the reporter activity is measured. A variety of deletion mutants allows the identification of regions that are responsible for gene regulation. Even though a reporter assay is a useful tool for identifying transcription factors and binding DNA sequences controlling transcription, this method has a major disadvantage in that a reporter plasmid is free of chromatin structure and does not reflect “real” transcription machinery. In addition, changes in histone modifications cannot be determined by the system.
The development of the chromatin immunoprecipitation (ChIP) method was based on Jackson and Chalkley’s reports that “whole cell” fixation with formaldehyde preserved chromatin structure4,5. Since then, many related techniques have been developed and improved6. In ChIP assays, cells are fixed with formaldehyde to cross-link DNA and proteins. The chromatin is fragmented and then immunoprecipitated with antibodies of interest. The immune complex is washed, and DNA is purified. PCR amplification with primers targeted to a particular region of the genome reveals the occupancy of proteins of interest in the genome.
Although ChIP is a powerful tool to identify the interactions of proteins such as transcription factors and modified histones with DNA, the method involves some difficulties, such as a chromatin fragmentation step, in practice. Sonication has been widely used for shearing chromatin; however, it is cumbersome to identify reproducible conditions. Micrococcal nuclease (MNase) treatment is an alternative method for chromatin shearing. MNase is an endo-exonuclease that digests double-stranded, single-stranded, circular and linear DNA and RNA. It is relatively easy to determine the conditions, including the amounts of chromatin and enzyme, temperature, and incubation time, for optimum chromatin fragmentation. We modified and simplified the existing protocols, and we established a straightforward and reproducible method. This paper provides the protocol for a ChIP assay using MNase in mammalian cells.
1. Preparation of Reagents
2. Determination of MNase Digestion Conditions
NOTE: In the step 2 of protocol, an example using VCaP, human prostate cancer cells is presented. Any mammalian cell lines can be used; see Note at the steps.
3. ChromatinImmunoprecipitation
Digesting chromatin is one of the important steps for a ChIP assay. We used MNase to digest chromatin to obtain a mixture of nucleosome oligomers. In the MNase digestion step, MNase can go through the nuclear membrane and digest chromatin. However, the digested chromatin cannot go through the membrane and remains in the nuclei. To release the digested chromatin from the nuclei, brief sonication is needed. Figure 1A shows microphotographs before and after sonication of VCaP cell suspension. Without sonication, the cell structure remains intact, indicating that the chromatin is present in the nuclei. A brief sonication breaks the cell structure, and checking the cells in microphotographs helps to determine the brief sonication conditions. We also represented other examples for brief sonication in 293T cells (Figure 1B) the human B-cell acute lymphoblastic leukemia cell line, REH cells (Figure 1C) and the human prostate cancer cell line, LNCaP (Figure 1D).
Figure 2A shows chromatin fragmentation after treatment with different amounts of MNase in VCaP cells. We treated 6 x 106 crosslinked VCaP cell pellets with 0, 50, 100, 200 gel units of MNase in 300 µL of digestion buffer for 10 min at 37 °C. After purification of the digested chromatin, 500 ng of DNA was analyzed on 2% agarose gel and stained with ethidium bromide. Without adding MNase, a smear pattern with a very high molecular weight appeared (lane 1). The addition of MNase gave a ladder pattern (N; a mononucleosome unit), showing that MNase digests internucleosome (lanes 2-5). Figure 2B shows an inappropriate digestion pattern. Overdigestion mainly resulted in mononucleosome production (Figure 2B, lane 7). We should find the proper conditions that produce chromatin fragments up to 900 bp (one to five nucleosomes; e.g., lane 5).
To check whether the ChIP assay is performed properly, it is essential to have appropriate controls in the assay. For immunoprecipitation, nonimmune IgGs from the same species as the antibodies of interest are used as a control that shows nonspecific binding to the same region (see discussion). In addition, it is recommended to measure the binding of the proteins (occupancy) in both positive and negative regions. It has been widely accepted that H3K4me3 occupancy is distributed between approximately one kilobase (kb) upstream and downstream of transcription start sites10,11. We measured H3K4me3 occupancy in the AR genome spanning approximately 20 kb upstream through 12 kb downstream of the AR transcription start site (AR-TSS) in AR-positive VCaP cells. Digestion pattern of chromatin in VCaP cells used in this experiment was shown in Figure 3A, indicating the proper digestion of chromatin. The highest occupancy of H3K4me3 was observed around the AR-TSS and 0.5 kb and 1 kb upstream of the AR-TSS (Figure 3B). As long as genes are transcriptionally active, TSSs can be “positive regions”. Regions located at 19 kb and 8 kb upstream and 12 kb downstream of AR-TSS, however, had little occupancy of H3K4me3 (Figure 3B), indicating that these can be used as “negative regions”.
It has been shown that an androgen increases RNA polymerase II occupancy in the PSA promoter and enhancer in LNCaP cells using sheared chromatin by sonication12,13. We therefore tested the validity of our protocol by measuring active RNA polymerase II occupancy (phosphorylated RNA polymerase II at serine 5; PolII(pS5)) in the cells. We performed the same experiment to check the reproducibility of our method. LNCaP cells were cultured in steroid-starved medium for 3 days and stimulated with a vehicle or 10 nM dihydrotestosterone (DHT) for 4 h. Active RNA polymerase II occupancy was measured by immunoprecipitation with anti-PolII(pS5), followed by real-time PCR. Figure 4A shows a reproducible digestion pattern of chromatin from LNCaP cells in three independent experiments. As shown in Figure 4B, DHT significantly increased PolII(pS5) occupancy in the PSA promoter and enhancer when using percent input method. We also calculated the occupancy using fold enrichment method (Figure 4C) and found that no significant difference in PolII(pS5) in the PSA promoter was observed with or without DHT treatment. DHT did not affect occupancy in the GAPDH promoter as previously published14. Importantly, our data were similar to that obtained from sonication-sheared chromatin samples12,13.
Figure 1: Representative microphotographs of crosslinked cell pellets before and after sonication. Crosslinked VCaP (A), 293T (B), REH (C) and LNCaP (D) cell pellets were treated with MNase, and pellets were resuspended in ChIP dilution buffer. Before and after sonication, pictures of the suspensions were taken. Scale bar = 200 µm. Please click here to view a larger version of this figure.
Figure 2: Representative agarose gel analysis of digested chromatin. (A) Crosslinked chromatin was prepared from VCaP cells and digested with various amounts of MNase as described in step 2.2. Digested chromatin was reverse crosslinked, purified, and analyzed in a 2% agarose gel. N; a mononucleosome unit. (B) Chromatin of VCaP cells was digested with 250 gel units of MNase per 2 x 106 cells at 37 °C for 20 min and analyzed (as described in A). Larger amounts of MNase and longer incubation times caused almost complete digestion of chromatin to form mononucleosomes (150 bp). Please click here to view a larger version of this figure.
Figure 3: H3K4me3 occupancy in the AR genome. Digested chromatin was prepared from VCaP cells. (A) Digestion pattern was analyzed using an agarose gel. (B) 5 µg of digested chromatin was immunoprecipitated with 2 µg of either normal rabbit IgG or anti-H3K4me3 antibody as mentioned in step 3.1 and step 3.2. Immune complexes were washed and eluted from beads, and reverse crosslinked. Purified DNA fragments were analyzed using real-time PCR with the primer sets listed in Table 2. Please click here to view a larger version of this figure.
Figure 4: Androgen increased active RNA polymerase II occupancy in the PSA promoter and enhancer. Steroid-starved LNCaP cells were treated with or without 10 nM DHT for 4 h, and digested chromatin was prepared. (A) Digestion pattern of chromatin from LNCaP cells in three independent experiments. (B,C) Digested chromatin was immunoprecipitated with an anti-PolII(pS5) antibody, and DNA fragments were purified as described for Figure 3. The occupancy of active RNA polymerase II in the PSA promoter, enhancer, and GAPDH promoter as a percent input (B) and fold enrichment (C) was determined using real-time PCR with the primer sets listed in Table 2. The results shown are mean ± SE of three independent experiments. (*); p<0.05, (**); p < 0.01 versus 0 nM DHT treatment. NS; not significant versus 0 nM DHT. Please click here to view a larger version of this figure.
Cell line | gel units per two million cells in 100 µL of buffer, 37 °C for 10 min |
LNCaP | 267 |
VCaP | 66.7 |
293T | 450 |
REH | 134 |
22Rv1 | 400 |
Table 1: Optimum amount of micrococcal nuclease in various cell lines. The value represents the amounts of MNase per 2 x 106 cells in 100 µL of buffer, 37 °C for 10 min.
Primer name | Sequence | |
AR (-18.8kb) | FWD | ATTTGGAACTGGGAACATCT |
REV | CACCTTCTCTCCTCCACTCT | |
AR (-8.8kb) | FWD | TAACAGCTTTGCATCCAAGT |
REV | TGAAATCTGGGACTAAAGCA | |
AR (-8.2kb) | FWD | CAGTGCTATTCCCTTGTGAC |
REV | TTGGACTGGCTCTATCTTGA | |
AR-TSS (0 kb) | FWD | GCAAACTGTTGCATTTGCTC |
REV | GGCCCTTTTTCCCTCTGTC | |
AR (0.6 kb) | FWD | CACGACCCGCCTGGTTAG |
REV | TGAAGACCTGACTGCCTTTTC | |
AR (+1.0kb) | FWD | CCGCAAGTTTCCTTCTCTGG |
REV | CTTCCCAGCCCTAACTGCAC | |
AR (+11.8kb) | FWD | CCTTGCTTGTGGAACTGTAG |
REV | TTTATTGTCTGGTGCTAGGC | |
PSA promoter | FWD | CCTAGATGAAGTCTCCATGAGCTACA |
REV | GGGAGGGAGAGCTAGCACTTG | |
PSA enhancer | FWD | GCCTGGATCTGAGAGAGATATCATC |
REV | ACACCTTTTTTTTTCTGGATTGTTG | |
GAPDH | FWD | TACTAGCGGTTTTACGGGCG |
REV | TCGAACAGGAGGAGCAGAGAGCGA |
Table 2: Paired Primer sequences used for ChIP assay.
AR (-18.8kb) | Cq | SQ | Adjusted to one IP | % Input | Fold enrichment |
1% Input | 23.49 | 0.815 | 81.5 | 100 | |
IP with Control IgG | 27.68 | 0.051 | 0.051 | 0.062 | 1 |
IP with anti-H3K4me3 | 22.48 | 1.590 | 1.590 | 1.951 | 31.4 |
AR (-8.8kb) | Cq | SQ | Adjusted to one IP | % Input | Fold enrichment |
1% Input | 23.22 | 0.586 | 58.6 | 100 | |
IP with Control IgG | 26.81 | 0.052 | 0.052 | 0.088 | 1 |
IP with anti-H3K4me3 | 23.74 | 0.414 | 0.414 | 0.706 | 8.0 |
AR (-8.2kb) | Cq | SQ | Adjusted to one IP | % Input | Fold enrichment |
1% Input | 23.19 | 0.643 | 64.3 | 100 | |
IP with Control IgG | 26.99 | 0.048 | 0.048 | 0.075 | 1 |
IP with anti-H3K4me3 | 23.63 | 0.477 | 0.477 | 0.742 | 9.9 |
AR-TSS (0 kb) | Cq | SQ | Adjusted to one IP | % Input | Fold enrichment |
1% Input | 25.06 | 0.657 | 65.7 | 100 | |
IP with Control IgG | 28.63 | 0.050 | 0.050 | 0.077 | 1 |
IP with anti-H3K4me3 | 20.70 | 15.064 | 15.064 | 22.944 | 299.8 |
AR (0.6 kb) | Cq | SQ | Adjusted to one IP | % Input | Fold enrichment |
1% Input | 23.86 | 0.716 | 71.6 | 100 | |
IP with Control IgG | 26.67 | 0.106 | 0.106 | 0.147 | 1 |
IP with anti-H3K4me3 | 19.15 | 17.787 | 17.787 | 24.840 | 168.6 |
AR (+1.0kb) | Cq | SQ | Adjusted to one IP | % Input | Fold enrichment |
1% Input | 23.51 | 0.730 | 73.0 | 100 | |
IP with Control IgG | 25.94 | 0.125 | 0.125 | 0.171 | 1 |
IP with anti-H3K4me3 | 19.06 | 18.486 | 18.486 | 25.335 | 147.8 |
AR (+11.8kb) | Cq | SQ | Adjusted to one IP | % Input | Fold enrichment |
1% Input | 24.54 | 0.876 | 87.6 | 100 | |
IP with Control IgG | 29.14 | 0.033 | 0.033 | 0.037 | 1 |
IP with anti-H3K4me3 | 24.47 | 0.918 | 0.918 | 1.048 | 27.97 |
Table 3: Raw data from quantitative PCR analysis for Figure 3. Cq: Threshold cycle number, SQ: starting quantity calculated using a standard curve, Adjusted to one IP: multiply SQ in 1% input by 100 as 1% sample volume of one IP is used for PCR, % Input: divide SQ in IP sample by adjusted SQ in Input, Fold enrichment: divide SQ in IP with anti-H3K4me3 by SQ in IP with control IgG.
Although sonication is commonly used to obtain fragmented chromatin, it is time-consuming and cumbersome to identify reproducible conditions. In this protocol, we used MNase digestion because enzyme digestion should be easier to identify reproducible conditions. A brief sonication step after MNase digestion (see step 2.2) was necessary to break the cell membrane and to release the digested chromatin. Therefore, the sonication power in our protocol should be as low as possible. We use the same sonication conditions for all cells we employed (see Figure 1 and Table 1) to obtain complete breakdown of the membrane.
Digestion of chromatin by MNase is a critical step in our protocol, and thus we have exerted great effort to optimize the conditions for the digestion of chromatin inside cells. Digestion activity is determined by the amounts of enzyme and substrate (chromatin) and incubation time. In addition, since ploidy varies among different cells, the optimum conditions for MNase digestion must be identified for each cell type. Once established, the same conditions can be applied irrespective of the cell treatments. We always check the chromatin digestion patterns in each experiment to ensure that the digestion patterns are suitable for ChIP assays, as shown in Figure 2A, lane 5.
Some factors affect the results of ChIP assays. It is important to use the right amount of digested chromatin in our protocol. In many protocols, the cell number but not the amount of chromatin for one IP is shown15,16. These experimental conditions produce high variability in the amount of chromatin among cells due to ploidy differences. We use 5 µg of chromatin and 2 µg of antibody per one IP in the assay, thus our protocol is more straightforward and clearer than other protocols, although optimum amounts of antibody for IP may be needed. The selection of antibodies is also important; use ChIP assay-validated antibodies.
There are two methods to analyze ChIP PCR data: the percent input method and the fold enrichment method. In the percent input method, signals from IP samples are divided by signals from total chromatin in the IP sample. In the fold enrichment method, signals from IP with a specific antibody such as H3K4me3 are divided by signals from IP with the control IgG. The later method is only applicable when signals from IP with the control IgG are similar and reproducible at multiple targets or identical targets under the different physiological conditions. In practice, the signal levels vary so the fold enrichment value has high variability as shown in Figure 4C. Therefore, we do not recommend using the fold enrichment method to represent ChIP data in our method.
MNase favors an euchromatin ‘open’ environment and is not accessible to a heterochromatin structure, suggesting that MNase digestion may produce some bias. It has been reported that enrichment of lamin A-interacting chromatin domains is different in between sonication-sheared and MNase-digested chromatin preparations17. Thus, MNase digestion in ChIP assay may not be suitable to analyze nuclear structure-associated molecules such as lamin A/C and Special AT-rich Sequence Binding Protein 1 (SATB1).
In ChIP assay protocols designed for downstream microarray (ChIP-on-chip) or sequencing (ChIP-seq) analyses, RNase is used during the DNA purification step, although not in protocols designed for PCR analyses18. We have not tested whether our protocol is compatible with ChIP-on-chip and ChIP-seq analyses, but we assume that our protocol is applicable when samples are treated with RNase. If RNase treatment is needed, use 2 µL of 10 mg/mL DNase-free RNase A instead of proteinase K in step 3.3 and incubate at 65 °C overnight. Before purifying DNA (step 3.4), add proteinase K and incubate for an additional 1 h at 60 °C.
We routinely carry out ChIP assays with modified histone and transcription factors using the method described here and have shown that the chromatin remodeling factor, AT-rich interaction domain 5B, regulates AR gene expression by changing the occupancy of PolII(pS5) and H3K4me3 in the AR promoter19. We believe that our protocol is technically easier than other ChIP assays and is widely acceptable in molecular biology research.
The authors have nothing to disclose.
This research is supported by Genentech royalties to City of Hope. This work is not supported in whole or in part by the National Institutes of Health.
0.5 M EDTA (pH 8.0) | Thermo Scientific | AM9010 | |
2 M KCl | Thermo Scientific | AM9010 | |
2X iQ SYBR Green supermix | Bio-Rad | 1706862 | |
5 M NaCl | Thermo Scientific | AM9010 | |
50 bp DNA ladder | New England Biolabs | N3236S | |
Agarose | Research Product International | A20090 | |
Branched octylphenoxy poly(ethyleneoxy)ethanol | Millipore Sigma | I8896 | IGEPAL CA-630 |
ChIP-grade protein G magnetic beads | Cell signaling technology | 9006S | |
Chromatin Immunoprecipitation (ChIP) Dilution Buffer | Millipore Sigma | 20-153 | Buffer composition: 0.01% SDS, 1.1% Triton X- 100, 1.2mM EDTA, 16.7mM Tris-HCl, pH 8.1, 167mM NaCl. |
Gel Loading Dye Purple (6X) | New England Biolabs | B7024S | |
Glycine | Bio-Rad | 161-0724 | Electropheresis grade |
Glycogen | Millipore Sigma | G1767 | 19-22 mg/mL |
Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100x) | Thermo Scientific | 78445 | |
High Salt Immune Complex Wash Buffer | Millipore Sigma | 20-155 | Buffer composition: 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl. |
Histone H3K4me3 antibody (pAb) | Active Motif | 39915 | |
LiCl Immune Complex Wash Buffer | Millipore Sigma | 20-156 | Buffer composition: 0.25M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid (sodium salt), 1mM EDTA, 10mM Tris, pH 8.1. |
Low Salt Immune Complex Wash Buffer | Millipore Sigma | 20-154 | Buffer composition: 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl. |
Magna GrIP Rack (8 well) | Millipore Sigma | 20-400 | Any kind of magnetic separation stands that are compatible with a 1.5 mL tube is fine. |
Micrococcal nuclease | New England Biolabs | M0247S | comes with 10 x buffer (500 mM Tris-HCl, 50 mM CaCl2, pH 7.9 @ 25 °C) and 100 x BSA (10 mg/ml) |
NaHCO3 | JT Baker | 3506-01 | |
Normal rabbit IgG | Millipore Sigma | 12-370 | |
PIPES | Millipore Sigma | P6757 | |
Proteinase K | Millipore Sigma | 3115887001 | |
Real-time PCR system | Bio-Rad | CFX96, C1000 | |
RNA pol II CTD phospho Ser5 antibody | Active Motif | 39749 | |
SDS | Boehringer Mannheim | 100155 | Electropheresis grade |
sodium acetate | Millipore Sigma | S5636 | |
Sonicator equipped with a microtip probe | QSONICA | Q700 | Any kind of sonicators that are compatible with a 1.5 mL tube is fine. |
UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) | Thermo Scientific | 15593031 | pH 8.05 |