CHROMATIN
Balint L. Balint
University of
Debrecen, Medical and Health Science Center, Department of Biochemistry and
Molecular Biology, Life Science Building, Room 3-211, Egyetem ter 1, Debrecen,
H-4010, Hungary, Tel.: +36 52 416432, Fax: +36 52 314989
INTRODUCTION TO THE PROCEDURE
Chromatin immunoprecipitation (ChIP) is a method to study protein DNA interactions. It is a powerful technique to analyze the interaction of a specific protein with a known DNA sequence. Its main advantage is that the proteinDNA interaction is studied in the intact cell in its chromatin context. In contrast, the majority of other methods developed to study protein DNA interactions are either using purified proteins in vitro (as in the case of EMSA – electro/mobility shift assay) or overexpressed proteins (as in the case of mammalian two-hybrid techniques). In the case of ChIP-on-chip (chromatin IP combined with microarray analysis) even the whole genome, or a selected part of it, can be interrogated for a specific protein-DNA interaction.
The ChIP itself combines the classical technique of protein immunoprecipitation by using specific antibodies, with the sequence-specific detection of the bound DNA to the assessed protein. To allow co-immunoprecipitation of DNA with the investigated protein, a covalent cross-linking step is included. The in vivo protein-DNA interactions are stabilized by a reversible crosslink formed during a short (in our case for 10 minutes) mild (1 % formaldehyde) fixation. This crosslink is later reversed by incubation at 65 °C prior to the DNA purification.
For correct interpretation of the results, a number of controls are needed. The most important control is the No antibody control, which is showing us the background of the reaction. Another control is the input sample, which is the starting DNA before IP. Positive controls for genomic DNA regions that are known to be bound by the investigated protein, and negative controls for genomic regions that are not bound, also help interpretation of the results.
The technique is relatively laborious but can be divided in three blocks: 1. Soluble chromatin isolation, 2. Immunoprecipitation, 3. DNA purification and analysis. In our experimental system we will analyze HL60 cells in control and retinoid treated states.
OUTLINE OF THE EXPERIMENTS
In these experiments we will isolate soluble chromatin from naïve, primed and primed-retinoid treated HL60 cells to monitor the effects of DMSO priming and 9-cis retinoic acid treatment. This fixed soluble chromatin will be used to immunoprecipitate nucleosomes with two different antibodies raised against polyacetylated histone H4 and K4 dimethylated histone H3. Both are markers of active euchromatin state, but they are found on different regulatory regions. We will analyze the ChIP product by QPCR during Practical 8. A small portion of the ChIP product will be amplified to be analyzed on a Tiling Array platform. The platform used for this experiment is the Affymetrix Encode Tiling Array.
Practical 5 -
Chromatin preparation
Each group will receive a flask with naïve, primed or retinoid treated cells HL60 cells. During this practical we will crosslink the protein-DNA complexes, isolate nuclei and sonicate to have soluble chromatin.
Checklist:
At room temperature:
37 % Formaldehyde
Proteinase Inhibitor Cocktail
Sonication Buffer
On Ice:
1.67 M Glycine
1X PBS
Nuclear Isolation Lysis Buffer
Other:
Timer
Microcentrifuge tubes
50 ml Tubes
Prechilled centrifuge for 50 ml tubes
Prechilled benchtop centrifuge for 1.5 ml tubes
Ice
NOTE: For your experiments, cells were grown as in steps 1-2.
1. Grow enough cells to allow detection of a single copy gene (usually 2 x 107 cells minimum, depending on IP efficiency).
2. Use ~ 0.5 - 2 x 107 cells per IP
3. Fix cells by adding formaldehyde to a final concentration of 1 % (add 1100 ml of 37 % formaldehyde to 40 ml of cell suspension in culture medium).
4. Incubate at RT for 10 min, gently swirling the 40 ml culture occasionally to resuspend cells.
5. Add 1/12 volume of 1.67 M glycine (3.4 ml to 41 ml fixed culture). Place tube on ice, and perform the rest of the procedure on ice.
6. Pellet cells by centrifuging @ 4 °C, 700 g, 4 min - discard supernatant.
7. Wash pellet with 10 ml ice-cold 1X PBS.
8. Pellet cells by centrifuging @ 4 °C, 700 g, 4 min - discard supernatant.
9. Resuspend pellet in 1000 µl of Nuclear Isolation Lysis buffer.
10. Incubate on ice for 10 minutes, vortex several times during incubation.
11. Pellet nuclei in a benchtop centrifuge at 4 °C, 12000 g, 10 minutes.
12. Resuspend the nuclei in Sonication Buffer (500 µl).
13. Incubate on ice for 10 min and proceed to sonication.
Checklist:
Prechill the sonicator with ice and water
Prechill the benchtop centrifuge
Sonication is performed with a Bioruptor type sonicator in closed 1.5 ml tubes (maximum 500 µl). Settings of the sonicator are: High intensity, 0.5 min ON/0.5 min OFF. For HL60 CDM1 cells two cycles of 10+10-minute sonication is enough.
Fragment size can be checked after decrosslinking on an agarose gel (described later in the protocol).
NOTE: Fixation and sonication times may vary between different cell types!
1. Insert the 1.5 ml tubes in the appropriate tube holder and start the first cycle of sonication.
2. Add fresh ice to the sonication bath after the first 10-min cycle to cool it back and start the second 10 minutes cycle.
3. Centrifuge samples for 15 min at 4 °C using a benchtop centrifuge at 12 000 rpm.
4. Transfer the supernatant in a clean tube.
At this step soluble chromatin can be snap-frozen in liquid N2 and stored at –70 °C
OPTIONAL: How to check sonication efficiency?
Incubate 50 µl of chromatin at 65 °C overnight, then adjust the SDS concentration to 0.5% and add Proteinase K to final concentration of 0.5 mg/ml. Incubate at 42 °C for 3 hrs. Clean-up using Qiaquick PCR purification columns (described in Practical 6D). Load 2-20 µl on a 1% agarose gel to check size distribution – sonication efficiency.
During this part of the protocol the chromatin prepared previously will be diluted and precleared with Protein A agarose beads to lower the non specific binding of DNA during the next step of immunoprecipitation and we will continue with the immunoprecipitation. For the immunoprecipitation we will use H4-Acetylated and H3K4-methylated antibodies. One third of the sample will serve as a No Antibody Control. The immunoprecipitated complexes will be washed extensively and after decrosslinking and digesting of the proteins, the collected DNA will be purified.
Checklist:
On ice:
IP dilution buffer
Proteinase Inhibitor Cocktail (1000X)
Blocked Protein A sepharose beads (50 % slurry)
Other:
Prechilled centrifuge
Rotator in cold room (4 °C)
1. ( If the sample was frozen, spin again after thawing to remove precipitates that might have formed.)
2. Transfer 150 ml of soluble chromatin to a 1.5 ml Eppendorf tube and add 9 volumes (1350 ml) of IP dilution buffer (containing proteinase inhibitors at a 1X final concentration, approx. 1.5 ml).
3. Pre-clear chromatin by adding 70 µl blocked protein A sepharose beads (50% slurry)
4. Incubate on a rotating platform at 4 °C for 1 hour.
5. Centrifuge in a prechilled centrifuge at 2,000 rpm for 2 min.
6. Transfer supernatant to fresh tube, discard beads.
7. Remove 150 µl of pre-cleared samples as “input”, store at -20 °C until later use in the protocol
Checklist:
Antibodies (H4 acetylation and H3K4 dimethylation) (at
the instructor)
Blocked Protein A sepharose beads (50% slurry)
Prechilled centrifuge
1. Divide the precleared sample in three equal parts (each 450 ml).
2. Add 5 µl of the appropriate antibodies to the other two tubes (Anti-acetyl-Histone H4 Upstate #06-866 and Anti H3K4 dimethyl Ab Upstate # 07-030).
3. Incubate on rotating platform at 4 °C overnight.
Next morning:
1. Centrifuge the tubes on a tabletop centrifuge at 4 °C, 15 minutes, 13000 rpm. During this step non/specific precipitates are removed from the solution.
2. Transfer supernatant to a new tube.
3. Add 60 µl protein A bead to each sample and incubate at 4°C for one hour.
4. Pellet by centrifuging at 2000 rpm, 2 min, discard supernatant.
Practical 6C:
Washing of bead-bound DNA after IP
Checklist:
On ice:
Wash Buffer A
Wash Buffer B
Wash Buffer C
TE Buffer
Other:
Proteinase Inhibitor Cocktail (1000X)
Rotator at room temperature
Prechilled centrifuge
NOTE: Washing during the next steps is critical! Try to remove all the supernatant without touching the pelleted beads.
1. Resuspend beads in 1 ml IP dilution buffer + PROTEINASE INHIBITORS.
2. Pellet beads by centrifuging at 2000 rpm, 2 minutes - discard supernatant.
3. Wash beads for 2 min at RT on a rotator with 1 mL ChIP Wash Buffer A + PROTEINASE INHIBITORS.
4. Pellet beads - 2000 rpm, 2 minutes - discard supernatant.
5. Wash beads for 2 min at RT on a rotator with 1 mL ChIP Wash Buffer B + PROTEINASE INHIBITORS.
6. Pellet beads - 2000 rpm, 2 minutes - discard supernatant.
7. Wash beads for 2 min at RT on a rotator with 1 mL ChIP Wash Buffer C+ PROTEINASE INHIBITORS.
8. Pellet beads - 2000 rpm, 2 minutes - discard supernatant.
9. Wash beads for 2 min at RT on a rotator with 1 mL TE buffer + PROTEINASE INHIBITORS.
10. Pellet beads - 2000 rpm, 2 minutes - discard supernatant.
11. Repeat wash as in steps 9-10.
NOTE: If the No antibody control in your later experiments gives a high signal, you might try to wash twice with buffers A, B, and C.
Practical 6D:
De-crosslinking and DNA purification
Checklist:
On ice:
Proteinase K 10mg/ml
TE Buffer
At room temperature:
SDS (10%)
Qiaquick PCR purification column
Qiaquick Binding Buffer
Qiaquick Wash Buffer
Qiaquick Elution Buffer
Nuclease free water
1. Add 100 µl TE buffer to the beads. No need for proteinase inhibitors from now on.
2. Incubate at 65 °C for 5 hrs.
3. Adjust the SDS concentration to 0.5 % (add 5 µl of 10 % SDS).
4. Add Proteinase K to final concentration of 0.5 mg/ml (add 5 µl of 10 mg/ml Proteinase K).
5. Incubate at 42 °C for 3 hrs.
6. Stop the reaction by putting tubes to -20 °C (will be done by the course staff).
Next morning:
7. Clean-up using Qiaquick PCR columns:
This is the ChIP product. At this step you have 40 ml-s of product. Put 10 ml-s in a tube to use for the next protocol of DNA amplification (Practical 7). The remaining 30 ml-s will be diluted with 50 ml-s of Nuclease free water and put in 8 well strips to be later used at Practical 8A for QPCR measurements.
Practical 7 - DNA amplification after ChIP
A fraction of the previously purified DNA will be flagged with a linker by a primer extension reaction. The 3‘ end of the used primer is random, while the 5‘ end is specific. This material will be later amplified in a PCR reaction using a second primer that is complementary with the specific 5‘ end of the primer used in the previous primer extension reaction.
Checklist:
On ice:
Primer A: GTTTCCCAGTCACGATCNNNNNNNNN (HPLC purified)
Nuclease free water
5X sequenase buffer
dNTP 2.5mM
DTT 0.1M
BSA 0.5 mg/ml
SEQENASE 13U/ul (at the instructor)
1. Use 30 % (in our case use 7 µl from the 10 ml-s) of ChIP or input DNA for initial round of linear amplification.
2. Prepare first round reaction by mixing the following components in a 0.2 ml PCR tube on ice:
3. Primer annealing (performed in a PCR machine)
4. Add Reaction Mixture to sample:
TO ADD TOTAL 5.05 µl
5. Place tubes in PCR machine
6. Incubation program:
7. Prepare diluted Sequenase (1 µl per sample): 0.3 µl Sequenase+0.7 Sequenase dilution buffer
8. Place tubes on ice, and add 1.0 µl of diluted Sequenase per sample. Total volume now: 16 µl.
9. Place tubes in PCR machine
10. Incubation program:
11. Place tubes on ice
12. Dilute samples with 44 µl nuclease-free water, to a final volume of 60 µl
Practical 7B:
DNA Amplification
Checklist:
On ice:
Nuclease free water
MgCl2 25mM
10X PCR Buffer
Taq polymerase 5 U/ul (at the instructor)
Primer B (GTTTCCCAGTCACGATC) (100 µM)
1. Use the diluted sample from Practical 7A, Step 12 as template for the amplification.
2. Mix the following components in a 0.2 ml PCR tube on ice:
Final volume: 100 µl
Total 100ul (Mix to add at this step 85ul).
3. Place tubes in the PCR machine and begin amplification:
PCR cycle conditions:
1. 30 sec 94 °C
2. 30 sec 40 °C
3. 30 sec 50 °C
4. 2 min 72 °C
Repeat steps 2-4 35 times
5. Hold 4 °C
4. Check amplified DNA with gel electrophoresis on a 1% agarose gel.
If you want to send samples to a ChIP-on-chip Core facility, proceed to step 5.
During the course we stop at point 3, the PCR reaction. The amplification will be checked by the course staff by using agarose gel electrophoresis.
5. Purify amplified DNA with Qiaquick PCR purification columns:
You will need approximately 10 µg of amplified, purified material. If the yield is not enough, continue the amplification. Take into account that a Qiaquick PCR purification column has a maximum binding capacity of 10 µg or lower. Purify your amplified DNA on two Qiaquick columns!
Send at least 7.5 µg-s of this amplified product to a microarray core facility.
Tips and tricks
1. Use always siliconized tubes.
2. This protocol is using Protein A –Agarose beads for collecting of the antibody- protein complexes. Alternatively, magnetic secondary antibodies could be used as described by Ren et al. (see also http://www.chiponchip.org/).
3. Antibody selection: usually a good approach is to use verified ChIP grade antibodies. If no such antibody is available, the approach is to use antibodies that have been proved as working in immunhistohemistry (IHC) and immunoprecipitation (IP), and are polyclonal. Regarding good signal in immunhistochemistry: this is a sign that the epitope recognized by the antibody is not destroyed by formaldehyde fixation. Regarding immunoprecipitation: antibodies that work in IP have an intact Fc terminal and should work in Protein A based immunoprecipitation. If this is a problem, alternatively, magnetic secondary antibodies against the primary Ab-s can be used for collecting the complexes. Regarding polyclonals: due to the fact that polyclonals recognize several epitopes, there is a smaller chance to lose the epitopes during fixation. For histone modification studies native ChIP ( as described by Laura O’Neil - N-ChIP) can be used (in this case there is no need for fixation, and we found that solutions used for the preparation of chromatin during N-ChIP should be supplemented with 1 mM DTT).
4. If using Protein A for complex collection you might pay attention to the fact that there are slight species-differences between Protein A and Protein G regarding the binding to antibodies raised in different species (for more information consult Using Antibodies-Harlow and Lane, CSHL Press) (as a general rule for antibodies raised in rabbit work well with Protein A, those raised in mouse work well with Protein G).
5. During usage of Protein A-agarose beads, pay attention to the fact that these beads need to blocked. You can buy preblocked protein A beads, or you can block them yourself. For this use ultrapure BSA and sonicated salmon sperm DNA. Wash beads to remove any additives by washing with 2 volumes of IP buffer 5 times. Pay attention to always centrifuge the beads gently with maximum speed of 2500 rpm (0.5 rcf), because the beads are fragile. Use 1-5 mg/ml BSA and sonicated salmon sperm DNA in IP buffer and rotate at least 4-6 hours. For ChIP-on-chip experiments you might use yeast tRNA in place of salmon sperm DNA.
6. Fragmentation: you might use micrococcal nuclease digestion or sonication. Even restriction enzyme digestion could be used (this is used on fixed cells in the so-called chromosome conformation capture technique ). For details check the websites listed below.
7. Sonication: you might use other type of sonicators also. If using a sonicator with tip (e.g. Branson) sonicate enough to have a fragment size of 500-1500 bp-s.
8. For each sample prepare a ”No antibody” or “Beads only” control. This is showing the background. If the background is too high, this might be a sign of inappropriate blocking of the beads, not enough washing of the beads or sticky, non-siliconized tubes.
9. The last washing step with TE buffer: be careful not to lose the pellet. I would strongly suggest to use the 1 ml pipet for the removal of the last TE wash solution!!!
10. The bound complexes can be eluted from the beads (Elution Buffer: 25 mM Tris-Cl pH 7.5, 5M EDTA, 0.5% SDS). This might be useful if you want to use them in a Re-Chip experiment. If you are not doing Re-Chip, the most convenient approach is to add TE buffer and after incubation at 65 °C (4-6 hours till overnight) digest the proteins with Proteinase K at 45 °C for 1-3 hours. Use this solution to purify the DNA.
11. The most convenient way to purify the DNA is by column purification. Some protocols use phenol : chloroform purification by using glycogen as a carrier.
12. Use 30% of the ChIP sample to perform the primer extension reaction. Instead of Sequenase you might use high concentration Klenow exo minus enzyme.
13. The amplified material can be purified with Qiaquick PCR purification columns or other similar products.
14. Useful websites:
http://www.genome.gov/10005107
http://genome.ucsc.edu/ENCODE/
http://www.affymetrix.com/products/arrays/specific/encode.affx
Reagents/Solutions
SIGMA Prot Inh. Cocktail (PI)
P8340
1000X STOCK
NUCLEAR ISOLATION LYSIS BUFFER
5mM PIPES pH 8 (SIGMA P1851)
85 mM KCl
0.5% NP40 (IGEPAL CA 630, FLUKA 5674)
+PI add before use
SONICATION BUFFER
1% SDS (SIGMA L 4390)
10 mM EDTA
50 mM Tris Hcl pH 8.1
+PI add before use
IP DILUTION BUFFER
0.01% SDS
1.1% Triton-X 100 (SIGMA T8787)
1.2 mM EDTA
16.7 mM Tris pH 8.1
167 mM NaCl
+PI add before use
TE
0.1 mM Tris
1 mM EDTA pH 8.0
WASH BUFFER A
0.1% SDS
1% Triton X-100
2 mM EDTA
20 mM Tris-HCl, pH 8.1
150 mM NaCl
+PI add before use
WASH BUFFER B
0.1% SDS
1% Triton X-100
2 mM EDTA
20 mM Tris-HCl, pH 8.1
500 mM NaCl
+PI add before use
WASH BUFFER C
0.25 M LiCl (SIGMA L0505)
1% NP40
1% sodium deoxycholate (SIGMA D6750)
1 mM EDTA
10 mM Tris HCl pH 8.1
+PI add before use
Other Ordering Information
Proteinase K
Fermentas E00492
Siliconized Tubes
Axygen, microtubes MCT-150-SL-C
1.5 ML SILICONIZED, CLEAR
Protein A Agarose /Salmon Sperm DNA
Upstate 16-157
Protein G Agarose /Salmon Sperm DNA
Upstate 16-201
Antibodies used
Upstate:
H4 polyacetylated 06-866
H3K4 dimethylated 07-030
Primer A: GTTTCCCAGTCACGATCNNNNNNNNN (HPLC purified)
Primer B (GTTTCCCAGTCACGATC) standard purification
9 cis retinoic acid
Sigma R4643
For alternative blocking of Protein A:
Protein A-Sepharose CL-4B
Amersham Pharmacia (GE) cat 17-0780-01
BSA
Sigma A3059
ssDNA (Deoxyribonucleic acid from Salmon Testes)
Sigma D9156
bakers yeast tRNA (RIBONUCLEIC ACID TRANSFER YEAST)
Sigma R563