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

lbalint@indi.biochem.dote.hu

 

 

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.

 

 

 

 

 

 

CHROMATIN IMMUNOPRECIPITATION – PRACTICALS

 

 

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.

 

 

Practical 5A:Cross-linking with formaldehyde (Fixation)

 

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 l 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 l).

13.  Incubate on ice for 10 min and proceed to sonication.


Practical 5B: Sonication of cells after cross-linking chromatin

 

 

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 l). 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 l 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 l on a 1% agarose gel to check size distribution – sonication efficiency.

 

 


Practical 6 - Chromatin Immunoprecipitation

 

 

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.

 

 

Practical 6A: Preclearing of chromatin

 

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 l 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 l of pre-cleared samples as input, store at -20 C until later use in the protocol

 


Practical 6B: Immunoprecipitation

 

 

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 l 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 l protein A bead to each sample and incubate at 4C 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 l 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 l of 10 % SDS).

4.  Add Proteinase K to final concentration of 0.5 mg/ml (add 5 l 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.

 

Practical 7A: Primer extension

 

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 l 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 l

 

5.  Place tubes in PCR machine

6.  Incubation program:

 

7.  Prepare diluted Sequenase (1 l per sample): 0.3 l Sequenase+0.7 Sequenase dilution buffer

8.  Place tubes on ice, and add 1.0 l of diluted Sequenase per sample. Total volume now: 16 l.

9.  Place tubes in PCR machine

10.  Incubation program:

11.  Place tubes on ice

12.  Dilute samples with 44 l nuclease-free water, to a final volume of  60 l

 


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 M)

 

 

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 l

 

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 g 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 g or lower. Purify your amplified DNA on two Qiaquick columns!

 

 

Send  at least 7.5 g-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 ONeil - 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.abcam.com

http://www.diagenode.com/

http://www.upstate.com

 

http://www.chromatin.us

http://www.epigenome-noe.net/

http://www.chiponchip.org/

 

http://www.genome.gov/10005107

http://www.genecore.embl.de/

http://genome.ucsc.edu/

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