Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using BrdU-ChIP-Slot-Western Technique

Summary

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

Abstract

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.

Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

Introduction

Defective DNA repair and/or DNA replication are a major cause of genome instability, which can trigger cell death. A single unrepaired double strand break is sufficient to cause cell death¹. Chromatin organization is transiently altered during both replication and repair^2,3, and failure to maintain epigenetic information during these processes will result in a threat to genome integrity. Loss of HDAC3 or HDAC1,2 function impedes S-phase progression, DNA replication and repair leading to genotoxic stress (DNA damage) and cell death^4-9. It is therefore a practical strategy to use selective HDAC inhibitors to disrupt replication, repair and chromatin in cancer cells and cause DNA damage, which in turn can stop growth and induce cell death selectively in rapidly growing cancer cells.

During DNA replication, chromatin is rapidly disassembled and then reassembled following DNA duplication. Newly synthesized histone H4 is acetylated at K5 and K12 residues (H4K5K12ac) and following deposition onto chromatin, they are rapidly deacetylated by histone deacetylases (HDACs)^10,11. Failure of cells to maintain nascent chromatin integrity by histone deacetylation can lead to fork collapse, which in turn can result in DNA damage and cell death. We recently showed that selective inhibition of HDAC1,2 increases histone acetylation (H4K5ac, H4K12ac and H4K16ac) and inhibits SMARCA5 chromatin remodeler activity on nascent chromatin, which correlates with reduced replication fork progression, increased fork collapse and increased replication stress-induced DNA damage⁶. Thus, HDAC inhibitor treatment can alter nascent chromatin structure to trigger DNA damage and death very quickly in cancer cells, as these cells cycle rapidly and pass many times through the S-phase. It is therefore important to understand how HDACs function to regulate histone acetylation and protein binding to maintain DNA replication in mammalian cells.

To quantitatively measure the amount of histone acetylation and SMARCA5 chromatin remodeler associated with nascent DNA during DNA replication, we devised a modified ChIP assay called the BrdU-ChIP-Slot-Western technique. Following chromatin immunoprecipitation (ChIP) of a desired protein or histone modification, the amount of nascent DNA (labeled using thymidine analog, BrdU) in the ChIP sample can be detected using western analysis of BrdU-labeled ChIP DNA transferred onto a membrane using a Slot Blot apparatus. Using this technique, we showed that H4K16ac (a mark involved in chromatin packaging) and the ISWI family member SMARCA5 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin) chromatin remodeler are associated with nascent DNA in S-phase cells⁶. We also found that the nascent H4K16ac mark is deacetylated by HDAC1,2 during DNA replication⁶. H4K16ac inhibits chromatin remodeler activity of the ISWI family member SMARCA5¹². Hence, using the BrdU-CHIP-Slot-Western technique, we could connect the functions for HDAC1,2 in regulating chromatin remodeling during DNA replication. Hence, the BrdU-ChIP-Slot-Western Blot technique is a powerful approach to quantitatively measure the association and dissociation dynamics of proteins or their post-translational modifications that are bound to nascent DNA.

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Protocol

1. Chromatin immunoprecipitation (ChIP) following BrdU Labeling

1. Culture NIH3T3 cells in the presence of either DMSO or HDAC1,2-selective inhibitors as described previously⁶.
2. Following treatment with DMSO or HDAC1,2 selective inhibitors, add BrdU to the cells in a sterile tissue culture hood. Incubate 2 x 10⁶ NIH3T3 cells plated in 10 ml NIH3T3 media with a 20 µM final concentration of bromodeoxyuridine (BrdU) for 60 min in a sterile 37 °C tissue culture incubator.
3. Crosslink intracellular proteins to DNA by adding formaldehyde directly to the culture media at a final concentration of 1%. Incubate 2 x 10⁶ NIH3T3 cells with formaldehyde at RT for 10 min on a shaker.
4. Quench the cross linking by the addition of glycine to a final concentration of 125 mM. Incubate at RT for 5 min on a shaker.
5. Remove the media and collect cells in PBS in a 15 ml centrifuge tube. Spin cells at 956 x g for 5 min at 4 °C. Wash the cell pellet twice with ice-cold PBS. Remove PBS from the tube. Store the cross-linked cell pellet without PBS (the supernatant) at -80 °C until use.
6. Prepare the lysate from the crosslinked cell pellet by adding 500 µl FA140 buffer (50 mM of HEPES KOH pH 7.5, 140 mM of NaCl, 1 mM of EDTA (pH 8.0), 1% Triton-X-100 and 0.1% sodium deoxycholate) supplemented with protease inhibitor cocktail.
7. Shear the chromatin by sonicating the samples five times at 35% power and with a 15 sec pulse, 0.9 sec on and 0.1 sec off setting. Keep the tubes on ice after every round of sonication to avoid heating of the samples.
   1. Check the shearing on a DNA gel as the shearing efficiency will vary between different sonicators. To check sonication, reverse crosslink by adding NaCl to a concentration of 0.16 M and perform steps 1.17.1. - 1.22. Run eluted 10 µl DNA on a 1.5% agarose gel to check whether the shearing is between 300 - 700 bp with an average intensity at 500 bp.
8. Following sonication, centrifuge samples for 10 min at 17,949 x g at 4 °C and transfer the supernatant to a new tube.
   1. Perform protein estimation using the commercial protein assay kit according to manufacturer's protocol. Use equal amount of lysate for ChIP of control and experimental samples. Generally, use 1 mg of total lysate per ChIP reaction.
9. Prepare 50% slurry of protein A-agarose using FA140 buffer containing protease inhibitor cocktail. Add 1/10 volume of the protease inhibitor cocktail. Calculate the volume of beads needed based on the number of samples in total.
   1. Wash Protein A-agarose beads with FA140 buffer twice to remove the storage buffer and add an equal volume to FA140 buffer to the beads to make 50% slurry.
   2. To remove proteins that non-specifically bind to the beads, add 50 µl of 50% slurry of Protein A-agarose and incubate the samples at 4 °C with constant end-over-end rotation in a rotisserie mixer for 30 min.
10. Spin the samples at 956 x g at 4 °C for a minute. Collect the supernatant and perform immunoprecipitation by adding either 8 μl H4K16ac antibody or 5 μl (5 μg) of 1 mg/ml SMARCA5 antibody to the supernatant. Incubate at 4 °C O/N with constant end-over-end rotation in a rotisserie mixer.
11. Save 1/10th of the extract for 'input' control and set it as aside at -20 °C. The input needs to be reverse cross-linked along with the IP samples.
12. Use equal amount of rabbit IgG (5 μl of 1 mg/ml) as the negative control immunoprecipitation sample.
13. The following day, add 50 μl of 50% protein A-agarose slurry to the extract and incubate for 1 hr at 4 °C with rotation.
14. Pellet agarose beads by centrifugation at 956 x g for 2 min at 4 °C, carefully remove the supernatant and wash the beads with the following buffers sequentially. In buffer preparations, add protease inhibitors right before use.
    1. Wash with Low Salt Immune Complex Wash Buffer (FA140; final concentration of 50 mM of HEPES KOH pH 7.5, 140 mM of NaCl, 1 mM of EDTA (pH 8.0), 1% Triton-X-100 and 0.1% Sodium deoxycholate) for 5 min at 4 °C with end-over-end rotation.
    2. Wash with High Salt Immune Complex Wash Buffer (FA500; final concentration of 50 mM of HEPES KOH pH 7.5, 500 mM of NaCl, 1 mM of EDTA (pH 8.0), 1% Triton-X-100 and 0.1% Sodium deoxycholate) for 5 min at 4 °C with end-over-end rotation.
    3. Wash with LiCl Immune Complex Wash Buffer (final concentration of 10 mM of Tris-Cl pH 8.0, 250 mM of LiCl, 0.5% NP40 and 0.5% Sodium deoxycholate) for 5 min at 4 °C with end-over-end rotation.
15. Following washes as described above, add 200 µl elution solution (final concentration of 1% SDS and 100 mM Sodium bicarbonate) and incubate at RT for 15 min with end-over-end rotation at RT.
16. Spin the samples at 956 x g at RT and transfer the eluate to a new 1.5 ml tube. Repeat step 1.15 with additional 200 µl elution solution.
17. To 400 µl of the eluate, add NaCl to a concentration of 0.16 M and mix well.
    1. In addition, add 400 µl elution solution to 10 µl input that was set aside (step 1.11) and add NaCl to a concentration of 0.16 M to reverse crosslink input samples as well. Reverse crosslink the samples by incubating in 65 °C water bath for 5 hr.
18. Add 1 ml of 100% EtOH to the reverse-crosslinked eluate. Mix well and incubate at -80 °C for 2 - 3 hr or at -20 °C O/N. Spin the samples at 17,949 x g for 15 min and discard ethanol.
19. Add 800 µl 70% ethanol to wash the pellet. Spin at 17,949 x g for 15 min at 4 °C and discard ethanol. Spin briefly to remove any residual ethanol. Air-dry the samples at 37 °C for 10 min to remove residual ethanol.
20. Dissolve DNA in 90 µl RNase and DNase free water and add 2 µl 10 mg/ml RNase at a 0.2 mg/ml final concentration. Incubate at 37 °C in water bath for 30 min.
21. Add 10 µl 10x Proteinase K Buffer (100 mM Tris-Cl pH 8.0, 50 mM EDTA pH 8.0, 5% SDS). Mix well and add 1 µl Proteinase K. Incubate at 42 °C for 1 hr.
22. Purify input and ChIP DNA using commercial PCR purification kit according to manufacturer's protocol and elute DNA in 50 µl water. Store DNA at -20 °C until further use.

2. Slot Blot Analysis of ChIP DNA

1. Measure the yield of input and ChIP DNA eluted in 50 µl water as described in step 1.22 using a spectrophotometer at 280 nm according to manufacturer's protocol.
2. Take equal volumes (50 µl) of input or immunoprecipitated DNA that are within the linear range of detection.
   1. For instance, if input DNA yield is 1.5 ng/µl, then take 30 µl (or 45 ng total DNA) and make volume to 50 µl with water. For factor ChIP, take the entire 50 µl eluate from Step 2.1 and proceed to step 2.3.
3. Denature 50 µl input or immunoprecipitated DNA by adding 2.5 volumes of 0.4 N NaOH, briefly vortex and centrifuge for 956 x g for 10 sec, and incubate at RT for 30 min.
4. Neutralize the denatured DNA by adding 175 µl of 1 M Tris-HCl, pH 6.8, vortex, centrifuge for 956 x g for 10 sec and place the samples on ice.
5. Prepare a serial dilution of input and immunoprecipitated DNA for the Slot Blot assay as described below.
   1. Follow steps 2.3 - 2.4 to get a total sample volume of 350 µl (i.e., 50 µl Input/ChIP DNA, 125 µl 0.4 N NaOH, 175 µl 1 M Tris-HCl, pH 6.8).
      Note: For the instance stated above, 45 ng of input DNA will end up being 0.129 ng/ µl.
   2. For input DNA, label three tubes as 100, 50 and 25 and add 100 µl, 50 µl and 25 µl of the denatured and neutralized DNA sample. Make the final volume to 100 µl with nuclease-free water. For ChIP DNA, label three tubes as 200, 100 and 50 and add 200 µl, 100 µl and 50 µl of the denatured and neutralized DNA sample. Make final volume to 200 µl with nuclease free water.
6. Cut the membrane to the size of the Slot Blot apparatus. Pre-wet the membrane and blotting papers in 20x SSC before and prior to adding DNA. Assemble them into the Slot Blot apparatus according to the manufacturer's protocol.
7. Pipet DNA from step 2.5.2 into appropriate slots. Apply vacuum slowly to pull the DNA onto the zeta-probe membrane. Stop vacuum after all samples have passed through the apparatus.
   1. Disassemble the apparatus and immediately immobilize the DNA onto the membrane using a UV crosslinker. Have the DNA side up in the UV crosslinker and use the autocrosslink setting in the crosslinker. Follow manufacturer's protocol.
8. Detect BrdU on the slot blot by performing western blotting.
   1. Block the membrane with 5% non-fat dry milk in PBS containing 0.1% Tween-20 (PBST) for 1 hr at RT to prevent the non-specific binding of antibodies to the membrane.
   2. Add primary anti-BrdU antibody (1:500 dilution) diluted in 1% non-fat dry milk in PBST and incubate the blot with the primary antibody for 3 hr at RT on a shaker. Wash the membrane with PBST three times after the primary antibody incubation.
   3. Add HRP-conjugated anti-mouse secondary antibody to the blot (1:5,000 dilution) and incubate at RT for 1 hr. Wash the membrane with PBST three times after the secondary antibody incubation.
      NOTE: Use enough volume of the antibodies to completely cover the membrane during primary and secondary antibody incubations.
9. Prepare the ECL mix and add it to the blot according to the manufacturer's protocol. Incubate the membrane with ECL mix for 5 min at RT.
   1. Place the membrane in an X-ray cassette, expose the membrane to the X-ray film and develop the blot using the manufacturer's protocol.
   2. Quantify the signal obtained for input and ChIPed DNA after scanning the blots using the Image J software according to manufacturer's protocol. For quantitation, use the signal that is within the linear range of detection in order to ensure accuracy of measurement.

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Results

To determine the specificity of HDAC1,2-selective inhibitors, Hdac1,2^FL/FL and Hdac3^FL/FL fibrosarcoma cells were used. Adenovirus-containing Cre recombinase (Ad-Cre) was used to delete Hdac1,2 and Hdac3 in these cells. Following Ad-Cre infection of Hdac1,2^FL/FL cells, a robust increase in H4K5ac was observed. Treatment of Hdac1,2 knockout cells with 233 or 898 did not result in...

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Discussion

The protocol described in this manuscript is a relatively quick method to demonstrate the presence of proteins or their post-translationally modified forms on newly replicated or nascent DNA. Additionally, this technique permits one to measure the association-dissociation kinetics of a protein or its modified form with nascent DNA. This technique is complementary to the elegant iPOND technology¹³. In the iPOND technology, newly synthesized DNA is labeled with ethyl deoxyuridine (EdU). A biotin conjugate is the...

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Materials

Name	Company	Catalog Number	Comments
Anti-BrdU (westerns)	BD Biosciences	B555627
Anti-SMARCA5	Abcam	ab3749
Anti-H4K16ac	Active Motif	39167
Zeta-Probe GT Membrane	Bio-Rad	162-0197
Formaldehyde	Fisher	BP531-500
Protein A agarose	Millipore	16-156
Rnase A	Qiagen	19101
Proteinase K	Sigma	P4850
PCR Purification Kit	Qiagen	28106
Glycine	Sigma	G7403
Protease inhibitor cocktail	Roche	11836170001
BrdU	Sigma	B9285
Rabbit IgG	Millipore	12-370
HEPES	Sigma	H3375
NaCl	Sigma	S-3014
Triton-X-100	Sigma	93443
NP40	USB Corporation	19628
Sodium deoxycholate	Sigma	D6750
Sodium bicarbonate	Sigma	S-4019
Ethanol	Decon Laboratories	04-355-222
DEPC-treated water	Sigma	95284
Tris	Fisher	BP-152-5
EDTA	Invitrogen	15575-020
SDS	Ambion	AM9820
Sodium hydroxide	Mallinckrodt GenAR	MAL7772-06
20x SSC	Life Technologies	15557-036
Non-fat dry milk	Lab Scientific Inc	M0841
ECL	Thermo Fisher	80196
X-ray film	Genesee Sci	30-101
Developer	Konica Minolta	SRX-101A
UV Crosslinker	Stratagene	XLE-1000
Sonicator	Branson Sonifier  Digital Ultrasonic Cell Disruptor	Model: 450
Centrifuge	Eppendorf	Model: 5810R
Water bath	Fisher Scientific Isotemp	Model: 2322
NanoDrop 1000 spectrophotometer	ThermoScientific	Model: 1000
Slot Blot apparatus	Schleicher and Schuell Minifold II	44-27570
Tissue Culture Incubator	Thermo Scientific	Series II 3110 Water-Jacketed CO2 Incubators