Biology
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.
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.
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 repair2,3, 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 celldeath4-9. It is therefore a practical strategy to use selective HDAC inhibitors to disrupt replication....
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1. Chromatin immunoprecipitation (ChIP) following BrdU Labeling
- Culture NIH3T3 cells in the presence of either DMSO or HDAC1,2-selective inhibitors as described previously6.
- 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.
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To determine the specificity of HDAC1,2-selective inhibitors, Hdac1,2FL/FL and Hdac3FL/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 Hdac1,2FL/FL cells, a robust increase in H4K5ac was observed. Treatment of Hdac1,2knockout cells with 233 or 898 did not result in.......
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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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The work in this manuscript was supported by funds from Dept. of Radiation Oncology and Huntsman Cancer Institute and the National Institute of Health grant (R01-CA188520) to SB. I thank Danielle Johnson and Steven Bennett in my lab for demonstrating the protocol and explaining its benefits. I am grateful to Dr. Mahesh Chandrasekharan (Huntsman Cancer Institute) for critical comments on the manuscript.
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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 co*cktail | 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 |
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