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....

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 10

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 any .......

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.......

Materials

List of materials used in this article

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