3 RAG1/2 introduces double-stranded breaks at non-Ig loci | 57 them to circumvent the STI571-induces cell-death19. The following v-Abl/Bcl2 cell lines were used: A70 wildtype (WT), Rag2-/- (R2K3), Artemis-/- (AH2), and Rag2-/-/Artemis-/- (R2K. Art) as a model for developing lymphocytes, cultured under the conditions as previously described20. The cell lines were kindly provided by Dr. Craig Bassing (University of Pennsylvania School of Medicine, Philadelphia, PA). The cells were cultured in RPMI1640 (Life Technologies, Bleiswijk, the Netherlands) supplemented with 2 mM of L-glutamine, 100 U/ ml penicillin, 100 μg/ml streptomycin, and 10% FCS. To introduce RAG1/2 expression, and the subsequent RAG1/2 endonuclease activity leading to Igk recombination, the cells were treated with 5µM STI571 (imatinib methanesulfonate; LC Laboratories, Woburn, MA). The formation of Vκ6-23 to Jκ1 coding joins was determined by semiquantitative polymerase chain reaction (PCR) by using previously published primers19. Chromatin immunoprecipitation (ChIP) and Real-time quantitative PCR Cells were seeded at 2x106 cell/mL density and cultured for 72h following an addition of 5µM STI571 to allow sufficient V(D)J recombination and accumulation of DSBs. At this timepoint the cell death is still minimal19. After 72h, the viable cells were isolated using 3mL Lympholyte M spun at 1200rcf/20min. The cells were washed in RPMI1640 containing a cocktail of protease and phosphatase inhibitors: 100 mM PMSF, 1 mM sodium ortho-vanadate, 2mM sodium fluoride, and 1.25mM b-glycero-phosphate. Crosslinking of the chromatin was achieved by adding 37% formaldehyde (1% final formaldehyde concentration) to the resuspended cells in RPMI1640 at 2x106 cell/mL density, and incubating in a 37°C water bath for 5min. The crosslinking reaction was terminated by adding glycine to a final concentration of 125mM using 2.5M stock solution. The cells were washed in PBS and stored at -80°C for at least overnight. The cells were re-suspended at a concentration of 40x106 cell/mL in sonication buffer (2% SDS, 10 mM EDTA, 50mM Tris-Cl at pH=8, and containing the cocktail of protease and phosphatase inhibitors as mentioned above) and subjected to ultrasound sonication to obtain DNA fragments of average length of 250bp. Following the sonication, the cells were diluted in a dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-Cl pH=8, 167 mM NaCl) and incubated with 5mg of an antibody (rabbit anti-NBS1 antibody, Abcam; rabbit anti-histone H4 antibody, Millipore) per sample for 2.5h at 4°C, after with Protein A on beads was added to each sample and the incubation continued for another 2.5h at 4°C. The beads were washed with series of buffers, the low salt buffer (0.1% SDS, 1%, 2 mM EDTA, 20 mM Tris-Cl, pH=8, 150 mM NaCl), high-salt buffer (0.1% SDS, 1%, 2 mM EDTA, 20 mM Tris-Cl, pH=8, 500 mM NaCl), LiCl buffer (250 mM LiCl, 1% deoxycholate, 1% NP-40, 1mM EDTA, 10 mM Tris-Cl pH=8), TE buffer (10 mM Tris-Cl pH=8, 1mM EDTA) and finally the elution buffer (0.5% SDS, 5 mM EDTA, 10 mM Tris-Cl, pH=8, 300 mM NaCl) to elute the protein-chromatin complexes off the beads. The samples were heated overnight at 65°C to reverse the crosslinking, followed by the addition of Protease K and RNase A and subsequent overnight incubation at 55°C to destroy the remaining protein and RNA.
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