Fibroblast and lymphoblastoid cell lines used for ChIP were obtained from the Coriell Cell Repositories: Normal human lung fibroblasts AG06814 (WI-38), BS skin fibroblasts GM02932B (blmAsh
), normal lymphoblastoid cell lines GM00103 and GM00536. The Tet-responsive SV40-transformed BS cell line HG2551a and cell lines expressing GFP-BLM fusion genes were described before [19
]. HG1522 and HG1525 cell lines were isolated and described previously [28
Fibroblast cell lines were grown as described before [4
] in T-75 flasks. Normal and BS fibroblasts were split and seeded out 24 hours before fixation. The medium was removed by aspiration, followed by the addition of PBS + 1% formaldehyde (Sigma) to the flasks (5 mls) After 10 minutes of incubation at room temperature glycine pH 7.0 was added to 0.125 M and the flasks were further incubated for 5 minutes. The fixed cells were washed with PBS twice and harvested by scraping with 1 ml cold lysis buffer (RIPA buffer (1% SDS, 0.1% NP40, 1% triton X-100, 0.5% sodium deoxycholate, 1 mM EDTA) + 1 mM PMSF + 1 Roche Biomedical Complete protease inhibitor tablet). Lymphoblastoid cell lines were harvested by centrifugation and fixed in 5 mls of PBS + 1% formaldehyde. After washing with PBS the cells were lysated on ice by addition of 1 ml of the cold lysis buffer. The lysate was collected on ice and sonicated with a Braun sonicator fitted with a microtip for 5 times for 20 seconds at the maximum setting for the microtip. The lysate was centrifuged at 13 k rpm in a Sorval SS-34 rotor for 10 minutes. The final lysate preparation was aliquoted into siliconized sterile 1 ml microfuge tubes and stored at -70°C. The protein concentration of the lysates and the affinity-purified BLM antibody [4
] were determined using the Bradford reagent from BioRad. Immunoprecipitations were performed by mixing 1 mg of lysate with 10 μg of polyclonal BLM antibody and overnight incubation at 4°C on a Clay Adams Nutator platform. Protein A beads (Pierce) were equilibrated with the lysis buffer and 50 μl of a 50% suspension was added to the immunoprecipitate and mixed on the Nutator at 4°C for 1 hour. The beads were collected by spinning at 10 k rpm in an Eppendorf microfuge for 15 seconds, washed twice in lysis buffer and resuspended in 50 μl 2% SDS. The eluted chromatin fragments were processed as described [26
] using the linkers listed below and PCR amplification with one primer [26
] using the Promega PCR Master Mix. Qiagen PCR purification kits were used to purify the DNA fragments before and after amplification as described [26
DNA cloning, sequence analysis and websites
The purified DNA fragments were visualized on a 1% agarose gel to verify recovery of 200–600 bp products and cloned into the pCR 2.1 TOPO vector using the TOPO TA Cloning kit (Invitrogen). Plasmid inserts were sequenced directly or after PCR amplification with the M13 primers supplied in the cloning kit using an ABI BigDye Terminator v3.0 kit and an ABI377 sequencer.
Linkers and primers
Invitrogen synthesized oligonucleotides. Linkers for ChIP clones:
5'-TACGTGCGATAACGAGCGAGTATCGTC-3'/5'-GACGATACTCGC-3' or 5'-ATGCACGCTATTGCTCGCTCATAGCAG-3' / 5'-CTGCTATGAGCG-3'. Primers for amplifying plasmid inserts:
5'-CAGCAAACAGCTATCACC-3' / 5'-TGTAAAACGACGGCCAGT-3'.
Ribosomal DNA primers:
1 kb 5'-CTCTGCCGCGATCCTTCT-3'/5'-GGGAGCCGGAAGCATTTT-3',
16 kb 5'-ATGTATCGAAGCCCCATTTC-3'/5'-GGCCCCAAAAGAAAAGAAAA-3',
19 kb 5'-GTGTGTTGGGGCTGAAAACT-3'/5'-CCTCCTCAAACGCAAGAAAG-3',
27 kb 5'-TGTTGGCTTGTTTTGTTTCG-3'/5'-AAACATCAACCGGCTCTCAC-3',
34 kb 5'-GACAACTCACGCCCTAATGC-3'/5'-ATCCTCCGAAAGAGGTTGCT-3',
37 kb 5'-AGGAGTCCCCTGGTCTGTCT-3'/5'-GTCAAGGTCCAAACCGAAAA-3',
42 kb 5'-GATGGTGGCGTTTTTGGG-3'/5'-CGACTCGGAGCGAAAGATATA-3'
4 kb primers were supplied by Perkin-Elmer and included in the kit for rRNA quantitation (see below). Oligonucleotide primers for PCR were chosen using the primer3 Website at MIT http://www.genome.wi.mit.edu/
The molecular beacon and primers for quantitating the recovery of genomic DNA using the Alu-Sb2 elements were made by Research Genetics and used as described [27
]. The relative amounts of rDNA were measured from 200 ng of total genomic DNA prepared previously [28
] isolated from each cell line using a kit from Perkin-Elmer (ABI TaqMan Ribosomal RNA Control Reagents) containing primers and a VIC probe to the 18S rRNA coding region in an ABI 7700. Membrane-transfer hybridizations were performed using Hybond N+ membranes (Amersham-Pharmacia) and a dot blot transfer apparatus and protocols supplied by the manufacturer (Schleicher and Schuell Minifold I Spot Blot System). Radiolabeled oligonucleotide probes were made as described [10
]. Relative hybridization units were measured using a Molecular Dynamics Storm Phosphorimager and Imagequant. Relative total rDNA and telomere DNA are reported by normalizing values to the cell line with the lowest amount.
BLM alleles were constructed by replacing fragments of the full-length normal cDNA construction with the deleted ones described before [19
]. Co-transfection of the GFP-fusion plasmids and linear Bac-N-Blue AcMNPV DNA into Sf9 cells was according the to supplier's recommendations (Invitrogen). Recombinant baculoviruses were isolated by limiting dilution and inspection by immunofluorescent microscopy to confirm nuclear localization [19
Purification of GFP-fusion proteins and helicase assays
Full length and deleted BLM proteins were expressed and purified as before [19
] except that a 5 ml Hi-trap heparin column (Amersham-Pharmacia) was substituted for the ion-exchange step. Standard helicase assays were performed as previously [4
Cell cycle analysis
SV40-transformed BS fibroblasts containing different BLM alleles [19
] were grown for 5 days in DMEM + 10% fetal bovine serum (Clontech tetracycline-free) + 0.2 mg/ml G418 (Geneticin, Invitrogen) + 0.1 mg/ml hygromycin (Roche Molecular Biochemicals) and 5% CO2
with 1 μg/ml doxycycline. Cells were harvested and fixed in PBS + 50% ethanol on ice and stored at 4°C. Fixed cells were resuspended in PBS + PI according to a protocol supplied by Becton-Dickinson. Cell cycle populations were calibrated using control cells and software purchased from Becton-Dickinson (FACSCalibur flow cytometer and CellQuest).