Cell lines, culture and treatments
Early passage (PD25) IMR90 HDFs (ATCC) were grown in Glutamax-DMEM (Invitrogen) with 15% (v/v) fetal bovine serum, at 7.5% (v/v) CO2
and 3% (v/v) O2
. Cells were passaged for an additional 60 population doublings where the new PD was calculated as PD= PD at plating + [ln (#harvested/#seeded)/ln 2]. Cells from young (GM00038) and old (AG09602) individuals (Coriell Cell Repository) were cultured as above. Cells were counted with the Z1 Coulter Counter (Beckman Coulter). Retroviruses were produced and cells were infected as described 53
. Cells to be used for flow cytometry were fixed in 70% (v/v) EtOH and stained according to standard protocols. For cell sorting, asynchronous logarithmically growing cultures of IMR90 were labeled for 1 hour prior to harvest with 5ug ml−1
lipophilic Hoechst 33342. Cells were sorted into pure G1 and S-G2/M populations as described 12
. Bleomycin treatment was done for six days at concentrations that did not inhibit cell growth (500, 50 and 5 ng ml−1
IMR90 cells were plated at 1×106 cells in 15cm dishes and grown overnight. Cells were washed twice in DPBS and new SILAC medium containing 30mg l−1 L-methionine-13CD3 (Sigma Isotec) and 63mg l−1 L-Cystine DiHydrochloride (Sigma) added. Cells were incubated for 12hrs and harvested by trypsin treatment, washed in DPBS and lysed in NuPage LDS sample buffer (Invitrogen) at 104 cells per µ.
Histones were separated by 8–12% (v/v) SDS-PAGE and stained with Coomassie. The histone region was excised and digested by a modified in-gel digestion protocol. Briefly, gel bands were destained overnight with 50% (w/v) acetonitrile/50mM NH4HCO3. Then free primary and secondary amines were propionylated: To the gel pieces 20µl 50mM NH4HCO3 were added followed by 50µl 30% (v/v) propionic anhydride in methanol. This procedure was repeated twice. Then gel bands were washed with 100mM NH4HCO3, 50mM NH4HCO3 and 50% (w/v) acetonitrile/50mM NH4HCO3. Cysteines were reduced with 100µl 10mM dithiothreitol (DTT) at 56 ° C for 20min and then carbamidomethylated by addition of 200µl 5mM iodoacetamide in 100mM NH4HCO3 for 20min at RT, protected from light. Samples were washed twice with 100mM NH4HCO3 and twice with 50mM NH4HCO3. Then 200µl acetonitrile were added and the gel pieces shrunk on a speedvac. Enough trypsin (12.5ng µl−1 in 50mM NH4HCO3) was added so that the gel pieces were covered and incubated for 5min. Then the supernatant was removed, 20µl 50mM NH4HCO3 were added and the samples incubated over night at 37°C. Peptides were extracted by removal of the supernatant from the gel pieces followed by two extractions with 20µl 5% (v/v) formic acid on an ultrasonic bath for 10min. The three supernatants were combined and analyzed by mass spectrometry.
Peptide samples were analyzed on a ThermoFisher Scientific Orbitrap mass spectrometer coupled to an Agilent 1100 nano-LC pump fitted with a 12.5cm × 75µm column pulled and packed in house with Reprosil C18-AQ 3µm packing material. For quantification, mass chromatograms corresponding to expected masses of 1+ and 2+ ions [2+ and 3+ ions for H3(27–40) peptides] were analyzed using Xcalibur software (Thermo Electron Corp., Waltham, MA, USA). Mass accuracy was set to 20ppm. Peak areas were calculated and corrected for different mass spectrometric responses as determined by synthetic peptide standards. The total abundance of any modification was calculated from the sum of all corrected peak areas of differentially modified fragments covering a particular amino acid sequence.
Cell cycle synchronization
Cells were synchronized by growing IMR90 HDFs to 50% confluence in 15cm plates and incubated overnight with 2mM thymidine (Sigma). The cells were washed three times with PBS at 37°C, and fresh medium at 37°C was added. After 9–10 hr in the incubator, 1 µg ml−1 aphidicolin (AG Scientific) was added for 12hrs. Finally, the cells were washed three times with PBS at 37°C, and fresh media were added.
Cell Cycle Immunoblotting
Following release from aphidicolin block cells were harvested every 2 hr by trypsin treatment, washed in DPBS and lysed in NuPage LDS sample buffer (Invitrogen) at 104 cells per µl. Proteins were homogenized using benzonase (Novagen), denatured for 10 minutes at 68°C and resolved by SDS-PAGE, transferred to nitrocellulose, blocked in 2–5% (w/v) BSA and 0.1% (v/v) Tween20 for 20 minutes and probed. As secondary antibodies, HPR-linked anti-rabbit or anti-mouse (Amersham) was used, and the HPR signal was visualized with Supersignal West Pico Sensitivity substrate (Pierce). Abundance of post-translational modifications was calculated relative to the total amount of that particular protein. For other proteins the enrichment was calculated relative to γ-tubulin. All comparative western images shown were processed in parallel and membranes were exposed on the same film for equal time.
Synchronized Chromatin Immunoprecipitation
After release, cells were harvested every 2 hrs, washed with PBS, and fixed with 1% (v/v) formaldehyde in PBS for 30 min at RT. Chromatin was sonicated to an average fragment size of 500bp and precipitations were performed as described 55
. DNA was then used in dot-blot southern hybridization with TTAGGG and 17P (sub-telomeric) probes. For the quantifications, the enrichment of histone modifications was calculated as a percentage of total H3 signal. For other proteins, the enrichment was calculated as a percentage of input signal.
BrdU DNA Immunoprecipitation
IMR90 cells were synchronized as described. Cells were incubated with 50 µM Br-dUTP (Sigma) for 1 hr prior to harvesting by trypsin treatment. Immunoprecipitation of BrdU labeled DNA was performed according to protocols outlined in 56
. DNA was purified by Phenol-Chloroform extraction and used for dot-blot southern hybridization with TTAGGG and 17P probes.
Figures were generated using Adobe Photoshop and Adobe Illustrator.