Preparation of anaphase cells and mechanically extended chromatin fibers
Immuno-fiber FISH was used for detecting specific DNA sequences on extended chromatin fibers from anaphase cells. For labeling DNA with bromo-deoxyurdine (BrdU), cells were incubated with BrdU (BrdU-labeling reagent, diluted 1/500 in normal medium; Zymed Laboratories, South San Francisco, CA) for 10 h before Noscapine treatment. To enrich for anaphase cells, HeLaS3 cells were arrested in mitosis by 20 μM Noscapine for 18 h and then released into normal medium for 30 min. Anaphase cells were collected by mitotic shake-off, spun down (1,000 rpm, 5 min), and resuspended for 10 min into 75 mM KCl at a concentration of 2
cells/ml. Fifty microliters of cells were then spotted on positively charged microscope slides (SuperFrost Ultra Plus; Menzel Gläser), air dried, and immersed for 15 min at room temperature (RT) in salt detergent buffer (25 mM Tris pH 7.5, 500 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS]). Slides were then removed slowly from this solution and placed for 10 min into fixation buffer as described above. Subsequently, slides were washed twice in phosphate-buffered saline (PBS), 0.1% Twin-20, incubated for 2 min in 0.1 N HCl, rinsed twice in 2× sodium chloride–sodium citrate (SSC), and then used for FISH, followed by immunostaining with antibodies, as described below.
Biotin-labeled DNA probes for alphoid pan-centromere DNA, ribosomal DNA (rDNA), and telomeric DNA were prepared as described previously (Ijdo et al. 1991
; Dunham et al. 1992
; Langer et al. 2001
Antibodies against PICH, Hec1, and CREST antiserum have been described (Baumann et al. 2007
). Rabbit anti histone H3 (ab1791), H3 tri-methyl K9 (3mH3K9, ab8898), H3 di-methyl K4 (2mH3K4, ab7766), H3 acetyl K18 (ab1191), as well as mouse monoclonal anti-CENP-A (ab13939) were obtained from Abcam (Cambridge, UK). Mouse anti-BrdU (RPN20AB) was from BD Biosciences Pharmingen, NJ, and mouse monoclonal anti-TRF2 (4A794) from Upstate (Lake Placid, NY). The FISH secondary antibodies avidine-Cy3.5 was from Rockland Immunochemicals, Gilbertsville, PA. The following secondary antibodies were used in immunofluorescence (IF) staining: donkey anti-rat Cy3 and Cy2 conjugated (Jackson ImmunoResearch, West Grove, PA), Alexa Fluor 488 conjugated donkey anti-mouse or anti-rabbit, and Alexa Fluor 647 conjugated donkey anti-rabbit IgG (Molecular Probes, Invitrogen).
Preparation of cells for immunofluorescence and in situ hybridization To achieve staining with both antibodies and DNA probes, a fixation–permeabilization protocol aimed at maximizing both preservation of protein antigenicity and accessibility of nucleic acid to DNA probes was used. Asynchronous HCT116 cells grown on coverslips were rinsed three times in PBS and fixed for 10 min with fixation buffer (0.2% Triton X-100, 20 mM Pipes pH 6.8, 1 mM MgCl2, 10 mM ethylene glycol tetraacetic acid [EGTA], 4% formaldehyde) at RT. Slides were then washed for three times 3 min with PBS containing 0.01% Triton X-100 and incubated for 5 min in 0.1 M HCl at RT. Cells were rinsed in 2× SSC and incubated in 2× SSC with 50% formamide for at least 1 h at RT before hybridization.
Immuno-FISH and immuno-fiber FISH HCT116 cells (on coverslips) or HeLaS3 chromatin fibers (on glass slides) were brought into contact with DNA probes, and samples were sealed with coverslips and rubber cement. Thus, the following treatments denatured both probe and target DNA simultaneously. All DNA fiber preparations were denatured for 3 min, using 75°C for centromere DNA and rDNA probes and 80°C for telomeric probes. For cells, denaturation time (at the same temperatures) was increased to 5 min. Samples were subsequently incubated overnight in a wet chamber at 42°C. Then, the rubber cement was removed, and coverslips (carrying cells) or glass slides (carrying DNA fibers) were rinsed in 4× SSC/0.2% Tween and washed for three times 5 min in the same solution at 42°C, followed by three times 5 min in 1× SSC at 60°C. After rinsing in 4× SSC/0.2% Tween, coverslips were blocked for 30 min in 3% bovine serum albumin (BSA) in 4× SSC/0.2% Tween in a wet chamber at 42°C. Detection of DNA probes was performed by incubating with appropriate antibodies diluted in 3% BSA in 4× SSC/0.2% Tween for 60 min in a wet chamber at 42°C. Samples were then washed for three times 5 min in 4× SSC/0.2% Tween at 42°C and rinsed in PBS for the subsequent IF procedure. Primary antibodies were diluted in PBS and 3% BSA and added to the samples for 90 min at RT. Next, samples were washed for three times 5 min in PBS and incubated for 60 min with secondary antibodies diluted in PBS, 3% BSA. After washing for three times 5 min in PBS, samples were rinsed in 2× SSC and counterstained for 5 min with 4′,6-diamidino-2-phenylindole (DAPI; 1 μg/ml).
Microscopy and image analysis Images were captured using a Deltavision microscope on a Nikon Eclipse TE200 base (Applied Precision, Issaquah, WA) with an Plan Apo 60/1.4 oil immersion objective and a CoolSnap HQ camera (Photometrics) or on a Nikon Eclipse TE2000 equipped with the same objective and a VDS COOL-1300Q camera (Vosskuehler). Images taken at different focal planes were processed with a deconvolution algorithm and projected into one picture using the Softworx software (Applied Precision). The projected images were opened in Adobe Photoshop and then sized and placed in figures using Adobe Illustrator CS (Adobe Systems, Mountain View, CA).
ChIP assay and Southern blotting
For ChIP assays, approximately 2
HeLaS3 anaphase cells (prepared as described above) were used per experiment. Anaphase cells were first treated with 5 mM dimethyl 3,3′-dithiobispropionimidate·2 HCl/PBS for 30 min on ice, washed in PBS, and cross-linked in 1% formaldehyde/PBS at RT for 10 min. The cross-linking was then quenched by adding 10% of 1.375 M glycine to the cell suspension. After two washes with ice-cold PBS, cells were resuspended in 500 μl of SDS-lysis buffer (0.1% SDS, 0.1% sodiumdeoxycholate, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 mM CaCl2
, 1 mM phenylmethylsulfonyl fluoride) and incubated on ice for 10 min. Then, cells were lysed by sonication for 30 s and treated with 300 U/ml of micrococcal nuclease at 37°C for 10 min to produce chromatin fragments of less than 1 kb (<5 nucleosomes), followed by centrifugation for 15 min at 14,000 rpm. The resulting chromatin fractions (supernatants) were diluted 10× in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM ethylenediamine tetraacetic acid [EDTA], 16.7 mM Tris–HCl pH 8.0, 167 mM NaCl), and precleared with 50 μl of salmon sperm DNA-saturated protein Sepharose G beads (200 μg/ml salmon sperm DNA, 50% slurry). Then, 5-ml chromatin fractions were first incubated with 5 μg of antibody for 2 h at 4°C, followed by an overnight incubation with 100 μl of salmon sperm DNA saturated Sepharose G beads. Beads were washed twice in low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8.0, 150 mM NaCl), twice in high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8.0, 500 mM NaCl), twice in LiCl wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxychlorate, 1 mM EDTA, 10 mM Tris–HCl pH 8.0), and twice in TE buffer (10 mM Tris–HCl pH 8.0, 1 mM EDTA). Bound nucleosomes were then eluted for 15 min by shaking in 1 ml elution buffer (50 mM Tris pH 8.0, 1% SDS, 1 mM EDTA, 0.1 M NaHCO3
). Cross-links were reversed by adding 100 μl of 1 M dithiothreitol and incubation at 37°C for 30 min, followed by the addition of 40 μl 5 M NaCl, 20 μl 0.5 M EGTA, 40 μl 1 M Tris pH 6.5, 10 μl 10 mg/ml proteinase K, and 10 μl 10 mg/ml RNase, and incubation at 65°C for 4–6 h. DNA was recovered by phenol/chloroform extraction, precipitated by isopropanol, washed in 70% ethanol, and then dissolved in 50 μl TE buffer.For detection of centromere DNA by Southern blotting, 10 μl of ChIP DNA was resolved on a 1% agarose gel. DNA was transferred onto Biodyne®
B membrane (KPL, Gaithersburg, MD) and hybridized for 18 h at 42°C with biotinylated pan-centromere probe or rDNA probe at concentration of 50 ng/ml. The detection of biotinylated probes was done according to the manufacturer’s instructions (Detector™ AP chemiluminescent blotting kit, KPL).