Ramos and Raji cells were purchased from the American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 medium (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS). 293T lentiviral packaging cells were maintained in DMEM/F12 medium (Life Technologies) supplemented with 10% FBS.
Short hairpin RNA–mediated depletion of CHD4 and MBD3
Generation of lentivirus was performed as previously described (Lai et al., 2010
), using the following vectors: pGIPZ shCHD4 (V2LHS_14657), shMBD3 (V3LHS_392211), and pGIPZ empty (RHS4349) (Open Biosystems, Thermo Biosystems, Huntsville, AL). Fluorescence-activated cell sorting (FACS) based on green fluorescent protein (GFP) intensity was used (FACSVantage with Digital Option; BD Biosciences, San Diego, CA).
Total protein was extracted from GFP-positive cells using lysis buffer (8 M urea, 1% SDS, 0.125 M Tris, pH 6.8), and phosphorylated proteins were enriched using a PhosphoProtein Purification Kit (Qiagen, Valencia, CA). Total protein extract was analyzed by resolving 10 μg of protein or 50 μg of phosphoprotein lysate on SDS–PAGE gels and immunoblotted using the following antibodies: CHD4 (A301-081A; Bethyl Laboratories, Montgomery, TX), MBD3 (sc-9402; Santa Cruz Biotechnology, Santa Cruz, CA), actin (MAB1501; Chemicon, Millipore, Billerica, MA), ATR (sc-1887; Santa Cruz Biotechnology), H3 general (ab1791; Abcam, Cambridge, MA), H3 acetylation (06-599; Upstate, Millipore), γH2AX (05-636; Upstate, Millipore), H3K56Ac (39281; Active Motif, Carlsbad, CA), MTA3, cyclin B1 (sc-594, Santa Cruz Biotechnology), H3S10P (sc-8656-R; Santa Cruz Biotechnology), phospho-CHK2 (2197, Cell Signaling Technology, Beverly, MA), phospho-KAP-1 (S824, A300-767A-1; Bethyl Laboratories), CDC25A (ab2357; Abcam,), H2BS14P (07-191; Upstate, Millipore), and H2B general (07-371, Upstate, Millipore).
Immunofluorescence, fluorescence in situ hybridization, and imaging
Immunofluorescence and immuno-FISH were performed as previously described (Helbling Chadwick et al., 2009
), using the following antibodies and probes: H3K9me3 (07-523; Millipore), MTA3 (Fujita et al., 2004
), BrdU (347580; BD Biosciences), H3S10P (sc-8656-R; Santa Cruz Biotechnology), H4K20me3 (39180; Active Motif), Rad51 (ab213; Abcam), P-ATM (S1981) (10H11.E12; Cell Signaling Technology), Classical Satellite (D1Z1) (LPE-001; Rainbow Scientific, Windsor, CT), and Chromosome 10 alpha satellite (LPE-010; Rainbow Scientific). To expose BrdU antigens for detection of actively replicating genomic regions, fixed and blocked cells were immersed in 4 N HCl for 10 min, followed by extensive washing in phosphate-buffered saline prior to the addition of BrdU antibody.
Images were collected on a Zeiss Axiovert 200 imaging system equipped with an Axiocam MR digital camera controlled by AxioVision software (Zeiss, Thornwood, NY). Confocal images were collected on a Zeiss LSM 510 UV laser scanning confocal microscope controlled by Zeiss LSM software. Maximum-intensity projections were derived from Z-series images taken at 1-μm steps and rendered around a single x-axis plane. Images were analyzed using Photoshop CS2 (Adobe, San Jose, CA) and Zeiss LSM software. H3K9me3 and H4K20me3 foci analysis was performed using MetaMorph imaging software (version 7.7.1; Molecular Devices, Downington, PA) to count the number of nuclei in an image in addition to the number of H3K9me3 or H4K20me3 foci in each cell. First nuclei were counted with the Integrated Morphology Analysis feature using a threshold (>50 units) to separate each nucleus. In instances in which multiple nuclei overlapped, manual separation was performed with the cut tool. The threshold was then reset, and a morphological TopHat filter (100 pixels2) was applied to identify the heterochromatin. Before measuring the number of heterochromatin spots by “integrated morphometry,” a low threshold (<10 units) was applied to filter low-intensity noise. Log data were then transferred to Excel sheets (Microsoft, Redmond, WA). Student's t tests were used to determine statistical significance of all images analyzed.
Cell synchronization and FACS analysis
Two days after transduction, Ramos cells were synchronized as previously described (Helbling Chadwick et al., 2009
). After synchronization, cells were released into medium either in the presence or absence of 0.5 mM caffeine or 10 μM TSA (Sigma-Aldrich, St. Louis, MO). At each time point, cells were pulsed with 10 μM BrdU for 15 min, fixed, and stained using the APC BrdU Flow Kit (BD Biosciences). Cells were analyzed using the BD LSR II Flow Cytometer System. Analysis of flow data were performed using Flowjo software (TreeStar, Ashland, OR).
Nucleic acid extraction
Total RNA was extracted from GFP-positive cells 5 d after transduction using the RNeasy Mini Kit (Qiagen). cDNA was synthesized as described (Fujita et al., 2004
). Expression analysis of NuRD complex members was performed using Quantitect primer assays (Qiagen): MTA3 (QT00064988), CHD4 (QT00025522), CHD3 (QT00007063), MBD2 (QT00007084), MBD3 (QT00035252), and GAPDH (QT01192646). Total genomic DNA was extracted from GFP-positive cells 7 d after transduction using the DNeasy Mini Kit (Qiagen). Copy-number analysis was performed as described (Quivy et al., 2008
), using the following primer sets: Satellite 2, CATCGAATGGAAATGAAAGGAGTC and ACCATTGGATGATTGCAGTCAA; GAPDH, GCCCCCGGTTTCTATAAATTG and GGCGACGCAAAAGAAGATG
Cells were incubated in nuclear isolation buffer (150 mM NaCl, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 0.5% Nonidet P-40, 0.5 mM dithiothreitol) at 4°C to release nuclei. Nuclei were resuspended in MNase digestion buffer (0.32 M sucrose, 50 mM Tris-HCl, pH 7.4, 4 mM MgCl2, 1 mM CaCl2) to a final concentration of 1 μg/ml as determined by A260. Amounts of 0.01, 0.05, 0.1, and 0.5 μg/ml MNase were added, and nuclei were digested for 5 min at 37°C before quenching with 10 mM EDTA. Amounts of 0.02% SDS, 1 μg of proteinase K, and 1 μg of RNase A were added prior to phenol extraction and ethanol precipitation of genomic DNA. A total of 1 μg of DNA was fractionated by agarose electrophoresis.