Materials and Methods

Generation of Chaf1a mutant mice and embryos.
Using PCR, we amplified two genomic fragments (about 3 kb each) flanking Chaf1a exon 3. These DNA fragments were assembled by conventional cloning with the neomycin and DT (diphtheria toxin) cassettes, as described in Figure 1A. The construct was transfected in ES cells by electroporation. We identified recombinant ES cells carrying the mutant Chaf1a tm1Ger (abbreviated Chaf1a − in the manuscript) allele by Southern blot (Figure 1A). We derived Chaf1a +/− mice by injecting recombined ES cells into C57BL/6N blastocysts. E4 embryos obtained from the intercross of Chaf1a +/− mice were genotyped by nested-PCR amplification. Embryos were collected in a PCR reaction mix containing oligonucleotide primers 1, 2, and 3 (Figure 1A) that amplify the wild-type and mutant alleles. After 30 cycles of amplification, 1 μl of each PCR reaction was used in a second round of PCR amplification with a new set of oligonucleotide primers. After 30 cycles of amplification, PCR reactions were run onto an agarose gel, which revealed the presence of the wild-type (150 bp) and recombinant (200 bp) alleles.

P150CAF-1 depletion by RNAi.
The RNAi plasmid vector that we used in this study contains the mouse H1 promoter and a puromycin selection gene (SB and MG, unpublished data). We characterized in detail the properties of this new vector using a GFP target gene and FACS: Extinction of target genes in ES cells is highly efficient in 70% of the cells, of intermediate efficiency in 15%, and inefficient in the remaining 15% of cells analysis. The sequence of p150CAF-1 siRNA (short interfering RNA) duplex [16] was cloned into this plasmid. The control vector expressed a siRNA that targets GFP RNA degradation. ES cells were transfected by electroporation with the RNA plasmid vector, seeded onto gelatin-coated slides, and cultured for 24 h in the absence of selection. Puromycin (2 μg/ml) was added to the culture medium and cells were cultured for an additional 48-h period. Using the p150CAF-1 RNAi vector, IF microscopy quantification reveals a complete CAF-1 depletion in most cells (Figure S1B). Western blot analysis (Figure S1A) reveals residual p150CAF-1 expression, as expected from the 15% of cells that do not inhibit the target gene.
MEFs (passage 3, 90% confluence) were transfected during 4 h with the RNAi plasmid vector using Lipofectamine 2000 (Invitrogene, Carlsbad, California, United States) according to manufacturer's conditions. Cells were trypsinized, plated at 1/3 dilution, and cultured for 48 h in the presence of puromycin.

Cell lines.
AT-1 ES cells [42] (a gift of M. Vernet) were used for gene targeting. LTM7 ES cells were used in all RNAi experiments. We derived this cell line from (C57BL/6 × 129) F1 females bred with C3H/HeJ males. LTM7 are XX ES cells, competent for germ line transmission. Primary MEFs were derived from E13 embryos as described in [43].

Immunofluorescence.
Cells were fixed for 20 min in PBS with 4% paraformaldehyde, and immunodetection was performed as previously described [44]. E4 embryos were collected and treated with tyrode acid to remove the zona pellucida, deposited onto microscope slides, and processed for immunostaining as described for ES cells. Antibodies anti-HP1α (2HP1H5, Euromedex, France), anti-H4K20me3 (Abcam, Cambridge, United Kingdom), anti-BrdU (DakoCytomation, Glostrup, Denmark), anti-PCNA (DakoCytomation), and anti-PML (Upstate Biotechnology, Lake Placid, New York, United States) were all used at 1/1000 dilution. Antibodies anti-mouse p150CAF1 [16] and anti-H3K9me3 (Upstate Biotechnology) were used at 1/250 and 1/500, respectively. All secondary antibodies were purchased from Molecular Probes (Sunnyvale, California, United States).

Western blot analysis.
Cells were lysed in Laemli buffer and run onto a 4%–12% SDS PAGE gradient gel. We used antibodies against p150CAF-1 ([16]; dilution 1/500), HP1α (2G9, Euromedex; 1/500), Histone H3 (Abcam; 1/500), H3K9me3 (Upstate; 1/500) and β-actin (Upstate; 1/40 000).

DNA FISH.
Probes were described previously [20]. Biotin-16-dUTP or Digoxigenin-11-dUTP (Roche, Basel, Switzerland) labeled probes were generated by nick translation (Roche) and FISH performed as described [20]. Image acquisition was performed with the Deltavision RT microscope (100×, 1.4 NA objective), images were deconvoluted, and fluorescence profiles measured along an arbitrary line using SoftWorx.

Nuclei preparation, nuclease digestion, and biochemical analysis of chromatin.
ES cells were incubated on ice for 10 min in buffer 1 (15 mM Tris-HCl [pH 7.5], 0.3 M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA) with 0.15% IGEPAL (Sigma, St. Louis, Missouri, United States). Nuclei were purified by centrifugation (10,000 × g for 30 min, at 4 °C) on sucrose cushions (buffer 1 with 1.2 M sucrose) and resuspended in nuclease buffer (50 mM Tris-HCl [pH 7.5], 20 mM NaCl, 0.32 M sucrose, 4 mM MgCl2, 1 mM CaCl2). About 2.106 nuclei were incubated with increasing quantities of MNase (0.04–1.6 units) or DNase I (0.25–16 units). Digestion time at 37 °C was 10 min for MNase and 2 min for DNase I. Digestions were stopped by adding SDS to 1% and EDTA to 50 mM. DNAs were prepared by proteinase K digestion followed by phenol-chloroform extraction and isopropanol precipitation. To test the association of histone H3 with chromatin in control and p150-depleted cells, isolated nuclei were incubated on ice for 30 min in buffer 2 (50 mM Hepes [pH7.9], 20% Glycerol, 3 mM MgCl2, 0.1% IGEPAL, 0.5 mM DTT, 0.5 mM PMSF) supplemented with either 0.1 M, 0.3 M, 0.45 M, 0.7 M, or 1 M NaCl. After centrifugation at 15,000 g, pellets and supernants were analyzed by Western blot using a histone H3 antibody (Abcam).

ChIP.
We prepared native chromatin fragments of two to six nucleosomes in length as described [28]. 5 μg of chromatin were incubated overnight with 10 μl of commercial antibodies. After incubation with protein-G sepharose and three washes, immunoprecipitated DNA was purified, sized onto an agarose gel, and analyzed by Southern blot. Hybridization signals were quantified using an Instant Imager (PerkinElmer, Wellesley, California, United States). Antibodies specific for H4K20me3 were purchased from Abcam. Antibodies for H3K9me3 were purchased from both Abcam and Upstate Biotechnology and gave similar results.
For Southern blot analysis, we used a 240-bp EcoRI/BamHI fragment from pSAT (major satellite probe) [45–47] and a 360-bp EcoRI/HindIII fragment from R198, corresponding to three copies of the 120-bp minor satellite repeat [49]. Probes used in FISH experiments were described in [46–48]. The probe for IAP elements was described in [50].