borth et al, 2001) and harvested 72 h post-transfection. For immunofluorescence with the p150CAF-1 antibody, cells were extracted with the nonionic detergent Triton X-100 to remove soluble proteins (Martini et al, 1998). Immunodetection and RNase treatment were performed as described (Maison et al, 2002). Methanol fixation was used for PCNA staining. IdU and CldU (10 μM) were added to the cell medium for 5 min.
Image acquisition, 3D reconstruction and modeling, and colocalization
We used a DMR-HC (Leica) epifluorescence microscope with a × 63 objective and a CCD camera (CoolSnap Fx, Photometrics) the resolution limit of which is about 200 nm. For 3D reconstruction, using a DMIRE 2 (Leica) epifluorescence photo-microscope with a DG4 illumination device (Sutter Instruments), a × 100 objective and a piezoelectric objective positioning device (PIFOC, Physics instruments), stacks of images (200 nm step) were recorded using a chilled CCD camera (CoolSnap HQ, Photometrics) and deconvolved with Metamorph software (Universal Imaging). 3D modeling of nuclei was achieved by building a 3D texture from an iso-surface reconstruction of the deconvolved stack of images with the Amira software (TGS,). At least 1000 nuclei were examined and representative images are shown.
Antibodies
For immunofluorescence, we used the following antibodies: anti-HP1α 2HP1H5 (Euromedex), anti-HP1β 1MOD1A9 (Euromedex), and anti-HP1γ 2MOD1G6 (Euromedex), anti-PCNA PC10 (DAKO), anti-mouse p150CAF-1 from rabbit serum immunized with recombinant mouse p150 (AgroBio), anti-tri-Me H3-K9 (Abcam), anti-AcH4-K5 (Turner et al, 1989), and secondary antibodies coupled to Texas red, FITC, Cy-3, and Cy-5 (Jackson Immuno Research Laboratories Inc.). After 10 min denaturation in 4 M HCl, the following antibodies were used: anti-BrdU OBT0030 (AbCys) to detect BrdU, anti-BrdU supernatant OBT0030S (AbCys) for CldU, anti-BrdU (clone B44, Becton Dickinson) for IdU. For Western blots, we used anti-human p150CAF-1 H300 (Santa Cruz) or anti-human p150CAF-1 Ab765 (Abcam), anti-human p60CAF-1 (Agrobio), anti-HA tag 12CA5 (Roche), anti-H3 06-0755, anti-H4 07-108, and anti-HP1α 07-333 (Upstate).
siRNA synthesis
siRNA duplexes were constructed with the Silencer siRNA construction kit (Ambion) using phenol/chloroform and a G-25 spin column RNA grade (Roche) for purification. The sequence of p150CAF-1 duplex was 5′AAGGAGAAGGCGGAGAAGCAG3′ and 5′AAGCTGGAGTACAACTACAACCCTGTCTC3′ for GFP control duplex.
Supplementary Material
Supplementary Figure S1
Supplementary Figure S2
Supplementary Figure S3
Acknowledgments
We thank members of UMR218 for discussions, P Le Baccon and S Huart for 3D reconstruction, S Ross for the mouse p150 antibody, D Rouillard for FACS analysis, F Sangrado for technical help, A Verreault for recombinant CAF-1 and mouse p150 cDNA, T Jenuwein for the Suv39h double null MEFs, B Turner for AcH4-K5 antibodies, M Guenatri for early observations, and E Heard, P Hanawalt, and PA Defossez for critical reading. DK was supported by a European network grant to GA. (Disclaimer: the European Commission is not responsible for any views or results expressed.) GA is supported by la Ligue Nationale contre le Cancer (Equipe labellisée la Ligue), Euratom (FIGH-CT-1999-00010, FIGH-CT-2002-00207), Commissariat à l'Energie Atomique (LRC #. 26), and Curie Program on epigenetic parameters and YN by grants from NIH (GM065939-02).
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