Molecular cloning, cell culture, transfection, and immunocytochemistry. The CBX1 chromodomain (either WT or the triple mutant) was cloned into a pcDNA3 vector and fused to the N terminus of the photoactivatable mCherry (PAmCherry) protein. HeLa and mouse embryonic fibroblast (MEF) cells were cultured using standard protocols and plated on 3.5-cm glass-bottom dishes for imaging. For live-cell photoactivated localization microscopy (PALM) imaging, HeLa and MEF cells were transiently transfected using either Lipofectamine 2000 (Thermo Fisher Scientific) or Polyjet (SignaGen). For fixed-cell stochastic optical reconstruction microscopy (STORM) imaging, cells were fixed with 4% paraformaldehyde (PFA) for 20 min and then washed with 100 mM glycine in Hanks’ balanced salt solution (HBSS) to quench the free PFA. Cells were permeabilized and blocked in a permeabilization solution with 0.1% Triton X-100, 0.2% bovine serum albumin, 5% goat serum, and 0.01% sodium azide in HBSS. The cells were then incubated overnight at 4°C with an anti-H3K9me3 antibody (Abcam, ab8898) at a 1:500 dilution, followed by 1 to 2 hours with goat anti-rabbit Alexa 647–conjugated antibodies at 1:1000 dilution. The cells were then postfixed again in 4% PFA, quenched with 100 mM glycine in HBSS, and washed with HBSS to prepare for imaging. Immediately before imaging, the medium was changed to STORM-compatible buffer [50 mM tris-HCl (pH 8.0), 10 mM NaCl, and 10% glucose) with glucose oxidase (560 μg/ml), catalase (170 μg/ml), and mercaptoethylamide (7.7 mg/ml).

For the two-color STORM imaging, the cells were incubated overnight at 4°C with a mouse anti-FLAG antibody (for detection of FLAG-tagged CBX1; Sigma, F1804) at a 1:200 dilution and with a rabbit anti-H3K9me3 antibody (Abcam, 8898) at a 1:500 dilution. They were then incubated 1 to 2 hours with goat anti-rabbit and anti-mouse antibodies, labeled with Alexa 647 and Alexa 568, respectively, at a 1:1000 dilution.

Super-resolution imaging and image analysis. STORM and PALM images were obtained using a Nikon Ti total internal reflection fluorescence (TIRF) microscope with N-STORM, an Andor IXON3 Ultra DU897 EMCCD, and a 100× oil immersion TIRF objective. Photoactivation was driven by a Coherent 405-nm laser, while excitation was driven either with a Coherent 561-nm laser or a 647-nm laser. Illumination was done in a “near-TIRF” format, in which the TIRF angle was adjusted so that molecules in the nucleus were illuminated. All image analysis and image reconstruction were performed using the Localizer software (27) in the Igor Pro 6.3 environment. STORM and PALM images were segmented using the GLRT algorithm (27), and localizations were fit using Gaussian fitting. Reconstructed bitmap images were created in which intensity corresponds to the number of localizations in each box in a 0.2 pixel wide grid. For two-color STORM imaging colocalization analysis, the protocol can be found in the Supplementary Methods and Materials.

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