MET-2 and LIN-65, but not SET-25, drive the perinuclear association of heterochromatin. (A) Confocal images of GFP::LacI tagged gwIs4 heterochromatic array in wild-type, met-2(n4256), or set-25(n5021) embryonic nuclei. Neither met-2 nor set-25 deletion delocalize the array, whereas met-2 set-25 double mutants do (Towbin et al., 2012). Bar = 2 µm. (B) LEM-2 ChIP in wild-type (gray), met-2(n4256) (orange), or set-25(n5021) (green), on mixed embryos from the indicated mutant lines grown at 20°C (averaged from three biological replicates) for chromosomes I, II, and X. See Fig. S5 for other chromosomes. Mean signals averaged over 100-kb bins. (C) Left: Scheme of C. elegans chromosomes with FISH probe position. Right: Scheme of three-zone assay used to score FISH probe location; see Fig. 2 and Meister et al. (2010a). (D) Top: Confocal images of a single focal plane of wild-type, met-2(n4256), lin-65(gw1465), or set-25(n5021) nuclei from mid-stage embryos, probed by FISH for the indicated loci (red) and DAPI (blue). Bottom: Quantitation of FISH signals by three-zone scoring; gray line = random distribution or 33%. The χ² test compares changes in zonal enrichments. Foci were scored from multiple nuclei and embryos in >2 replicates (for probe 1: wt, n = 563; nmet-2, n = 394; lin-65, n = 593; set-25, n = 676; for probe 2: wt, n = 423; met-2, n = 282; lin-65, n = 502; set-25, n = 276; for probe 3: wt, n = 504; met-2, n = 256; lin-65, n = 391; set-25, n = 349. Bar = 2 µm.