Figure S4. Cell-cycle Dependent Localization of DPY-21 to X in Wild-type and dpy-21
(JmjC) Mutant Embryos. Related to Figure 3.(A) Immunofluorescence of 3xFLAG-tagged DPY-21 confirmed the cell-cycle dependent recruitment of DPY-21 to X chromosomes. Enlargements of individual nuclei at different stages of the cell cycle from a confocal image of a dpy-21
(5'-FLAG) XX embryo at 277-cell stage stained with DAPI and antibodies against SDC-3, DPY-27 and FLAG. 3xFLAG-tagged DPY-21 colocalizes with SDC-3 and DPY-27 on X during interphase but dissociates from X during mitosis, while SDC-3 and DPY-27 remain on X throughout the cell cycle. Scale bar, 1 um.(B) Immunofluorescence of the 3FLAG-tagged dpy-21
(JmjC) mutant confirmed that the JmjC demethylase mutation does not affect the recruitment of DPY-21 to X chromosomes in interphase nuclei. Enlargements of individual nuclei at different stages of the cell cycle from confocal images of a dpy-21
JmjC) XX embryo at the 396-cell stage co-stained with DAPI and antibodies against SDC-3, DPY-27, and FLAG. Scale bar, 1 um.(C) Immunofluorescence of the dpy-21
(JmjC) using DPY-21 antibodies also showed that the JmjC mutation does not affect the cell-cycle dependent recruitment of DPY-21 to X chromosomes of interphase nuclei but eliminates H4K20me1 enrichment. Individual nuclei at different stages of the cell cycle from the dpy-21
JmjC) mutant 335-cell embryo shown in Figure 3 stained with DAPI and antibodies to H4K20me1, SDC-3, and DPY-21. In interphase, DPY-21 and SDC-3 colocalize on X. During mitosis, DPY-21 dissociates from X, while SDC-3 remains on X. The JmjC mutation eliminates H4K20me1 enrichment on X during interphase, but does not affect the H4K20me1 level during mitosis. Scale bar, 1 um.