JCS03451 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. H2A_T120ph and H2B_T119ph and meiotic silencing of unpaired chromatin (MSUC). (A) Double immunostaining of pachytene nuclei isolated from T/T’ mice with anti-H2A_T120ph (green) and anti-SYCP3 (red). Arrowhead indicates the XY body, and the arrow indicates the 1¹³ translocation bivalent. (B) As in A, but these nuclei were stained with anti-H2B_T119ph (green) and anti-SYCP3 (red). Bar, 10 μm.

• Supplemental Figure S2 -

  Fig. S2. Increased dimethylation of H3_K4 on X and Y chromatin of Hr6b-knockout meiotic metaphase nuclei. Immunostaining with anti-H3_K4m2 (green, upper panels) combined with X chromosome FISH (red, lower panels) on spread chromosomes of MI (one primary spermatocyte) or MII (two secondary spermatocytes) nuclei from wild-type and Hr6b-knockout mice. Chromosomes are stained with Dapi (blue). Arrowhead indicates the X chromosome. Bar, 10 μm.

• Supplemental Figure S3 -

  Fig. S3. Histone variants in wild-type and Hr6b-knockout spermatocytes. (A) Triple immunostaining with anti-H3.1 (green), anti-SYCP3 (red) and anti-H2A_T120ph (blue) in early (eP) and late (lP) pachytene spermatocytes. H3.1 is present on all chromosomes in early pachytene, but has disappeared from the XY body in late pachytene. Concomitantly with the loss of H3.1, H2A_T120p decreases. In Hr6b-knockout pachytene spermatocytes (B), H3.1 disappears normally from the XY body, but H2A_T120p remains high. Arrowheads indicate the XY body. (C,D) Immunostaining with anti-macroH2A1 (green) and anti-SYCP3 (red) of wild-type (left panels) and Hr6b-knockout (right panels) pachytene (P) and diplotene (D) spermatocytes. In Hr6b-knockout diplotene spermatocytes, the overall level of macroH2A1 is higher compared with that in wild-type spermatocytes. The immunofluorescent macroH2A1 signal was quantified (D) and arbitrary units per μm² were calculated for the area covering the XY body and for the area covering the autosomes. Error bars represent the s.e.m. of 20 nuclei measured for each genotype from two different animals. (E) Western blot analysis of H3_K4m2, H3_K9m2 and macroH2A1 in basic nuclear protein extracts from spermatocytes (spc) and spermatids (spt) isolated from wild-type (wt) and Hr6b-knockout (ko) mice. Arrowhead indicates the specific protein bands. Equal amounts of protein were present in each lane, as verified by Ponceau S staining of the blot (not shown). All modifications show approximately equal levels in wild-type and Hr6b-knockout samples. The differences between wild-type and Hr6b-knockout diplotene spermatocytes that were observed using immunocytochemistry may not appear on western blots because diplotene spermatocytes represent only ∼10% of the spermatocyte population. For round spermatids, the immunocytochemical differences in H3_K4m2 and H3_K9m2 levels are also observed in only a subfraction of the cells, and only in a subregion of the nucleus, and likewise may not appear in western blot analysis of the whole cell population. Bars, 10 μm.