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First published online 8 May 2007
doi: 10.1242/jcs.03451

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Increased phosphorylation and dimethylation of XY body histones in the Hr6b-knockout mouse is associated with derepression of the X chromosome

¹ Department of Reproduction and Development, Erasmus MC–University Medical Center, Rotterdam, The Netherlands
² Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA

View larger version (21K): [in this window] [in a new window]   Fig. 1. HR6B is not required for histone ubiquitylation in spermatocytes. (A) Basic nuclear proteins were isolated from purified spermatocytes, and analyzed on western blots using anti-H2B antibody. The antibody recognizes unmodified H2B and H2BK120ub1 (as indicated by arrows). In the control lane (Ctr), the first antibody was omitted from the incubation mixture. The boxed area is presented for each immunostaining in B. (B) Histone ubiquitylation was analyzed in spermatocytes (spc) and spermatids (spt) isolated from wild-type (+/+) and Hr6b-knockout (–/–) mice. From top to bottom, antibodies were targeting ubiquitin, H2AK119ub1 and H2B as indicated on the right of the blots, and the modified histones are indicated by arrows. Representative results are shown; each experiment was repeated at least twice with germ cell preparations isolated from a different pool of mice. Equal amounts of protein were present in each lane, as verified by Ponceau S staining of the blot (not shown).

View larger version (53K): [in this window] [in a new window]   Fig. 2. H2BT119ph on the XY body and on meiotic and mitotic metaphase chromosomes. (A) Double immunostaining of pachytene and diplotene spermatocyte nuclei with anti-SYCP3 (red) and anti-H2BT119ph (green). The insert shows a larger magnification of the area containing the XY body. Arrowheads, pseudoautosomal synapsed region; arrows, patches of synaptonemal complex lateral elements that are H2BT119ph negative. (B) H2BT119ph (green) and SYCP3 (red) on meiotic metaphase (left) and H2BT119ph (green) on DAPI-stained (blue) mitotic HeLa cell nucleus (right). Bars, 10 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 3. H2AT120ph is increased in Hr6b-knockout spermatocyte nuclei. Double immunostaining of wild-type (A) and Hr6b-knockout (B) spermatocyte and spermatid nuclei with anti-SYCP3 (red) and anti-H2AT120ph (green) antibodies. For spermatids, separate images of the H2AT120ph (green) and DAPI (blue) staining are shown. Arrowhead, XY body; Z, zygotene; eP, early pachytene; lP, late pachytene; eD, early diplotene; lD, late diplotene; MI, Metaphase I; eT, early round spermatid; lT, late round spermatid. Bar, 10 µm. (C) Quantification of H2AT120ph immunofluorescent signal in early and late diplotene nuclei of wild-type (wt) and Hr6b-knockout (ko) mouse testes. 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. for 20 nuclei that were measured for each genotype from two different animals. (D) Quantification of H2AK119ub1 immunofluorescent signal in pachytene nuclei of wild-type (wt) and Hr6b-knockout (ko) mouse testes. 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. for 20 nuclei that were measured for each genotype from two different animals.

View larger version (67K): [in this window] [in a new window]   Fig. 4. H2AT120 phosphorylation in association with H2AK119 ubiquitylation in Hr6b-knockout pachytene spermatocytes. Triple immunostaining of wild-type (A) and Hr6b-knockout (B) early (eP), mid (mP) and late (lP) spread pachytene nuclei with anti-H2AT120ph (blue), anti-H2AK119ub1 (green) and anti-SYCP3 (red). The left panels show the merge of the H2AT120ph signal and SYCP3 signal, and right panels show the merge of the H2AK119ub1 (green) signal and SYCP3 signal (red). The arrowheads indicate the XY body. Bar, 10 µm. (C) Western blot analysis of H2AT120ph in basic nuclear protein extracts from total testis (C). Specificity of the antibody reaction is shown by competition of the signal with the phosphorylated H2A peptide (+p) but not with the nonphosphorylated peptide (–p). The identities of protein bands are indicated. Equal amounts of protein were present in each lane, as verified by Ponceau S staining of the blot (not shown). (D) Western blot analyses of H2AT120ph and ubiquitylated histones (ubi-his) in basic nuclear protein extracts from spermatocytes (spc) and spermatids (spt) isolated from wild-type (wt) and Hr6b-knockout (ko) mice. Asterisk indicates a non-specific protein band enriched in germ cell extracts compared with total testis extracts; the localization of H2AK119ub1T120ph was verified using the localization of ubiquitylated histones visible on the same blot that was stripped and reprobed with anti-ubiquitin antibody as shown. Equal amounts of protein were present in each lane, as verified by Ponceau S staining of the blot (not shown).

View larger version (57K): [in this window] [in a new window]   Fig. 5. Increased dimethylation of H3K4 on XY chromatin of Hr6b-knockout spermatocytes and spermatids. (A) Double immunostaining of wild-type and Hr6b-knockout diplotene nuclei with anti-H3K4m2 (green) and anti-SYCP3 (red). Arrowhead indicates the XY body. (B) Quantification of H3K4m2 immunofluorescent signal on autosomal and XY body chromatin of wild-type (wt) and Hr6b-knockout (ko) diplotene spermatocytes. 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. for 20 nuclei that were measured for each genotype from two different animals. (C) Immunostaining with anti-H3K4m2 (green) combined with X chromosome FISH (red) on wild-type spermatocyte and spermatid nuclei (left panel). The right panels show immunostaining of wild-type and Hr6b-knockout spermatid nuclei with anti-H3K4m2 (green). DNA is stained with DAPI (blue). White arrows in the left panel indicate the position of the X chromosome, and the pink arrow denotes the Y chromosome. The arrowhead indicates an XY body of a pachytene spermatocyte. Bars, 10 µm.

View larger version (42K): [in this window] [in a new window]   Fig. 6. Loss of H3K9m2 from centromeric heterochromatin, but not from X and Y in Hr6b-knockout diplotene spermatocytes and spermatids. Immunostaining with anti-H3K9m2 (green) and anti-SYCP3 (red) of wild-type (A) and Hr6b-knockout (B) early (eD) and late (lD) diplotene spermatocytes (upper panels), and round spermatids (spt) (lower panels). DNA is stained with DAPI (blue). Arrowheads indicate the XY body. Bar, 10 µm.

View larger version (28K): [in this window] [in a new window]   Fig. 7. Derepression of X chromosomal genes in Hr6b-knockout spermatids. Real-time RT-PCR quantification of mRNA levels of four autosomal genes (A) and six X chromosomal genes (B) in a mixed spermatocyte/spermatid (spc/spt) and purified spermatid (spt) cell preparation from wild-type (wt) and Hr6b-knockout (ko) mice. The amount of PCR products was normalized to Actb (-actin) mRNA (x100). Results from two independent experiments are shown. For each experiment, the value represents the average of a duplicate real-time PCR experiment.

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