HP1γ function is required for male germ cell survival and spermatogenesis
© Brown et al; licensee BioMed Central Ltd. 2010
Received: 19 December 2009
Accepted: 27 April 2010
Published: 27 April 2010
HP1 proteins are conserved components of eukaryotic constitutive heterochromatin. In mammals, there are three genes that encode HP1-like proteins, termed HP1α, HP1β and HP1γ, which have a high degree of homology This paper describes for the first time, to our knowledge, the physiological function of HP1γ using a gene-targeted mouse.
While targeting the Cbx3 gene (encoding the HP1γ protein) with a conditional targeting vector, we generated a hypomorphic allele (Cbx3 hypo ), which resulted in much reduced (barely detectable) levels of HP1γ protein. Homozygotes for the hypomorphic allele (Cbx3hypo/hypo) are rare, with only 1% of Cbx3hypo/hypoanimals reaching adulthood. Adult males exhibit a severe hypogonadism that is associated with a loss of germ cells, with some seminiferous tubules retaining only the supporting Sertoli cells (Sertoli cell-only phenotype). The percentage of seminiferous tubules that are positive for L1 ORF1 protein (ORF1p) in Cbx3hypo/hypotestes is greater than that for wild-type testes, indicating that L1 retrotransposon silencing is reversed, leading to ectopic expression of ORF1p in Cbx3hypo/hypogerm cells.
The Cbx3 gene product (the HP1γ protein) has a non-redundant function during spermatogenesis that cannot be compensated for by the other two HP1 isotypes. The Cbx3hypo/hypospermatogenesis defect is similar to that found in Miwi2 and Dnmt3L mutants. The Cbx3 gene-targeted mice generated in this study provide an appropriate model for the study of HP1γ in transposon silencing and parental imprinting.
The presence of methylated lysine 9 of histone H3 (H3K9ME) and structural heterochromatin protein (HP) 1 proteins are characteristic evolutionarily conserved hallmarks of heterochromatin . In mammals, there are three HP1 isotypes, which have a high degree of homology, termed HP1α (encoded by the Cbx5 gene), HP1β (encoded by the Cbx1 gene) and HP1γ (encoded by the Cbx3 gene) [2, 3]. Despite the significant degree of sequence conservation shared between the mammalian HP1 isotypes, several studies have indicated that they are likely to have non-redundant functions. First, their nuclear localization patterns are different: HP1α and HP1β are usually found enriched at sites of constitutive heterochromatin, whereas HP1γ has a more uniform distribution [4–6]. Second, biochemical assays have identified isotype-specific binding partners  and, third, targeted deletion of the Cbx1 gene has shown that it is essential, and that its loss of function cannot be compensated for by the products of the Cbx5 and Cbx3 genes .
Analysis of the Cbx1 null mutant has shown that the Cbx1 gene product, HP1β, is required for proper development of the brain, with Cbx1-/- neurospheres cultured in vitro showing a dramatic genomic instability that is indicative of a defect in centromere function . Interestingly, the lethality of the Cbx1 mutation compared with the observed viability of the Suv(3)9h1/h2 histone methyl transferase (HMTase) double mutant  shows that the essential function(s) of HP1β lies outside its interaction with the heterochromatic H3K9ME3 determinant generated by the Suv(3)9h1/h2 HMTases . By contrast, homozygous Cbx5-/- mutants are indistinguishable from wild-type littermates, indicating that its function is redundant  (Singh PB: unpublished data). To date, nothing is known about the biological function of the Cbx3 gene.
The mouse Cbx3 gene lies on chromosome 6, and is tightly linked to the Hnrnpa2b1 gene . Both genes, which are divergently transcribed, share a 3 kb CpG island that is conserved in the syntenic HNRNPA2B1-CBX3 region in humans . Fragments from the CpG-rich HNRNPA2B1-CBX3 region have been shown to be able to confer high-level expression of linked transgenes in the mouse and thus, it has been termed a ubiquitously acting chromatin opening element (UCOE) [12, 13].
In a first attempt to elucidate the biological function of the Cbx3 gene, we undertook a gene-targeting experiment using a conditional targeting vector. During production of this conditional mutation, we fortuitously generated a hypomorphic allele of Cbx3 (Cbx3 hypo ), which results in a dramatic reduction in HP1γ protein expression to barely detectable levels; expression of the Hnrnpa2b1 protein was not affected. The number of Cbx3hypo/hypohomozygotes that survive to adulthood is low, with adult males exhibiting a severe spermatogenic defect. This result confirms the non-redundant functions of mammalian HP1 proteins, and provides the first insight into the function of HP1γ during development. We also observed a dramatic reduction in the number of germ cells in Cbx3hypo/hypo, with a concomitant increase in expression of the ORF1 protein encoded by the LINE-1 (L1) retrotransposon. These data indicate that HP1γ might be part of a Miwi2-HP1γ silencing pathway that is required for proper germ-cell renewal and survival in the testes.
Results and discussion
To test the hypothesis that the neo-tk selection cassette was interfering with Cbx3 expression, we took advantage of the fact that neo-tk cassette is flanked by FRT sites that allow its excision by FlpE expression (Figure 1). When the neo-tk cassette was excised after electroporation of FlpE mRNA into Cbx3hypo/hypoMEFs, the HP1γ expression levels returned to normal wild-type levels (Figure 2a, top row: FlpE treated) indicating that it was indeed the presence of the neo-tk cassette that resulted in the reduced HP1γ levels. As a control, the Cbx3 gene was disrupted by Cre expression, resulting in Cbx3-/- MEFs and complete loss of HP1γ expression (Figure 2, top row: -/-). The Western blot analysis was complemented with immunofluorescence experiments, which confirmed that the presence of the neo-tk cassette affected Cbx3 expression (Figure 2b). The reduced levels of HP1γ protein in Cbx3hypo/hypoMEFs did not affect the expression of HP1α and HP1β as measured by immunofluorescence and Western blot analysis (see Additional files 1 and 2, Figures s1 and s2). Western blot analysis also revealed that there were no significant changes between Cbx3hypo/hypoand wild-type MEFs in the levels of three different histone post-translational modifications, H3K9ME3, H4K20ME3 and H3K9AC (see Additional file 2, Figure s2). When Cbx3-/- MEFs were included into this analysis, we observed an increase in H4K20me3 levels compared with wild-type and Cbx3hypo/hypoMEFs (see Additional file 2, Figure s2), indicating that complete loss of HP1γ in Cbx3-/-cells might affect the activity of enzymes involved in regulating this determinant of the histone code.
Spermatid stages at which HP1α, HP1β and HP1γ protein expression was extinguished.
Spermatid stage number
The similarity of the Cbx3hypo/hypospermatogenesis defect to Dnmt3L and Miwi2 mutants [21, 22] prompted us to investigate whether there were any changes in the expression of retrotransposon expression in the mutant testes. For this, we used a polyclonal antibody to the L1-encoded ORF1 protein . ORF1p is required for L1 transposition, and its levels of expression are increased in germ cells, as the L1 transposons become de-repressed [24, 25]. Using this antibody, we found that 45% of the tubules in Cbx3hypo/hypotestes that contained germ cells were positive by immunohistochemistry for ORF1 protein expression, compared with 5% in wild-type testes (see Additional file 6 and 7, Figure s6 and Figure s7). Again, this indicates that the Cbx3hypo/hypomutation may affect the same silencing pathway that is affected in the Dnmt3L and Miwi2 mutants [21, 22].
We next investigated whether the Cbx3 hypo mutation affects the expression of the other two HP1 isotypes, HP1α and HP1β. Accordingly, we stained wild-type and Cbx3hypo/hypotestes with HP1α and HP1β antibodies, and compared the cell types and levels of staining for the two proteins on the different genetic backgrounds. For HP1α, most cells of the wild-type testes were HP1α-positive (see Additional file 8, Figure s8). Sertoli cells were stained with anti-HP1α (see Additional file 8, Figure s8, black arrows) as were pachytene spermatocytes, where HP1α was enriched within a few bright foci which probably represent heterochromatic regions (see Additional file 8, Figure s8b, blue arrows). The round (stage 2-6) spermatids were also stained and exhibited a single spot of staining in the nucleus, which is characteristic of the heterochromatic chromocenter found in these cell types (see Additional file 8, Figure s8c, white arrows). Meiotic chromosomes were not stained but, unlike HP1γ staining in wild-type testes, very little staining was observed in the meiotic cytoplasm (see Additional file 8, Figure s8c, arrowheads). In addition, unlike with HP1γ, there are some cells, probably spermatogonia, which were not stained by the anti-HP1α antibody (see in Additional file 8, Figure s8b, yellow arrows). In wild-type testes, HP1α staining was lost at an earlier stage than HP1γ staining, with stage 7 spermatids (see Additional file 8, Figure s8b, arrowheads) being the last stage at which HP1α was still seen (Table 1). In the Cbx3hypo/hypotestes, the cell types stained were the same as those found in wild-type cells, notwithstanding the obvious suppression of spermatogenesis seen in the Cbx3hypo/hypotestes (see Additional file 8, Figure s8d, Figure s8e). The levels of HP1α staining were also unchanged in the Cbx3hypo/hypotestes, as evidenced by the typical staining of the round (stage 2-6) spermatids in Cbx3hypo/hypotestes (see Additional file 8, Figure s8e, white arrows). HP1β staining of wild-type testes (see Additional file 9, Figure s9) was similar to that for HP1α (see Additional file 8, Figure s8), with the only difference being that the staining of HP1β was still visible at a later stage, in stage 10 spermatids, as was seen for HP1γ (Table 1) . The levels of HP1β staining and the cell types stained in Cbx3hypo/hypotestes (see Additional file 9, Figure s9d, Figure s9e) were not significantly different to those in the wild-type testes. These data indicate that the defects seen in the Cbx3hypo/hypomutation are unlikely to operate through changes in the expression of the other two isotypes, HP1α and HP1β.
The clear differences between wild-type and Cbx3hypo/hypoadult testes were not observed in embryonic E17 testes. The morphology of the seminiferous tubules and numbers of gonocytes in wild-type and Cbx3hypo/hypotestes were similar (see Additional file 10, Figure s10), indicating that the suppression of spermatogenesis seen in the adult Cbx3hypo/hypotestes (Figure 4) probably occur at later stages, after meiosis has been initiated.
Housing the one adult Cbx3hypo/hypofemale mouse with wild-type males also resulted in no litters. Although it is difficult to conclude from a single Cbx3hypo/hypoanimal that Cbx3hypo/hypofemales are sterile, we nevertheless decided to examine sections of wild-type and Cbx3hypo/hypoovaries. Examination of the sections revealed no obvious morphological difference between wild-type and Cbx3hypo/hypoovaries; all stages of folliculogenesis were observed in Cbx3hypo/hypoovaries, including corpora lutea, indicating that ovulation was normal in the Cbx3hypo/hypofemale (data not shown).
The similarity of the Cbx3hypo/hypophenotype in the testes with those observed in the testes of Miwi2  and Dnmt3L mutants  is suggestive. Both Miwi2 and Dnmt3L are involved in DNA methylation of interspersed repeats during spermatogenesis, and mutation of either Miwi2 or Dnmt3L results in a SCO phenotype and the loss of DNA methylation of transposons, resulting in their ectopic expression [21, 22]. Our analysis of the Cbx3 hypo mutation indicates that HP1γ might also be involved in a Miwi2-HP1 silencing pathway, as observed for HP1a-PIWI pathway in Drosophila . These data, in conjunction with the generation of a Cre-inducible Cbx3 allele from the Cbx3 hypo allele (unpublished), form a sound basis for investigating the role of HP1γ in transposon silencing and parental imprinting.
HP1γ has a non-redundant function that cannot be rescued by the other HP1 isotypes, HP1α and HP1β. This function is essential for male germ cell survival and proper spermatogenesis.
The experimental research on mice was carried out in accordance with German animal protection law, and the study has been approved by the Ministerium für Landwirtschaft, Umwelt und ländliche Räume of Schleswig-Holstein in Kiel (Germany).
Staining of testes sections
Testes were fixed in Bouin's fixative (saturated aqueous solution of picric acid, 37% formaldehyde, and glacial acetic acid, 15:5:1) overnight, embedded in paraffin wax and cut into sections 2 μm thick. Subsequent antigen retrieval by pressure cooker and indirect immunoperoxidase staining was performed as described previously . In addition, blocking solution (Image-iT FX Signal Enhancer; Invitrogen, Carlsbad, CA, USA) was applied for 30 minutes to reduce background staining. All antibodies were diluted in Tris-buffered saline with 10% bovine serum albumin (BSA). Endogenous peroxidase was inactivated with 3% H2O2, and diaminobenzidine (DAB; Sigma, St. Louis, MO, USA) was used to detect peroxidase activity. Primary antibodies used in this study were anti-GCNA1 (mAB 10D9G11, kind gift of Professor G C Enders), anti-HP1α, anti-HP1β  and anti-HP1γ (all Chemicon, Temecula, CA, USA). Species-specific horseradish-peroxidase coupled secondary antibodies were purchased from Dianova (Hamburg, Germany). Images were photographed with a microscope and camera (DMLB2 microscope and DFC320 camera; Leica, Basel, Switzerland).
For L1 ORF1p staining, paraffin wax-embedded sections were dewaxed and subsequently incubated for 15 minutes with 1% peroxide followed by 15 minutes with signal enhancer (Image-iT FX Signal Enhancer; Invitrogen). For antigen retrieval, pancreatic trypsin (1 mg/ml in phosphate-buffered saline (PBS)) was applied for 2 minutes. The samples were incubated with anti-mouse ORF1p antibody (kind gift of Professor S L Martin) at 1:500 dilution in PBS with 10% BSA overnight at 4°C, with secondary antibody and peroxidase detection with DAB performed as described above. Finally, the sections were incubated with haematoxylin for 6 minutes. Images were taken with a Olympus (Hamburg, Germany) DS-Ri1 microscope, an Nikon (Melville, NY, USA) BX41 camera and NIS-Elements documentation software. Tubules were scored negative for ORF1p if the resident germ cells exhibited haematoxylin staining only with no brown staining (see Additional file 6, Figure s6c, Figure s6e). Tubules that were scored positive for ORF1p contained germ cells with robust brown staining of the nucleus and cytoplasm (see Additional file 6, Figure 6c, Figure s6e). Cbx3hypo/hypotubules that had no germ cells (see Additional file 6, Figure s6b, asterisks) were not included in the analysis.
Western blotting was performed essentially as described previously . For histone isolation, tissues were cut to pieces and further disintegrated in buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT) and Complete Mini EDTA-free Protease Inhibitor (Roche, Mannheim, Germany)) with a Dounce homogenizer (50 strokes with a tight pestle). Nuclei were pelleted and then resuspended in buffer S1 (0.25 M sucrose, 10 mM MgCl2 and protease inhibitor) and layered over an equal volume of buffer S3 (0.88 M saccharose, 0.5 mM MgCl2 and protease inhibitor). After separation by centrifugation (2,800 g for 10 minutes at 4°C) the pellet was resuspended in extraction buffer (1 M HCl, 0.02% β-βmercaptoethanol and protease inhibitor) and incubated at 4°C overnight. Pellet was extracted twice. The supernatants were pooled and treated with 10 volumes of acetone for precipitation (- 20°C overnight). After separation by centrifugation (10,000 g g for 4 minutes at 4°C), the pellet was reconstituted in water and finally denatured in Laemmli buffer (5 minutes at 95°C) for SDS-PAGE.
For Western blotting, primary antibodies for HP1α, HP1β, HP1γ, histone 3, histone 4, H3K9AC (all Chemicon), β-Actin (Sigma), hnRNP A2B1 (Biozol, Eching, Germany), H3K9ME3  and H4K20ME3  were used. Detection of these antibodies was carried out either with colorimetric reaction through species-specific AP-coupled secondary antibodies (Dianova, Germany) or by incubation with infrared dye-coupled secondary antibodies (Alexa Fluor 680-coupled goat anti-mouse (Invitrogen) and IRDye 800 CW anti-rabbit (LI-COR, Lincoln, NE, USA)) and subsequent scanning with an infrared imager (Odyssey; LI-COR).
Immunofluorescence was performed as described previously . Primary antibodies used in this study were against HP1α, HP1β and HP1γ (all Chemicon). Nucleic acids were stained with 4',6-diamidino-2-phenylindole (1 μg/ml). Confocal images were acquired, processed and assembled as described previously .
We thank Dmitris Kioussis and Ursula Menzel for production of chimeras from the targeted ES cells and Vladimir Shteyn for the hnrnpa2b1 blot. We thank Professors S L Martin and G C Enders for providing antibodies.
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