Correction: HP1gamma function is required for male germ cell survival and spermatogenesis

Correction After the publication of this work [1] it was brought to the authors' attention that Figure three (Figure 1 here) contained a duplication error, where the HP1gamma staining for wild-type thymus and brain are identical. The correct figure is given below. Acknowledgement We regret any inconvenience that this inaccuracy may have caused.


Background
The presence of methylated lysine 9 of histone H3 (H3K9ME) and structural heterochromatin protein (HP) 1 proteins are characteristic evolutionarily conserved hallmarks of heterochromatin [1]. 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 dis-tribution [4][5][6]. Second, biochemical assays have identified isotype-specific binding partners [7] 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 [8].
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 [8]. Interestingly, the lethality of the Cbx1 mutation compared with the observed viability of the Suv(3)9h1/h2 histone methyl transferase (HMTase) double mutant [9] 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 [3]. By contrast, homozygous Cbx5 -/mutants are indistinguishable from wild-type littermates, indicating that its function is redundant [8] (Singh PB: unpublished data).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 [10]. Both genes, which are divergently transcribed, share a 3 kb CpG island that is conserved in the syntenic HNRNPA2B1-CBX3 region in humans [11]. Fragments from the CpGrich 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 Cbx3 hypo/hypo homozygotes 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 Cbx3 hypo/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
The conditional targeting vector used to disrupt the Cbx3 gene was designed with a FlpE-excisable neo-tk cassette placed in the intron separating exons 3 and 4 ( Figure 1, second and third rows). Disruption of Cbx3 gene function, after excision of the neo-tk by FlpE, would be effected by Cre excision of exons 2 and 3, which contain the alternative ATG start codons of the Cbx3 gene ( Figure  1, third and fourth rows). Targeting frequency with the neo-tk-containing conditional vector (Figure 1, second row) was approximately 1:100. Germline transmission of the targeted allele ( Figure 1, third row) was achieved, and intercrossing of the heterozygotes for the targeted allele resulted in a normal mendelian ratio of wild-type to targeted alleles at E19 (not shown) the day before birth. However, the number of adult mice that were homozygous for the targeted allele was low, with only 3 of 216 adult males and 1 of 193 adult females being homozygous for the targeted allele ( Figure 1, third row), which was designated Cbx3 hypo . The nature and timing of the attrition of Cbx3 hypo/hypo homozygotes that takes place after birth is not known, and the data presented below on Cbx3 hypo/hypo adult animals are based on the three males and one female that reached adulthood.
The greatly reduced numbers of Cbx3 hypo/hypo adults prompted us to investigate whether the targeting event itself had affected Cbx3 expression, and if this was the likely cause of the reduced numbers of Cbx3 hypo/hypo adults. To explore this hypothesis further, we generated primary mouse fibroblasts from E13.5 wild-type and Cbx3 hypo/hypo littermates and compared the expression levels of HP1γ by Western blotting. As shown in Figure 2, there was a dramatic reduction in HP1γ expression levels in Cbx3 hypo/hypo compared with wild-type mouse embryonic fibroblasts (MEFs) (Figure 2a, top row: wild-type to hypo/hypo) indicating that the Cbx3 hypo allele was a hypomorph. The effect of the targeting event was specific to the Cbx3 gene, as protein expression of the closely linked Hnrnpa2b1 gene was not changed (Figure 2a, middle row). Given this unexpected result, we were prompted to investigate whether the presence of the neo-tk selection cassette itself was interfering with Cbx3 expression. Previous work has shown that knockdown of target gene expression can result from the presence of a neo gene in the targeting vector [14]. The mechanism for such a knockdown is not fully understood but may involve transcriptional interference, by which the presence of one transcriptional unit interferes with another that is in cis [15].
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 Cbx3 hypo/hypo MEFs, 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  Figure  s2). When Cbx3 -/-MEFs were included into this analysis, we observed an increase in H4K20me3 levels compared with wild-type and Cbx3 hypo/hypo MEFs (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.
A dramatic reduction of HP1γ levels was also observed in the Cbx3 hypo/hypo mouse tissues; Western blot analysis revealed that HP1γ levels were reduced to almost undetectable levels in all tissues examined ( Figure 3). There was no effect of the Cbx3 hypo/hypo mutation on the protein levels of HP1α (see Additional file 3, Figure s3a) and HP1β (see Additional file 3, Figure s3b) or on the same three histone post-translational modifications H4K20ME3, H3K9ME3 and H3K9AC (see Additional file 3, Figures s3c to s3e).
Housing the three Cbx3 hypo/hypo males with wild-type females resulted in no litters, indicating a possible problem with male fertility. The males were killed and the testes removed, which revealed severe hypogonadism compared with wild-type males (Additional file 4, Figure  s4). Examination of wild-type testes showed that expression of the Cbx3 gene product, HP1γ, was present in vir-tually all cells ( Figure 4a) and is distinct from the staining pattern of transcriptional intermediary factor (TIF)1β, a HP1γ-interacting protein, in wild-type testes, where preleptotene and spermatogonia are TIF1β-negative [16]. Sertoli cells are prominently stained, as are round (stage 2-6) spermatids (Figure 4b), with the latter showing an enriched spot of HP1γ staining in the nucleus (Figure 4b; see also Additional file 5, Figure s5b), which probably represents the characteristic heterochromatic chromocenter found in these nuclei [17]. HP1γ is excluded from meiotic metaphase chromosomes (Figure 4c), which is similar to the known expulsion of the bulk of HP1 proteins from metaphase chromosomes by the so-called serine 10 phosphorylation switch [18]. It may well be that the chromosome condensation during meiosis or mitosis requires the removal of the bulk of HP1 proteins from the chromosomes. In pachytene spermatocytes, HP1γ is found throughout the nucleus but is enriched at a few sites that probably represent heterochromatic chromocenters (Figures 4d; see also Additional file 5, figure s5a). HP1γ staining of spermatogenic cell types is detectable until elongating spermatid stage 10 ( Figure 4d; Table 1), which  is around the time that the bulk of the histones are removed and replaced by the protamines [19].
By contrast, HP1γ staining was almost undetectable in sections taken from Cbx3hypo/hypo testes (Figure 4e, f). Histological examination revealed a severe impairment of spermatogenesis in Cbx3hypo/hypo testes. The diameter of the tubules in Cbx3hypo/hypo testes was smaller (0.13 mm) than that in wild-type testes (0.2 mm). Of 70 tubules examined, 22 were almost completely devoid of germ cells (for example, Figure 4e, upper right tubule) and 48 tubules had impaired spermatogenesis. Tubules in which mature sperm could be observed were rare (Figure 4f, especially the magnified inset). The loss of germ cells was confirmed using a germ-specific antibody (anti-germ cell nuclear antigen (GCNA)) [20], which revealed a dramatic reduction in germ cells (GCNA-positive cells) in Cbx3hypo/hypo testes (Figure 5c, Figure 5d) compared with wild-type mice ( Figure 5, Figure 5b). Some of the tubules in Cbx3hypo/hypo testes exhibited a Sertoli cellonly (SCO) phenotype, reminiscent of the tubules seen in the Dnmt3L and Miwi2 mutants [21,22]. Thus, the Cbx3hypo/hypo mutation results in the general suppression of spermatogenesis, which can vary from tubule to tubule; in some tubules suppression is complete, resulting in a SCO phenotype, whereas in others, spermatogenesis can proceed and give rise to some mature sperms. This variation across the tubules might reflect the fact that the Cbx3hypo mutation is 'leaky', that is, the interference of Cbx3 by the neo-tk is variable giving rise to leaky expression of Cbx3. Some Cbx3hypo/hypo germ cells and their progeny might have closer to wild-type levels of HP1γ, thus enabling greater likelihood of survival with more normal development.
The similarity of the Cbx3 hypo/hypo spermatogenesis 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 L1encoded ORF1 protein [23]. 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 Cbx3 hypo/hypo testes 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 Cbx3 hypo/hypo mutation 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 Cbx3 hypo/hypo testes 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 Figure 3 HP1γ protein expression was dramatically reduced in Cbx3 hypo/hypo tissues. Protein expression was reduced to almost undetectable levels in testis, kidney, lung, brain, liver spleen and thymus tissues from the Cbx3 hypo/hypo mice. 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 Cbx3 hypo/hypo testes, the cell types stained were the same as those found in wild-type cells, notwithstanding the obvious suppression of spermatogenesis seen in the Cbx3 hypo/hypo testes (see Additional file 8, Figure s8d, Figure s8e). The levels of HP1α staining were also unchanged in the Cbx3 hypo/hypo testes, as evidenced by the typical staining of the round (stage 2-6) spermatids in Cbx3 hypo/hypo testes (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) [26]. The levels of HP1β staining and the cell types stained in Cbx3 hypo/hypo testes (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 Cbx3 hypo/hypo mutation are unlikely to operate through changes in the expression of the other two isotypes, HP1α and HP1β. The clear differences between wild-type and Cbx3 hypo/ hypo adult testes were not observed in embryonic E17 testes. The morphology of the seminiferous tubules and numbers of gonocytes in wild-type and Cbx3 hypo/hypo tes- In wild-type tubules, Sertoli cells stained strongly for HP1γ (black arrows) as did round spermatids (stage 2-6) (white arrows). Many round spermatid nuclei possessed an enriched focus of HP1γ staining that was characteristic of the single block of heterochromatin observed at this stage (see Additional file 5, Figures 5b). (c) HP1γ was largely excluded from meiotic metaphase chromosomes and was found surrounding the condensed chromosomes in the meiotic cytoplasm (black arrows). Mature sperm (white arrows) were negative for HP1γ. (d) Stage 10 spermatids, at around the time they elongated, were either positive (black arrows) or negative (white arrows) for HP1γ, indicating that it was during this stage that levels of HP1γ proteins decrease. Pachytene spermatocytes (yellow arrows) showed a few brightly stained spots that represent sites of constitutive heterochromatin (see Additional file 5, Figures 5a). (e) In Cbx3 hypo/hypo testes, HP1γ staining was very weak and the tubules showed severely impaired spermatogenesis with greatly reduced numbers of cells and some tubules exhibiting a Sertoli cells-only phenotype (see upper right tubule). (f) In some tubules of Cbx3 hypo/hypo testes, mature sperm could be observed (inset, black arrowhead). Bar = 100 μm. tes were similar (see Additional file 10, Figure s10), indicating that the suppression of spermatogenesis seen in the adult Cbx3 hypo/hypo testes ( Figure 4) probably occur at later stages, after meiosis has been initiated.
Housing the one adult Cbx3 hypo/hypo female mouse with wild-type males also resulted in no litters. Although it is difficult to conclude from a single Cbx3 hypo/hypo animal that Cbx3 hypo/hypo females are sterile, we nevertheless decided to examine sections of wild-type and Cbx3 hypo/ hypo ovaries. Examination of the sections revealed no obvious morphological difference between wild-type and Cbx3 hypo/hypo ovaries; all stages of folliculogenesis were observed in Cbx3 hypo/hypo ovaries, including corpora lutea, indicating that ovulation was normal in the Cbx3 hypo/hypo female (data not shown).
The similarity of the Cbx3 hypo/hypo phenotype in the testes with those observed in the testes of Miwi2 [21] and Dnmt3L mutants [21] 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 [27]. 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.

Conclusion
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.

Animal studies
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 [28]. 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% H 2 O 2 , 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 [4] 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). Cbx3 hypo/hypo tubules that had no germ cells (see Additional file 6, Figure s6b, asterisks) were not included in the analysis.

Western blotting
Western blotting was performed essentially as described previously [8]. For histone isolation, tissues were cut to pieces and further disintegrated in buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl 2 , 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 MgCl 2 and protease inhibitor) and layered over an equal volume of buffer S3 (0.88 M saccharose, 0.5 mM MgCl 2 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.