BIOLOGY OF REPRODUCTION (2014) 90(6):138, 1–7 Published online before print 23 April 2014. DOI 10.1095/biolreprod.113.116624
Phosphorylation of CDK2 on Threonine 160 Influences Silencing of Sex Chromosome During Male Meiosis1 Lu Wang,3 Wenjing Liu,3,4 Weidong Zhao,5 Gendi Song,5 Guishuan Wang,3 Xiaorong Wang,3 and Fei Sun2,3 3
Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China 4 College of Life Science and Technology, Southwest University of Science and Technology, Mianyang, Sichuan, China 5 Engineering College of Animal Husbandry and Veterinary Science, Henan Agricultural University, Zhengzhou, Henan, China
In mammalian meiosis, the X and Y chromosomes are largely unsynapsed and transcriptionally silenced during the pachytene stage of meiotic prophase (meiotic sex chromosome inactivation), forming a specialized nuclear territory called sex or XY body. An increasing number of proteins and noncoding RNAs were found to localize to the sex body and take part in influencing expression of sex chromosome genes. Cyclindependent kinase 2 (Cdk2 /) spermatocytes show incomplete sex chromosome pairing. Here, we further showed that phosphorylation of CDK2 isoform 1 (p-CDK239 [39 kDa]) on threonine 160 localizes to the sites of asynapsis and the sex body, interacting with phosphorylated gamma-H2AX. Meanwhile, p-CDK239 is frequently mislocalized throughout the sex body, and meiotic sex chromosome inactivation is disrupted in PWK3C57BL/6J hybrid mice. Furthermore, pachytene spermatocytes treated with mevastatin (an inhibitor of p-CDK2) showed overexpression of sex chromosome-linked genes. Our results highlight an important role for p-CDK239 in influencing silencing of the sex chromosomes during male meiosis by interacting with gamma-H2AX. histone modifications, kinases, meiosis, MSCI, p-CDK239, spermatocyte, X chromosome, Y chromosome, c-H2AX
INTRODUCTION During meiosis, homologous chromosomes form synapses and recombine, generating crossovers which are required to ensure accurate segregation of homologous chromosomes [1]. In contrast, the X and Y chromosomes are largely unsynapsed and do pair along a spatially restricted region (the pseudoautosomal region), and this triggers meiotic silencing of X- and Y-linked genes. This phenomenon is known as meiotic sex chromosome inactivation (MSCI) [2–7], in which the X and Y sex chromosomes are packaged into a compact subnuclear structure known as the XY or sex body [8–10]. The XY
MATERIALS AND METHODS Animals Strain ICR/C57BL/6 mice and PB6F1 mice (PWK 3 c57BL/6 F1) were respectively obtained from the Animal Center of University of Science and Technology of China (USTC) and Henan Agricultural University, and maintained under a 14L:10D cycle at room temperature (23 6 28C). Mice were provided with food and water ad libitum. PB6F1 mice showed infertility with spermatogenic arrest at mid-pachytene stage of meiosis (Supplemental Fig. S1; all supplemental data are available online at www.biolreprod.org). This study received ethical approval from the institutional review boards of USTC.
1 Supported by National Natural Science Foundation of China grant 81125005 and National Basic Research Program of China grant 2014CB943100 to F.S. 2 Correspondence: Dr. Fei Sun, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, P.R. China. E-mail: [email protected]
Antibodies
Received: 9 December 2013. First decision: 7 January 2014. Accepted: 25 March 2014. Ó 2014 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363
Antisera were generated in two rabbits against the synthetic polypeptide CSYLEVAASQGG, corresponding to the biologically active specific peptide of mouse CDK2 isoform 1 (39KD; henceforth, CDK239). Rabbit polyclonal CDK239 antibody was produced at Abgent Biotechnology Co. Western blotting analysis revealed that in mouse testes, the CDK239 antibody detected one positive protein band whose estimated molecular mass was 39 kDa
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chromosome territory is typically found in association with the nuclear lamina, where the paired distal ends of X and Y are anchored but are not membrane bound [10]. Likewise, unsynapsed autosomal chromatin during pachytene stage is also silenced (meiotic silencing of unsynapsed chromatin, MSUC), and MSCI is acknowledged as an example of the ancient fundamental mechanism specifically affecting the sex chromosomes [11–13]. Failure of MSCI initiation is associated with meiotic arrest at the pachytene stage, for example, in hybrid male sterility [14, 15]. An increasing number of proteins have been localized to the XY body, including modified histone variants and proteins involved in DNA double-strand break (DSB) repair such as cH2AX, BRCA1, MDC1, and macroH2A1.2 [16–19]. Recruitment of the checkpoint kinase ATR by the BRCA1 protein and subsequent phosphorylation of H2AX at Ser139 by ATR have been shown to be essential for MSCI [20–22]. Moreover, noncoding RNAs including selective miRNAs and piRNAs were also found to localize to the sex body [23]. However, the mechanistic aspects of MSCI initiation and maintenance remain largely unknown. Cyclin-dependent kinase 2 (CDK2) plays an important role in mammalian cell cycle progression, both at the transition from G1 to S and through S phase. In addition, cdk2/ spermatocytes show incomplete chromosome pairing and formation of sex body and extensive nonhomologous synapsis [24]. Phosphorylation of CDK2 at threonine 160 (henceforth referred to as p-CDK2) is essential for its kinase activity [25]. Here, the roles of p-CDK2 in influencing expression of sex chromosome-linked genes and then in MSCI were investigated
ABSTRACT
WANG ET AL. (Supplementary Fig. S2). Anti-CDK2 antibody and anti-p-CDK2 antibody were purchased from Abcam. The primary and secondary antibodies used in the present study are listed in Supplementary Tables S1 and S2, respectively.
Fluorescence Immunostaining Slides with cryosections, chromosome squash, and chromosome spreads were subjected to immunofluorescence staining as described previously [26– 28]. Primary antibodies against the following proteins were used: rabbit antiSYCP3 (1:200 dilution; Abcam), mouse anti-SYCP3 (1:500 dilution; Abcam), rabbit anti-CDK2 (1:100 dilution; Abcam), rabbit anti-p-CDK2 (1:200 dilution; Abcam), mouse anti-RNA polymerase II (1:200 dilution; Millipore), rabbit anti-c-H2AX (1:800 dilution; Abcam), mouse anti-c-H2AX (1:800 dilution; Abcam), and rabbit anti-CDK239 (1:200 dilution). These primary antibodies were detected using a cocktail of secondary antibodies (donkey antisera) conjugated with different fluorochrome compounds: green Alexa 488 and red Alexa 555 (Invitrogen). Nuclei were stained with Hoechst 33342 dye (Sigma). Fluorescent signals were examined using an Eclipse 80i model epifluorescence microscope (Nikon).
Plasmid Construction Full-length cDNAs for mouse CDK239, CDK233, CDK239 mutants (T160), and H2AX were obtained by reverse transcription (RT)-PCR from total RNA of mouse testis by using primers containing specific restriction sites. For the construction of expression vectors, CDK233 cDNA was cloned into pEGFP-C1 (Clontech) vector at EcoRI and SalI sites; and CDK239 and CDK239 mutants and H2AX cDNA were cloned into p3XFLAG-myc-CMV-24 (Sigma) and pEGFP-C1 (Clontech) vectors at EcoRI and SalI sites. Primer sequences are listed in Supplementary Table S3. All plasmids generated were verified by sequencing.
Western Blotting Primary Sertoli cell cultures were prepared from testes of 18-day-old mice, as described previously [29]. The nuclear and cytoplasmic extracts were isolated using NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology) according to the manufacturer’s instructions. Cells were lysed in RIPA buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% SDS, 1% sodium deoxycholate, and 1 mM EDTA) containing complete EDTAfree protease inhibitor cocktail (Roche), 1 mM PMSF, and phosphatase inhibitors (5 mM sodium orthovanadate). Protein lysates were loaded onto SDS-polyacrylamide gels, electroblotted onto Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane (GE Healthcare), and immunoblotted with antibodies and visualized using ECL (Kodak).
Cell Culture and Transfection Human embryonic kidney (HEK)-293T cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBSLife Technologies) and 1% antibiotics (100 units/ml penicillin and 100 lg/ml streptomycin) at 378C under a 5% CO2 atmosphere. HEK-293T cells were transfected using Lipofectamine 2000 (Invitrogen), following the manufacturer’s instructions. Isolation of pachytene spermatocytes from the testes was performed using previously described techniques [30, 31]. Primary pachytene spermatocytes were cultured in DMEM supplemented with 5% FBS and 1% antibiotics at 338C under a 5% CO2 atmosphere. Pachytene spermatocytes were treated with 10 lM mevastatin (Sigma) for 4 h and harvested and then processed for nuclear spreads, protein blotting, and real-time PCR analysis.
CDK239 Interacts with c-H2AX To further validate the interaction between p-CDK2 and cH2AX (a marker of the XY body and the sites of meiotic DSB), we performed immunostaining and IP assays in the testis. Phosphorylated-CDK2 was clearly distributed at the sex body and asynapsed regions of chromatin and colocalized with c-H2AX (Figs. 1E and 2, A and B, and Supplementary Fig. S4A) from early pachytene stage to diplotene stage. IP of testis lysates with the CDK2 antibody and CDK239-specific antibody demonstrated that only CDK239 could pull down c-H2AX (Fig. 2C). The association of CDK239 with H2AX was also validated by immunoblotting assay in vitro (Supplementary Fig. S4B). To confirm the requirement of phosphorylation of CDK2 on Thr160 for binding to c-H2AX, point mutant versions, namely Flag-CDK239 (m) and green fluorescent protein (GFP)CDK239 (m), in which the ACU codon (at Thr160) was mutated to AAU, was transfected in vitro for IP. The results showed that mutation of the ACU codon in the CDK239 protein substantially reduced its interaction with c-H2AX (Fig. 2D).
Immunoprecipitation Assay and Real-Time PCR Immunoprecipitation (IP) and real-time PCR analysis were performed as described previously [29]. Primer sequences for real-time PCR are listed in Supplementary Table S4.
RESULTS Subcellular Localization and Expression of CDK2/p-CDK2 During Spermatogenesis The localization of p-CDK2 in spermatocytes was analyzed by immunostaining of frozen sections (Fig. 1A), chromosome squash (Fig. 1B), and chromosome spread preparations (Fig. 2
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1C) together with staining of the synaptonemal complex, using antibodies against the lateral element protein SYCP3 from prophase I spermatocytes. In the nuclei of primary spermatocytes, p-CDK2 showed marked localization in the subnuclear region of mouse testicular cross-sections (Fig. 1, A and B). pCDK2 staining was evident in the nucleus, with the most intense staining around the XY synaptonemal complex from early pachytene stage, when the sex body forms, to diplotene stage (Fig. 1C). Interestingly, p-CDK2 staining was first found in late zygotene stage, embracing the single X and Y chromosomes (Fig. 1C); but we did not found any staining on autosomes in zygotene stage, which indicated that p-CDK2 might participate in sex chromosome paring and sex body formation. p-CDK2 staining was also found at the sex body in rat spermatocytes (Supplementary Fig. S3A). To determine whether the meiotic localization of p-CDK2 to the X and Y chromosomes was a consequence of their lack of pairing, we examined the localization of CDK2/p-CDK2 in the hybrid offspring, PB6F1, of two divergent strains (Fig. 1, D and E). CDK2/p-CDK2 localized to all sites of autosomal asynapsis and the axis of sex chromosomes and colocalized with cH2AX to these locations. Given that asynapsed regions of chromatin are silenced during prophase I [11, 22], these observations suggest that p-CDK2 may participate in meiotic silencing. To further determine the cellular localization of CDK2/pCDK2, cytoplasmic and nuclear fractions from male testes undergoing the meiotic stages of prophase I were analyzed (Fig. 1F). Western blotting showed weak expression of CDK239 from 8 to 16 days postpartum (dpp) and higher expression at 18 dpp in the nuclei, but there were no differences in the cytoplasm. Conversely, the level of pCDK2 isoform 1 (henceforth, p-CDK239 [39 kDa]) protein was found to be different from the total protein level, which increased from 14 dpp in the nuclei. However, both CDK2 isoform 2 (henceforth, CDK233 [33 kDa]) and p-CDK233 were expressed at a constant level in both places. Furthermore, only CDK239 was detected in the insoluble nuclear fraction (Fig. 1G), which suggests that it is CDK239 that associates with X and Y chromosomes. In addition, CDK239 was not expressed in Sertoli cells (Fig. 1H). By using the antibodies specifically against CDK239 instead of CDK233, CDK239 staining was shown mainly in the nucleus and cytoplasm of spermatocytes (Supplementary Fig. S3B).
PHOSPHORYLATION OF CDK2 IN MALE MEIOSIS
Downloaded from www.biolreprod.org. FIG. 1. Subcellular localization and expression of CDK2/p-CDK2 during the first wave of spermatogenesis. A–E) Cryosections, chromosome squash, and chromosome spreads from testicular germ cells of 2-month-old male mice were stained with Hoechst (blue) and immunolabeled using antibodies against p-CDK2, CDK2, c-H2AX, and the synaptonemal complex lateral element protein SYCP3. C) White arrowheads point to X and Y chromosomes. D and E) White arrowheads point to aggregates of CDK2 and p-CDK2 that localize at the sites of autosomal asynapsis. F and G) Western blots of equal volumes of testicular fractions from 8-, 14-, 16-, and 18-day-old mice showing the subcellular distribution of CDK2 and p-CDK2, with GAPDH and lamin A/C used as cytoplasmic and nuclear markers, respectively, to assess the purity of the fractionation. C ¼ cytoplasmic fraction; N ¼ nuclear fraction; Ns ¼ supernatant of the nuclear fraction; Np ¼ pellet of the nuclear fraction. H) Western blotting analysis of CDK2 protein in testicular Sertoli cells (SC) and germ cells (GC) isolated from 18-day-old mice; SYCP3 and c-H2AX were used as controls for germ cells, and b-actin was used as a protein loading control. Bar ¼ 50 lm for all images.
localization (Fig. 3A). Analysis of cytoplasmic and nuclear fractions from the testes of PB6F1 mice, and 16-day-old normal mice showed that CDK239 was severely downregulated in the nucleus of PB6F1 mice (Fig. 3B). A lack of staining of RNA polymerase II (a marker of transcriptional elongation) at the XY body indicated transcriptional silencing
Absence of p-CDK2 in the Sex Body Leads to Failure of Meiotic Sex Chromosome Inactivation In PB6F1 pachytene cells, p-CDK2 is frequently mislocalized, with aberrant staining patterns, including a complete signal loss at the sex body and reduced sex chromosome core 3
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Downloaded from www.biolreprod.org. FIG. 2. Colocalization and interaction between CDK239 and c-H2AX at the sex body. A and B) Localization of c-H2AX and p-CDK2 was determined by immunofluorescence analysis of cryosections and chromosomal spreads from the meiotic stages of prophase I of adult mouse testes. A) Hoechst (blue), pCDK2 (green), and c-H2AX (red) stains are shown. B) p-CDK2 (green), c-H2AX (red), and SYCP3 (blue) stains are shown. C) Co-immunoprecipitation (IP) using antibodies to CDK2 and CDK239, followed by Western blotting using an antibody to c-H2AX in adult mouse testes. D) Co-IP using an antibody to FLAG, followed by Western blotting using antibodies to FLAG and GFP. Flag-CDK2 (m) and GFP-CDK2 (39 kDa); m ¼ mutation of CDK2 on threonine 160. Bar ¼ 50 lm for all images.
in normal mice (Fig. 3C). This exclusion was no longer maintained in a significant fraction of pachytene-stage spermatocytes from PB6F1 males (Fig. 3, C and D). In addition, expression was increased in the sex-linked genes Ccnb3 (5.75-fold), Suv39h1 (2.07-fold), Map7d2 (1.97-fold), H2bfm (6.45-fold), Rhox13 (2.10-fold), Rbmy (4.33-fold), Zfy1 (2.77-fold), Sry (2.56-fold), and Ube1y (2.89-fold) in PB6F1 mice compared with those in normal fertile mice (Fig. 3E). However, the expression levels of the autosomal genes Ndufa4, Rrbp1, Stx12, and Nop58 were not altered between the two groups (Fig. 3E). Furthermore, spermatocytes treated with mevastatin, an inhibitor of p-CDK2 [25, 32], inhibited the level of p-CDK2 at the XY body (Fig. 4A), reduced levels of both of the two isoforms of p-CDK2 (Fig. 4B), and up-regulated
expression of sex chromosome genes (Rbmy, Rhox13, and Ube1y) (Fig. 4C). Interestingly, pachytene-stage spermatocytes treated with mevastatin did not change the localization of cH2AX at the sex body (Supplementary Fig. S5). Meanwhile, RNA polymerase II staining was visible on XY chromosomes in mevastatin-treated spermatocytes (Fig. 4, D and E). These data suggest that p-CDK2 influences transcriptional activity of sex chromosome genes. DISCUSSION In this study, p-CDK2 co-localized with c-H2AX at the sites of asynapsis and the sex body during meiosis. Loss of p-CDK2 at the sex body caused an influx of RNA polymerase II and an upregulation of sex-linked transcripts. Our data suggest the 4
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Downloaded from www.biolreprod.org. FIG. 3. p-CDK2 is frequently mislocalized at the sex body in PB6F1 pachytene cells, associated with disruption of MSCI. A) Localization of p-CDK2 was determined by immunofluorescence analysis of chromosomal spreads from adult C57 and PB6F1 mice. p-CDK2 (green), SYCP3 (red) are shown. B) Western blotting of equal volumes of testicular fractions from 16-day-old C57 and PB6F1 mice, with GAPDH and lamin A/C used as cytoplasmic and nuclear markers, respectively. C ¼ cytoplasmic fraction; N ¼ nuclear fraction. C and D) RNA polymerase II staining of chromosomal spreads from 16.5day-old C57 and PB6F1 mice. RNA polymerase II (green), SYCP3 (red) are shown (n ¼ 100). **P , 0.01, t-test. White arrowheads point to the sex body. E) Real-time PCR analysis of XY-linked gene expression in total RNAs from the testes of 16.5-day-old C57 (n ¼ 3) and PB6F1 (n ¼ 3) mice. *P , 0.05, **P , 0.01, t-test. Bar ¼ 50 lm for all images.
important role of p-CDK2 in MSCI by interaction with cH2AX. Sex body is not formed in Cdk2/ spermatocytes [24, 33]. Phosphorylated CDK2 is essential for its kinase activity [25, 32]. Here, we found distinct localization patterns and expression levels for two (39-kDa and 33-kDa) isoforms of p-CDK2 within the nuclei of mouse spermatogenic cells, and it was not until pachytene stage that the level of p-CDK239 protein suddenly increased. It is only CDK239 that is expressed in male germ cells, instead of Sertoli cells, and diffuses out into XY chromatin and all sites of autosomal asynapsis, in interaction with c-H2AX (a marker of the XY body and DSBs). Given the localization of p-CDK239 to asynapsed regions and the sex body in pachynema, together with its colocalization and interaction with c-H2AX (a mediator of
MSCI), our results indicate a role for p-CDK2 in the initiation and/or maintenance of transcriptional silencing during meiotic prophase I. MSCI, silencing of sex chromatin in male meiosis, is one case of MSUC [6, 12, 20]. It is reported that once BRCA1 localizes to the axial elements of the sex chromosomes, it recruits ATR, which phosphorylates H2AX, and it subsequently diffuses into XY chromatin to trigger MSCI [21]. Here, by using PB6F1 mice and spermatocytes treated with inhibitors of p-CDK2, we detected many misexpressed sex chromosomelinked genes. CDK2 is able to phosphorylate BRCA1 [34, 35]. In addition, when CDK2 was absent, BRCA1 was not loaded on sex chromosomes, and then ATR was not recruited to the sex body. In turn, H2AX was not phosphorylated at the site of serine 139, so that a sex body was not formed. In this study, p5
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Downloaded from www.biolreprod.org. FIG. 4. Mevastatin inhibits p-CDK2 at the sex body in spermatocytes, leading to failure of MSCI. Spermatocytes were isolated from 18-day-old mouse testes. A) p-CDK2 staining was determined by immunofluorescence analysis of spermatocytes treated with dimethyl sulfoxide (DMSO) and mevastatin. pCDK2 (green), SYCP3 (red). B) Western blotting analysis of p-CDK2 protein level from spermatocytes treated with DMSO and mevastatin. C) Real-time PCR analysis of XY-linked gene expression in total RNA from spermatocytes treated with DMSO (n ¼ 3) and mevastatin (n ¼ 3). **P , 0.01, t-test. D and E) RNA polymerase II staining from spermatocytes treated with DMSO and mevastatin: RNA polymerase II (green), SYCP3 (red); n ¼ 100. *P , 0.05, t-test. Bar ¼ 50 lm for all images.
CDK2 interacted with c-H2AX in the XY chromatin region instead of the axis of the XY chromosome, which suggests that p-CDK2 may mediate the initiation and/or maintenance of the sex body. Although condensation of the X and Y chromosomes in meiotic cells was observed nine decades ago [36] and more and more proteins and noncoding RNAs have been found to be distributed at the XY body, the function and molecular basis of sex body formation and the associated MSCI remain an enigma. Here, we add p-CDK2 as a potential regulator for
MSCI in male mice. Further studies are required to elucidate the specific mechanism of p-CDK2 involvement in the regulation of sex chromosome genes. ACKNOWLEDGMENT We thank Dr. Mian Wu for the HEK-293T cell line.
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Phosphorylation of CDK2 on Threonine 160 Influences Silencing of Sex Chromosome During Male Meiosis1 Lu Wang,3 Wenjing Liu,3,4 Weidong Zhao,5 Gendi Song,5 Guishuan Wang,3 Xiaorong Wang,3 and Fei Sun2,3 3
Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China 4 College of Life Science and Technology, Southwest University of Science and Technology, Mianyang, Sichuan, China 5 Engineering College of Animal Husbandry and Veterinary Science, Henan Agricultural University, Zhengzhou, Henan, China
In mammalian meiosis, the X and Y chromosomes are largely unsynapsed and transcriptionally silenced during the pachytene stage of meiotic prophase (meiotic sex chromosome inactivation), forming a specialized nuclear territory called sex or XY body. An increasing number of proteins and noncoding RNAs were found to localize to the sex body and take part in influencing expression of sex chromosome genes. Cyclindependent kinase 2 (Cdk2 /) spermatocytes show incomplete sex chromosome pairing. Here, we further showed that phosphorylation of CDK2 isoform 1 (p-CDK239 [39 kDa]) on threonine 160 localizes to the sites of asynapsis and the sex body, interacting with phosphorylated gamma-H2AX. Meanwhile, p-CDK239 is frequently mislocalized throughout the sex body, and meiotic sex chromosome inactivation is disrupted in PWK3C57BL/6J hybrid mice. Furthermore, pachytene spermatocytes treated with mevastatin (an inhibitor of p-CDK2) showed overexpression of sex chromosome-linked genes. Our results highlight an important role for p-CDK239 in influencing silencing of the sex chromosomes during male meiosis by interacting with gamma-H2AX. histone modifications, kinases, meiosis, MSCI, p-CDK239, spermatocyte, X chromosome, Y chromosome, c-H2AX
INTRODUCTION During meiosis, homologous chromosomes form synapses and recombine, generating crossovers which are required to ensure accurate segregation of homologous chromosomes [1]. In contrast, the X and Y chromosomes are largely unsynapsed and do pair along a spatially restricted region (the pseudoautosomal region), and this triggers meiotic silencing of X- and Y-linked genes. This phenomenon is known as meiotic sex chromosome inactivation (MSCI) [2–7], in which the X and Y sex chromosomes are packaged into a compact subnuclear structure known as the XY or sex body [8–10]. The XY
MATERIALS AND METHODS Animals Strain ICR/C57BL/6 mice and PB6F1 mice (PWK 3 c57BL/6 F1) were respectively obtained from the Animal Center of University of Science and Technology of China (USTC) and Henan Agricultural University, and maintained under a 14L:10D cycle at room temperature (23 6 28C). Mice were provided with food and water ad libitum. PB6F1 mice showed infertility with spermatogenic arrest at mid-pachytene stage of meiosis (Supplemental Fig. S1; all supplemental data are available online at www.biolreprod.org). This study received ethical approval from the institutional review boards of USTC.
1 Supported by National Natural Science Foundation of China grant 81125005 and National Basic Research Program of China grant 2014CB943100 to F.S. 2 Correspondence: Dr. Fei Sun, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, P.R. China. E-mail: [email protected]
Antibodies
Received: 9 December 2013. First decision: 7 January 2014. Accepted: 25 March 2014. Ó 2014 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363
Antisera were generated in two rabbits against the synthetic polypeptide CSYLEVAASQGG, corresponding to the biologically active specific peptide of mouse CDK2 isoform 1 (39KD; henceforth, CDK239). Rabbit polyclonal CDK239 antibody was produced at Abgent Biotechnology Co. Western blotting analysis revealed that in mouse testes, the CDK239 antibody detected one positive protein band whose estimated molecular mass was 39 kDa
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chromosome territory is typically found in association with the nuclear lamina, where the paired distal ends of X and Y are anchored but are not membrane bound [10]. Likewise, unsynapsed autosomal chromatin during pachytene stage is also silenced (meiotic silencing of unsynapsed chromatin, MSUC), and MSCI is acknowledged as an example of the ancient fundamental mechanism specifically affecting the sex chromosomes [11–13]. Failure of MSCI initiation is associated with meiotic arrest at the pachytene stage, for example, in hybrid male sterility [14, 15]. An increasing number of proteins have been localized to the XY body, including modified histone variants and proteins involved in DNA double-strand break (DSB) repair such as cH2AX, BRCA1, MDC1, and macroH2A1.2 [16–19]. Recruitment of the checkpoint kinase ATR by the BRCA1 protein and subsequent phosphorylation of H2AX at Ser139 by ATR have been shown to be essential for MSCI [20–22]. Moreover, noncoding RNAs including selective miRNAs and piRNAs were also found to localize to the sex body [23]. However, the mechanistic aspects of MSCI initiation and maintenance remain largely unknown. Cyclin-dependent kinase 2 (CDK2) plays an important role in mammalian cell cycle progression, both at the transition from G1 to S and through S phase. In addition, cdk2/ spermatocytes show incomplete chromosome pairing and formation of sex body and extensive nonhomologous synapsis [24]. Phosphorylation of CDK2 at threonine 160 (henceforth referred to as p-CDK2) is essential for its kinase activity [25]. Here, the roles of p-CDK2 in influencing expression of sex chromosome-linked genes and then in MSCI were investigated
ABSTRACT
WANG ET AL. (Supplementary Fig. S2). Anti-CDK2 antibody and anti-p-CDK2 antibody were purchased from Abcam. The primary and secondary antibodies used in the present study are listed in Supplementary Tables S1 and S2, respectively.
Fluorescence Immunostaining Slides with cryosections, chromosome squash, and chromosome spreads were subjected to immunofluorescence staining as described previously [26– 28]. Primary antibodies against the following proteins were used: rabbit antiSYCP3 (1:200 dilution; Abcam), mouse anti-SYCP3 (1:500 dilution; Abcam), rabbit anti-CDK2 (1:100 dilution; Abcam), rabbit anti-p-CDK2 (1:200 dilution; Abcam), mouse anti-RNA polymerase II (1:200 dilution; Millipore), rabbit anti-c-H2AX (1:800 dilution; Abcam), mouse anti-c-H2AX (1:800 dilution; Abcam), and rabbit anti-CDK239 (1:200 dilution). These primary antibodies were detected using a cocktail of secondary antibodies (donkey antisera) conjugated with different fluorochrome compounds: green Alexa 488 and red Alexa 555 (Invitrogen). Nuclei were stained with Hoechst 33342 dye (Sigma). Fluorescent signals were examined using an Eclipse 80i model epifluorescence microscope (Nikon).
Plasmid Construction Full-length cDNAs for mouse CDK239, CDK233, CDK239 mutants (T160), and H2AX were obtained by reverse transcription (RT)-PCR from total RNA of mouse testis by using primers containing specific restriction sites. For the construction of expression vectors, CDK233 cDNA was cloned into pEGFP-C1 (Clontech) vector at EcoRI and SalI sites; and CDK239 and CDK239 mutants and H2AX cDNA were cloned into p3XFLAG-myc-CMV-24 (Sigma) and pEGFP-C1 (Clontech) vectors at EcoRI and SalI sites. Primer sequences are listed in Supplementary Table S3. All plasmids generated were verified by sequencing.
Western Blotting Primary Sertoli cell cultures were prepared from testes of 18-day-old mice, as described previously [29]. The nuclear and cytoplasmic extracts were isolated using NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology) according to the manufacturer’s instructions. Cells were lysed in RIPA buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% SDS, 1% sodium deoxycholate, and 1 mM EDTA) containing complete EDTAfree protease inhibitor cocktail (Roche), 1 mM PMSF, and phosphatase inhibitors (5 mM sodium orthovanadate). Protein lysates were loaded onto SDS-polyacrylamide gels, electroblotted onto Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane (GE Healthcare), and immunoblotted with antibodies and visualized using ECL (Kodak).
Cell Culture and Transfection Human embryonic kidney (HEK)-293T cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBSLife Technologies) and 1% antibiotics (100 units/ml penicillin and 100 lg/ml streptomycin) at 378C under a 5% CO2 atmosphere. HEK-293T cells were transfected using Lipofectamine 2000 (Invitrogen), following the manufacturer’s instructions. Isolation of pachytene spermatocytes from the testes was performed using previously described techniques [30, 31]. Primary pachytene spermatocytes were cultured in DMEM supplemented with 5% FBS and 1% antibiotics at 338C under a 5% CO2 atmosphere. Pachytene spermatocytes were treated with 10 lM mevastatin (Sigma) for 4 h and harvested and then processed for nuclear spreads, protein blotting, and real-time PCR analysis.
CDK239 Interacts with c-H2AX To further validate the interaction between p-CDK2 and cH2AX (a marker of the XY body and the sites of meiotic DSB), we performed immunostaining and IP assays in the testis. Phosphorylated-CDK2 was clearly distributed at the sex body and asynapsed regions of chromatin and colocalized with c-H2AX (Figs. 1E and 2, A and B, and Supplementary Fig. S4A) from early pachytene stage to diplotene stage. IP of testis lysates with the CDK2 antibody and CDK239-specific antibody demonstrated that only CDK239 could pull down c-H2AX (Fig. 2C). The association of CDK239 with H2AX was also validated by immunoblotting assay in vitro (Supplementary Fig. S4B). To confirm the requirement of phosphorylation of CDK2 on Thr160 for binding to c-H2AX, point mutant versions, namely Flag-CDK239 (m) and green fluorescent protein (GFP)CDK239 (m), in which the ACU codon (at Thr160) was mutated to AAU, was transfected in vitro for IP. The results showed that mutation of the ACU codon in the CDK239 protein substantially reduced its interaction with c-H2AX (Fig. 2D).
Immunoprecipitation Assay and Real-Time PCR Immunoprecipitation (IP) and real-time PCR analysis were performed as described previously [29]. Primer sequences for real-time PCR are listed in Supplementary Table S4.
RESULTS Subcellular Localization and Expression of CDK2/p-CDK2 During Spermatogenesis The localization of p-CDK2 in spermatocytes was analyzed by immunostaining of frozen sections (Fig. 1A), chromosome squash (Fig. 1B), and chromosome spread preparations (Fig. 2
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1C) together with staining of the synaptonemal complex, using antibodies against the lateral element protein SYCP3 from prophase I spermatocytes. In the nuclei of primary spermatocytes, p-CDK2 showed marked localization in the subnuclear region of mouse testicular cross-sections (Fig. 1, A and B). pCDK2 staining was evident in the nucleus, with the most intense staining around the XY synaptonemal complex from early pachytene stage, when the sex body forms, to diplotene stage (Fig. 1C). Interestingly, p-CDK2 staining was first found in late zygotene stage, embracing the single X and Y chromosomes (Fig. 1C); but we did not found any staining on autosomes in zygotene stage, which indicated that p-CDK2 might participate in sex chromosome paring and sex body formation. p-CDK2 staining was also found at the sex body in rat spermatocytes (Supplementary Fig. S3A). To determine whether the meiotic localization of p-CDK2 to the X and Y chromosomes was a consequence of their lack of pairing, we examined the localization of CDK2/p-CDK2 in the hybrid offspring, PB6F1, of two divergent strains (Fig. 1, D and E). CDK2/p-CDK2 localized to all sites of autosomal asynapsis and the axis of sex chromosomes and colocalized with cH2AX to these locations. Given that asynapsed regions of chromatin are silenced during prophase I [11, 22], these observations suggest that p-CDK2 may participate in meiotic silencing. To further determine the cellular localization of CDK2/pCDK2, cytoplasmic and nuclear fractions from male testes undergoing the meiotic stages of prophase I were analyzed (Fig. 1F). Western blotting showed weak expression of CDK239 from 8 to 16 days postpartum (dpp) and higher expression at 18 dpp in the nuclei, but there were no differences in the cytoplasm. Conversely, the level of pCDK2 isoform 1 (henceforth, p-CDK239 [39 kDa]) protein was found to be different from the total protein level, which increased from 14 dpp in the nuclei. However, both CDK2 isoform 2 (henceforth, CDK233 [33 kDa]) and p-CDK233 were expressed at a constant level in both places. Furthermore, only CDK239 was detected in the insoluble nuclear fraction (Fig. 1G), which suggests that it is CDK239 that associates with X and Y chromosomes. In addition, CDK239 was not expressed in Sertoli cells (Fig. 1H). By using the antibodies specifically against CDK239 instead of CDK233, CDK239 staining was shown mainly in the nucleus and cytoplasm of spermatocytes (Supplementary Fig. S3B).
PHOSPHORYLATION OF CDK2 IN MALE MEIOSIS
Downloaded from www.biolreprod.org. FIG. 1. Subcellular localization and expression of CDK2/p-CDK2 during the first wave of spermatogenesis. A–E) Cryosections, chromosome squash, and chromosome spreads from testicular germ cells of 2-month-old male mice were stained with Hoechst (blue) and immunolabeled using antibodies against p-CDK2, CDK2, c-H2AX, and the synaptonemal complex lateral element protein SYCP3. C) White arrowheads point to X and Y chromosomes. D and E) White arrowheads point to aggregates of CDK2 and p-CDK2 that localize at the sites of autosomal asynapsis. F and G) Western blots of equal volumes of testicular fractions from 8-, 14-, 16-, and 18-day-old mice showing the subcellular distribution of CDK2 and p-CDK2, with GAPDH and lamin A/C used as cytoplasmic and nuclear markers, respectively, to assess the purity of the fractionation. C ¼ cytoplasmic fraction; N ¼ nuclear fraction; Ns ¼ supernatant of the nuclear fraction; Np ¼ pellet of the nuclear fraction. H) Western blotting analysis of CDK2 protein in testicular Sertoli cells (SC) and germ cells (GC) isolated from 18-day-old mice; SYCP3 and c-H2AX were used as controls for germ cells, and b-actin was used as a protein loading control. Bar ¼ 50 lm for all images.
localization (Fig. 3A). Analysis of cytoplasmic and nuclear fractions from the testes of PB6F1 mice, and 16-day-old normal mice showed that CDK239 was severely downregulated in the nucleus of PB6F1 mice (Fig. 3B). A lack of staining of RNA polymerase II (a marker of transcriptional elongation) at the XY body indicated transcriptional silencing
Absence of p-CDK2 in the Sex Body Leads to Failure of Meiotic Sex Chromosome Inactivation In PB6F1 pachytene cells, p-CDK2 is frequently mislocalized, with aberrant staining patterns, including a complete signal loss at the sex body and reduced sex chromosome core 3
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Downloaded from www.biolreprod.org. FIG. 2. Colocalization and interaction between CDK239 and c-H2AX at the sex body. A and B) Localization of c-H2AX and p-CDK2 was determined by immunofluorescence analysis of cryosections and chromosomal spreads from the meiotic stages of prophase I of adult mouse testes. A) Hoechst (blue), pCDK2 (green), and c-H2AX (red) stains are shown. B) p-CDK2 (green), c-H2AX (red), and SYCP3 (blue) stains are shown. C) Co-immunoprecipitation (IP) using antibodies to CDK2 and CDK239, followed by Western blotting using an antibody to c-H2AX in adult mouse testes. D) Co-IP using an antibody to FLAG, followed by Western blotting using antibodies to FLAG and GFP. Flag-CDK2 (m) and GFP-CDK2 (39 kDa); m ¼ mutation of CDK2 on threonine 160. Bar ¼ 50 lm for all images.
in normal mice (Fig. 3C). This exclusion was no longer maintained in a significant fraction of pachytene-stage spermatocytes from PB6F1 males (Fig. 3, C and D). In addition, expression was increased in the sex-linked genes Ccnb3 (5.75-fold), Suv39h1 (2.07-fold), Map7d2 (1.97-fold), H2bfm (6.45-fold), Rhox13 (2.10-fold), Rbmy (4.33-fold), Zfy1 (2.77-fold), Sry (2.56-fold), and Ube1y (2.89-fold) in PB6F1 mice compared with those in normal fertile mice (Fig. 3E). However, the expression levels of the autosomal genes Ndufa4, Rrbp1, Stx12, and Nop58 were not altered between the two groups (Fig. 3E). Furthermore, spermatocytes treated with mevastatin, an inhibitor of p-CDK2 [25, 32], inhibited the level of p-CDK2 at the XY body (Fig. 4A), reduced levels of both of the two isoforms of p-CDK2 (Fig. 4B), and up-regulated
expression of sex chromosome genes (Rbmy, Rhox13, and Ube1y) (Fig. 4C). Interestingly, pachytene-stage spermatocytes treated with mevastatin did not change the localization of cH2AX at the sex body (Supplementary Fig. S5). Meanwhile, RNA polymerase II staining was visible on XY chromosomes in mevastatin-treated spermatocytes (Fig. 4, D and E). These data suggest that p-CDK2 influences transcriptional activity of sex chromosome genes. DISCUSSION In this study, p-CDK2 co-localized with c-H2AX at the sites of asynapsis and the sex body during meiosis. Loss of p-CDK2 at the sex body caused an influx of RNA polymerase II and an upregulation of sex-linked transcripts. Our data suggest the 4
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Downloaded from www.biolreprod.org. FIG. 3. p-CDK2 is frequently mislocalized at the sex body in PB6F1 pachytene cells, associated with disruption of MSCI. A) Localization of p-CDK2 was determined by immunofluorescence analysis of chromosomal spreads from adult C57 and PB6F1 mice. p-CDK2 (green), SYCP3 (red) are shown. B) Western blotting of equal volumes of testicular fractions from 16-day-old C57 and PB6F1 mice, with GAPDH and lamin A/C used as cytoplasmic and nuclear markers, respectively. C ¼ cytoplasmic fraction; N ¼ nuclear fraction. C and D) RNA polymerase II staining of chromosomal spreads from 16.5day-old C57 and PB6F1 mice. RNA polymerase II (green), SYCP3 (red) are shown (n ¼ 100). **P , 0.01, t-test. White arrowheads point to the sex body. E) Real-time PCR analysis of XY-linked gene expression in total RNAs from the testes of 16.5-day-old C57 (n ¼ 3) and PB6F1 (n ¼ 3) mice. *P , 0.05, **P , 0.01, t-test. Bar ¼ 50 lm for all images.
important role of p-CDK2 in MSCI by interaction with cH2AX. Sex body is not formed in Cdk2/ spermatocytes [24, 33]. Phosphorylated CDK2 is essential for its kinase activity [25, 32]. Here, we found distinct localization patterns and expression levels for two (39-kDa and 33-kDa) isoforms of p-CDK2 within the nuclei of mouse spermatogenic cells, and it was not until pachytene stage that the level of p-CDK239 protein suddenly increased. It is only CDK239 that is expressed in male germ cells, instead of Sertoli cells, and diffuses out into XY chromatin and all sites of autosomal asynapsis, in interaction with c-H2AX (a marker of the XY body and DSBs). Given the localization of p-CDK239 to asynapsed regions and the sex body in pachynema, together with its colocalization and interaction with c-H2AX (a mediator of
MSCI), our results indicate a role for p-CDK2 in the initiation and/or maintenance of transcriptional silencing during meiotic prophase I. MSCI, silencing of sex chromatin in male meiosis, is one case of MSUC [6, 12, 20]. It is reported that once BRCA1 localizes to the axial elements of the sex chromosomes, it recruits ATR, which phosphorylates H2AX, and it subsequently diffuses into XY chromatin to trigger MSCI [21]. Here, by using PB6F1 mice and spermatocytes treated with inhibitors of p-CDK2, we detected many misexpressed sex chromosomelinked genes. CDK2 is able to phosphorylate BRCA1 [34, 35]. In addition, when CDK2 was absent, BRCA1 was not loaded on sex chromosomes, and then ATR was not recruited to the sex body. In turn, H2AX was not phosphorylated at the site of serine 139, so that a sex body was not formed. In this study, p5
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Downloaded from www.biolreprod.org. FIG. 4. Mevastatin inhibits p-CDK2 at the sex body in spermatocytes, leading to failure of MSCI. Spermatocytes were isolated from 18-day-old mouse testes. A) p-CDK2 staining was determined by immunofluorescence analysis of spermatocytes treated with dimethyl sulfoxide (DMSO) and mevastatin. pCDK2 (green), SYCP3 (red). B) Western blotting analysis of p-CDK2 protein level from spermatocytes treated with DMSO and mevastatin. C) Real-time PCR analysis of XY-linked gene expression in total RNA from spermatocytes treated with DMSO (n ¼ 3) and mevastatin (n ¼ 3). **P , 0.01, t-test. D and E) RNA polymerase II staining from spermatocytes treated with DMSO and mevastatin: RNA polymerase II (green), SYCP3 (red); n ¼ 100. *P , 0.05, t-test. Bar ¼ 50 lm for all images.
CDK2 interacted with c-H2AX in the XY chromatin region instead of the axis of the XY chromosome, which suggests that p-CDK2 may mediate the initiation and/or maintenance of the sex body. Although condensation of the X and Y chromosomes in meiotic cells was observed nine decades ago [36] and more and more proteins and noncoding RNAs have been found to be distributed at the XY body, the function and molecular basis of sex body formation and the associated MSCI remain an enigma. Here, we add p-CDK2 as a potential regulator for
MSCI in male mice. Further studies are required to elucidate the specific mechanism of p-CDK2 involvement in the regulation of sex chromosome genes. ACKNOWLEDGMENT We thank Dr. Mian Wu for the HEK-293T cell line.
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