Proteasome subunit α4s is essential for formation of spermatoproteasomes and histone degradation during meiotic DNA repair in spermatocytes
2020; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1074/jbc.ra120.016485
ISSN1083-351X
AutoresZihui Zhang, Tian-Xia Jiang, Lian-Bin Chen, Wenhui Zhou, Yixun Liu, Fei Gao, Xiao‐Bo Qiu,
Tópico(s)Chromosomal and Genetic Variations
ResumoMeiosis, which produces haploid progeny, is critical to ensuring both faithful genome transmission and genetic diversity. Proteasomes play critical roles at various stages of spermatogenesis, including meiosis, but the underlying mechanisms remain unclear. The atypical proteasomes, which contain the activator PA200, catalyze the acetylation-dependent degradation of the core histones in elongated spermatids and DNA repair in somatic cells. We show here that the testis-specific proteasome subunit α4s/PSMA8 is essential for male fertility by promoting proper formation of spermatoproteasomes, which harbor both PA200 and constitutive catalytic subunits. Immunostaining of a spermatocyte marker, SYCP3, indicated that meiosis was halted at the stage of spermatocytes in the α4s-deficient testes. α4s stimulated the in vitro degradation of the acetylated core histones, instead of nonacetylated histones, by the PA200-proteasome. Deletion of α4s blocked degradation of the core histones at DNA damage loci in spermatocytes, leading to meiotic arrest at metaphase I. Thus, α4s is required for histone degradation at meiotic DNA damage loci, proper progression of meiosis, and fertility in males by promoting proper formation of spermatoproteasomes. These results are important for understanding male infertility and might provide potential targets for male contraception or treatment of male infertility. Meiosis, which produces haploid progeny, is critical to ensuring both faithful genome transmission and genetic diversity. Proteasomes play critical roles at various stages of spermatogenesis, including meiosis, but the underlying mechanisms remain unclear. The atypical proteasomes, which contain the activator PA200, catalyze the acetylation-dependent degradation of the core histones in elongated spermatids and DNA repair in somatic cells. We show here that the testis-specific proteasome subunit α4s/PSMA8 is essential for male fertility by promoting proper formation of spermatoproteasomes, which harbor both PA200 and constitutive catalytic subunits. Immunostaining of a spermatocyte marker, SYCP3, indicated that meiosis was halted at the stage of spermatocytes in the α4s-deficient testes. α4s stimulated the in vitro degradation of the acetylated core histones, instead of nonacetylated histones, by the PA200-proteasome. Deletion of α4s blocked degradation of the core histones at DNA damage loci in spermatocytes, leading to meiotic arrest at metaphase I. Thus, α4s is required for histone degradation at meiotic DNA damage loci, proper progression of meiosis, and fertility in males by promoting proper formation of spermatoproteasomes. These results are important for understanding male infertility and might provide potential targets for male contraception or treatment of male infertility. Proteasomes are responsible for degradation of most cellular proteins, and their inhibitors, such as bortezomib and carfilzomib, are clinically used to treat multiple myeloma and mantle cell lymphoma (1Richardson P.G. Xie W. Mitsiades C. Chanan-Khan A.A. Lonial S. Hassoun H. Avigan D.E. Oaklander A.L. Kuter D.J. Wen P.Y. Kesari S. Briemberg H.R. Schlossman R.L. Munshi N.C. Heffner L.T. et al.Single-agent bortezomib in previously untreated multiple myeloma: efficacy, characterization of peripheral neuropathy, and molecular correlations with response and neuropathy.J. Clin. Oncol. 2009; 27: 3518-3525Crossref PubMed Scopus (215) Google Scholar). Proteasomes usually contain one 20S catalytic core particle (CP) and one or two regulatory particles, which serve as activators, including the 19S regulatory particle, PA28α/β, PA28γ, and PA200 (2Stadtmueller B.M. Hill C.P. Proteasome activators.Mol. Cell. 2011; 41: 8-19Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). The typical 26S proteasome contains the 19S regulatory particle and the 20S CP with constitutive catalytic subunits (including β1, β2, and β5) and promotes degradation of the ubiquitinated proteins. The immunoproteasome contains the 20S CP with the variants of catalytic subunits (including β1i, β2i, and β5i) (3Jiang T.X. Zhao M. Qiu X.B. Substrate receptors of proteasomes.Biol. Rev. Camb. Philos. Soc. 2018; 93: 1765-1777Crossref PubMed Scopus (11) Google Scholar). Certain fraction of 20S CPs in the PA200-containing proteasomes in testes harbors the catalytic subunits of the immunoproteasome, rather than regular catalytic subunits (4Qian M.X. Pang Y. Liu C.H. Haratake K. Du B.Y. Ji D.Y. Wang G.F. Zhu Q.Q. Song W. Yu Y. Zhang X.X. Huang H.T. Miao S. Chen L.B. Zhang Z.H. et al.Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis.Cell. 2013; 153: 1012-1024Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). The PA200-containing proteasomes promote the acetylation-dependent degradation of the core histones during somatic DNA repair and spermiogenesis (4Qian M.X. Pang Y. Liu C.H. Haratake K. Du B.Y. Ji D.Y. Wang G.F. Zhu Q.Q. Song W. Yu Y. Zhang X.X. Huang H.T. Miao S. Chen L.B. Zhang Z.H. et al.Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis.Cell. 2013; 153: 1012-1024Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 5Mandemaker I.K. Geijer M.E. Kik I. Bezstarosti K. Rijkers E. Raams A. Janssens R.C. Lans H. Hoeijmakers J.H. Demmers J.A. Vermeulen W. Marteijn J.A. DNA damage-induced replication stress results in PA200-proteasome-mediated degradation of acetylated histones.EMBO Rep. 2018; 19e45566Crossref PubMed Scopus (22) Google Scholar). In testes, proteasomes are largely specialized into spermatoproteasomes, which contain the testis-specific 20S subunit α4s/PSMA8 and/or the catalytic subunits of the immunoproteasome in addition to PA200 (4Qian M.X. Pang Y. Liu C.H. Haratake K. Du B.Y. Ji D.Y. Wang G.F. Zhu Q.Q. Song W. Yu Y. Zhang X.X. Huang H.T. Miao S. Chen L.B. Zhang Z.H. et al.Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis.Cell. 2013; 153: 1012-1024Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). α4s is specifically expressed in pachytene spermatocytes and the cells derived from them, including spermatids and spermatozoa (6Uechi H. Hamazaki J. Murata S. Characterization of the testis-specific proteasome subunit alpha4s in mammals.J. Biol. Chem. 2014; 289: 12365-12374Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Meiosis includes 2 cell divisions to produce haploid progeny. Spermatogenesis is a complex process in which primary spermatocytes progress through leptotene, zygotene, pachytene, and diplotene stages at prophase I of meiosis. After completion of meiosis I, secondary spermatocytes rapidly go through meiosis II to form haploid spermatids, which undergo spermiogenesis to differentiate into spermatozoa (7Yin Y. Lin C. Kim S.T. Roig I. Chen H. Liu L. Veith G.M. Jin R.U. Keeney S. Jasin M. Moley K. Zhou P. Ma L. The E3 ubiquitin ligase cullin 4A regulates meiotic progression in mouse spermatogenesis.Dev. Biol. 2011; 356: 51-62Crossref PubMed Scopus (57) Google Scholar, 8Bao J. Bedford M.T. Epigenetic regulation of the histone-to-protamine transition during spermiogenesis.Reproduction. 2016; 151: R55-R70Crossref PubMed Scopus (93) Google Scholar, 9Barral S. Morozumi Y. Tanaka H. Montellier E. Govin J. de Dieuleveult M. Charbonnier G. Coute Y. Puthier D. Buchou T. Boussouar F. Urahama T. Fenaille F. Curtet S. Hery P. et al.Histone variant H2A.L.2 guides transition protein-dependent protamine assembly in male germ cells.Mol. Cell. 2017; 66: 89-101.e108Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). During meiosis I, homologous chromosomes undergo genetic recombination by which DNA double-strand breaks (DSBs) are generated and then repaired, allowing them to exchange some of the genetic information. The subsequent repair of DNA DSBs is also critical to successful meiosis (10Phadnis N. Hyppa R.W. Smith G.R. New and old ways to control meiotic recombination.Trends Genet. 2011; 27: 411-421Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 11Lange J. Yamada S. Tischfield S.E. Pan J. Kim S. Zhu X. Socci N.D. Jasin M. Keeney S. The landscape of mouse meiotic double-strand break formation, processing, and repair.Cell. 2016; 167: 695-708.e616Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The X and Y chromosomes share homology only in a small segment, the pseudoautosomal region. This asynapsis leads to the prolonged DNA damage response. Thus, male sex chromosomes are associated with many DNA damage response proteins, including γH2AX (a phosphorylated form of the histone variant H2AX), at the XY body (12Hunter N. Meiotic recombination: the essence of heredity.Cold Spring Harb. Perspect. Biol. 2015; 7a016618PubMed Google Scholar). The pseudoautosomal region forms DSBs at a higher frequency than typical autosome segments (11Lange J. Yamada S. Tischfield S.E. Pan J. Kim S. Zhu X. Socci N.D. Jasin M. Keeney S. The landscape of mouse meiotic double-strand break formation, processing, and repair.Cell. 2016; 167: 695-708.e616Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 13Kauppi L. Barchi M. Baudat F. Romanienko P.J. Keeney S. Jasin M. Distinct properties of the XY pseudoautosomal region crucial for male meiosis.Science. 2011; 331: 916-920Crossref PubMed Scopus (156) Google Scholar). SPO11 makes DSBs through a topoisomerase-like reaction (12Hunter N. Meiotic recombination: the essence of heredity.Cold Spring Harb. Perspect. Biol. 2015; 7a016618PubMed Google Scholar). We demonstrate here that α4s/PSMA8 is required for the removal of the core histones at DNA damage loci, the proper progression of meiosis, and fertility in males by promoting formation of the properly assembled spermatoproteasome, which harbors both PA200 and regular constitutive catalytic subunits. During the preparation of this article, two independent works on the role of α4s/PSMA8 in male meiosis have been published (14Go'mez-H L. Felipe-Medina N. Condezo Y.B. Garcia-Valiente R. The PSMA8 subunit of the spermatoproteasome is essential for proper meiotic exit and mouse fertility.PLoS Genet. 2019; 15e1008316Crossref PubMed Scopus (9) Google Scholar, 15Zhang Q. Ji S.Y. Busayavalasa K. Shao J. Meiosis I progression in spermatogenesis requires a type of testis-specific 20S core proteasome.Nat. Commun. 2019; 10: 3387Crossref PubMed Scopus (13) Google Scholar). Although they also demonstrated that the deletion of α4s/PSMA8 leads to male infertility in mice, the underlying mechanisms we each provide are complementary. Given that proteasomes are the known drug targets (1Richardson P.G. Xie W. Mitsiades C. Chanan-Khan A.A. Lonial S. Hassoun H. Avigan D.E. Oaklander A.L. Kuter D.J. Wen P.Y. Kesari S. Briemberg H.R. Schlossman R.L. Munshi N.C. Heffner L.T. et al.Single-agent bortezomib in previously untreated multiple myeloma: efficacy, characterization of peripheral neuropathy, and molecular correlations with response and neuropathy.J. Clin. Oncol. 2009; 27: 3518-3525Crossref PubMed Scopus (215) Google Scholar), our results might provide potential targets for male contraception or treatment of male infertility. To investigate the role of α4s in spermatogenesis, we generated the mutant mice with global deletion of α4s gene (Fig. 1A and Fig. S1A). Homozygous deletion of α4s led to the reduced size and weight of testes in adult mice and caused male infertility but had no obvious adverse effects on female fertility or other male organs/tissues (Fig. 1, B–C and Fig. S1, B–C). There were few, if any, spermatids or spermatozoa in the seminiferous tubule and the epididymis from the α4s-deficient mice (Fig. 1D). Actually, a small fraction of haploid population (i.e., 1C) of cells was observed in the α4s-deficient testes (Fig. S1D), suggesting that a relatively small number of spermatids survived after meiosis. Synaptonemal complex proteins (SYCP) 1, 2, and 3 are meiosis-specific scaffolds in spermatocytes (16Meuwissen R.L. Offenberg H.H. Dietrich A.J. Riesewijk A. van Iersel M. Heyting C. A coiled-coil related protein specific for synapsed regions of meiotic prophase chromosomes.EMBO J. 1992; 11: 5091-5100Crossref PubMed Scopus (329) Google Scholar, 17Dobson M.J. Pearlman R.E. Karaiskakis A. Spyropoulos B. Moens P.B. Synaptonemal complex proteins: occurrence, epitope mapping and chromosome disjunction.J. Cell Sci. 1994; 107: 2749-2760Crossref PubMed Google Scholar). Immunostaining of SYCP3 indicated that meiosis, which is not yet completed at postnatal day (pnd) 20, was halted at the stage of spermatocytes in the α4s-deficient testes (Fig. 1, E–F). As specifically marked by SOX9 (18Gao F. Maiti S. Alam N. Zhang Z. Deng J.M. Behringer R.R. Lecureuil C. Guillou F. Huff V. The Wilms tumor gene, Wt1, is required for Sox9 expression and maintenance of tubular architecture in the developing testis.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11987-11992Crossref PubMed Scopus (161) Google Scholar), the formation of Sertoli cells (nurse cells) in testes was not affected by α4s deletion (Fig. S1E). The typical 26S proteasome contains three constitutive catalytic subunits (i.e., β1, β2, and β5) in addition to the 19S regulatory particle, whereas the immunoproteasome contains different catalytic subunits (i.e., β1i, β2i, and β5i) (2Stadtmueller B.M. Hill C.P. Proteasome activators.Mol. Cell. 2011; 41: 8-19Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Deletion of α4s markedly decreased the protein levels of PA200 and all three constitutive catalytic subunits (i.e., β1, β2, and β5) but increased the protein levels of the catalytic subunits of the immunoproteasome (e.g., β1i and β5i) and the proteasome activators PA28α and PA28β in adult testes (Fig. 2, A–B). The protein levels of PA28γ and the other subunits from the typical 26S proteasome (e.g., Rpt2, α4, and β7) had little, if any, change in the α4s-deficient testes (Fig. 2, A–B). To understand the mechanisms for these effects of α4s deficiency on the levels of various proteasome subunits, we demonstrated that the mRNA levels had similar changes to their protein levels (Fig. 2C), suggesting that α4s deficiency causes these changes in various proteasome subunits at least partially by altering their transcription or mRNA stability. Considering the difference in cell types, we purified 4C spermatocytes, which contain four times monoploid number of chromosomes (i.e., 4C spermatocytes) at stages after DNA replication and before nuclear separation in the process of meiosis I, and haploid spermatids. Isolation of these types of cells was successful as indicated by the presence of protamine and SYCP3 (Fig. 2D and Fig. S1D), which mark spermatids and spermatocytes, respectively (16Meuwissen R.L. Offenberg H.H. Dietrich A.J. Riesewijk A. van Iersel M. Heyting C. A coiled-coil related protein specific for synapsed regions of meiotic prophase chromosomes.EMBO J. 1992; 11: 5091-5100Crossref PubMed Scopus (329) Google Scholar, 19Balhorn R. The protamine family of sperm nuclear proteins.Genome Biol. 2007; 8: 227Crossref PubMed Scopus (425) Google Scholar). α4s deficiency had similar effects on the levels of the above-mentioned proteasome subunits in 4C spermatocytes in comparison with those in testicle homogenates (Fig. 2, A and D). Native PAGE and glycerol gradient analyses demonstrated that changes in the levels of these subunits were similar to those in the proteasomal complexes at or after pnd 28 (Fig. 3, A–B and Fig. S2A). Although a dramatic decrease in the levels of PA200 was not observed until pnd 28, replacement of constitutive catalytic subunits with immunoproteasome counterparts was obvious at pnd 23 in the α4s-deficient testes, as evidenced by the reciprocal changes in the levels of β5 and β5i (Fig. S2A). Using peptide substrates for proteasomes (i.e., LLVY-amc, LLE-amc, and LRR-amc), we showed that deletion of α4s decreased all three peptidase activities of proteasomes in testes after pnd 23 but had no effect on these activities in young testes from mice at pnd 18, adult liver with the typical 26S proteasome, and adult spleen with the immunoproteasome (Fig. 3C and Fig. S2, A–B). Thus, α4s is required for the formation of the properly assembled spermatoproteasome, which contains both PA200 and regular catalytic subunits, in adult testes.Figure 3Deletion of α4s reduces the amount of PA200 and regular catalytic subunits in proteasomes of mature testes. A, immunoblotting and peptidase activity analyses were performed following native PAGE of the extracts from the testes of the wildtype and the α4s-deficient mice. Proteasomal peptidase activity was analyzed by incubating the gel with LLVY-amc in the absence or presence of 0.02% SDS, which activates the 20S proteasome. B, immunoblotting of the fractions of glycerol gradient ultracentrifugation of the extracts from the testes of the wildtype and the α4s-deficient mice. Proteasomal peptidase activities were assayed using LLVY-amc as a substrate. C, the proteasomal peptidase activities of three tissue extracts of the wildtype and the α4s-deficient mice. All mice were 84 days old (mean ± SEM, n = 6). Data are representative of one experiment with at least two independent biological replicates. ∗∗p < 0.01 (two-tailed unpaired t test).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The TUNEL assay can usually detect apoptotic cells by attaching the fluorescently labeled nucleotides to the exposed 3' ends of DSBs. Under low-resolution microscopy (20×) in the tissue sections, the TUNEL assay could show the condensed chromatin or apoptotic bodies, hallmarks for apoptotic cells. Deletion of α4s sharply increased the number of the apoptotic bodies-positive spermatocytes in the sections of testes (Fig. 4, A–B). Fluorescent annexin V conjugates provide reliable detection of the externalized phosphatidylserine, another indicator of apoptosis (20Tong C. Shi B. Xiao X. Liao H. Zheng Y. Shen G. Tang D. Liu X. An annexin V-based biosensor for quantitatively detecting early apoptotic cells.Biosens. Bioelectron. 2009; 24: 1777-1782Crossref PubMed Scopus (20) Google Scholar). Deletion of α4s also dramatically increased the number of the annexin V-positive spermatocytes in the sections of testes (Fig. S3, A–B). When normalized to that in the wildtype testes, the rate for the increased number of apoptotic spermatocytes was much higher than that for γH2AX- or SYCP3-positive cells during mouse development (Fig. 4, C–F). SYCP1 and SYCP3 are present in autosomes only and all chromosomes, respectively. Deletion of α4s had not affected synapsis formation of either autosomes or sex chromosomes, which were differentially marked by SYCP1 and SYCP3 (Fig. S3C). In early pachytene spermatocytes, γH2AX is present only as small foci. In mid-pachytene to late diplotene spermatocytes, γH2AX is restricted solely to the XY body (21Lyndaker A.M. Lim P.X. Mleczko J.M. Diggins C.E. Holloway J.K. Holmes R.J. Kan R. Schlafer D.H. Freire R. Cohen P.E. Weiss R.S. Conditional inactivation of the DNA damage response gene Hus1 in mouse testis reveals separable roles for components of the RAD9-RAD1-HUS1 complex in meiotic chromosome maintenance.PLoS Genet. 2013; 9e1003320Crossref PubMed Scopus (34) Google Scholar). Although deletion of α4s increased the number of the γH2AX-positive cells in testes (Fig. 4G and Fig. S4A), it did not affect the chromosomal distribution of γH2AX at various stages of prophase I of meiosis in spermatocyte nuclei (Fig. S4B). Transcription in the XY body is repressed, leading to meiotic sex chromosome inactivation (MSCI) in the asynapsed sex chromosome regions, and sex chromosomes are depleted from active histone marks, especially acetylation (22Goudarzi A. Zhang D. Huang H. Barral S. Kwon O.K. Qi S. Tang Z. Buchou T. Vitte A.L. He T. Cheng Z. Montellier E. Gaucher J. Curtet S. Debernardi A. et al.Dynamic competing histone H4 K5K8 acetylation and butyrylation are hallmarks of highly active gene promoters.Mol. Cell. 2016; 62: 169-180Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). DSBs are required for this meiotic silencing. For example, mice with a mutation in Spo11 are defective in MSCI (23Bellani M.A. Romanienko P.J. Cairatti D.A. Camerini-Otero R.D. SPO11 is required for sex-body formation, and Spo11 heterozygosity rescues the prophase arrest of Atm-/- spermatocytes.J. Cell Sci. 2005; 118: 3233-3245Crossref PubMed Scopus (144) Google Scholar, 24Barchi M. Mahadevaiah S. Di Giacomo M. Baudat F. de Rooij D.G. Burgoyne P.S. Jasin M. Keeney S. Surveillance of different recombination defects in mouse spermatocytes yields distinct responses despite elimination at an identical developmental stage.Mol. Cell. Biol. 2005; 25: 7203-7215Crossref PubMed Scopus (151) Google Scholar). Accordingly, H2AX-null male mice display meiotic arrest with MSCI failure (25Fernandez-Capetillo O. Mahadevaiah S.K. Celeste A. Romanienko P.J. Camerini-Otero R.D. Bonner W.M. Manova K. Burgoyne P. Nussenzweig A. H2AX is required for chromatin remodeling and inactivation of sex chromosomes in male mouse meiosis.Dev. Cell. 2003; 4: 497-508Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). The X-linked gene, Hprt1, and the Y-linked gene, Rbmy1a1, are usually silenced during the MSCI in the wildtype testes (26Date S. Nozawa O. Inoue H. Hidema S. Nishimori K. Impairment of pachytene spermatogenesis in Dmrt7 deficient mice, possibly causing meiotic arrest.Biosci. Biotechnol. Biochem. 2012; 76: 1621-1626Crossref PubMed Scopus (9) Google Scholar) but were not silenced in the α4s-deficient testes. Their backup genes (Cetn1, Pdha2) located on autosomes are usually activated during MSCI but were silenced in the α4s-deficient testes (Fig. S4C). Thus, deletion of α4s disrupts MSCI, hinting an essential role of α4s in meiotic DNA repair. Natural generation and subsequent repair of DNA DSBs are critical to mammalian meiosis and genetic diversity (10Phadnis N. Hyppa R.W. Smith G.R. New and old ways to control meiotic recombination.Trends Genet. 2011; 27: 411-421Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 11Lange J. Yamada S. Tischfield S.E. Pan J. Kim S. Zhu X. Socci N.D. Jasin M. Keeney S. The landscape of mouse meiotic double-strand break formation, processing, and repair.Cell. 2016; 167: 695-708.e616Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). MLH1, a DNA mismatch repair protein, plays an important role in the formation of meiotic crossover in mid-pachynema (27Baker S.M. Plug A.W. Prolla T.A. Bronner C.E. Harris A.C. Yao X. Christie D.M. Monell C. Arnheim N. Bradley A. Ashley T. Liskay R.M. Involvement of mouse Mlh1 in DNA mismatch repair and meiotic crossing over.Nat. Genet. 1996; 13: 336-342Crossref PubMed Scopus (654) Google Scholar). Deletion of α4s had no influence in the recruitment of MLH1, because the number of MLH1 foci did not change in the nuclei of the α4s-deficient spermatocytes (Fig. 5A and Fig. S5). Rad51 is required for heteroduplex formation in meiotic DNA repair and forms foci along the axial element from leptonema on. These foci are located along synaptonemal complexes in zygonema, and gradually disappear in pachynema (28Romanienko P.J. Camerini-Otero R.D. The mouse Spo11 gene is required for meiotic chromosome synapsis.Mol. Cell. 2000; 6: 975-987Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar, 29de Vries F.A. de Boer E. van den Bosch M. Baarends W.M. Ooms M. Yuan L. Liu J.G. van Zeeland A.A. Heyting C. Pastink A. Mouse Sycp1 functions in synaptonemal complex assembly, meiotic recombination, and XY body formation.Genes Dev. 2005; 19: 1376-1389Crossref PubMed Scopus (291) Google Scholar). In the α4s-deficient spermatocytes, the pattern for RAD51 foci was similar to that in the wildtype throughout prophase I (Fig. 5, B–C and Fig. S6, A–B), suggesting that deletion of α4s did not increase the recruitment of RAD51 onto chromosomes during prophase I. To directly examine the role of α4s in meiotic DNA repair, we employed the TUNEL assay to monitor DSBs in the nuclei of nonapoptotic cells under a microscope with high resolution (100×) as reported (30Mets D.G. Meyer B.J. Condensins regulate meiotic DNA break distribution, thus crossover frequency, by controlling chromosome structure.Cell. 2009; 139: 73-86Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The numbers of TUNEL foci in the spread nuclei were similar between the wildtype and the α4s-deficient spermatocytes at prophase I (Fig. S6, C–E). In comparison with those in the wildtype testes, the levels of SYCP3 and γH2AX increased, but the levels of the acetylated histones, including H4K16ac, decreased, whereas the levels of the core histones (such as H2B, H3, and H4) did not change in the homogenates of the α4s-deficient testes (Fig. S6F). The decreased levels of acetylated histones in the α4s-deficient testes were apparently due to the disappearance of round spermatids, where acetylation of the core histones is known to be associated with histone displacement or degradation during the elongation of spermatids (4Qian M.X. Pang Y. Liu C.H. Haratake K. Du B.Y. Ji D.Y. Wang G.F. Zhu Q.Q. Song W. Yu Y. Zhang X.X. Huang H.T. Miao S. Chen L.B. Zhang Z.H. et al.Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis.Cell. 2013; 153: 1012-1024Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 31Meistrich M.L. Trostle-Weige P.K. Lin R. Bhatnagar Y.M. Allis C.D. Highly acetylated H4 is associated with histone displacement in rat spermatids.Mol. Reprod. Dev. 1992; 31: 170-181Crossref PubMed Scopus (136) Google Scholar). To exclude the influence of the cell types, we purified 4C spermatocytes and haploid spermatids. Indeed, deletion of α4s increased the levels of H4K16ac in the lysates of 4C spermatocytes in addition to the levels of SYCP3 and γH2AX (Fig. 5D, Figs. S6F and S7A). If DNA repair is normal, the levels of DNA repair proteins would increase, since the ratio of spermatocyte increased in the α4s-deficient testes (Fig. 1). However, the levels of MLH1, RAD51, and RPA1 all remained constant in both the homogenates of α4s-deficient testes and the lysates of 4C spermatocytes (Fig. 5D, Figs. S6F and S7A), hinting that deletion of α4s might block the repair of DNA breaks, resulting in eventual failure of meiosis and the ensuing apoptosis. As revealed by staining of α-tubulin, deletion of α4s sharply reduced the numbers of spermatocytes at metaphase I and the ensuing phases, such as anaphase I and telophase I (Fig. 5E). γH2AX can mark chromatin domains with DNA breaks (32Blanco-Rodriguez J. gammaH2AX marks the main events of the spermatogenic process.Microsc. Res. Tech. 2009; 72: 823-832Crossref PubMed Scopus (41) Google Scholar). Although there was no detectable colocalization between γH2AX and DNA at metaphase I in the wildtype testes, γH2AX colocalized with DNA in almost all spermatocytes at metaphase I in the α4s-deficient testes (Fig. 5F). In accord, deletion of α4s also caused the staining of DNA breaks by TUNEL assay at metaphase I of spermatocytes (Fig. 5G). These results suggest that deletion of α4s suppresses the repair of DSBs at metaphase I in spermatocytes. Chromatins with meiotic DSB sites are sensitive to DNases in both yeast and mice, hinting that histones might be removed during meiotic recombination repair (33Wu T.C. Lichten M. Meiosis-induced double-strand break sites determined by yeast chromatin structure.Science. 1994; 263: 515-518Crossref PubMed Scopus (347) Google Scholar, 34Mizuno K. Koide T. Sagai T. Moriwaki K. Shiroishi T. Molecular analysis of a recombinational hotspot adjacent to Lmp2 gene in the mouse MHC: fine location and chromatin structure.Mamm. Genome. 1996; 7: 490-496Crossref PubMed Scopus (23) Google Scholar). But, unlike that in the wildtype testes, H4K16ac partially colocalized with DSBs as marked by TUNEL staining and γH2AX in the α4s-deficient testes (Fig. 6, A–B), hinting that degradation of the acetylated histones was insufficient at DSB loci in the α4s-deficient testes. Recently, we have demonstrated that the PA200-containing proteasomes degrade the acetylated core histones during somatic DNA repair and spermiogenesis (4Qian M.X. Pang Y. Liu C.H. Haratake K. Du B.Y. Ji D.Y. Wang G.F. Zhu Q.Q. Song W. Yu Y. Zhang X.X. Huang H.T. Miao S. Chen L.B. Zhang Z.H. et al.Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis.Cell. 2013; 153: 1012-1024Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). To test the role of α4s in histone degradation, we incubated the acetylated core histones with the 20S catalytic particles from testes, which contain α4s, in the absence or presence of PA200. The 20S particle from muscle, which does not contain any α4s, served as a control. α4s stimulated the in vitro degradation of the acetylated core histones, instead of nonacetylated histones (e.g., H2B), by the PA200-proteasome (Fig. 6C). Finally, deletion of α4s suppressed the degradation of the acetylated core histones in testis lysates from mature mice (Fig. S7B). Taken together, our results suggest that α4s mediates degradation of the core histones during meiotic DNA repair, and is required for proper progression of meiosis and fertility in male mice. The repair of DNA DSBs is critical to the completion of meiosis (10Phadnis N. Hyppa R.W. Smith G.R. New and old ways to control meiotic recombination.Trends Genet. 2011; 27: 411-421Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 11Lange J
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