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The Zinc-Finger Protein Basonuclin 2 Is Required for Proper Mitotic Arrest, Prevention of Premature Meiotic Initiation and Meiot

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The Zinc-Finger Protein Basonuclin 2 Is Required for Proper Mitotic Arrest, Prevention of Premature Meiotic Initiation and Meiot © 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 4298-4310 doi:10.1242/dev.112888 RESEARCH ARTICLE The zinc-finger protein basonuclin 2 is required for proper mitotic arrest, prevention of premature meiotic initiation and meiotic progression in mouse male germ cells Amandine Vanhoutteghem1,Sébastien Messiaen2, Françoise Hervé1, Brigitte Delhomme1, Delphine Moison2, Jean-Maurice Petit3, Virginie Rouiller-Fabre2, Gabriel Livera2 and Philippe Djian1,* ABSTRACT germ cells enter meiosis shortly after they reach the fetal gonad, Absence of mitosis and meiosis are distinguishing properties of whereas male germ cells become quiescent in the fetal gonad and male germ cells during late fetal and early neonatal periods. initiate meiosis after birth, when spermatogonia start forming Repressors of male germ cell meiosis have been identified, but spermatocytes. Transplantation studies have shown that bipotential mitotic repressors are largely unknown, and no protein repressing germ cells are intrinsically programmed to differentiate following both meiosis and mitosis is known. We demonstrate here that the the female pathway and to undergo early meiosis (McLaren and zinc-finger protein BNC2 is present in male but not in female germ Southee, 1997). Prevention of meiosis in fetal testis is achieved cells. In testis, BNC2 exists as several spliced isoforms and through synthesis in male Sertoli cells of CYP26B1, an enzyme that presumably binds to DNA. Within the male germ cell lineage, degrades the retinoic acid necessary for meiosis induction (Bowles BNC2 is restricted to prospermatogonia and undifferentiated et al., 2006; Koubova et al., 2006). When the level of testicular spermatogonia. Fetal prospermatogonia that lack BNC2 multiply CYP26B1 decreases, prevention of meiosis is taken over by excessively on embryonic day (E)14.5 and reenter the cell cycle NANOS2, a protein that is synthesized by fetal male germ cells in prematurely. Mutant prospermatogonia also engage in abnormal the absence of retinoic acid signaling (Suzuki and Saga, 2008). meiosis; on E17.5, Bnc2−/− prospermatogonia start synthesizing During spermatogenesis, spermatogonia undergo meiosis, in the synaptonemal protein SYCP3, and by the time of birth, the first division of which homologous chromosomes pair, many Bnc2−/− prospermatogonia have accumulated large recombine and synapse. Disturbance of any of these processes amounts of nonfilamentous SYCP3, thus appearing to be blocked leads to massive apoptosis-mediated death of the spermatocytes at at leptonema.Bnc2−/− prospermatogonia do not undergo proper a specific check point in stage IV of the seminiferous epithelial male differentiation, as they lack almost all the mRNA for the male- cycle (Hamer et al., 2008). specific methylation protein DNMT3L and have increased levels of Basonuclin 2 (BNC2) is an extremely conserved zinc-finger mRNAs that encode meiotic proteins, including STRA8. Bnc2−/− protein (Romano et al., 2004; Vanhoutteghem and Djian, 2004). It is prospermatogonia can produce spermatogonia, but these enter orthologous to BNC1, a protein that is essential for germ cells meiosis prematurely and undergo massive apoptotic death during (Mahoney et al., 1998; Tseng and Green, 1992; Yang et al., 1997; meiotic prophase. This study identifies BNC2 as a major regulator of Zhang et al., 2012). Mice with a homozygous disruption of Bnc2 die male germ stem cells, which is required for repression of meiosis shortly after birth from a cleft palate (Hervé et al., 2012; and mitosis in prospermatogonia, and for meiosis progression Vanhoutteghem et al., 2011, 2009). during spermatogenesis. In view of the extreme evolutionary Because of its abundance in testis (Vanhoutteghem and Djian, conservation of BNC2, the findings described here are likely to 2004), we thought that BNC2 might have a function in male apply to many species. reproduction in addition to its function in palatal mesenchymal cells. Direct evidence for a function of BNC2 as a DNA-binding KEY WORDS: BNC2, Gonocytes, Mouse, Spermatogonia, protein that regulates meiosis and mitosis in male germ cells is Transcription described below. INTRODUCTION RESULTS In mammals, primordial germ cells remain sexually bipotential In mouse testis, BNC2 is presumably a DNA-binding protein during their migration across the embryo; they become sexually and exists as several splicing isoforms differentiated upon reaching the fetal gonad. Sexual differentiation In this study, we have used the previously reported Ayu21:18 mouse is correlated with the timing of entry into meiosis. In mice, female line, the Bnc2 gene of which is disrupted by a gene-trap insertion. The disrupted allele is likely to be null because all major Bnc2 mRNA isoforms are absent from the homozygous mutant 1Laboratoire de physiologie cérébrale, Centre National de la Recherche (Vanhoutteghem et al., 2009). 2 Scientifique, UniversitéParis Descartes, UMR 8118, Paris, France. Laboratoire de We have previously described, in human keratinocytes, an développement des gonades, UniversitéParis Diderot, Sorbonne Paris Cité, INSERM U967, CEA/DSV/iRCM/SCSR, Fontenay-aux-Roses F-92265, France. insoluble form of human BNC2, which colocalizes with the splicing 3Service central de microscopie, Centre Universitaire des Saints-Peres,̀ Université factor SC35 in nuclear speckles and has a presumed function in Paris Descartes, Paris, France. nuclear RNA processing. The apparent molecular mass of insoluble *Author for correspondence ([email protected]) BNC2 is 145 kDa (Vanhoutteghem and Djian, 2006). We also found that, in human keratinocytes, there exists a second BNC2 Received 16 May 2014; Accepted 11 September 2014 isoform with a molecular mass of 160 kDa and a homogenous DEVELOPMENT 4298 RESEARCH ARTICLE Development (2014) 141, 4298-4310 doi:10.1242/dev.112888 distribution throughout the nucleus. The two human isoforms result Because alternative promoters and alternative splicing are from alternative splicing of the transcript. prominent features of the human BNC2 gene, we searched mouse As shown in Fig. 1A, when the testes of newborn mice were testis for alternative Bnc2 promoters and alternatively spliced Bnc2 analyzed by western blotting with an antibody against amino acid transcripts. For promoter mapping, the 5′ end of the mRNA was residues 722-849 of BNC2 (herein referred to as BNC2722-849), amplified by 5′ rapid amplification of cDNA ends (RACE), resolved three protein bands in the range of 120-160 kDa were detected in by using agarose gel electrophoresis and then sequenced. A single Bnc2+/+ and Bnc2+/− testis, but not in Bnc2−/− testis. These proteins transcription start site in exon 1 was found (supplementary material were present in the nuclear extract and absent from the cytoplasm. Fig. S2A,B). Alternative exons were identified by using RT-PCR The major protein ran at ∼160 kDa, and we thought it probable that with primers in either exons 1 and 3, or 4 and 6. The unfractionated it was the mouse equivalent of human soluble BNC2. The identity products were cloned and sequenced. Because a large number of of the 160-kDa protein was confirmed by its staining with another clones were sequenced, we could obtain an estimate of the relative antibody against BNC2 (supplementary material Fig. S1A). abundance of each Bnc2 mRNA isoform. We found a total of 11 Testicular BNC2 was found in the soluble nuclear extract and in exons, of which five were constitutive (exons 1, 3, 4, 5 and 6) and six association with chromatin. No BNC2 was detected in either the were alternative (1a, 2, 2a, 2c, 5cS,5cL and 5b) (supplementary insoluble nuclear fraction or the cytoplasm (Fig. 1B). We conclude material Fig. S2C). The 5′ alternative exons (1a, 2, 2a and 2c) were that in mouse testis, BNC2 is a largely soluble nuclear protein with all in frame with the rest of the coding region and caused variations an apparent molecular mass of ∼160 kDa. in the N-terminal part of the protein, whereas the 3′ alternative exons 722-849 Both of the antibodies against BNC2 and amino acid (5cS,5cL and 5b) resulted in either premature termination and residues 661-782 of BNC2 produced homogenous nuclear staining suppression of the last three zinc-fingers or disruption of finger 4 in newborn testis (supplementary material Fig. S1B-E). Such a with conservation of fingers 5 and 6. As a result, testis contained a diffuse nuclear distribution is in keeping with the presence of family of BNC2 proteins with a constant central sequence that BNC2 in the soluble nuclear extract and its association with included the three N-terminal zinc-fingers and the nuclear chromatin. This suggests that BNC2 is a DNA-binding protein localization signal, but variable N-terminal regions and variable in testis. numbers of C-terminal zinc-fingers (Fig. 1C). Fig. 1. BNC2 exists as multiple isoforms, the major of which is soluble and associated with chromatin. (A,B) Western blot analysis of newborn testis. − (A) BNC2 runs as a 160-kDa protein in nuclear extracts (NE) of Bnc2+/+ and Bnc2+/ testes (arrows). The protein is neither found in cytoplasm (Cyt) nor in the mutant. Two weaker bands with an electrophoretic mobility of ∼140 and ∼120 kDa might correspond to splicing or degradation products of BNC2. Proteins with an − − electrophoretic mobility of 100 kDa or less are stained nonspecifically, as they are found in Bnc2 / testis. (B) Subnuclear fractionation. BNC2 (arrows) is found in nuclear (NE) and chromatin (Chr) extracts; it is absent from the insoluble nuclear fraction (Nins) and cytoplasm (Cyt). (C) Map of the Bnc2 gene and the splicing isoforms detected by using RT-PCR. Exons are represented by green boxes and designated by Arabic numerals. Intron sizes (kb) are shown in between the green boxes. The arrowhead indicates the most abundant RNA isoform. BNC2 protein isoforms resulting from alternative splicing are shown in the schematic to the right. Black and red vertical bars represent zinc-fingers and nuclear localization signal, respectively. Protein isoforms vary by their N-terminal regions and/or number of C-terminal zinc-fingers. (D) Agarose gel electrophoresis of RT-PCR products.
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