José Luis Barbero

Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice

We targeted the locus encoding the cyclin-dependent kinase 2 (CDK2) by homologous recombination in mouse embryonic stem (ES) cells. Embryonic fibroblasts lacking CDK2 proliferate normally and become immortal after continuous passage in culture. Elimination of a conditional Cdk2 allele in immortal cells does not have a significant effect on proliferation. Cdk2−/− mice are viable and survive for up to two years, indicating that CDK2 is also dispensable for proliferation and survival of most cell types. But CDK2 is essential for completion of prophase I during meiotic cell division in male and female germ cells, an unforeseen role for this cell cycle kinase.

Mammalian STAG3 is a cohesin specific to sister chromatid arms in meiosis I

Cohesins, which have been characterized in budding yeast and Xenopus, are multisubunit protein complexes involved in sister chromatid cohesion. Regulation of the interactions among different cohesin subunits and the assembly/disassembly of the cohesin complex to chromatin are key steps in chromosome segregation. We previously characterized the mammalian STAG3 protein as a component of the synaptonemal complex that is specifically expressed in germinal cells, although its function in meiosis remains unknown. Here we show that STAG3 has a role in sister chromatid arm cohesion during mammalian meiosis I. Immunofluorescence results in prophase I cells suggest that STAG3 is a component of the axial/lateral element of the synaptonemal complex. In metaphase I, STAG3 is located at the interchromatid domain and is absent from the chiasma region. In late anaphase I and the later stages of meiosis, STAG3 is not detected. STAG3 interacts with the structural maintenance chromosome proteins SMC1 and SMC3, which have been reported to be subunits of the mitotic cohesin complex. We propose that STAG3 is a sister chromatid arm cohesin that is specific to mammalian meiosis I.

STAG3, a novel gene encoding a protein involved in meiotic chromosome pairing and location of STAG3-related genes flanking the Williams-Beuren syndrome deletion

Chromatin rearrangements in the meiotic prophase are characterized by the assembly and disassembly of synaptonemal complexes (SC), a protein structure that stabilizes the pairing of homologous chromosomes in prophase. We report the identification of human and mouse cDNA coding for stromalin 3 (STAG3), a new mammalian stromalin member of the synaptonemal complex. The stroma‐lins are a group of highly conserved proteins, represented in several organisms from yeast to humans. Stromalins are characterized by the stromalin conservative domain (SCD), a specific motif found in all proteins of the family described to date. STAG3 is expressed specifically in testis, and immunolocalization experiments show that STAG3 is associated to the synaptonemal complex. As the protein encoded by the homologous gene (Scc3p) in Saccharomyces cerevisiae was found to be a subunit of a cohesin complex that binds chromosomes until the onset of anaphase, our data suggest that STAG3 is involved in chromosome pairing and maintenance of synaptone‐mal complex structure during the pachytene phase of meiosis in a cohesin‐like manner. We have mapped the human STAG3 gene to the 7q22 region of chromosome 7; six human STAG3‐related genes have also been mapped: two at 7q22 near the functional gene, one at 7qll.22, and three at 7qll.23, two of them flanking the breakpoints commonly associated with the Williams‐Beuren syndrome (WBS) deletion. Since the WBS deletion occurs as a consequence of unequal meiotic crossing over, we suggest that STAG3 duplications predispose to germline chromosomal rearrangement within this region.—Pezzi, N., Prieto, I., Kremer, L., Pérez Jurado, L. A., Valero, C., del Mazo, J., Martínez‐A., C., Barbero, J. L. STAG3, a novel gene encoding a protein involved in meiotic chromosome pairing and location of STAG3‐related genes flanking the Williams‐Beuren syndrome deletion. FASEB J. 14, 581–592 (2000)

Shugoshin-2 is essential for the completion of meiosis but not for mitotic cell division in mice

Shugoshin-2 (SGOL2) is one of the two mammalian orthologs of the Shugoshin/Mei-S322 family of proteins that regulate sister chromatid cohesion by protecting the integrity of the multiprotein cohesin complexes. This protective system is essential for faithful chromosome segregation during mitosis and meiosis, which is the physical basis of Mendelian inheritance. Regardless of its evolutionary conservation from yeast to mammals, little is known about the in vivo relevance and specific role that SGOL2 plays in mammals. Here we show that disruption of the gene encoding mouse SGOL2 does not cause any alteration in sister chromatid cohesion in embryonic cultured fibroblasts and adult somatic tissues. Moreover, mutant mice develop normally and survive to adulthood without any apparent alteration. However, both male and female Sgol2-deficient mice are infertile. We demonstrate that SGOL2 is necessary for protecting centromeric cohesion during mammalian meiosis I. In vivo, the loss of SGOL2 promotes a premature release of the meiosis-specific REC8 cohesin complexes from anaphase I centromeres. This molecular alteration is manifested cytologically by the complete loss of centromere cohesion at metaphase II leading to single chromatids and physiologically with the formation of aneuploid gametes that give rise to infertility.

Cohesin component dynamics during meiotic prophase I in mammalian oocytes.

Cohesins are chromosomal proteins that form complexes involved in the maintenance of sister chromatid cohesion during division of somatic and germ cells. Three meiosis-specific cohesin subunits have been reported in mammals, REC8, STAG3 and SMC1β; their expression in mouse spermatocytes has also been described. Here we studied the localization of different meiotic and mitotic cohesin components during prophase I in human and murine female germ cells. In normal and atretic human fetal oocytes, from leptotene to diplotene stages, REC8 and STAG3 colocalize in fibers. In murine oocytes, SMC1β, SMC3 and STAG3 are localized along fibers that correspond first to the chromosome axis and then to the synaptonemal complex in pachytene. Mitotic cohesin subunit RAD21 is also found in fibers that decorate the SC during prophase I in mouse oocytes, suggesting a role for this cohesin in mammalian sister chromatid cohesion in female meiosis. We observed that, unlike human oocytes, murine synaptonemal complex protein SYCP3 localizes to nucleoli throughout prophase I stages, and centromeres cluster in discrete locations from leptotene to dictyate. At difference from meiosis in male mice, the cohesin axis is progressively lost during the first week after birth in females with a parallel destruction of the axial elements at dictyate arrest, demonstrating sexual dimorphism in sister chromatid cohesion in meiosis.

Mutant cohesin in premature ovarian failure

Premature ovarian failure is a major cause of female infertility. The genetic causes of this disorder remain unknown in most patients. Using whole-exome sequence analysis of a large consanguineous family with inherited premature ovarian failure, we identified a homozygous 1-bp deletion inducing a frameshift mutation in STAG3 on chromosome 7. STAG3 encodes a meiosis-specific subunit of the cohesin ring, which ensures correct sister chromatid cohesion. Female mice devoid of Stag3 are sterile, and their fetal oocytes are arrested at early prophase I, leading to oocyte depletion at 1 week of age.

Depletion of Drad21/Scc1 in Drosophila Cells Leads to Instability of the Cohesin Complex and Disruption of Mitotic Progression

BACKGROUND The coordination of cell cycle events is necessary to ensure the proper duplication and dissemination of the genome. In this study, we examine the consequences of depleting Drad21 and SA, two non-SMC subunits of the cohesin complex, by dsRNA-mediated interference in Drosophila cultured cells. RESULTS We have shown that a bona fide cohesin complex exists in Drosophila embryos. Strikingly, the Drad21/Scc1 and SA/Scc3 non-SMC subunits associate more intimately with one another than they do with the SMCs. We have observed defects in mitotic progression in cells from which Drad21 has been depleted: cells delay in prometaphase with normally condensed, but prematurely separated, sister chromatids and with abnormal spindle morphology. Much milder defects are observed when SA is depleted from cells. The dynamics of the chromosome passenger protein, INCENP, are affected after Drad21 depletion. We have also made the surprising observation that SA is unstable in the absence of Drad21; however, we have shown that the converse is not true. Interference with Drad21 in living Drosophila embryos also has deleterious effects on mitotic progression. CONCLUSIONS We conclude that Drad21, as a member of a cohesin complex, is required in Drosophila cultured cells and embryos for proper mitotic progression. The protein is required in cultured cells for chromosome cohesion, spindle morphology, dynamics of a chromosome passenger protein, and stability of the cohesin complex, but apparently not for normal chromosome condensation. The observation of SA instability in the absence of Drad21 implies that the expression of cohesin subunits and assembly of the cohesin complex will be tightly regulated.

The cohesin subunit RAD21L functions in meiotic synapsis and exhibits sexual dimorphism in fertility.

The cohesin complex is a ring‐shaped proteinaceous structure that entraps the two sister chromatids after replication until the onset of anaphase when the ring is opened by proteolytic cleavage of its α‐kleisin subunit (RAD21 at mitosis and REC8 at meiosis) by separase. RAD21L is a recently identified α‐kleisin that is present from fish to mammals and biochemically interacts with the cohesin subunits SMC1, SMC3 and STAG3. RAD21L localizes along the axial elements of the synaptonemal complex of mouse meiocytes. However, its existence as a bona fide cohesin and its functional role awaits in vivo validation. Here, we show that male mice lacking RAD21L are defective in full synapsis of homologous chromosomes at meiotic prophase I, which provokes an arrest at zygotene and leads to total azoospermia and consequently infertility. In contrast, RAD21L‐deficient females are fertile but develop an age‐dependent sterility. Thus, our results provide in vivo evidence that RAD21L is essential for male fertility and in females for the maintenance of fertility during natural aging.

STAG2 and Rad21 mammalian mitotic cohesins are implicated in meiosis

STAG/SA proteins are specific cohesin complex subunits that maintain sister chromatid cohesion in mitosis and meiosis. Two members of this family, STAG1/SA1 and STAG2/SA2, ‡ are classified as mitotic cohesins, as they are found in human somatic cells and in Xenopus laevis as components of the cohesinSA1 and cohesinSA2 complexes, in which the shared subunits are Rad21/SCC1, SMC1 and SMC3 proteins. A recently reported third family member, STAG3, is germinal cell‐specific and is a subunit of the meiotic cohesin complex. To date, the meiosis‐specific cohesin complex has been considered to be responsible for sister chromatid cohesion during meiosis. We studied replacement of the mitotic by the meiotic cohesin complex during mouse germinal cell maturation, and we show that mammalian STAG2 and Rad21 are also involved in several meiosis stages. Immunofluorescence results suggest that a cohesin complex containing Rad21 and STAG2 cooperates with a STAG3‐specific complex to maintain sister chromatid cohesion during the diplotene stage of meiosis.

Drosophila grim induces apoptosis in mammalian cells

Genetic studies have shown that grim is a central genetic switch of programmed cell death in Drosophila; however, homologous genes have not been described in other species, nor has its mechanism of action been defined. We show here that grim expression induces apoptosis in mouse fibroblasts. Cell death induced by grim in mammalian cells involves membrane blebbing, cytoplasmic loss and nuclear DNA fragmentation. Grim‐induced apoptosis is blocked by both natural and synthetic caspase inhibitors. We found that grim itself shows caspase‐dependent proteolytic processing of its C‐terminus in vitro. Grim‐induced death is antagonized by bcl‐2 in a dose‐dependent manner, and neither Fas signalling nor p53 are required for grim pro‐apoptotic activity. Grim protein localizes both in the cytosol and in the mitochondria of mouse fibroblasts, the latter location becoming predominant as apoptosis progresses. These results show that Drosophila grim induces death in mammalian cells by specifically acting on mitochondrial apoptotic pathways executed by endogenous caspases. These findings advance our knowledge of the mechanism by which grim induces apoptosis and show the conservation through evolution of this crucial programmed cell death pathway.