Cohesin Removal along the Chromosome Arms during the First Meiotic Division Depends on a NEK1-PP1γ-WAPL Axis in the Mouse.

Mammalian NIMA-like kinase-1 (NEK1) is a dual-specificity kinase highly expressed in mouse germ cells during prophase I of meiosis. Loss of NEK1 induces retention of cohesin on chromosomes at meiotic prophase I. Timely deposition and removal of cohesin is essential for accurate chromosome segregation. Two processes regulate cohesin removal: a non-proteolytic mechanism involving WAPL, sororin, and PDS5B and direct cleavage by separase. Here, we demonstrate a role for NEK1 in the regulation of WAPL loading during meiotic prophase I, via an interaction between NEK1 and PDS5B. This regulation of WAPL by NEK1-PDS5B is mediated by protein phosphatase 1 gamma (PP1γ), which both interacts with and is a phosphotarget of NEK1. Taken together, our results reveal that NEK1 phosphorylates PP1γ, leading to the dephosphorylation of WAPL, which, in turn, results in its retention on chromosome cores to promote loss of cohesion at the end of prophase I in mammals.

An Unusual Role for doublesex in Sex Determination in the Dipteran Sciara.

The gene doublesex, which is placed at the bottom of the sex-determination gene cascade, plays the ultimate discriminatory role for sex determination in insects. In all insects where this gene has been characterized, the dsx premessenger RNA (pre-mRNA) follows a sex-specific splicing pattern, producing male- and female-specific mRNAs encoding the male-DSXM and female-DSXF proteins, which determine male and female development, respectively. This article reports the isolation and characterization of the gene doublesex of dipteran Sciara insects. The Sciara doublesex gene is constitutively transcribed during development and adult life of males and females. Sciara had no sex-specific doublesex mRNAs but the same transcripts, produced by alternative splicing of its primary transcript, were present in both sexes, although their relative abundance is sex specific. However, only the female DSXF protein, but not the male DSXM protein, was produced at similar amounts in both sexes. An analysis of the expression of female and male Sciara DSX proteins in Drosophila showed that these proteins conserved female and male function, respectively, on the control of Drosophila yolk-protein genes. The molecular evolution of gene doublesex of all insects where this gene has been characterized revealed that Sciara doublesex displays a considerable degree of divergence in its molecular organization and its splicing pattern with respect to the rest of dipterans as suggested by its basal position within the doublesex phylogeny. It is suggested that the doublesex gene is involved in Sciara sex determination although it appears not to play the discriminatory role performed in other insects.

Genetic basis of cohesinopathies.

Cohesin is a ring-form multifunctional protein complex, which was discovered during a search for molecules that keep sister chromatids together during segregation of chromosomes during cell division. In the past decade, a large number of results have also demonstrated a need for the cohesin complex in other crucial events in the life cycle of the cell, including DNA duplication, heterochromatin formation, DNA double-strand break repair, and control of gene expression. The dynamics of the cohesin ring are modulated by a number of accessory and regulatory proteins, known as cohesin cofactors. Loss of function of the cohesin complex is incompatible with life; however, mutations in the genes encoding for cohesin subunits and/or cohesin cofactors, which have very little or a null effect on chromosome segregation, represent a newly recognized class of human genetic disorders known as cohesinopathies. A number of genetic, biochemical, and clinical approaches, and importantly, animal models, can help us to determine the underlying mechanisms for these human diseases.

Essential role of the N-terminal region of TFII-I in viability and behavior.

BACKGROUND: GTF2I codes for a general intrinsic transcription factor and calcium channel regulator TFII-I, with high and ubiquitous expression, and a strong candidate for involvement in the morphological and neuro-developmental anomalies of the Williams-Beuren syndrome (WBS). WBS is a genetic disorder due to a recurring deletion of about 1,55-1,83 Mb containing 25-28 genes in chromosome band 7q11.23 including GTF2I. Completed homozygous loss of either the Gtf2i or Gtf2ird1 function in mice provided additional evidence for the involvement of both genes in the craniofacial and cognitive phenotype. Unfortunately nothing is now about the behavioral characterization of heterozygous mice. METHODS: By gene targeting we have generated a mutant mice with a deletion of the first 140 amino-acids of TFII-I. mRNA and protein expression analysis were used to document the effect of the study deletion. We performed behavioral characterization of heterozygous mutant mice to document in vivo implications of TFII-I in the cognitive profile of WBS patients. RESULTS: Homozygous and heterozygous mutant mice exhibit craniofacial alterations, most clearly represented in homozygous condition. Behavioral test demonstrate that heterozygous mutant mice exhibit some neurobehavioral alterations and hyperacusis or odynacusis that could be associated with specific features of WBS phenotype. Homozygous mutant mice present highly compromised embryonic viability and fertility. Regarding cellular model, we documented a retarded growth in heterozygous MEFs respect to homozygous or wild-type MEFs. CONCLUSION: Our data confirm that, although additive effects of haploinsufficiency at several genes may contribute to the full craniofacial or neurocognitive features of WBS, correct expression of GTF2I is one of the main players. In addition, these findings show that the deletion of the fist 140 amino-acids of TFII-I altered it correct function leading to a clear phenotype, at both levels, at the cellular model and at the in vivo model.

CDK2 is required for proper homologous pairing, recombination and sex-body formation during male mouse meiosis.

Cyclin-dependent kinase 2 (CDK2) was assumed to be essential in the mammalian cell cycle both at the G1-S transition and throughout the S phase. Interestingly, ablation of Cdk2 in mice does not have substantial consequences for embryonic or postnatal development, but both males and females are infertile. In the present study, we have analysed the meiotic alterations leading to infertility in Cdk2-/- male mice. We have studied the distribution and dynamics of several proteins related to meiosis progression, such as synaptonemal complex proteins, cohesin complexes, and centromere-, telomere- and recombination-related proteins. Cdk2-/- spermatocytes show an incomplete chromosome pairing, an extensive non-homologous synapsis and arrest at a pachytene-like stage with unrepaired programmed double-strand breaks. In these spermatocytes, some telomeres do not attach to the nuclear envelope, and sex chromosomes do not form a sex body. Our data demonstrate an unpredicted participation of CDK2 in the accurate pairing and recombination between homologues during mammalian meiosis.

Cohesins: chromatin architects in chromosome segregation, control of gene expression and much more.

Cells have evolved to develop molecules and control mechanisms that guarantee correct chromosome segregation and ensure the proper distribution of genetic material to daughter cells. In this sense, the establishment, maintenance, and removal of sister chromatid cohesion is one of the most fascinating and dangerous processes in the life of a cell because errors in the control of these processes frequently lead to cell death or aneuploidy. The main protagonist in this mechanism is a four-protein complex denominated the cohesin complex. In the last 10 years, we have improved our understanding of the key players in the regulation of sister chromatid cohesion during cell division in mitosis and meiosis. The last 2 years have seen an increase in evidence showing that cohesins have important functions in non-dividing cells, revealing new, unexplored roles for these proteins in the control of gene expression, development, and other essential cell functions in mammals.

Sequential loading of cohesin subunits during the first meiotic prophase of grasshoppers.

The cohesin complexes play a key role in chromosome segregation during both mitosis and meiosis. They establish sister chromatid cohesion between duplicating DNA molecules during S-phase, but they also have an important role during postreplicative double-strand break repair in mitosis, as well as during recombination between homologous chromosomes in meiosis. An additional function in meiosis is related to the sister kinetochore cohesion, so they can be pulled by microtubules to the same pole at anaphase I. Data about the dynamics of cohesin subunits during meiosis are scarce; therefore, it is of great interest to characterize how the formation of the cohesin complexes is achieved in order to understand the roles of the different subunits within them. We have investigated the spatio-temporal distribution of three different cohesin subunits in prophase I grasshopper spermatocytes. We found that structural maintenance of chromosome protein 3 (SMC3) appears as early as preleptotene, and its localization resembles the location of the unsynapsed axial elements, whereas radiation-sensitive mutant 21 (RAD21) (sister chromatid cohesion protein 1, SCC1) and stromal antigen protein 1 (SA1) (sister chromatid cohesion protein 3, SCC3) are not visualized until zygotene, since they are located in the synapsed regions of the bivalents. During pachytene, the distribution of the three cohesin subunits is very similar and all appear along the trajectories of the lateral elements of the autosomal synaptonemal complexes. However, whereas SMC3 also appears over the single and unsynapsed X chromosome, RAD21 and SA1 do not. We conclude that the loading of SMC3 and the non-SMC subunits, RAD21 and SA1, occurs in different steps throughout prophase I grasshopper meiosis. These results strongly suggest the participation of SMC3 in the initial cohesin axis formation as early as preleptotene, thus contributing to sister chromatid cohesion, with a later association of both RAD21 and SA1 subunits at zygotene to reinforce and stabilize the bivalent structure. Therefore, we speculate that more than one cohesin complex participates in the sister chromatid cohesion at prophase I.

Mammalian SGO2 appears at the inner centromere domain and redistributes depending on tension across centromeres during meiosis II and mitosis.

Shugoshin (SGO) is a family of proteins that protect centromeric cohesin complexes from release during mitotic prophase and from degradation during meiosis I. Two mammalian SGO paralogues - SGO1 and SGO2 - have been identified, but their distribution and function during mammalian meiosis have not been reported. Here, we analysed the expression of SGO2 during male mouse meiosis and mitosis. During meiosis I, SGO2 accumulates at centromeres during diplotene, and colocalizes differentially with the cohesin subunits RAD21 and REC8 at metaphase I centromeres. However, SGO2 and RAD21 change their relative distributions during telophase I when sister-kinetochore association is lost. During meiosis II, SGO2 shows a striking tension-dependent redistribution within centromeres throughout chromosome congression during prometaphase II, as it does during mitosis. We propose a model by which the redistribution of SGO2 would unmask cohesive centromere proteins, which would be then released or cleaved by separase, to trigger chromatid segregation to opposite poles.

Drosophila cohesins DSA1 and Drad21 persist and colocalize along the centromeric heterochromatin during mitosis.

Sister chromatid cohesion in eukaryotes is maintained mainly by a conserved multiprotein complex termed cohesin. Drad21 and DSA1 are the Drosophila homologues of the yeast Scc1 and Scc3 cohesin subunits, respectively. We recently identified a Drosophila mitotic cohesin complex composed of Drad21/DSA1/DSMC1/DSMC3. Here we study the contribution of this complex to sister chromatid cohesion using immunofluorescence microscopy to analyze cell cycle chromosomal localization of DSA1 and Drad21 in S2 cells. We observed that DSA1 and Drad21 colocalize during all cell cycle stages in cultured cells. Both proteins remain in the centromere until metaphase, colocalizing at the centromere pairing domain that extends along the entire heterochromatin; the centromeric cohesion protein MEI-S332 is nonetheless reported in a distinct centromere domain. These results provide strong evidence that DSA1 and Drad21 are partners in a cohesin complex involved in the maintenance of sister chromatid arm and centromeric cohesion during mitosis in Drosophila.

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 SMC1 beta; 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, SMC1beta, 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.

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.

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,double dagger are classified as mitotic cohesins, as they are found in human somatic cells and in Xenopus laevis as components of the cohesin(SA1) and cohesin(SA2) 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.