Condensin dysfunction is a reproductive isolating barrier in mice.

Reproductive isolation occurs when the genomes of two populations accumulate genetic incompatibilities that prevent interbreeding1,2. Understanding of hybrid incompatibility at the cell biology level is limited, particularly in the case of hybrid female sterility3. Here we find that species divergence in condensin regulation and centromere organization between two mouse species, Mus musculus domesticus and Mus spretus, drives chromosome decondensation and mis-segregation in their F1 hybrid oocytes, reducing female fertility. The decondensation in hybrid oocytes was especially prominent at pericentromeric major satellites, which are highly abundant at M. m. domesticus centromeres4-6, leading to species-specific chromosome mis-segregation and egg aneuploidy. Consistent with the condensation defects, a chromosome structure protein complex, condensin II7,8, was reduced on hybrid oocyte chromosomes. We find that the condensin II subunit NCAPG2 was specifically reduced in the nucleus in prophase and that overexpressing NCAPG2 rescued both the decondensation and egg aneuploidy phenotypes. In addition to the overall reduction in condensin II on chromosomes, major satellites further reduced condensin II levels locally, explaining why this region is particularly prone to decondensation. Together, this study provides cell biological insights into hybrid incompatibility in female meiosis and demonstrates that condensin misregulation and pericentromeric satellite expansion can establish a reproductive isolating barrier in mammals.

The 3D Topography of Mitotic Chromosomes.

A long-standing conundrum is how mitotic chromosomes can compact, as required for clean separation to daughter cells, while maintaining close parallel alignment of sister chromatids. Pursuit of this question, by high resolution 3D fluorescence imaging of living and fixed mammalian cells, has led to three discoveries. First, we show that the structural axes of separated sister chromatids are linked by evenly spaced "mini-axis" bridges. Second, when chromosomes first emerge as discrete units, at prophase, they are organized as co-oriented sister linear loop arrays emanating from a conjoined axis. We show that this same basic organization persists throughout mitosis, without helical coiling. Third, from prophase onward, chromosomes are deformed into sequential arrays of half-helical segments of alternating handedness (perversions), accompanied by correlated kinks. These arrays fluctuate dynamically over <15 s timescales. Together these discoveries redefine the foundation for thinking about the evolution of mitotic chromosomes as they prepare for anaphase segregation.

ATR function is indispensable to allow proper mammalian follicle development.

Mammalian female fertility relies on the proper development of follicles. Right after birth in the mouse, oocytes associate with somatic ovarian cells to form follicles. These follicles grow during the adult lifetime to produce viable gametes. In this study, we analyzed the role of the ATM and rad3-related (ATR) kinase in mouse oogenesis and folliculogenesis using a hypomorphic mutation of the Atr gene (Murga et al. 2009). Female mice homozygotes for this allele have been reported to be sterile. Our data show that female meiotic prophase is not grossly altered when ATR levels are reduced. However, follicle development is substantially compromised, since Atr mutant ovaries present a decrease of growing follicles. Comprehensive analysis of follicular cell death and proliferation suggest that wild-type levels of ATR are required to achieve optimal follicular development. Altogether, these findings suggest that reduced ATR expression causes sterility due to defects in follicular progression rather than in meiotic recombination. We discuss the implications of these findings for the use of ATR inhibitors such as anti-cancer drugs and its possible side-effects on female fertility.

Distinct prophase arrest mechanisms in human male meiosis.

To prevent chromosomal aberrations being transmitted to the offspring, strict meiotic checkpoints are in place to remove aberrant spermatocytes. However, in about 1% of males these checkpoints cause complete meiotic arrest leading to azoospermia and subsequent infertility. Here, we unravel two clearly distinct meiotic arrest mechanisms that occur during prophase of human male meiosis. Type I arrested spermatocytes display severe asynapsis of the homologous chromosomes, disturbed XY-body formation and increased expression of the Y chromosome-encoded gene ZFY and seem to activate a DNA damage pathway leading to induction of p63, possibly causing spermatocyte apoptosis. Type II arrested spermatocytes display normal chromosome synapsis, normal XY-body morphology and meiotic crossover formation but have a lowered expression of several cell cycle regulating genes and fail to silence the X chromosome-encoded gene ZFX Discovery and understanding of these meiotic arrest mechanisms increases our knowledge of how genomic stability is guarded during human germ cell development.

DEK over-expression promotes mitotic defects and micronucleus formation.

The DEK gene encodes a nuclear protein that binds chromatin and is involved in various fundamental nuclear processes including transcription, RNA splicing, DNA replication and DNA repair. Several cancer types characteristically over-express DEK at the earliest stages of transformation. In order to explore relevant mechanisms whereby DEK supports oncogenicity, we utilized cancer databases to identify gene transcripts whose expression patterns are tightly correlated with that of DEK. We identified an enrichment of genes involved in mitosis and thus investigated the regulation and possible function of DEK in cell division. Immunofluorescence analyses revealed that DEK dissociates from DNA in early prophase and re-associates with DNA during telophase in human keratinocytes. Mitotic cell populations displayed a sharp reduction in DEK protein levels compared to the corresponding interphase population, suggesting DEK may be degraded or otherwise removed from the cell prior to mitosis. Interestingly, DEK overexpression stimulated its own aberrant association with chromatin throughout mitosis. Furthermore, DEK co-localized with anaphase bridges, chromosome fragments, and micronuclei, suggesting a specific association with mitotically defective chromosomes. We found that DEK over-expression in both non-transformed and transformed cells is sufficient to stimulate micronucleus formation. These data support a model wherein normal chromosomal clearance of DEK is required for maintenance of high fidelity cell division and chromosomal integrity. Therefore, the overexpression of DEK and its incomplete removal from mitotic chromosomes promotes genomic instability through the generation of genetically abnormal daughter cells. Consequently, DEK over-expression may be involved in the initial steps of developing oncogenic mutations in cells leading to cancer initiation.

Aberrant expression of the CHFR prophase checkpoint gene in human B-cell non-Hodgkin lymphoma.

Checkpoint with FHA and Ring Finger (CHFR) is a checkpoint protein that reportedly initiates a cell cycle delay in response to microtubule stress during prophase in mitosis, which has become an interesting target for understanding cancer pathogenesis. Recently, aberrant methylation of the CHFR gene associated with gene silencing has been reported in several cancers. In the present study, we examined the expression of CHFR in B-cell non-Hodgkin lymphoma (B-NHL) in vitro and in vivo. Our results showed that the expression level of CHFR mRNA and protein was reduced in B-NHL tissue samples and B cell lines. Furthermore, CHFR methylation was detected in 39 of 122 B-NHL patients, which was not found in noncancerous reactive hyperplasia of lymph node (RH) tissues. CHFR methylation correlated with the reduced expression of CHFR, high International Prognostic Index (IPI) scores and later pathologic Ann Arbor stages of B-NHL. Treatment with demethylation reagent, 5-Aza-dC, could eliminate the hypermethylation of CHFR, enhance CHFR expression and cell apoptosis and inhibit the cell proliferation of Raji cells, which could be induced by high expression of CHFR in Raji cells. Our results indicated that aberrant methylation of CHFR may be associated with the pathogenesis, progression for B-NHL, which might be a novel molecular marker as prognosis and treatment for B-NHL.

Variation in genome-wide levels of meiotic recombination is established at the onset of prophase in mammalian males.

Segregation of chromosomes during the first meiotic division relies on crossovers established during prophase. Although crossovers are strictly regulated so that at least one occurs per chromosome, individual variation in crossover levels is not uncommon. In an analysis of different inbred strains of male mice, we identified among-strain variation in the number of foci for the crossover-associated protein MLH1. We report studies of strains with "low" (CAST/EiJ), "medium" (C3H/HeJ), and "high" (C57BL/6J) genome-wide MLH1 values to define factors responsible for this variation. We utilized immunofluorescence to analyze the number and distribution of proteins that function at different stages in the recombination pathway: RAD51 and DMC1, strand invasion proteins acting shortly after double-strand break (DSB) formation, MSH4, part of the complex stabilizing double Holliday junctions, and the Bloom helicase BLM, thought to have anti-crossover activity. For each protein, we identified strain-specific differences that mirrored the results for MLH1; i.e., CAST/EiJ mice had the lowest values, C3H/HeJ mice intermediate values, and C57BL/6J mice the highest values. This indicates that differences in the numbers of DSBs (as identified by RAD51 and DMC1) are translated into differences in the number of crossovers, suggesting that variation in crossover levels is established by the time of DSB formation. However, DSBs per se are unlikely to be the primary determinant, since allelic variation for the DSB-inducing locus Spo11 resulted in differences in the numbers of DSBs but not the number of MLH1 foci. Instead, chromatin conformation appears to be a more important contributor, since analysis of synaptonemal complex length and DNA loop size also identified consistent strain-specific differences; i.e., crossover frequency increased with synaptonemal complex length and was inversely related to chromatin loop size. This indicates a relationship between recombination and chromatin compaction that may develop as DSBs form or earlier during establishment of the meiotic axis.

A Wisp3 Cre-knockin allele produces efficient recombination in spermatocytes during early prophase of meiosis I.

Individuals with the autosomal recessive skeletal disorder Progressive Pseudorheumatoid Dysplasia have loss-of-function mutations in WISP3, and aberrant WISP3 expression has been detected in tumors from patients with colon and breast cancer. In mice however, neither absence nor over-expression of WISP3 was found to cause a phenotype, and endogenous Wisp3 expression has been difficult to detect. To confirm that Wisp3 knockout mice have no phenotype and to identify potential sites of endogenous Wisp3 expression, we generated mice with a knockin allele (Wisp3 (GFP-Cre)) designed to express Green Fluorescent Protein (GFP) and Cre-recombinase instead of WISP3. Heterozygous and homozygous knockin mice were fertile and indistinguishable from their wild-type littermates, confirming that mice lacking Wisp3 have no phenotype. We could not detect GFP-expression from the knockin allele, but we could detect Cre-expression after crossing mice with the knockin allele to Cre-reporter mice; the double heterozygous offspring had evidence of Cre-mediated recombination in several tissues. The only tissue that had high levels of Cre-mediated recombination was the testis, where recombination in spermatocytes occurred by early prophase of meiosis I. As a consequence, males that were double heterozygous for a Wisp3 (GFP-Cre) and a floxed allele only contributed a recombined allele to their offspring. We detected no evidence of Cre-mediated recombination in the female ovary, although when double heterozygous females contributed the reporter allele to their offspring it had recombined ~7% of the time. Wisp3 (GFP-Cre) expression therefore occurs less frequently and most likely at a later stage of oocyte development in female mice compared to male mice. We conclude that although WISP3 is dispensable in mice, male mice with a Wisp3 (GFP-Cre) allele (Jackson Laboratory stock # 017685) will be useful for studying early prophase of meiosis I and for efficiently recombining floxed alleles that are passed to offspring.

Cardiac glycosides block cancer growth through HIF-1α- and NF-κB-mediated Plk1.

Cardiac glycosides as inhibitors of the sodium/potassium adenosine triphosphatase (sodium pump) have been reported to block cancer growth by inducing G2/M phase arrest in many cancer cells. However, no detailed studies have been performed to distinguish between these two phases of cardiac glycoside-arrested cells. Furthermore, the underlying mechanisms involved in this cell cycle arrest process are still not known. Here, we report that bufalin and other cardiac glycosides potently induce mitotic arrest by the downregulation of polo-like kinase 1 (Plk1) expression. Live-cell imaging results demonstrate that bufalin-treated cells exhibit a marked delay in entering prophase at an early stage and are then arrested at prometaphase or induced entry into apoptosis. This phenotypic change is attributed to the downregulation of Plk1. We also show that bufalin and the knockdown of sodium pump reduce Plk1, at least in part, through downregulation of the nuclear transcription factors, hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-κB). These findings suggest that cardiac glycosides induce mitotic arrest and apoptosis through HIF-1α- and NF-κB-mediated downregulation of Plk1 expression, demonstrating that HIF-1α and NF-κB are critical targets of cardiac glycosides in exerting their anticancer action.

Actomyosin-dependent cortical dynamics contributes to the prophase force-balance in the early Drosophila embryo.

BACKGROUND: The assembly of the Drosophila embryo mitotic spindle during prophase depends upon a balance of outward forces generated by cortical dynein and inward forces generated by kinesin-14 and nuclear elasticity. Myosin II is known to contribute to the dynamics of the cell cortex but how this influences the prophase force-balance is unclear. PRINCIPAL FINDINGS: Here we investigated this question by injecting the myosin II inhibitor, Y27632, into early Drosophila embryos. We observed a significant increase in both the area of the dense cortical actin caps and in the spacing of the spindle poles. Tracking of microtubule plus ends marked by EB1-GFP and of actin at the cortex revealed that astral microtubules can interact with all regions of these expanded caps, presumably via their interaction with cortical dynein. In Scrambled mutants displaying abnormally small actin caps but normal prophase spindle length in late prophase, myosin II inhibition produced very short spindles. CONCLUSIONS: These results suggest that two complementary outward forces are exerted on the prophase spindle by the overlying cortex. Specifically, dynein localized on the mechanically firm actin caps and the actomyosin-driven contraction of the deformable soft patches of the actin cortex, cooperate to pull astral microtubules outward. Thus, myosin II controls the size and dynamic properties of the actin-based cortex to influence the spacing of the poles of the underlying spindle during prophase.

Flow cytometry and GISH reveal mixed ploidy populations and Spartina nonaploids with genomes of S. alterniflora and S. maritima origin.

BACKGROUND: The genus Spartina exhibits extensive hybridization and includes classic examples of recent speciation by allopolyploidy. In the UK there are two hexaploid species, S. maritima and S. alterniflora, as well as the homoploid hybrid S. x townsendii (2n = 60) and a derived allododecaploid S. anglica (2n = 120, 122, 124); the latter two are considered to have originated in Hythe, southern England at the end of the 19th century. METHODS: Genomic in situ hybridization (GISH) and flow cytometry were used to characterize the genomic composition and distribution of these species and their ploidy levels at Eling Marchwood and Hythe, both near Southampton, southern England. KEY RESULTS: GISH identified approx. 60 chromosomes each of S. maritima and S. alterniflora origin in S. anglica and 62 chromosomes from S. alterniflora and 30 chromosomes from S. maritima in a nonaploid individual from Eling Marchwood, UK. GISH and flow cytometry also revealed that most (94 %) individuals examined at Hythe were hexaploid (the remaining two individuals (6 %) were dodedcaploid; n = 34), whereas hexaploid (approx. 36 % of plants), nonaploid (approx. 27 %) and dodecaploid (approx. 36 %) individuals were found at Eling Marchwood (n = 22). CONCLUSIONS: Nonaploid individuals indicate the potential for introgression between hexaploid and dodecaploid species, complicating the picture of polyploid-induced speciation within the genus. Despite the aggressive ecological habit of S. anglica, it has not out-competed S. x townsendii at Hythe (homoploid hybrids at a frequency of 94 %, n = 34), despite >100 years of coexistence. The success of GISH opens up the potential for future studies of polyploid-induced genome restructuring in this genus.

Systematic kinetic analysis of mitotic dis- and reassembly of the nuclear pore in living cells.

During mitosis in higher eukaryotes, nuclear pore complexes (NPCs) disassemble in prophase and are rebuilt in anaphase and telophase. NPC formation is hypothesized to occur by the interaction of mitotically stable subcomplexes that form defined structural intermediates. To determine the sequence of events that lead to breakdown and reformation of functional NPCs during mitosis, we present here our quantitative assay based on confocal time-lapse microscopy of single dividing cells. We use this assay to systematically investigate the kinetics of dis- and reassembly for eight nucleoporin subcomplexes relative to nuclear transport in NRK cells, linking the assembly state of the NPC with its function. Our data establish that NPC assembly is an ordered stepwise process that leads to import function already in a partially assembled state. We furthermore find that nucleoporin dissociation does not occur in the reverse order from binding during assembly, which may indicate a distinct mechanism.

Caenorhabditis elegans prom-1 is required for meiotic prophase progression and homologous chromosome pairing.

A novel gene, prom-1, was isolated in a screen for Caenorhabditis elegans mutants with increased apoptosis in the germline. prom-1 encodes an F-box protein with limited homology to the putative human tumor suppressor FBXO47. Mutations in the prom-1 locus cause a strong reduction in bivalent formation, which results in increased embryonic lethality and a Him phenotype. Furthermore, retarded and asynchronous nuclear reorganization as well as reduced homologous synapsis occur during meiotic prophase. Accumulation of recombination protein RAD-51 in meiotic nuclei suggests disturbed repair of double-stranded DNA breaks. Nuclei in prom-1 mutant gonads timely complete mitotic proliferation and premeiotic replication, but they undergo prolonged delay upon meiotic entry. We, therefore, propose that prom-1 regulates the timely progression through meiotic prophase I and that in its absence the recognition of homologous chromosomes is strongly impaired.

Artemis links ATM to G2/M checkpoint recovery via regulation of Cdk1-cyclin B.

Artemis is a phospho-protein that has been shown to have roles in V(D)J recombination, nonhomologous end-joining of double-strand breaks, and regulation of the DNA damage-induced G(2)/M cell cycle checkpoint. Here, we have identified four sites in Artemis that are phosphorylated in response to ionizing radiation (IR) and show that ATM is the major kinase responsible for these modifications. Two of the sites, S534 and S538, show rapid phosphorylation and dephosphorylation, and the other two sites, S516 and S645, exhibit rapid and prolonged phosphorylation. Mutation of both of these latter two residues results in defective recovery from the G(2)/M cell cycle checkpoint. This defective recovery is due to promotion by mutant Artemis of an enhanced interaction between unphosphorylated cyclin B and Cdk1, which in turn promotes inhibitory phosphorylation of Cdk1 by the Wee1 kinase. In addition, we show that mutant Artemis prevents Cdk1-cyclin B activation by causing its retention in the centrosome and inhibition of its nuclear import during prophase. These findings show that ATM regulates G(2)/M checkpoint recovery through inhibitory phosphorylations of Artemis that occur soon after DNA damage, thus setting a molecular switch that, hours later upon completion of DNA repair, allows activation of the Cdk1-cyclin B complex. These findings thus establish a novel function of Artemis as a regulator of the cell cycle in response to DNA damage.

Mutations that affect meiosis in male mice influence the dynamics of the mid-preleptotene and bouquet stages.

Meiosis pairs and segregates homologous chromosomes and thereby forms haploid germ cells to compensate the genome doubling at fertilization. Homologue pairing in many eukaryotic species depends on formation of DNA double strand breaks (DSBs) during early prophase I when telomeres begin to cluster at the nuclear periphery (bouquet stage). By fluorescence in situ hybridization criteria, we observe that mid-preleptotene and bouquet stage frequencies are altered in male mice deficient for proteins required for recombination, ubiquitin conjugation and telomere length control. The generally low frequencies of mid-preleptotene spermatocytes were significantly increased in male mice lacking recombination proteins SPO11, MEI1, MLH1, KU80, ubiquitin conjugating enzyme HR6B, and in mice with only one copy of the telomere length regulator Terf1. The bouquet stage was significantly enriched in Atm(-/-), Spo11(-/-), Mei1(m1Jcs/m1Jcs), Mlh1(-/-), Terf1(+/-) and Hr6b(-/-) spermatogenesis, but not in mice lacking recombination proteins DMC1 and HOP2, the non-homologous end-joining DNA repair factor KU80 and the ATM downstream effector GADD45a. Mice defective in spermiogenesis (Tnp1(-/-), Gmcl1(-/-), Asm(-/-)) showed wild-type mid-preleptotene and bouquet frequencies. A low frequency of bouquet spermatocytes in Spo11(-/-)Atm(-/-) spermatogenesis suggests that DSBs contribute to the Atm(-/-)-correlated bouquet stage exit defect. Insignificant changes of bouquet frequencies in mice with defects in early stages of DSB repair (Dmc1(-/-), Hop2(-/-)) suggest that there is an ATM-specific influence on bouquet stage duration. Altogether, it appears that several pathways influence telomere dynamics in mammalian meiosis.

The meiotic bouquet promotes homolog interactions and restricts ectopic recombination in Schizosaccharomyces pombe.

Chromosome architecture undergoes extensive, programmed changes as cells enter meiosis. A highly conserved change is the clustering of telomeres at the nuclear periphery to form the "bouquet" configuration. In the fission yeast Schizosaccharomyces pombe the bouquet and associated nuclear movement facilitate initial interactions between homologs. We show that Bqt2, a meiosis-specific protein required for bouquet formation, is required for wild-type levels of homolog pairing and meiotic allelic recombination. Both gene conversion and crossing over are reduced and exhibit negative interference in bqt2Delta mutants, reflecting reduced homolog pairing. While both the bouquet and nuclear movement promote pairing, only the bouquet restricts ectopic recombination (that between dispersed repetitive DNA). We discuss mechanisms by which the bouquet may prevent deleterious translocations by restricting ectopic recombination.

Mouse emi1 has an essential function in mitotic progression during early embryogenesis.

For successful mitotic entry and spindle assembly, mitosis-promoting factors are activated at the G(2)/M transition stage, followed by stimulation of the anaphase-promoting complex (APC), an E3 ubiquitin ligase, to direct the ordered destruction of several critical mitotic regulators. Given that inhibition of APC activity is important for preventing premature or improper ubiquitination and destruction of substrates, several modulators and their regulation mechanisms have been studied. Emi1, an early mitotic inhibitor, is one of these regulatory factors. Here we show, by analyzing Emi1-deficient embryos, that Emi1 is essential for precise mitotic progression during early embryogenesis. Emi1(-/-) embryos were found to be lethal due to a defect in preimplantation development. Cell proliferation appeared to be normal, but mitotic progression was severely defective during embryonic cleavage. Moreover, multipolar spindles and misaligned chromosomes were frequently observed in Emi1 mutant cells, possibly due to premature APC activation. Our results collectively suggest that the late prophase checkpoint function of Emi1 is essential for accurate mitotic progression and embryonic viability.

Temporal progression of recombination in human males.

To date, immunocytology has been used in humans to detect a limited number of meiotic proteins: components of the synaptonemal complex (SCP1 and SCP3) and some proteins known to participate in recombination events, such as MLH1 or RAD51. However, the colocalization or coexistence of proteins known to participate during the different stages of human meiosis remains largely unstudied, and these studies could provide important clues about the mechanics of recombination. This work reports the relative timing and localization of five different meiotic proteins that have previously been implicated in human homologous recombination [RAD51, replication protein A (RPA), MSH4, MLH1 and MLH3]. MSH4 foci appear concurrently with synapsis initiation at zygotene, shortly after the first RAD51 foci are detected. The presence of RPA in MSH4 foci was noted, suggesting that these two proteins may act co-operatively. Both RPA and MSH4 foci reach maximal numbers at the end of zygotene, when synapsis is concluding. From this point, RPA foci all but disappear by the end of pachytene, whereas MSH4 foci decline to a stable number at mid-pachytene, where they localize with MLH1/MLH3 recombination sites. We discuss a possible role for MSH4 in synapsis initiation and/or maintenance.

Aberrant quantity and localization of Aurora-B/AIM-1 and survivin during megakaryocyte polyploidization and the consequences of Aurora-B/AIM-1-deregulated expression.

Megakaryocytes skip late anaphase and cytokinesis during endomitosis. We found normal expression and localization of a fundamental regulator of mitosis, Aurora-B/AIM-1, during prophase in polyploidizing mouse bone marrow megakaryocytes. At late anaphase, however, Aurora-B/AIM-1 is absent or mislocalized. Megakaryocytes treated with a proteasome inhibitor display Aurora-B/AIM-1 properly expressed and localized to the midzone, suggesting that protein degradation contributes to this atypical appearance. In contrast, survivin, an Aurora-B/AIM-1 coregulator of mitosis, is not detected at any stage of the endomitotic cell cycle, and in most megakaryocytes proteasome inhibition does not rescue this phenotype. To further explore the importance of reduced Aurora-B/AIM-1 for polyploidization, it was overexpressed in megakaryocytes of transgenic mice. The phenotype includes increased transgenic mRNA, but not protein, in polyploidy megakaryocytes, further suggesting that Aurora-B/AIM-1 is regulated at the protein level. Aurora-B/AIM-1 protein is, however, elevated in diploid transgenic megakaryocytes. Transgenic mice also exhibit enhanced numbers of megakaryocytes with increased proliferative potential, and some mice exhibit mild decreases in ploidy level. Hence, the molecular programming involved in endomitosis is characterized by the mislocalization or absence of at least 2 critical mitotic regulators, Aurora-B/AIM-1 and survivin. Future studies will examine the impact of survivin restoration on mouse megakaryocyte polyploidization.