Tripolar chromosome segregation drives the association between maternal genotype at variants spanning PLK4 and aneuploidy in human preimplantation embryos.

Aneuploidy is prevalent in human embryos and is the leading cause of pregnancy loss. Many aneuploidies arise during oogenesis, increasing with maternal age. Superimposed on these meiotic aneuploidies are frequent errors occurring during early mitotic divisions, contributing to widespread chromosomal mosaicism. Here we reanalyzed a published dataset comprising preimplantation genetic testing for aneuploidy in 24 653 blastomere biopsies from day-3 cleavage-stage embryos, as well as 17 051 trophectoderm biopsies from day-5 blastocysts. We focused on complex abnormalities that affected multiple chromosomes simultaneously, seeking insights into their formation. In addition to well-described patterns such as triploidy and haploidy, we identified 4.7% of blastomeres possessing characteristic hypodiploid karyotypes. We inferred this signature to have arisen from tripolar chromosome segregation in normally fertilized diploid zygotes or their descendant diploid cells. This could occur via segregation on a tripolar mitotic spindle or by rapid sequential bipolar mitoses without an intervening S-phase. Both models are consistent with time-lapse data from an intersecting set of 77 cleavage-stage embryos, which were enriched for the tripolar signature among embryos exhibiting abnormal cleavage. The tripolar signature was strongly associated with common maternal genetic variants spanning the centrosomal regulator PLK4, driving the association we previously reported with overall mitotic errors. Our findings are consistent with the known capacity of PLK4 to induce tripolar mitosis or precocious M-phase upon dysregulation. Together, our data support tripolar chromosome segregation as a key mechanism generating complex aneuploidy in cleavage-stage embryos and implicate maternal genotype at a quantitative trait locus spanning PLK4 as a factor influencing its occurrence.

Ipl1/Aurora B kinase coordinates synaptonemal complex disassembly with cell cycle progression and crossover formation in budding yeast meiosis.

Several protein kinases collaborate to orchestrate and integrate cellular and chromosomal events at the G2/M transition in both mitotic and meiotic cells. During the G2/M transition in meiosis, this includes the completion of crossover recombination, spindle formation, and synaptonemal complex (SC) breakdown. We identified Ipl1/Aurora B kinase as the main regulator of SC disassembly. Mutants lacking Ipl1 or its kinase activity assemble SCs with normal timing, but fail to dissociate the central element component Zip1, as well as its binding partner, Smt3/SUMO, from chromosomes in a timely fashion. Moreover, lack of Ipl1 activity causes delayed SC disassembly in a cdc5 as well as a CDC5-inducible ndt80 mutant. Crossover levels in the ipl1 mutant are similar to those observed in wild type, indicating that full SC disassembly is not a prerequisite for joint molecule resolution and subsequent crossover formation. Moreover, expression of meiosis I and meiosis II-specific B-type cyclins occur normally in ipl1 mutants, despite delayed formation of anaphase I spindles. These observations suggest that Ipl1 coordinates changes to meiotic chromosome structure with resolution of crossovers and cell cycle progression at the end of meiotic prophase.

Monopolin subunit Csm1 associates with MIND complex to establish monopolar attachment of sister kinetochores at meiosis I.

Sexually reproducing organisms halve their cellular ploidy during gametogenesis by undergoing a specialized form of cell division known as meiosis. During meiosis, a single round of DNA replication is followed by two rounds of nuclear divisions (referred to as meiosis I and II). While sister kinetochores bind to microtubules emanating from opposite spindle poles during mitosis, they bind to microtubules originating from the same spindle pole during meiosis I. This phenomenon is referred to as mono-orientation and is essential for setting up the reductional mode of chromosome segregation during meiosis I. In budding yeast, mono-orientation depends on a four component protein complex referred to as monopolin which consists of two nucleolar proteins Csm1 and Lrs4, meiosis-specific protein Mam1 of unknown function and casein kinase Hrr25. Monopolin complex binds to kinetochores during meiosis I and prevents bipolar attachments. Although monopolin associates with kinetochores during meiosis I, its binding site(s) on the kinetochore is not known and its mechanism of action has not been established. By carrying out an imaging-based screen we have found that the MIND complex, a component of the central kinetochore, is required for monopolin association with kinetochores during meiosis. Furthermore, we demonstrate that interaction of monopolin subunit Csm1 with the N-terminal domain of MIND complex subunit Dsn1, is essential for both the association of monopolin with kinetochores and for monopolar attachment of sister kinetochores during meiosis I. As such this provides the first functional evidence for a monopolin-binding site at the kinetochore.

Smc5/6 coordinates formation and resolution of joint molecules with chromosome morphology to ensure meiotic divisions.

During meiosis, Structural Maintenance of Chromosome (SMC) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and the regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of a subset of joint-molecule intermediates. In this regard, Smc5/6 functions independently of the major crossover pathway defined by the MutLγ complex. Furthermore, we show that Smc5/6 is required for stable chromosomal localization of the XPF-family endonuclease, Mus81-Mms4(Eme1). Our data suggest that the Smc5/6 complex is required for specific recombination and chromosomal processes throughout meiosis and that in its absence, attempts at cell division with unresolved joint molecules and residual cohesin lead to severe recombination-induced meiotic catastrophe.

The synaptonemal complex protein, Zip1, promotes the segregation of nonexchange chromosomes at meiosis I.

Crossing over establishes connections between homologous chromosomes that promote their proper segregation at the first meiotic division. However, there exists a backup system to ensure the correct segregation of those chromosome pairs that fail to cross over. We have found that, in budding yeast, a mutation eliminating the synaptonemal complex protein, Zip1, increases the meiosis I nondisjunction rate of nonexchange chromosomes (NECs). The centromeres of NECs become tethered during meiotic prophase, and this tethering is disrupted by the zip1 mutation. Furthermore, the Zip1 protein often colocalizes to the centromeres of the tethered chromosomes, suggesting that Zip1 plays a direct role in holding NECs together. Zip3, a protein involved in the initiation of synaptonemal complex formation, is also important for NEC segregation. In the absence of Zip3, both the tethering of NECs and the localization of Zip1 to centromeres are impaired. A mutation in the MAD3 gene, which encodes a component of the spindle checkpoint, also increases the nondisjunction of NECs. Together, the zip1 and mad3 mutations have an additive effect, suggesting that these proteins act in parallel pathways to promote NEC segregation. We propose that Mad3 promotes the segregation of NECs that are not tethered by Zip1 at their centromeres.

Advances in Anti-Cancer Immunotherapy: Car-T Cell, Checkpoint Inhibitors, Dendritic Cell Vaccines, and Oncolytic Viruses, and Emerging Cellular and Molecular Targets.

Unlike traditional cancer therapies, such as surgery, radiation and chemotherapy that are typically non-specific, cancer immunotherapy harnesses the high specificity of a patient's own immune system to selectively kill cancer cells. The immune system is the body's main cancer surveillance system, but cancers may evade destruction thanks to various immune-suppressing mechanisms. We therefore need to deploy various immunotherapy-based strategies to help bolster the anti-tumour immune responses. These include engineering T cells to express chimeric antigen receptors (CARs) to specifically recognise tumour neoantigens, inactivating immune checkpoints, oncolytic viruses and dendritic cell (DC) vaccines, which have all shown clinical benefit in certain cancers. However, treatment efficacy remains poor due to drug-induced adverse events and immunosuppressive tendencies of the tumour microenvironment. Recent preclinical studies have unveiled novel therapies such as anti-cathepsin antibodies, galectin-1 blockade and anti-OX40 agonistic antibodies, which may be utilised as adjuvant therapies to modulate the tumour microenvironment and permit more ferocious anti-tumour immune response.

Chromosome errors in human eggs shape natural fertility over reproductive life span.

Chromosome errors, or aneuploidy, affect an exceptionally high number of human conceptions, causing pregnancy loss and congenital disorders. Here, we have followed chromosome segregation in human oocytes from females aged 9 to 43 years and report that aneuploidy follows a U-curve. Specific segregation error types show different age dependencies, providing a quantitative explanation for the U-curve. Whole-chromosome nondisjunction events are preferentially associated with increased aneuploidy in young girls, whereas centromeric and more extensive cohesion loss limit fertility as women age. Our findings suggest that chromosomal errors originating in oocytes determine the curve of natural fertility in humans.

Generation of meiomaps of genome-wide recombination and chromosome segregation in human oocytes.

We have developed a protocol for the generation of genome-wide maps (meiomaps) of recombination and chromosome segregation for the three products of human female meiosis: the first and second polar bodies (PB1 and PB2) and the corresponding oocyte. PB1 is biopsied and the oocyte is artificially activated by exposure to calcium ionophore, after which PB2 is biopsied and collected with the corresponding oocyte. The whole genomes of the polar bodies and oocytes are amplified by multiple displacement amplification and, together with maternal genomic DNA, genotyped for ∼300,000 single-nucleotide polymorphisms (SNPs) genome-wide by microarray. Informative maternal heterozygous SNPs are phased using a haploid PB2 or oocyte as a reference. A simple algorithm is then used to identify the maternal haplotypes for each chromosome, in all of the products of meiosis for each oocyte. This allows mapping of crossovers and analysis of chromosome segregation patterns. The protocol takes a minimum of 3-5 d and requires a clinical embryologist with micromanipulation experience and a molecular biologist with basic bioinformatic skills. It has several advantages over previous methods; importantly, the use of artificial oocyte activation avoids the creation of embryos for research purposes. In addition, compared with next-generation sequencing, targeted SNP genotyping is cost-effective and it simplifies the bioinformatic analysis, as only one haploid reference sample is required to establish phase for maternal haplotyping. Finally, meiomapping is more informative than copy-number analysis alone for analysis of chromosome segregation patterns. Using this protocol, we have provided new insights that may lead to improvements in assisted reproduction for the treatment of infertility.

Genome-wide maps of recombination and chromosome segregation in human oocytes and embryos show selection for maternal recombination rates.

Crossover recombination reshuffles genes and prevents errors in segregation that lead to extra or missing chromosomes (aneuploidy) in human eggs, a major cause of pregnancy failure and congenital disorders. Here we generate genome-wide maps of crossovers and chromosome segregation patterns by recovering all three products of single female meioses. Genotyping >4 million informative SNPs from 23 complete meioses allowed us to map 2,032 maternal and 1,342 paternal crossovers and to infer the segregation patterns of 529 chromosome pairs. We uncover a new reverse chromosome segregation pattern in which both homologs separate their sister chromatids at meiosis I; detect selection for higher recombination rates in the female germ line by the elimination of aneuploid embryos; and report chromosomal drive against non-recombinant chromatids at meiosis II. Collectively, our findings show that recombination not only affects homolog segregation at meiosis I but also the fate of sister chromatids at meiosis II.

Ipl1/Aurora kinase suppresses S-CDK-driven spindle formation during prophase I to ensure chromosome integrity during meiosis.

Cells coordinate spindle formation with DNA repair and morphological modifications to chromosomes prior to their segregation to prevent cell division with damaged chromosomes. Here we uncover a novel and unexpected role for Aurora kinase in preventing the formation of spindles by Clb5-CDK (S-CDK) during meiotic prophase I and when the DDR is active in budding yeast. This is critical since S-CDK is essential for replication during premeiotic S-phase as well as double-strand break induction that facilitates meiotic recombination and, ultimately, chromosome segregation. Furthermore, we find that depletion of Cdc5 polo kinase activity delays spindle formation in DDR-arrested cells and that ectopic expression of Cdc5 in prophase I enhances spindle formation, when Ipl1 is depleted. Our findings establish a new paradigm for Aurora kinase function in both negative and positive regulation of spindle dynamics.

Characterization of the Escherichia coli K-12 ydhYVWXUT operon: regulation by FNR, NarL and NarP.

In Escherichia coli K-12 the expression of many genes is controlled by the oxygen-responsive transcription factor FNR and the nitrate- and nitrite-responsive two-component systems NarXL and NarPQ. Here, the ydhY gene is shown to be the first gene of a six-gene operon (ydhYVWXUT) that encodes proteins predicted to be components of an oxidoreductase. Mapping the ydhY-T transcript start and site-directed mutagenesis confirmed that the ydhY-T genes are transcribed from an FNR-dependent class II promoter and showed that the FNR site is centred at -42.5. In the presence of nitrate or nitrite, NarXL and NarPQ repressed ydhY-T expression. Analysis of the DNA sequence of the ydhY promoter region (PydhY) revealed the presence of four heptameric sequences similar to NarL/P binding sites centred at -42, -16, +6 and +15. The latter heptamers are arranged as a 7-2-7 inverted repeat, which is required for recognition by NarP. Accordingly, NarP protected the 7-2-7 region in DNase I footprints, and mutation of either heptamer +6 or heptamer +15 impaired nitrite-mediated repression, whereas mutation of heptamer -42 and heptamer -16 did not affect the response to nitrite. The NarL protein also protected the 7-2-7 region, but in contrast to NarP, the NarL footprint extended further upstream to encompass the -16 heptamer. The extended NarL footprint was consistent with the presence of multiple NarL-PydhY complexes in gel retardation assays. Mutation of heptamer -42, which is located within the FNR binding site, or heptamer +6 (but not heptamers -16 or +15) impaired nitrate-mediated repression. Thus, although the region of the ydhY-T promoter containing the -16 and +15 heptamers was recognized by NarL in vitro, mutation of these heptamers did not affect NarL-mediated repression in vivo.