Centromeres are characterized by the centromere-specific H3 variant CENP-A, embedded in chromatin with a pattern characteristic of active transcription that is required for centromere identity. It is unclear how centromeres remain transcriptionally active despite being flanked by repressive pericentric heterochromatin. To further understand centrochromatin’s response to repressive signals, we nucleated a polycomb-like chromatin state within the centromere of a HAC (Human Artificial Chromosome) by tethering the methyltransferase EZH2. This led to deposition of the H3K27me3 mark and PRC1 repressor binding. Surprisingly, this state did not abolish HAC centromere function or transcription, and this apparent resistance was not observed on a non-centromeric locus, where transcription was silenced. Directly tethering the reader/repressor PRC1 bypassed this resistance, inactivating the centromere. We observed analogous responses when tethering the heterochromatin Editor Suv39h1-methyltransferase-domain (centromere resistance) or reader HP1α (centromere inactivation), respectively. Our results reveal that the HAC centromere can resist repressive pathways driven by H3K9me3/H3K27me3 and may help to explain how centromeres are able to resist inactivation by flanking heterochromatin.
Recent studies have revealed the importance of Ki-67 and the chromosome periphery in chromosome structure and segregation, but little is known about this elusive chromosome compartment. Here we used correlative light and serial block-face scanning electron microscopy, which we term 3D-CLEM, to model the entire mitotic chromosome complement at ultra-structural resolution. Prophase chromosomes exhibit a highly irregular surface appearance with a volume smaller than metaphase chromosomes. This may be because of the absence of the periphery, which associates with chromosomes only after nucleolar disassembly later in prophase. Indeed, the nucleolar volume almost entirely accounts for the extra volume found in metaphase chromosomes. Analysis of wild-type and Ki-67-depleted chromosomes reveals that the periphery comprises 30%-47% of the entire chromosome volume and more than 33% of the protein mass of isolated mitotic chromosomes determined by quantitative proteomics. Thus, chromatin makes up a surprisingly small percentage of the total mass of metaphase chromosomes.
Aneuploidy in human eggs increases with maternal age and can result in infertility, miscarriages, and birth defects. The molecular mechanisms leading to aneuploidy, however, are largely unknown especially in the human where eggs are exceedingly rare and precious. We obtained human eggs from subjects ranging from 16.4 to 49.7 years old following in vitro maturation of oocyte-cumulus complexes isolated directly from surgically removed ovarian tissue. A subset of these eggs was used to investigate how age-associated aneuploidy occurs in the human. The inter-kinetochore distance between sister chromatids increased significantly with maternal age, indicating weakened cohesion. Moreover, we observed unpaired sister chromatids from females of advanced age. We conclude that loss of cohesion with increasing maternal age likely contributes to the well-documented increased incidence of aneuploidy.
The cyclin-dependent kinase CDK11(p58) is specifically expressed at G2/M phase. CDK11(p58) depletion leads to different cell cycle defects such as mitotic arrest, failure in centriole duplication and centrosome maturation, and premature sister chromatid separation. We report that upon CDK11 depletion, loss of sister chromatid cohesion occurs during mitosis but not during G2 phase. CDK11(p58) depletion prevents Bub1 and Shugoshin 1 recruitment but has no effect on the dimethylation of histone H3 lysine 4 at centromeres. We also report that a construct expressing a kinase dead version of CDK11(p58) fails to prevent CDK11 depletion-induced sister chromatid separation, showing that CDK11(p58) kinase activity is required for protection of sister chromatid cohesion at centromeres during mitosis. Thus, CDK11(p58) kinase activity appears to be involved in early events in the establishment of the centromere protection machinery.
The spindle assembly checkpoint (SAC) ensures the accurate segregation of sister chromatids during mitosis. Activation of the SAC occurs through a series of ordered molecular events that result in recruitment of Mad1:Mad2 complexes to improperly attached kinetochores. The current model involves sequential phospho-dependent recruitment of Bub3:Bub1 to KNL1 followed by binding of Mad1:Mad2 to Bub1. Here, we show in non-transformed diploid human cells that the KNL1-Bub3-Bub1 (KBB) pathway is required during normal mitotic progression when kinetochores are misaligned but is nonessential for SAC activation and Mad2 loading when kinetochores are unattached from microtubules. We provide evidence that the Rod-ZW10-Zwilch (RZZ) complex is necessary to recruit Mad1:Mad2 to, and delay anaphase onset in response to, unattached kinetochores independently of the KBB pathway. These data suggest that the KBB and RZZ complexes provide two distinct kinetochore receptors for Mad1:Mad2 and reveal mechanistic differences between SAC activation by unattached and improperly attached kinetochores.
The pairing behaviour of the individual chromosome arms of Hordeum vulgare (Hv) with their homoeologous arms of H. bulbosum (Hb) at metaphase I of meiosis in tetraploid Hb × Hv hybrids and the frequencies of recombined Hv chromosome arms in selfed offspring were studied on differentially visualized chromosomes after fluorescent in situ hybridisation. The frequencies of paired Hv-Hb arms in the F2 and F3 hybrids were correlated with the frequencies of recombined Hv chromosomes in progenies. Self-generation of hybrids, the number of Hv and Hb chromosomes, and the number of recombined Hv chromosomes of the hybrids strongly influenced the Hv-Hb pairing frequency in meiosis. Within the offspring of F2 and F3 hybrids both Hv plants and hybrids were detected. In contrast, all progenies of the F4 hybrid were hybrids which exhibited centromere misdivisions. The highest frequencies of homoeologous pairing in hybrids and most recombinants were obtained for the barley chromosome 1HL. Recombinants for 4HL, 5HS, 6HS, and 7HS were rarely found. Meiotic pairing and recombinants involving chromosome 1HS were never observed. The results of this study demonstrate that fertile tetraploid interspecific hybrids with a high intergenomic pairing at meiosis are valuable basic material for introgression breeding in barley.
Species within the human pathogenic Cryptococcus species complex are major threats to public health, causing approximately 1 million infections globally annually. Cryptococcus amylolentus is the most closely known related species of the pathogenic Cryptococcus species complex, and it is non-pathogenic. Additionally, while pathogenic Cryptococcus species have bipolar mating systems with a single large mating type (MAT) locus that represents a derived state in Basidiomycetes, C. amylolentus has a tetrapolar mating system with 2 MAT loci (P/R and HD) located on different chromosomes. Thus, studying C. amylolentus will shed light on the transition from tetrapolar to bipolar mating systems in the pathogenic Cryptococcus species, as well as its possible link with the origin and evolution of pathogenesis. In this study, we sequenced, assembled, and annotated the genomes of 2 C. amylolentus isolates, CBS6039 and CBS6273, which are sexual and interfertile. Genome comparison between the 2 C. amylolentus isolates identified the boundaries and the complete gene contents of the P/R and HD MAT loci. Bioinformatic and chromatin immunoprecipitation sequencing (ChIP-seq) analyses revealed that, similar to those of the pathogenic Cryptococcus species, C. amylolentus has regional centromeres (CENs) that are enriched with species-specific transposable and repetitive DNA elements. Additionally, we found that while neither the P/R nor the HD locus is physically closely linked to its centromere in C. amylolentus, and the regions between the MAT loci and their respective centromeres show overall synteny between the 2 genomes, both MAT loci exhibit genetic linkage to their respective centromere during meiosis, suggesting the presence of recombinational suppressors and/or epistatic gene interactions in the MAT-CEN intervening regions. Furthermore, genomic comparisons between C. amylolentus and related pathogenic Cryptococcus species provide evidence that multiple chromosomal rearrangements mediated by intercentromeric recombination have occurred during descent of the 2 lineages from their common ancestor. Taken together, our findings support a model in which the evolution of the bipolar mating system was initiated by an ectopic recombination event mediated by similar repetitive centromeric DNA elements shared between chromosomes. This translocation brought the P/R and HD loci onto the same chromosome, and further chromosomal rearrangements then resulted in the 2 MAT loci becoming physically linked and eventually fusing to form the single contiguous MAT locus that is now extant in the pathogenic Cryptococcus species.
Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation in mitosis and meiosis . Rec8-containing cohesin, bound to Smc3/Smc1α or Smc3/Smc1β, maintains bivalent cohesion in mammalian meiosis [2-6]. In females, meiotic DNA replication and recombination occur in fetal oocytes. After birth, oocytes arrest at the prolonged dictyate stage until recruited to grow into mature oocytes that divide at ovulation. How cohesion is maintained in arrested oocytes remains a pivotal question relevant to maternal age-related aneuploidy. Hypothetically, cohesin turnover regenerates cohesion in oocytes. Evidence for post-replicative cohesion establishment mechanism exists, in yeast and invertebrates [7, 8]. In mouse fetal oocytes, cohesin loading factor Nipbl/Scc2 localizes to chromosome axes during recombination [9, 10]. Alternatively, cohesion is maintained without turnover. Consistent with this, cohesion maintenance does not require Smc1β transcription, but unlike Rec8, Smc1β is not required for establishing bivalent cohesion [11, 12]. Rec8 maintains cohesion without turnover during weeks of oocyte growth . Whether the same applies to months or decades of arrest is unknown. Here, we test whether Rec8 activated in arrested mouse oocytes builds cohesion revealed by TEV cleavage and live-cell imaging. Rec8 establishes cohesion when activated during DNA replication in fetal oocytes using tamoxifen-inducible Cre. In contrast, no new cohesion is detected when Rec8 is activated in arrested oocytes by tamoxifen despite cohesin synthesis. We conclude that cohesion established in fetal oocytes is maintained for months without detectable turnover in dictyate-arrested oocytes. This implies that women’s fertility depends on the longevity of cohesin proteins that established cohesion in utero.
The transition of an ancestral circular genome to multiple linear chromosomes was crucial for eukaryogenesis because it allowed rapid adaptive evolution through aneuploidy. Here, we propose that the ends of nascent linear chromosomes should have had a dual function in chromosome end-protection (capping) and chromosome segregation to give rise to the “proto-telomeres”. Later on proper centromeres evolved at subtelomeric regions. We also propose that both non-canonical structures based on guanine-guanine interactions and the end-protection proteins recruited by the emergent telomeric heterochromatin have been required for telomere maintenance through evolution. We further suggest that the origin of Drosophila telomeres may be reminiscent of how the first telomeres arose.
Centromeres are essential for chromosome segregation and are specified epigenetically by the presence of the histone H3 variant CENP-A. In flies and humans, replenishment of the centromeric mark is uncoupled from DNA replication and requires the removal of H3 “placeholder” nucleosomes. Although transcription at centromeres has been previously linked to the loading of new CENP-A, the underlying molecular mechanism remains poorly understood. Here, we usedDrosophila melanogastertissue culture cells to show that centromeric presence of actively transcribing RNA polymerase II temporally coincides with de novo deposition of dCENP-A. Using a newly developed dCENP-A loading system that is independent of acute transcription, we found that short inhibition of transcription impaired dCENP-A incorporation into chromatin. Interestingly, initial targeting of dCENP-A to centromeres was unaffected, revealing two stability states of newly loaded dCENP-A: a salt-sensitive association with the centromere and a salt-resistant chromatin-incorporated form. This suggests that transcription-mediated chromatin remodeling is required for the transition of dCENP-A to fully incorporated nucleosomes at the centromere.