Heat stress impairs centromere structure and segregation of meiotic chromosomes in Arabidopsis.

Heat stress is a major threat to global crop production, and understanding its impact on plant fertility is crucial for developing climate-resilient crops. Despite the known negative effects of heat stress on plant reproduction, the underlying molecular mechanisms remain poorly understood. Here, we investigated the impact of elevated temperature on centromere structure and chromosome segregation during meiosis in Arabidopsis thaliana. Consistent with previous studies, heat stress leads to a decline in fertility and micronuclei formation in pollen mother cells. Our results reveal that elevated temperature causes a decrease in the amount of centromeric histone and the kinetochore protein BMF1 at meiotic centromeres with increasing temperature. Furthermore, we show that heat stress increases the duration of meiotic divisions and prolongs the activity of the spindle assembly checkpoint during meiosis I, indicating an impaired efficiency of the kinetochore attachments to spindle microtubules. Our analysis of mutants with reduced levels of centromeric histone suggests that weakened centromeres sensitize plants to elevated temperature, resulting in meiotic defects and reduced fertility even at moderate temperatures. These results indicate that the structure and functionality of meiotic centromeres in Arabidopsis are highly sensitive to heat stress, and suggest that centromeres and kinetochores may represent a critical bottleneck in plant adaptation to increasing temperatures.

Functions of HP1 in preventing chromosomal instability.

Chromosomal instability (CIN), caused by errors in the segregation of chromosomes during mitosis, is a hallmark of many types of cancer. The fidelity of chromosome segregation is governed by a sophisticated cellular signaling network, one crucial orchestrator of which is Heterochromatin protein 1 (HP1). HP1 dynamically localizes to distinct sites at various stages of mitosis, where it regulates key mitotic events ranging from chromosome-microtubule attachment to sister chromatid cohesion to cytokinesis. Our evolving comprehension of HP1's multifaceted role has positioned it as a central protein in the orchestration of mitotic processes.

Determinants of minor satellite RNA function in chromosome segregation in mouse embryonic stem cells.

The centromere is a fundamental higher-order structure in chromosomes ensuring their faithful segregation upon cell division. Centromeric transcripts have been described in several species and suggested to participate in centromere function. However, low sequence conservation of centromeric repeats appears inconsistent with a role in recruiting highly conserved centromeric proteins. Here, we hypothesized that centromeric transcripts may function through a secondary structure rather than sequence conservation. Using mouse embryonic stem cells (ESCs), we show that an imbalance in the levels of forward or reverse minor satellite (MinSat) transcripts leads to severe chromosome segregation defects. We further show that MinSat RNA adopts a stem-loop secondary structure, which is conserved in human α-satellite transcripts. We identify an RNA binding region in CENPC and demonstrate that MinSat transcripts function through the structured region of the RNA. Importantly, mutants that disrupt MinSat secondary structure do not cause segregation defects. We propose that the conserved role of centromeric transcripts relies on their secondary RNA structure.

The noncanonical function of borealin, a component of chromosome passenger complex, promotes glycolysis via stabilization of survivin in squamous cell carcinoma cells.

The chromosome passenger complex (CPC) is a kinase complex formed by Aurora B, borealin, survivin and inner centromere protein (INCENP). The CPC is active during mitosis and contributes to proper chromosome segregation via the phosphorylation of various substrates. Overexpression of each CPC component has been reported in most cancers. However, its significance remains unclear, as only survivin is known to confer chemoresistance. This study showed that the overexpression of borealin, a CPC component, stabilized survivin protein depending on its interaction with survivin. Unexpectedly, the accumulation of survivin by borealin overexpression did not affect the well-characterized functions of survivin, such as chemoresistance and cell proliferation. Interestingly, the overexpression of borealin promoted lactate production but not the overexpression of the deletion mutant that lacks the ability to bind to survivin. Consistent with these findings, the expression levels of glycolysis-related genes were enhanced in borealin-overexpressing cancer cells. Meanwhile, the overexpression of survivin alone did not promote lactate production. Overall, the accumulation of the borealin-survivin complex promoted glycolysis in squamous cell carcinoma cells. This mechanism may contribute to cancer progression via excessive lactate production.

Efficient formation of single-copy human artificial chromosomes.

Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through ~125-base pair DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule upon introduction to cells. We describe an approach that efficiently forms single-copy HACs. It employs a ~750-kilobase construct that is sufficiently large to house the distinct chromatin types present at the inner and outer centromere, obviating the need to multimerize. Delivery to mammalian cells is streamlined by employing yeast spheroplast fusion. These developments permit faithful chromosome engineering in the context of metazoan cells.

Cdc48 and its co-factor Ufd1 extract CENP-A from centromeric chromatin and can induce chromosome elimination in the fission yeast Schizosaccharomyces pombe.

CENP-A determines the identity of the centromere. Because the position and size of the centromere and its number per chromosome must be maintained, the distribution of CENP-A is strictly regulated. In this study, we have aimed to understand mechanisms to regulate the distribution of CENP-A (Cnp1SP) in fission yeast. A mutant of the ufd1+ gene (ufd1-73) encoding a cofactor of Cdc48 ATPase is sensitive to Cnp1 expressed at a high level and allows mislocalization of Cnp1. The level of Cnp1 in centromeric chromatin is increased in the ufd1-73 mutant even when Cnp1 is expressed at a normal level. A preexisting mutant of the cdc48+ gene (cdc48-353) phenocopies the ufd1-73 mutant. We have also shown that Cdc48 and Ufd1 proteins interact physically with centromeric chromatin. Finally, Cdc48 ATPase with Ufd1 artificially recruited to the centromere of a mini-chromosome (Ch16) induce a loss of Cnp1 from Ch16, leading to an increased rate of chromosome loss. It appears that Cdc48 ATPase, together with its cofactor Ufd1 remove excess Cnp1 from chromatin, likely in a direct manner. This mechanism may play a role in centromere disassembly, a process to eliminate Cnp1 to inactivate the kinetochore function during development, differentiation, and stress response.

Combining single-molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level.

In this work, we have developed an expansion microscopy (ExM) protocol that combines ExM with photoactivated localization microscopy (ExPALM) for yeast cell imaging, and report a robust protocol for single-molecule and expansion microscopy of fission yeast, abbreviated as SExY. Our optimized SExY protocol retains about 50% of the fluorescent protein signal, doubling the amount obtained compared to the original protein retention ExM (proExM) protocol. It allows for a fivefold, highly isotropic expansion of fission yeast cells, which we carefully controlled while optimizing protein yield. We demonstrate the SExY method on several exemplary molecular targets and explicitly introduce low-abundant protein targets (e.g. nuclear proteins such as cbp1 and mis16, and the centromere-specific histone protein cnp1). The SExY protocol optimizations increasing protein yield could be beneficial for many studies, when targeting low abundance proteins, or for studies that rely on genetic labelling for various reasons (e.g. for proteins that cannot be easily targeted by extrinsic staining or in case artefacts introduced by unspecific staining interfere with data quality).

Localization and quantitative distribution of a chromatin structural protein Topoisomerase II on plant chromosome using HVTEM and UHVTEM.

Topoisomerase II (TopoII) is an essential structural protein of the metaphase chromosome. It maintains the axial compaction of chromosomes during metaphase. It is localized at the axial region of chromosomes and accumulates at the centromeric region in metaphase chromosomes. However, little is known about TopoII localization and distribution in plant chromosomes, except for several publications. We used high voltage transmission electron microscopy (HVTEM) and ultra-high voltage transmission electron microscopy (UHVTEM) in conjunction with immunogold labeling and visualization techniques to detect TopoII and investigate its localization, alignment, and density on the barley chromosome at 1.4 nm scale. We found that HVTEM and UHVTEM combined with immunogold labeling is suitable for the detection of structural proteins, including a single molecule of TopoII. This is because the average size of the gold particles for TopoII visualization after silver enhancement is 8.9 ± 3.9 nm, which is well detected. We found that 31,005 TopoII molecules are distributed along the barley chromosomes in an unspecific pattern at the chromosome arms and accumulate specifically at the nucleolus organizer regions (NORs) and centromeric region. The TopoII density were 1.32-fold, 1.58-fold, and 1.36-fold at the terminal region, at the NORs, and the centromeric region, respectively. The findings of TopoII localization in this study support the multiple reported functions of TopoII in the barley metaphase chromosome.

Regional centromere configuration in the fungal pathogens of the Pneumocystis genus.

Centromeres are constricted chromosomal regions that are essential for cell division. In eukaryotes, centromeres display a remarkable architectural and genetic diversity. The basis of centromere-accelerated evolution remains elusive. Here, we focused on Pneumocystis species, a group of mammalian-specific fungal pathogens that form a sister taxon with that of the Schizosaccharomyces pombe, an important genetic model for centromere biology research. Methods allowing reliable continuous culture of Pneumocystis species do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that the Pneumocystis CENP-A ortholog is functionally equivalent to CENP-ACnp1 of S. pombe. Using organisms from a short-term in vitro culture or infected animal models and chromatin immunoprecipitation (ChIP)-Seq, we identified CENP-A bound regions in two Pneumocystis species that diverged ~35 million years ago. Each species has a unique short regional centromere (<10 kb) flanked by heterochromatin in 16-17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. These features suggest an epigenetic specification of centromere function. Analysis of centromeric DNA across multiple Pneumocystis species suggests a vertical transmission at least 100 million years ago. The common ancestry of Pneumocystis and S. pombe centromeres is untraceable at the DNA level, but the overall architectural similarity could be the result of functional constraint for successful chromosomal segregation.IMPORTANCEPneumocystis species offer a suitable genetic system to study centromere evolution in pathogens because of their phylogenetic proximity with the non-pathogenic yeast S. pombe, a popular model for cell biology. We used this system to explore how centromeres have evolved after the divergence of the two clades ~ 460 million years ago. To address this question, we established a protocol combining short-term culture and ChIP-Seq to characterize centromeres in multiple Pneumocystis species. We show that Pneumocystis have short epigenetic centromeres that function differently from those in S. pombe.

Fluorescence complementation-based FRET imaging reveals centromere assembly dynamics.

Visualization of specific molecules and their assembly in real time and space is essential to delineate how cellular dynamics and signaling circuit are orchestrated during cell division cycle. Our recent studies reveal structural insights into human centromere-kinetochore core CCAN complex. Here we introduce a method for optically imaging trimeric and tetrameric protein interactions at nanometer spatial resolution in live cells using fluorescence complementation-based Förster resonance energy transfer (FC-FRET). Complementary fluorescent protein molecules were first used to visualize dimerization followed by FRET measurements. Using FC-FRET, we visualized centromere CENP-SXTW tetramer assembly dynamics in live cells, and dimeric interactions between CENP-TW dimer and kinetochore protein Spc24/25 dimer in dividing cells. We further delineated the interactions of monomeric CENP-T with Spc24/25 dimer in dividing cells. Surprisingly, our analyses revealed critical role of CDK1 kinase activity in the initial recruitment of Spc24/25 by CENP-T. However, interactions between CENP-T and Spc24/25 during chromosome segregation is independent of CDK1. Thus, FC-FRET provides a unique approach to delineate spatiotemporal dynamics of trimerized and tetramerized proteins at nanometer scale and establishes a platform to report the precise regulation of multimeric protein interactions in space and time in live cells.

HJURP is recruited to double-strand break sites and facilitates DNA repair by promoting chromatin reorganization.

HJURP is overexpressed in several cancer types and strongly correlates with patient survival. However, the mechanistic basis underlying the association of HJURP with cancer aggressiveness is not well understood. HJURP promotes the loading of the histone H3 variant, CENP-A, at the centromeric chromatin, epigenetically defining the centromeres and supporting proper chromosome segregation. In addition, HJURP is associated with DNA repair but its function in this process is still scarcely explored. Here, we demonstrate that HJURP is recruited to DSBs through a mechanism requiring chromatin PARylation and promotes epigenetic alterations that favor the execution of DNA repair. Incorporation of HJURP at DSBs promotes turnover of H3K9me3 and HP1, facilitating DNA damage signaling and DSB repair. Moreover, HJURP overexpression in glioma cell lines also affected global structure of heterochromatin independently of DNA damage induction, promoting genome-wide reorganization and assisting DNA damage response. HJURP overexpression therefore extensively alters DNA damage signaling and DSB repair, and also increases radioresistance of glioma cells. Importantly, HJURP expression levels in tumors are also associated with poor response of patients to radiation. Thus, our results enlarge the understanding of HJURP involvement in DNA repair and highlight it as a promising target for the development of adjuvant therapies that sensitize tumor cells to irradiation.

Higher-order protein assembly controls kinetochore formation.

To faithfully segregate chromosomes during vertebrate mitosis, kinetochore-microtubule interactions must be restricted to a single site on each chromosome. Prior work on pair-wise kinetochore protein interactions has been unable to identify the mechanisms that prevent outer kinetochore formation in regions with a low density of CENP-A nucleosomes. To investigate the impact of higher-order assembly on kinetochore formation, we generated oligomers of the inner kinetochore protein CENP-T using two distinct, genetically engineered systems in human cells. Although individual CENP-T molecules interact poorly with outer kinetochore proteins, oligomers that mimic centromeric CENP-T density trigger the robust formation of functional, cytoplasmic kinetochore-like particles. Both in cells and in vitro, each molecule of oligomerized CENP-T recruits substantially higher levels of outer kinetochore components than monomeric CENP-T molecules. Our work suggests that the density dependence of CENP-T restricts outer kinetochore recruitment to centromeres, where densely packed CENP-A recruits a high local concentration of inner kinetochore proteins.

Interaction of histone H4 with Cse4 facilitates conformational changes in Cse4 for its sumoylation and mislocalization.

Mislocalization of overexpressed CENP-A (Cse4 in budding yeast, Cnp1 in fission yeast, CID in flies) contributes to chromosomal instability (CIN) in yeasts, flies, and human cells. Mislocalization of CENP-A is observed in many cancers and this correlates with poor prognosis. Structural mechanisms that contribute to mislocalization of CENP-A are poorly defined. Here, we show that interaction of histone H4 with Cse4 facilitates an in vivo conformational change in Cse4 promoting its mislocalization in budding yeast. We determined that Cse4 Y193A mutant exhibits reduced sumoylation, mislocalization, interaction with histone H4, and lethality in psh1Δ and cdc48-3 strains; all these phenotypes are suppressed by increased gene dosage of histone H4. We developed a new in vivo approach, antibody accessibility (AA) assay, to examine the conformation of Cse4. AA assay showed that wild-type Cse4 with histone H4 is in an 'open' state, while Cse4 Y193A predominantly exhibits a 'closed' state. Increased gene dosage of histone H4 contributes to a shift of Cse4 Y193A to an 'open' state with enhanced sumoylation and mislocalization. We provide molecular insights into how Cse4-H4 interaction changes the conformational state of Cse4 in vivo. These studies advance our understanding for mechanisms that promote mislocalization of CENP-A in human cancers.

DAXX promotes centromeric stability independently of ATRX by preventing the accumulation of R-loop-induced DNA double-stranded breaks.

Maintaining chromatin integrity at the repetitive non-coding DNA sequences underlying centromeres is crucial to prevent replicative stress, DNA breaks and genomic instability. The concerted action of transcriptional repressors, chromatin remodelling complexes and epigenetic factors controls transcription and chromatin structure in these regions. The histone chaperone complex ATRX/DAXX is involved in the establishment and maintenance of centromeric chromatin through the deposition of the histone variant H3.3. ATRX and DAXX have also evolved mutually-independent functions in transcription and chromatin dynamics. Here, using paediatric glioma and pancreatic neuroendocrine tumor cell lines, we identify a novel ATRX-independent function for DAXX in promoting genome stability by preventing transcription-associated R-loop accumulation and DNA double-strand break formation at centromeres. This function of DAXX required its interaction with histone H3.3 but was independent of H3.3 deposition and did not reflect a role in the repression of centromeric transcription. DAXX depletion mobilized BRCA1 at centromeres, in line with BRCA1 role in counteracting centromeric R-loop accumulation. Our results provide novel insights into the mechanisms protecting the human genome from chromosomal instability, as well as potential perspectives in the treatment of cancers with DAXX alterations.

CDK11 facilitates centromeric transcription to maintain centromeric cohesion during mitosis.

Actively-transcribing RNA polymerase (RNAP)II is remained on centromeres to maintain centromeric cohesion during mitosis, although it is largely released from chromosome arms. This pool of RNAPII plays an important role in centromere functions. However, the mechanism of RNAPII retention on mitotic centromeres is poorly understood. We here demonstrate that Cyclin-dependent kinase (Cdk)11 is involved in RNAPII regulation on mitotic centromeres. Consistently, we show that Cdk11 knockdown induces centromeric cohesion defects and decreases Bub1 on kinetochores, but the centromeric cohesion defects are partially attributed to Bub1. Furthermore, Cdk11 knockdown and the expression of its kinase-dead version significantly reduce both RNAPII and elongating RNAPII (pSer2) levels on centromeres and decrease centromeric transcription. Importantly, the overexpression of centromeric α-satellite RNAs fully rescues Cdk11-knockdown defects. These results suggest that the maintenance of centromeric cohesion requires Cdk11-facilitated centromeric transcription. Mechanistically, Cdk11 localizes on centromeres where it binds and phosphorylates RNAPII to promote transcription. Remarkably, mitosis-specific degradation of G2/M Cdk11-p58 recapitulates Cdk11-knockdown defects. Altogether, our findings establish Cdk11 as an important regulator of centromeric transcription and as part of the mechanism for retaining RNAPII on centromeres during mitosis.

A role for ß-1,6- and ß-1,3-glucans in kinetochore function in Saccharomyces cerevisiae.

Chromosome segregation is crucial for the faithful inheritance of DNA to the daughter cells after DNA replication. For this, the kinetochore, a megadalton protein complex, assembles on centromeric chromatin containing the histone H3 variant CENP-A, and provides a physical connection to the microtubules. Here, we report an unanticipated role for enzymes required for ß-1,6- and ß-1,3-glucan biosynthesis in regulating kinetochore function in Saccharomyces cerevisiae. These carbohydrates are the major constituents of the yeast cell wall. We found that the deletion of KRE6, which encodes a glycosylhydrolase/ transglycosidase required for ß-1,6-glucan synthesis, suppressed the centromeric defect of mutations in components of the kinetochore, foremost the NDC80 components Spc24, Spc25, the MIND component Nsl1, and Okp1, a constitutive centromere-associated network protein. Similarly, the absence of Fks1, a ß-1,3-glucan synthase, and Kre11/Trs65, a TRAPPII component, suppressed a mutation in SPC25. Genetic analysis indicates that the reduction of intracellular ß-1,6- and ß-1,3-glucans, rather than the cell wall glucan content, regulates kinetochore function. Furthermore, we found a physical interaction between Kre6 and CENP-A/Cse4 in yeast, suggesting a potential function for Kre6 in glycosylating CENP-A/Cse4 or another kinetochore protein. This work shows a moonlighting function for selected cell wall synthesis proteins in regulating kinetochore assembly, which may provide a mechanism to connect the nutritional status of the cell to cell-cycle progression and chromosome segregation.

The cysteine-rich domain in CENP-A chaperone Scm3HJURP ensures centromere targeting and kinetochore integrity.

Centromeric chromatin plays a crucial role in kinetochore assembly and chromosome segregation. Centromeres are specified through the loading of the histone H3 variant CENP-A by the conserved chaperone Scm3/HJURP. The N-terminus of Scm3/HJURP interacts with CENP-A, while the C-terminus facilitates centromere localization by interacting with the Mis18 holocomplex via a small domain, called the Mis16-binding domain (Mis16-BD) in fission yeast. Fungal Scm3 proteins contain an additional conserved cysteine-rich domain (CYS) of unknown function. Here, we find that CYS binds zinc in vitro and is essential for the localization and function of fission yeast Scm3. Disrupting CYS by deletion or introduction of point mutations within its zinc-binding motif prevents Scm3 centromere localization and compromises kinetochore integrity. Interestingly, CYS alone can localize to the centromere, albeit weakly, but its targeting is greatly enhanced when combined with Mis16-BD. Expressing a truncated protein containing both Mis16-BD and CYS, but lacking the CENP-A binding domain, causes toxicity and is accompanied by considerable chromosome missegregation and kinetochore loss. These effects can be mitigated by mutating the CYS zinc-binding motif. Collectively, our findings establish the essential role of the cysteine-rich domain in fungal Scm3 proteins and provide valuable insights into the mechanism of Scm3 centromere targeting.

Age-dependent loss of cohesion protection in human oocytes.

Aneuploid human eggs (oocytes) are a major cause of infertility, miscarriage, and chromosomal disorders. Such aneuploidies increase greatly as women age, with defective linkages between sister chromatids (cohesion) in meiosis as a common cause. We found that loss of a specific pool of the cohesin protector protein, shugoshin 2 (SGO2), may contribute to this phenomenon. Our data indicate that SGO2 preserves sister chromatid cohesion in meiosis by protecting a "cohesin bridge" between sister chromatids. In human oocytes, SGO2 localizes to both sub-centromere cups and the pericentromeric bridge, which spans the sister chromatid junction. SGO2 normally colocalizes with cohesin; however, in meiosis II oocytes from older women, SGO2 is frequently lost from the pericentromeric bridge and sister chromatid cohesion is weakened. MPS1 and BUB1 kinase activities maintain SGO2 at sub-centromeres and the pericentromeric bridge. Removal of SGO2 throughout meiosis I by MPS1 inhibition reduces cohesion protection, increasing the incidence of single chromatids at meiosis II. Therefore, SGO2 deficiency in human oocytes can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. Our data show that impaired SGO2 localization weakens cohesion integrity and may contribute to the increased incidence of aneuploidy observed in human oocytes with advanced maternal age.

Centromere-proximal suppression of meiotic crossovers in Drosophila is robust to changes in centromere number, repetitive DNA content, and centromere-clustering.

Accurate segregation of homologous chromosomes during meiosis depends on both the presence and the regulated placement of crossovers (COs). The centromere effect, or CO exclusion in pericentromeric regions of the chromosome, is a meiotic CO patterning phenomenon that helps prevent nondisjunction, thereby protecting against chromosomal disorders and other meiotic defects. Despite being identified nearly a century ago, the mechanisms behind this fundamental cellular process remain unknown, with most studies of the Drosophila centromere effect focusing on local influences of the centromere and pericentric heterochromatin. In this study, we sought to investigate whether dosage changes in centromere number and repetitive DNA content affect the strength of the centromere effect, using phenotypic recombination mapping. Additionally, we studied the effects of repetitive DNA function on centromere effect strength using satellite DNA-binding protein mutants displaying defective centromere-clustering in meiotic nuclei. Despite what previous studies suggest, our results show that the Drosophila centromere effect is robust to changes in centromere number, repetitive DNA content, as well as repetitive DNA function. Our study suggests that the centromere effect is unlikely to be spatially controlled, providing novel insight into the mechanisms behind the Drosophila centromere effect.

Defining a core configuration for human centromeres during mitosis.

The centromere components cohesin, CENP-A, and centromeric DNA are essential for biorientation of sister chromatids on the mitotic spindle and accurate sister chromatid segregation. Insight into the 3D organization of centromere components would help resolve how centromeres function on the mitotic spindle. We use ChIP-seq and super-resolution microscopy with single particle averaging to examine the geometry of essential centromeric components on human chromosomes. Both modalities suggest cohesin is enriched at pericentromeric DNA. CENP-A localizes to a subset of the α-satellite DNA, with clusters separated by ~562 nm and a perpendicular intervening ~190 nM wide axis of cohesin in metaphase chromosomes. Differently sized α-satellite arrays achieve a similar core structure. Here we present a working model for a common core configuration of essential centromeric components that includes CENP-A nucleosomes, α-satellite DNA and pericentromeric cohesion. This configuration helps reconcile how centromeres function and serves as a foundation to add components of the chromosome segregation machinery.