Centromere Research Articles

Variation of homologous recombination in Populus tomentosa with different genotypes.

Homologous recombination is an important source of biological genetic variation. Limited by detection methods, there are only a few reports on the homologous recombination in high plants and its product - heteroduplex DNA (hDNA). In the present study, applying the strategy of detecting hDNA by constructing populations from inhibited post-meiotic segregation, two hybrid triploid populations were constructed from two maternal parents inPopulus tomentosa by inhibiting post-meiotic segregation. One hundred and ten simple sequence repeat (SSR) markers were used to study the occurrence and variation of hDNA on nine chromosomes inP. tomentosa with different genotypes. The results showed that the frequencies of hDNA between two female parents inP. tomentosa ranged from 8.5% to 87.2%. The hDNA frequency was positively correlated to the distance from the centromere, but the average hDNA frequency on a chromosome had no correlation with the chromosome length. One to 3 times recombination events were detected on most chromosomes, and only a few four- or five-times recombination events were detected. The overall frequencies of hDNA on the same chromosome in two genotypic individuals were roughly similar, while the hDNA frequencies varied greatly at specific SSR loci. Compared withTacamahaca poplar hybrid,P. pseudo-simonii × P. nigra 'Zheyin3#', detection of homologous recombination times and the frequency and location of hDNA were largely different. This study is the first to describe the characteristics and variations of homologous recombination inP. tomentosa with two different genotypes, which will provide valuable insights for exploring the characteristics and variations of homologous recombination among interspecies and intraspecies in higher plant.

CBIO-24. KINETOCHORE MISREGULATION IN GLIOBLASTOMA AND OTHER CANCERS

Abstract Aneuploidy is a hallmark of most late stage cancers and contributes to tumor cell evolution, invasiveness, therapy resistance, and recurrence. A major cause of aneuploidy is lagging anaphase chromosomes that have merotelic kinetochore (KT) attachments in mitosis. In this scenario, a KT (i.e., the proteinaceous structure assembled onto centromeres in mitosis) is bound to microtubules (MTs) emanating from both spindle poles, causing chromosome mis-segregation. “Background” chromosome missegregation likely contributes to the initiation of multiple cancers, including glioblastoma (GBM), where chr 7 gain and chr 10 loss are early events. However, we have found that in GBM and transformed cells activation of the RAS signaling pathway significantly increases lagging anaphase chromosomes, and, as a result, aneuploidy and tumor heterogeneity. However, the underlying mechanism of RAS-induced chromosome instability has remained elusive. Here we present one possible cause: MAPK-driven KT stress. The hallmarks of KT stress include chronic KT-MT attachment instability in mitosis, dramatic changes in KT morphology, and increases in merotelic KT attachments and chromosome segregation errors. KT stress is lethal to cancer cells. However, stressed cells survive by relying on BUB1B/BubR1 activity to strengthen KT-MT attachments. We first observed KT stress in GBM patient isolates, where ~60–70% display its hallmarks. We subsequently found that RAS or MEK activity is sufficient to induce KT stress in both CNS and non-CNS derived cell types. We propose that KT stress is caused by aberrant mitotic RAS/MAPK activity, which likely targets one or more KT proteins resulting in KT-MT attachment defects and chromosome instability. In addition, Dr. Jun Zhu’s lab has also created a computational classifier that can identify KT stressed cells and tumors based on the expression of 838 genes associated with KT stress, which can be also used to identify orthogonal therapeutic sensitivities. We will present these findings as well at this meeting.

Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins.

Mitotic spindle microtubules (MTs) undergo continuous poleward flux, whose driving force and function in humans remain unclear. Here, we combined loss-of-function screenings with analysis of MT-dynamics in human cells to investigate the molecular mechanisms underlying MT-flux. We report that kinesin-7/CENP-E at kinetochores (KTs) is the predominant driver of MT-flux in early prometaphase, while kinesin-4/KIF4A on chromosome arms facilitates MT-flux during late prometaphase and metaphase. Both these activities work in coordination with kinesin-5/EG5 and kinesin-12/KIF15, and our data suggest that the MT-flux driving force is transmitted from non-KT-MTs to KT-MTs by the MT couplers HSET and NuMA. Additionally, we found that the MT-flux rate correlates with spindle length, and this correlation depends on the establishment of stable end-on KT-MT attachments. Strikingly, we find that MT-flux is required to regulate spindle length by counteracting kinesin 13/MCAK-dependent MT-depolymerization. Thus, our study unveils the long-sought mechanism of MT-flux in human cells as relying on the coordinated action of four kinesins to compensate for MT-depolymerization and regulate spindle length.

The whole is greater than the sum of its parts: at the intersection of order, disorder, and kinetochore function.

The kinetochore (KT) field has matured tremendously since Earnshaw first identified CENP-A, CENP-B, and CENP-C [1,2]. In the past 35 years, the accumulation of knowledge has included: defining the parts list, identifying epistatic networks of interdependence within the parts list, understanding the spatial organization of subcomplexes into a massive structure - hundreds of megadaltons in size, and dissecting the functions of the KT in its entirety as well as of its individual parts. Like nearly all cell and molecular biology fields, the structure-function paradigm has been foundational to advances in the KT field. A point nicely highlighted by the fact that we are at the precipice of the in vitro reconstitution of a functional KT holo complex. Yet conventional notions of structure cannot provide a complete picture of the KT especially since it contains an abundance of unstructured or intrinsically disordered constituents. The combination of structured and disordered proteins within the KT results in an assembled system that is functionally greater than the sum of its parts.

A Condensed View of the Chromosome Passenger Complex.

The inner centromere is a region on the mitotic chromosome that serves as a platform for mitotic signaling and possesses unique biophysical properties that enable it to withstand relatively large pulling forces that are generated by kinetochores (KTs) during chromosome segregation. The chromosomal passenger complex (CPC) localizes to and is the key regulator of inner centromere organization and function during mitosis. Recently, we demonstrated that in addition to its kinase and histone code-reading activities, the CPC also can undergo liquid-liquid phase separation (LLPS) and proposed that the inner centromere is a membraneless organelle scaffolded by the CPC. In this perspective, we explore mechanisms that can allow the formation and dissolution of this membraneless body. The cell-cycle-regulated spatially defined assembly and disassembly of the CPC condensate at the inner centromere can reveal general principles about how histone modifications control chromatin-bound membraneless organelles. We further explore how the ability of the CPC to undergo LLPS may contribute to the organization and function of the inner centromere during mitosis.

Kinetochore proteins suppress neuronal microtubule dynamics and promote dendrite regeneration.

Kinetochores connect centromeric chromatin to spindle microtubules during mitosis. Neurons are postmitotic, so it was surprising to identify transcripts of structural kinetochore (KT) proteins and regulatory chromosome passenger complex (CPC) and spindle assembly checkpoint (SAC) proteins in Drosophila neurons after dendrite injury. To test whether these proteins function during dendrite regeneration, postmitotic RNA interference (RNAi) was performed and dendrites or axons were removed using laser microsurgery. Reduction of KT, CPC, and SAC proteins decreased dendrite regeneration without affecting axon regeneration. To understand whether neuronal functions of these proteins rely on microtubules, we analyzed microtubule behavior in uninjured neurons. The number of growing plus, but not minus, ends increased in dendrites with reduced KT, CPC, and SAC proteins, while axonal microtubules were unaffected. Increased dendritic microtubule dynamics was independent of dual leucine zipper kinase (DLK)-mediated stress but was rescued by concurrent reduction of γ-tubulin, the core microtubule nucleation protein. Reduction of γ-tubulin also rescued dendrite regeneration in backgrounds containing kinetochore RNAi transgenes. We conclude that kinetochore proteins function postmitotically in neurons to suppress dendritic microtubule dynamics by inhibiting nucleation.

Effects of malleable kinetochore morphology on measurements of intrakinetochore tension: Nature of intrakinetochore tension

Effects of malleable kinetochore morphology on measurements of intrakinetochore tension: Nature of intrakinetochore tension

Gametophytic Abortion in Heterozygotes but Not in Homozygotes: Implied Chromosome Rearrangement during T-DNA Insertion at the ASF1 Locus in Arabidopsis.

T-DNA insertional mutations in Arabidopsis genes have conferred huge benefits to the research community, greatly facilitating gene function analyses. However, the insertion process can cause chromosomal rearrangements. Here, we show an example of a likely rearrangement following T-DNA insertion in the Anti-Silencing Function 1B (ASF1B) gene locus on Arabidopsis chromosome 5, so that the phenotype was not relevant to the gene of interest, ASF1B. ASF1 is a histone H3/H4 chaperone involved in chromatin remodeling in the sporophyte and during reproduction. Plants that were homozygous for mutant alleles asf1a or asf1b were developmentally normal. However, following self-fertilization of double heterozygotes (ASF1A/asf1a ASF1B/asf1b, hereafter AaBb), defects were visible in both male and female gametes. Half of the AaBb and aaBb ovules displayed arrested embryo sacs with functional megaspore identity. Similarly, half of the AaBb and aaBb pollen grains showed centromere defects, resulting in pollen abortion at the bi-cellular stage of the male gametophyte. However, inheritance of the mutant allele in a given gamete did not solely determine the abortion phenotype. Introducing functional ASF1B failed to rescue the AaBb- and aaBb- mediated abortion, suggesting that heterozygosity in the ASF1B gene causes gametophytic defects, rather than the loss of ASF1. The presence of reproductive defects in heterozygous mutants but not in homozygotes, and the characteristic all-or-nothing pollen viability within tetrads, were both indicative of commonly-observed T-DNA-mediated translocation activity for this allele. Our observations reinforce the importance of complementation tests in assigning gene function using reverse genetics.

SKA1/2/3 serves as a biomarker for poor prognosis in human lung adenocarcinoma.

BackgroundSpindle and kinetochore associated complex subunit 1/2/3 (SKA1/2/3), which stabilized spindle microtubules attaching to kinetochore (KT) in the middle stage of mitosis, were dysregulated, and closely related to prognosis in several malignant tumors. Nevertheless, the potential clinical value of SKA1/2/3, especially in terms of prognosis and development of NSCLC, had not been fully elucidated.MethodsONCOMINE, GEPIA, UALCAN, TCGA, STRING and other databases were used to analyze the expression of SKA1/2/3 in patients with lung adenocarcinoma (LUAD) and its clinical value, and to explore the possible regulatory mechanism of SKA in the occurrence and development of LUAD.ResultsIn patients with LUAD, SKA1/2/3 mRNA expression level was significantly up-regulated, and AUC was 0.9558, 0.7034 and 0.9775, respectively. Increased SKA 1/2/3 expression was associated with smoking, tissue typing, and poor prognosis in LUAD patients. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) showed that SKA1/2/3 was mainly enriched in DNA replication, cell cycle, homologous recombination, p53 signaling pathway, etc. Hub genes in protein-protein interactions are CDK1, BUB1, CCNA2, CDC20, CCNB2, CCNB1, BUB1B, AURKB, TOP2A and MAD2L1. Hub gene expression in LUAD is increased, and its increased expression is related to poor prognosis of LUAD patients. Finally, the expression of SKA1/2/3 and its correlation with clinicopathological features were verified in 30 clinical LUAD samples.ConclusionsSKA1/2/3 may serve as a potential prognostic biomarker and target for LUAD. In addition, SKA 1/2/3 may affect the prognosis of LUAD through DNA replication, cell cycle, homologous recombination and p53 signaling pathway.

Modulatory Effects of Vitamin C on Enrofloxacin Induced Chromosome Damage and AgNOR Counts in Chick Bone Marrow Nuclei

Quinolones are broad spectrum antibiotics and are widely used in veterinary practice. But because of continuous improvement in their bactericidal and metabolic properties, it resulted in an increased amount of genotoxicity among animals. Vitamin C is a well-known antioxidant and a free radical scavenger and protects the cell from endogenous oxidative DNA damage. The aim of the study was to find the modulatory effect of Vitamin C against the enrofloxacin induced chromosomal aberrations and nuclear damage. The toxicity includes a significant decrease in mitotic index and increases in the number of chromosomal aberrations like break, deletion, centromeric attenuation, centric fusion and AgNOR counts in chicken bone marrow nuclei. The combined administration of enrofloxacin and Vitamin C resulted has markedly increased the mitotic index and reduced the chromosomal aberrations and AgNOR counts in the bone marrow cells.

Cis- and Trans-chromosomal Interactions Define Pericentric Boundaries in the Absence of Conventional Heterochromatin.

The diploid budding yeast Candida albicans harbors unique CENPA-rich 3- to 5-kb regions that form the centromere (CEN) core on each of its eight chromosomes. The epigenetic nature of these CENs does not permit the stabilization of a functional kinetochore on an exogenously introduced CEN plasmid. The flexible nature of such centromeric chromatin is exemplified by the reversible silencing of a transgene upon its integration into the CENPA-bound region. The lack of a conventional heterochromatin machinery and the absence of defined boundaries of CENPA chromatin makes the process of CEN specification in this organism elusive. Additionally, upon native CEN deletion, C. albicans can efficiently activate neocentromeres proximal to the native CEN locus, hinting at the importance of CEN-proximal regions. In this study, we examine this CEN-proximity effect and identify factors for CEN specification in C. albicans We exploit a counterselection assay to isolate cells that can silence a transgene when integrated into the CEN-flanking regions. We show that the frequency of reversible silencing of the transgene decreases from the central core of CEN7 to its peripheral regions. Using publicly available C. albicans high-throughput chromosome conformation capture data, we identify a 25-kb region centering on the CENPA-bound core that acts as CEN-flanking compact chromatin (CFCC). Cis- and trans-chromosomal interactions associated with the CFCC spatially segregates it from bulk chromatin. We further show that neocentromere activation on chromosome 7 occurs within this specified region. Hence, this study identifies a specialized CEN-proximal domain that specifies and restricts the centromeric activity to a unique region.

Cell cycle-dependent association of polo kinase Cdc5 with CENP-A contributes to faithful chromosome segregation in budding yeast.

Evolutionarily conserved polo-like kinase, Cdc5 (Plk1 in humans), associates with kinetochores during mitosis; however, the role of cell cycle–dependent centromeric (CEN) association of Cdc5 and its substrates that exclusively localize to the kinetochore have not been characterized. Here we report that evolutionarily conserved CEN histone H3 variant, Cse4 (CENP-A in humans), is a substrate of Cdc5, and that the cell cycle–regulated association of Cse4 with Cdc5 is required for cell growth. Cdc5 contributes to Cse4 phosphorylation in vivo and interacts with Cse4 in mitotic cells. Mass spectrometry analysis of in vitro kinase assays showed that Cdc5 phosphorylates nine serine residues clustered within the N-terminus of Cse4. Strains with cse4-9SA exhibit increased errors in chromosome segregation, reduced levels of CEN-associated Mif2 and Mcd1/Scc1 when combined with a deletion of MCM21. Moreover, the loss of Cdc5 from the CEN chromatin contributes to defects in kinetochore integrity and reduction in CEN-associated Cse4. The cell cycle–regulated association of Cdc5 with Cse4 is essential for cell viability as constitutive association of Cdc5 with Cse4 at the kinetochore leads to growth defects. In summary, our results have defined a role for Cdc5-mediated Cse4 phosphorylation in faithful chromosome segregation.

Centromere and Pericentromere Transcription: Roles and Regulation … in Sickness and in Health.

The chromosomal loci known as centromeres (CEN) mediate the equal distribution of the duplicated genome between both daughter cells. Specifically, centromeres recruit a protein complex named the kinetochore, that bi-orients the replicated chromosome pairs to the mitotic or meiotic spindle structure. The paired chromosomes are then separated, and the individual chromosomes segregate in opposite direction along the regressing spindle into each daughter cell. Erroneous kinetochore assembly or activity produces aneuploid cells that contain an abnormal number of chromosomes. Aneuploidy may incite cell death, developmental defects (including genetic syndromes), and cancer (>90% of all cancer cells are aneuploid). While kinetochores and their activities have been preserved through evolution, the CEN DNA sequences have not. Hence, to be recognized as sites for kinetochore assembly, CEN display conserved structural themes. In addition, CEN nucleosomes enclose a CEN-exclusive variant of histone H3, named CENP-A, and carry distinct epigenetic labels on CENP-A and the other CEN histone proteins. Through the cell cycle, CEN are transcribed into non-coding RNAs. After subsequent processing, they become key components of the CEN chromatin by marking the CEN locus and by stably anchoring the CEN-binding kinetochore proteins. CEN transcription is tightly regulated, of low intensity, and essential for differentiation and development. Under- or overexpression of CEN transcripts, as documented for myriad cancers, provoke chromosome missegregation and aneuploidy. CEN are genetically stable and fully competent only when they are insulated from the surrounding, pericentromeric chromatin, which must be silenced. We will review CEN transcription and its contribution to faithful kinetochore function. We will further discuss how pericentromeric chromatin is silenced by RNA processing and transcriptionally repressive chromatin marks. We will report on the transcriptional misregulation of (peri)centromeres during stress, natural aging, and disease and reflect on whether their transcripts can serve as future diagnostic tools and anti-cancer targets in the clinic.

Positive Antinucleolar Antibodies on Direct Immunofluorescence of Lesional Skin of a Patient With Limited Systemic Sclerosis

To the Editor: Limited systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. Circulant anticentromere antibody is usually positive.1 Antinucleolar antibodies are positive less frequently (15%–40%),2 having prognostic significance on the overall course of the patients. We present a case of limited SSc with negative serologic test for extractable nuclear antigens in which direct immunofluorescence revealed positive antinuclear antibodies (ANA) with nucleolar pattern. A 54-year-old man was referred to the dermatology department because of a 5-year history of multiple asymptomatic telangiectasias on the face and neckline. Clinical examination revealed the presence of sclerodactyly and multiple scars of ulcers in the fingertips that appeared when the patient worked in a frigorific chamber. Capillaroscopy showed predominance of areas of angiogenesis and some small avascular areas. The patient complained about mild interphalangeal arthralgias without inflammatory changes. Unspecific gastrointestinal symptoms and dyspnea were also present. High-resolution chest computed tomography showed mild pulmonary fibrosis predominantly in peripheral areas and bases of the lungs. Echocardiography confirmed the clinical suspicion of pulmonary hypertension, and bosentan therapy was initiated. Circulant ANA resulted positive at 1:1280 dilution. The determinations of extractable nuclear antigens, antibodies against parietal cells, against smooth muscle, and against double-stranded DNA were all negative. A skin biopsy of the neckline area with telangiectasia revealed lymphocytic perifolliculitis, with no other alterations. Direct immunofluorescence study (DIF) performed on lesional skin showed the presence of IgG ANA with a nucleolar pattern (Fig. 1).FIGURE 1.: Direct immunofluorescence on a skin biopsy from an area with telangiectasia in the patient's neckline. IgG resulted positive with a nucleolar pattern (×100).Circulating autoantibodies are found in as many as 95% of patients with SSc and carry greater weight in the newly proposed ACR/EULAR classification system. Centromere (CEN) proteins (P), topoisomerase I, and RNA polymerase III autoantibodies closely reflect patterns of organ involvement and disease progression.3 Circulating anti-Th/To type antinucleolar antibodies are detectable mainly by immunoprecipitation, which is not performed routinely in most clinical laboratories. Its positivity is related to limited skin involvement and frequent fibrosis of internal organs (interstitial lung disease, pericarditis, and renal involvement). Frequency of negative circulating antibodies with positive DIF findings is largely unknown because the last is not routinely performed. In the study by Kulthanan et al,4 a subgroup of patients with SSc presented positive DIF findings in 77% of the cases with the dermoepidermal junction and epidermal nuclei as the most common site, and of these patients, 45% presented negative serum ANA. Shibeshi et al5 reported a DIF positivity rate of 25,7% in 63 patients with SSc, with only one case with no circulating autoantibodies at all, and another with positive serum ANA but no characteristic antibodies of SSc. Antinucleolar pattern has been rarely reported in DIF of patients with SSc. Only 6 cases were found in the literature, all of them with high ANA titles. As these cases date from the 1980s, only circulating anti-ribonucleoprotein was performed, resulting positive in one of the patients.6,7 Our patient had ANA in peripheral blood, but more specific antibodies were not detected (antinucleolar, Scl70, or anticentromere). However, a skin biopsy with DIF was allowed to demonstrate the fixation of antibodies with a nucleolar pattern. Nucleolar pattern ANA in DIF of a skin biopsy is an unusual finding. We believe this technique can help to confirm clinical diagnosis and assess prognosis on individual patients.

Centromeric signaling proteins boost G1 cyclin degradation and modulate cell size in budding yeast.

Cell size scales with ploidy in a great range of eukaryotes, but the underlying mechanisms remain unknown. Using various orthogonal single-cell approaches, we show that cell size increases linearly with centromere (CEN) copy number in budding yeast. This effect is due to a G1 delay mediated by increased degradation of Cln3, the most upstream G1 cyclin acting at Start, and specific centromeric signaling proteins, namely Mad3 and Bub3. Mad3 binds both Cln3 and Cdc4, the adaptor component of the Skp1/Cul1/F-box (SCF) complex that targets Cln3 for degradation, these interactions being essential for the CEN-dosage dependent effects on cell size. Our results reveal a pathway that modulates cell size as a function of CEN number, and we speculate that, in cooperation with other CEN-independent mechanisms, it could assist the cell to attain efficient mass/ploidy ratios.

Who Needs This Junk, or Genomic Dark Matter.

Centromeres (CEN), pericentromeric regions (periCEN), and subtelomeric regions (subTel) comprise the areas of constitutive heterochromatin (HChr). Tandem repeats (TRs or satellite DNA) are the main components of HChr forming no less than 10% of the mouse and human genome. HChr is assembled within distinct structures in the interphase nuclei of many species - chromocenters. In this review, the main classes of HChr repeat sequences are considered in the order of their number increase in the sequencing reads of the mouse chromocenters (ChrmC). TRs comprise ~70% of ChrmC occupying the first place. Non-LTR (-long terminal repeat) retroposons (mainly LINE, long interspersed nuclear element) are the next (~11%), and endogenous retroviruses (ERV; LTR-containing) are in the third position (~9%). HChr is not enriched with ERV in comparison with the whole genome, but there are differences in distribution of certain elements: while MaLR-like elements (ERV3) are dominant in the whole genome, intracisternal A-particles and corresponding LTR (ERV2) are prevalent in HChr. Most of LINE in ChrmC is represented by the 2-kb fragment at the end of the 2nd open reading frame and its flanking regions. Almost all tandem repeats classified as CEN or periCEN are contained in ChrmC. Our previous classification revealed 60 new mouse TR families with 29 of them being absent in ChrmC, which indicates their location on chromosome arms. TR transcription is necessary for maintenance of heterochromatic status of the HChr genome part. A burst of TR transcription is especially important in embryogenesis and other cases of radical changes in the cell program, including carcinogenesis. The recently discovered mechanism of epigenetic regulation with noncoding sequences transcripts, long noncoding RNA, and its role in embryogenesis and pluripotency maintenance is discussed.

Novel ESCRT functions at kinetochores.

Novel ESCRT functions at kinetochores.

HDAC2/3 binding and deacetylation of BubR1 initiates spindle assembly checkpoint silencing.

BubR1 acetylation is essential in spindle assembly checkpoint (SAC) signaling. Here we show that BubR1 deacetylation is a signal that initiates mitotic exit. Sustained BubR1 acetylation arrests the cells in metaphase, although chromosome congression is achieved. BubR1 deacetylation was coordinated with dephosphorylation in mitotic exit, suggesting the presence of a coordinated acetylation-phosphorylation code in mitotic signaling. Histone deacetylase (HDAC) 2 and 3 bound to acetylated BubR1 exclusively in mitosis and led to the polyubiquitination of BubR1. Subsequent degradation of BubR1 resulted in the disassembly of the mitotic checkpoint complex. Importantly, BRCA2 was required for HDAC2/3 association with acetylated BubR1 in nocodazole (Noc)-arrested cells. Plk1, PP2A, P300/CBP-associated factor (PCAF) and BubR1 were found in the mitotic BRCA2 complex, suggesting that BRCA2 acts as a signaling scaffold for BubR1 modification. Furthermore, we show that Plk1 is required for BRCA2 to localize at the prometaphase kinetochore (KT). Inhibition of Plk1 resulted in the loss of BRCA2 from the KT, and so did PCAF, consistent with the loss of BubR1 acetylation. Concordantly, BRCA2-dysfunctional cells exhibited resistance to trichostatin A, which was restored when BRCA2 was introduced. That loss of Brca2 conferred resistance to various HDAC inhibitors was corroborated by the experiments in mouse pancreatic organoids. These results suggest that the BRCA2-BubR1 acetylation-deacetylation pathway is an important decision-making point for the HDAC inhibitor response. Taken together, BRCA2 is a signaling platform for BubR1, and BubR1 deacetylation is a cue for SAC silencing.

The fission yeast nucleoporin Alm1 is required for proteasomal degradation of kinetochore components.

Kinetochores (KTs) are large multiprotein complexes that constitute the interface between centromeric chromatin and the mitotic spindle during chromosome segregation. In spite of their essential role, little is known about how centromeres and KTs are assembled and how their precise stoichiometry is regulated. In this study, we show that the nuclear pore basket component Alm1 is required to maintain both the proteasome and its anchor, Cut8, at the nuclear envelope, which in turn regulates proteostasis of certain inner KT components. Consistently, alm1-deleted cells show increased levels of KT proteins, including CENP-CCnp3, spindle assembly checkpoint activation, and chromosome segregation defects. Our data demonstrate a novel function of the nucleoporin Alm1 in proteasome localization required for KT homeostasis.

The tobacco carcinogen NNK disturbs mitotic chromosome alignment by interrupting p53 targeting to the centrosome

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent risk factor among tobacco-related carcinogens in lung cancer progression and outcomes. Although genetic mutations and chromosome instability have been detected in NNK-induced lung tumors, the oncogenic mechanisms of NNK are not fully understood. Here, we show that NNK increases chromosomal instability by disrupting spindle microtubule (MT) attachment to the kinetochore (KT) and spindle dynamics. Mechanistically, NNK blocks the targeting of p53 to the centrosome during mitosis, leading to chromosome alignment defects in metaphase. Therefore, lung cancer cells with wild-type p53, such as A594 and H226B, are more resistant to the NNK treatment than p53-mutant lung cancer cells, such as A1299 and H226Br. Although NNK does not affect the levels or transcriptional activity of p53, the reduction of the p53 level at the centrosome exacerbates the NNK-induced chromosome alignment defect in A549 and H226B cells. Therefore, p53 protects against NNK-induced chromosome instability by modulating the function of centrosome-localized p53 and not by modulating transcriptional activity. We conclude that NNK may increase the risk of lung cancer progression and poorer outcomes in patients with p53 mutations by perturbing proper mitotic progression and chromosome integrity.