The right place at the right time: Aurora B kinase localization to centromeres and kinetochores.

The fidelity of chromosome segregation during mitosis is intimately linked to the function of kinetochores, which are large protein complexes assembled at sites of centromeric heterochromatin on mitotic chromosomes. These key "orchestrators" of mitosis physically connect chromosomes to spindle microtubules and transduce forces through these connections to congress chromosomes and silence the spindle assembly checkpoint. Kinetochore-microtubule attachments are highly regulated to ensure that incorrect attachments are not prematurely stabilized, but instead released and corrected. The kinase activity of the centromeric protein Aurora B is required for kinetochore-microtubule destabilization during mitosis, but how the kinase acts on outer kinetochore substrates to selectively destabilize immature and erroneous attachments remains debated. Here, we review recent literature that sheds light on how Aurora B kinase is recruited to both centromeres and kinetochores and discuss possible mechanisms for how kinase interactions with substrates at distinct regions of mitotic chromosomes are regulated.

Cohesin Removal Reprograms Gene Expression upon Mitotic Entry.

As cells enter mitosis, the genome is restructured to facilitate chromosome segregation, accompanied by dramatic changes in gene expression. However, the mechanisms that underlie mitotic transcriptional regulation are unclear. In contrast to transcribed genes, centromere regions retain transcriptionally active RNA polymerase II (Pol II) in mitosis. Here, we demonstrate that chromatin-bound cohesin is necessary to retain elongating Pol II at centromeres. We find that WAPL-mediated removal of cohesin from chromosome arms during prophase is required for the dissociation of Pol II and nascent transcripts, and failure of this process dramatically alters mitotic gene expression. Removal of cohesin/Pol II from chromosome arms in prophase is important for accurate chromosome segregation and normal activation of gene expression in G1. We propose that prophase cohesin removal is a key step in reprogramming gene expression as cells transition from G2 through mitosis to G1.

Aurora B kinase is recruited to multiple discrete kinetochore and centromere regions in human cells.

Aurora B kinase has a critical role in regulating attachments between kinetochores and spindle microtubules during mitosis. Early in mitosis, kinase activity at kinetochores is high to promote attachment turnover, and in later mitosis, activity decreases to ensure attachment stabilization. Aurora B localizes prominently to inner centromeres, and a population of the kinase is also detected at kinetochores. How Aurora B is recruited to and evicted from these regions to regulate kinetochore-microtubule attachments remains unclear. Here, we identified and investigated discrete populations of Aurora B at the centromere/kinetochore region. An inner centromere pool is recruited by Haspin phosphorylation of histone H3, and a kinetochore-proximal outer centromere pool is recruited by Bub1 phosphorylation of histone H2A. Finally, a third pool resides ~20 nm outside of the inner kinetochore protein CENP-C in early mitosis and does not require either the Bub1/pH2A/Sgo1 or Haspin/pH3 pathway for localization or activity. Our results suggest that distinct molecular pathways are responsible for Aurora B recruitment to centromeres and kinetochores.

Self-organization of parS centromeres by the ParB CTP hydrolase.

ParABS systems facilitate chromosome segregation and plasmid partitioning in bacteria and archaea. ParB protein binds centromeric parS DNA sequences and spreads to flanking DNA. We show that ParB is an enzyme that hydrolyzes cytidine triphosphate (CTP) to cytidine diphosphate (CDP). parS DNA stimulates cooperative CTP binding by ParB and CTP hydrolysis. A nucleotide cocrystal structure elucidates the catalytic center of the dimerization-dependent ParB CTPase. Single-molecule imaging and biochemical assays recapitulate features of ParB spreading from parS in the presence but not absence of CTP. These findings suggest that centromeres assemble by self-loading of ParB DNA sliding clamps at parS ParB CTPase is not related to known nucleotide hydrolases and might be a promising target for developing new classes of antibiotics.

Division of labour between PP2A-B56 isoforms at the centromere and kinetochore.

PP2A-B56 is a serine/threonine phosphatase complex that regulates several major mitotic processes, including sister chromatid cohesion, kinetochore-microtubule attachment and the spindle assembly checkpoint. We show here that these key functions are divided between different B56 isoforms that localise to either the centromere or kinetochore. The centromeric isoforms rely on a specific interaction with Sgo2, whereas the kinetochore isoforms bind preferentially to BubR1 and other proteins containing an LxxIxE motif. In addition to these selective binding partners, Sgo1 helps to anchor PP2A-B56 at both locations: it collaborates with BubR1 to maintain B56 at the kinetochore and it helps to preserve the Sgo2/B56 complex at the centromere. A series of chimaeras were generated to map the critical region in B56 down to a small C-terminal loop that regulates the key interactions and defines B56 localisation. Together, this study describes how different PP2A-B56 complexes utilise isoform-specific interactions to control distinct processes during mitosis.

The N-Terminal Domain of cGAS Determines Preferential Association with Centromeric DNA and Innate Immune Activation in the Nucleus.

Cytosolic DNA activates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS), an innate immune sensor pivotal in anti-microbial defense, senescence, auto-immunity, and cancer. cGAS is considered to be a sequence-independent DNA sensor with limited access to nuclear DNA because of compartmentalization. However, the nuclear envelope is a dynamic barrier, and cGAS is present in the nucleus. Here, we identify determinants of nuclear cGAS localization and activation. We show that nuclear-localized cGAS synthesizes cGAMP and induces innate immune activation of dendritic cells, although cGAMP levels are 200-fold lower than following transfection with exogenous DNA. Using cGAS ChIP-seq and a GFP-cGAS knockin mouse, we find nuclear cGAS enrichment on centromeric satellite DNA, confirmed by imaging, and to a lesser extent on LINE elements. The non-enzymatic N-terminal domain of cGAS determines nucleo-cytoplasmic localization, enrichment on centromeres, and activation of nuclear-localized cGAS. These results reveal a preferential functional association of nuclear cGAS with centromeres.

The multifaceted allosteric regulation of Aurora kinase A.

The protein kinase Aurora A (AurA) is essential for the formation of bipolar mitotic spindles in all eukaryotic organisms. During spindle assembly, AurA is activated through two different pathways operating at centrosomes and on spindle microtubules. Recent studies have revealed that these pathways operate quite differently at the molecular level, activating AurA through multifaceted changes to the structure and dynamics of the kinase domain. These advances provide an intimate atomic-level view of the finely tuned regulatory control operating in protein kinases, revealing mechanisms of allosteric cooperativity that provide graded levels of regulatory control, and a previously unanticipated mechanism for kinase activation by phosphorylation on the activation loop. Here, I review these advances in our understanding of AurA function, and discuss their implications for the use of allosteric small molecule inhibitors to address recently discovered roles of AurA in neuroblastoma, prostate cancer and melanoma.

A mitosis-specific and R loop-driven ATR pathway promotes faithful chromosome segregation.

The ataxia telangiectasia mutated and Rad3-related (ATR) kinase is crucial for DNA damage and replication stress responses. Here, we describe an unexpected role of ATR in mitosis. Acute inhibition or degradation of ATR in mitosis induces whole-chromosome missegregation. The effect of ATR ablation is not due to altered cyclin-dependent kinase 1 (CDK1) activity, DNA damage responses, or unscheduled DNA synthesis but to loss of an ATR function at centromeres. In mitosis, ATR localizes to centromeres through Aurora A-regulated association with centromere protein F (CENP-F), allowing ATR to engage replication protein A (RPA)-coated centromeric R loops. As ATR is activated at centromeres, it stimulates Aurora B through Chk1, preventing formation of lagging chromosomes. Thus, a mitosis-specific and R loop-driven ATR pathway acts at centromeres to promote faithful chromosome segregation, revealing functions of R loops and ATR in suppressing chromosome instability.

An acetyltransferase-independent function of Eso1 regulates centromere cohesion.

Eukaryotes contain three essential Structural Maintenance of Chromosomes (SMC) complexes: cohesin, condensin, and Smc5/6. Cohesin forms a ring-shaped structure that embraces sister chromatids to promote their cohesion. The cohesiveness of cohesin is promoted by acetylation of N-terminal lysines of the Smc3 subunit by the acetyltransferases Eco1 in Saccharomyces cerevisiae and the homologue, Eso1, in Schizosaccharomyces pombe. In both yeasts, these acetyltransferases are essential for cell viability. However, whereas nonacetylatable Smc3 mutants are lethal in S. cerevisiae, they are not in S. pombe We show that the lethality of a temperature-sensitive allele of eso1 (eso1-H17) is due to activation of the spindle assembly checkpoint (SAC) and is associated with premature centromere separation. The lack of cohesion at the centromeres does not correlate with Psm3 acetylation or cohesin levels at the centromeres, but is associated ith significantly reduced recruitment of the cohesin regulator Pds5. The SAC activation in this context is dependent on Smc5/6 function, which is required to remove cohesin from chromosome arms but not centromeres. The mitotic defects caused by Smc5/6 and Eso1 dysfunction are cosuppressed in double mutants. This identifies a novel function (or functions) for Eso1 and Smc5/6 at centromeres and extends the functional relationships between these SMC complexes.

Polo kinase Cdc5 associates with centromeres to facilitate the removal of centromeric cohesin during mitosis.

Sister chromatid cohesion is essential for tension-sensing mechanisms that monitor bipolar attachment of replicated chromatids in metaphase. Cohesion is mediated by the association of cohesins along the length of sister chromatid arms. In contrast, centromeric cohesin generates intrastrand cohesion and sister centromeres, while highly cohesin enriched, are separated by >800 nm at metaphase in yeast. Removal of cohesin is necessary for sister chromatid separation during anaphase, and this is regulated by evolutionarily conserved polo-like kinase (Cdc5 in yeast, Plk1 in humans). Here we address how high levels of cohesins at centromeric chromatin are removed. Cdc5 associates with centromeric chromatin and cohesin-associated regions. Maximum enrichment of Cdc5 in centromeric chromatin occurs during the metaphase-to-anaphase transition and coincides with the removal of chromosome-associated cohesin. Cdc5 interacts with cohesin in vivo, and cohesin is required for association of Cdc5 at centromeric chromatin. Cohesin removal from centromeric chromatin requires Cdc5 but removal at distal chromosomal arm sites does not. Our results define a novel role for Cdc5 in regulating removal of centromeric cohesins and faithful chromosome segregation.

The activity regulation of the mitotic centromere-associated kinesin by Polo-like kinase 1.

The mitotic centromere-associated kinesin (MCAK), a potent microtubule depolymerase, is involved in regulating microtubule dynamics. The activity and subcellular localization of MCAK are tightly regulated by key mitotic kinases, such as Polo-like kinase 1 (Plk1) by phosphorylating multiple residues in MCAK. Since Plk1 phosphorylates very often different residues of substrates at different stages, we have dissected individual phosphorylation of MCAK by Plk1 and characterized its function in more depth. We have recently shown that S621 in MCAK is the major phosphorylation site of Plk1, which is responsible for regulating MCAK's degradation by promoting the association of MCAK with APC/CCdc20. In the present study, we have addressed another two residues phosphorylated by Plk1, namely S632/S633 in the C-terminus of MCAK. Our data suggest that Plk1 phosphorylates S632/S633 and regulates its catalytic activity in mitosis. This phosphorylation is required for proper spindle assembly during early phases of mitosis. The subsequent dephosphorylation of S632/S633 might be necessary to timely align the chromosomes onto the metaphase plate. Therefore, our studies suggest new mechanisms by which Plk1 regulates MCAK: the degradation of MCAK is controlled by Plk1 phosphorylation on S621, whereas its activity is modulated by Plk1 phosphorylation on S632/S633 in mitosis.

EB1 enables spindle microtubules to regulate centromeric recruitment of Aurora B.

The Aurora B kinase coordinates kinetochore-microtubule attachments with spindle checkpoint signaling on each mitotic chromosome. We find that EB1, a microtubule plus end-tracking protein, is required to enrich Aurora B at inner centromeres in a microtubule-dependent manner. This regulates phosphorylation of both kinetochore and chromatin substrates. EB1 regulates the histone phosphorylation marks (histone H2A phospho-Thr120 and histone H3 phospho-Thr3) that localize Aurora B. The chromosomal passenger complex containing Aurora B can be found on a subset of spindle microtubules that exist near prometaphase kinetochores, known as preformed K-fibers (kinetochore fibers). Our data suggest that EB1 enables the spindle microtubules to regulate the phosphorylation of kinetochores through recruitment of the Aurora B kinase.

Overexpression of Aurora-C interferes with the spindle checkpoint by promoting the degradation of Aurora-B.

The chromosomal passenger complex (CPC) plays a pivotal role in controlling accurate chromosome segregation and cytokinesis during cell division. Aurora-B, one of the chromosomal passenger proteins, is important for the mitotic spindle assembly checkpoint (SAC). Previous reports noted that Aurora-C is predominantly expressed in male germ cells and has the same subcellular localization as Aurora-B. Increasing evidence indicates that Aurora-C is overexpressed in many somatic cancers, although its function is uncertain. Our previous study showed that the aberrant expression of Aurora-C increases the tumorigenicity of cancer cells. Here, we demonstrate that overexpressed Aurora-C displaces the centromeric localization of CPCs, including INCENP, survivin, and Aurora-B. When cells were treated with nocodazole to turn on SAC, both the Aurora-B protein stability and kinase activity were affected by overexpressed Aurora-C. As a result, the activation of spindle checkpoint protein, BubR1, and phosphorylation of histone H3 and MCAK were also eliminated in Aurora-C-overexpressing cells. Thus, our results suggest that aberrantly expressed Aurora-C in somatic cancer cells may impair SAC by displacing the centromeric localization of CPCs.

Pds5B is required for cohesion establishment and Aurora B accumulation at centromeres.

Cohesin mediates sister chromatid cohesion and contributes to the organization of interphase chromatin through DNA looping. In vertebrate somatic cells, cohesin consists of Smc1, Smc3, Rad21, and either SA1 or SA2. Three additional factors Pds5, Wapl, and Sororin bind to cohesin and modulate its dynamic association with chromatin. There are two Pds5 proteins in vertebrates, Pds5A and Pds5B, but their functional specificity remains unclear. Here, we demonstrate that Pds5 proteins are essential for cohesion establishment by allowing Smc3 acetylation by the cohesin acetyl transferases (CoATs) Esco1/2 and binding of Sororin. While both proteins contribute to telomere and arm cohesion, Pds5B is specifically required for centromeric cohesion. Furthermore, reduced accumulation of Aurora B at the inner centromere region in cells lacking Pds5B impairs its error correction function, promoting chromosome mis-segregation and aneuploidy. Our work supports a model in which the composition and function of cohesin complexes differs between different chromosomal regions.

Bloom's syndrome and PICH helicases cooperate with topoisomerase IIα in centromere disjunction before anaphase.

Centromeres are specialized chromosome domains that control chromosome segregation during mitosis, but little is known about the mechanisms underlying the maintenance of their integrity. Centromeric ultrafine anaphase bridges are physiological DNA structures thought to contain unresolved DNA catenations between the centromeres separating during anaphase. BLM and PICH helicases colocalize at these ultrafine anaphase bridges and promote their resolution. As PICH is detectable at centromeres from prometaphase onwards, we hypothesized that BLM might also be located at centromeres and that the two proteins might cooperate to resolve DNA catenations before the onset of anaphase. Using immunofluorescence analyses, we demonstrated the recruitment of BLM to centromeres from G2 phase to mitosis. With a combination of fluorescence in situ hybridization, electron microscopy, RNA interference, chromosome spreads and chromatin immunoprecipitation, we showed that both BLM-deficient and PICH-deficient prometaphase cells displayed changes in centromere structure. These cells also had a higher frequency of centromeric non disjunction in the absence of cohesin, suggesting the persistence of catenations. Both proteins were required for the correct recruitment to the centromere of active topoisomerase IIα, an enzyme specialized in the catenation/decatenation process. These observations reveal the existence of a functional relationship between BLM, PICH and topoisomerase IIα in the centromere decatenation process. They indicate that the higher frequency of centromeric ultrafine anaphase bridges in BLM-deficient cells and in cells treated with topoisomerase IIα inhibitors is probably due not only to unresolved physiological ultrafine anaphase bridges, but also to newly formed ultrafine anaphase bridges. We suggest that BLM and PICH cooperate in rendering centromeric catenates accessible to topoisomerase IIα, thereby facilitating correct centromere disjunction and preventing the formation of supernumerary centromeric ultrafine anaphase bridges.

Aurora B dynamics at centromeres create a diffusion-based phosphorylation gradient.

Aurora B kinase is essential for successful cell division and regulates spindle assembly and kinetochore-microtubule interactions. The kinase localizes to the inner centromere until anaphase, but many of its substrates have distinct localizations, for example on chromosome arms and at kinetochores. Furthermore, substrate phosphorylation depends on distance from the kinase. How the kinase reaches substrates at a distance and how spatial phosphorylation patterns are determined are unknown. In this paper, we show that a phosphorylation gradient is produced by Aurora B concentration and activation at centromeres and release and diffusion to reach substrates at a distance. Kinase concentration, either at centromeres or at another chromosomal site, is necessary for activity globally. By experimentally manipulating dynamic exchange at centromeres, we demonstrate that the kinase reaches its substrates by diffusion. We also directly observe, using a fluorescence resonance energy transfer-based biosensor, phosphorylation spreading from centromeres after kinase activation. We propose that Aurora B dynamics and diffusion from the inner centromere create spatial information to regulate cell division.

Centromere-associated topoisomerase activity in bloodstream form Trypanosoma brucei.

Topoisomerase-II accumulates at centromeres during prometaphase, where it resolves the DNA catenations that represent the last link between sister chromatids. Previously, using approaches including etoposide-mediated topoisomerase-II cleavage, we mapped centromeric domains in trypanosomes, early branching eukaryotes in which chromosome segregation is poorly understood. Here, we show that in bloodstream form Trypanosoma brucei, RNAi-mediated depletion of topoisomerase-IIα, but not topoisomerase-IIß, results in the abolition of centromere-localized activity and is lethal. Both phenotypes can be rescued by expression of the corresponding enzyme from T. cruzi. Therefore, processes which govern centromere-specific topoisomerase-II accumulation/activation have been functionally conserved within trypanosomes, despite the long evolutionary separation of these species and differences in centromeric DNA organization. The variable carboxyl terminal region of topoisomerase-II has a major role in regulating biological function. We therefore generated T. brucei lines expressing T. cruzi topoisomerase-II truncated at the carboxyl terminus and examined activity at centromeres after the RNAi-mediated depletion of the endogenous enzyme. A region necessary for nuclear localization was delineated to six residues. In other organisms, sumoylation of topoisomerase-II has been shown to be necessary for regulated chromosome segregation. Evidence that we present here suggests that sumoylation of the T. brucei enzyme is not required for centromere-specific cleavage activity.

Micronucleus assay and labeling of centromeres with FISH technique.

The cytokinesis-block micronucleus (CBMN) assay has since many years been applied for in vitro genotoxicity testing and biomonitoring of human populations. The standard in vitro/ex vivo micronucleus test is usually performed on human lymphocytes and has become a comprehensive method to assess genetic damage, cytostasis, and cytotoxicity. The predictive association between the frequency of micronuclei (MN) in cytokinesis-blocked lymphocytes and cancer risk has recently been demonstrated. MN frequencies can be influenced by inherited (or acquired) genetic polymorphisms (or mutations) in genes responsible for the metabolic activation, detoxification of clastogens, and for the fidelity of DNA replication. An important advantage of the CBMN assay is its ability to detect both clastogenic and aneugenic events by centromere and kinetochore identification and contributes to the high sensitivity of the method. The objective of the present chapter is to review the mechanisms of induction of micronuclei, the method of the micronucleus assay and its combination with centromeric labeling in the FISH technique. Furthermore, an overview is given of recent results obtained by our laboratory by the application of the micronucleus assay.

MCAK is present at centromeres, midspindle and chiasmata and involved in silencing of the spindle assembly checkpoint in mammalian oocytes.

Mitotic centromere-associated kinesin (MCAK) is an ATP-dependent microtubule (MT) depolymerase regulated by Aurora kinase (AURK) phosphorylation and implicated in resolution of improper MT attachments in mitosis. Distribution of MCAK was studied in oocyte maturation by anti-MCAK antibody, anti-tubulin antibody, anti-AURKB antibody and anti-centromere antibody (ACA) and by the expression of MCAK-enhanced green fluorescent protein fusion protein in maturing mouse oocytes. Function was assessed by knockdown of MCAK and Mad2, by inhibiting AURK or the proteasome, by live imaging with polarization microscope and by chromosomal analysis. The results show that MCAK is transiently recruited to the nucleus and transits to spindle poles, ACA-positive domains and chiasmata at prometaphase I. At metaphase I and II, it is present at centrosomes and centromeres next to AURKB and checkpoint proteins Mad2 and BubR1. It is retained at centromeres at telophase I and also at the midbody. Knockdown of MCAK causes a delay in chromosome congression but does not prevent bipolar spindle assembly. MCAK knockdown also induces a meiosis I arrest, which is overcome by knockdown of Mad2 resulting in chiasma resolution, chromosome separation, formation of aberrant meiosis II spindles and increased hypoploidy. In conclusion, MCAK appears to possess a unique distribution and function in oocyte maturation. It is required for meiotic progression from meiosis I to meiosis II associated with silencing of the spindle assembly checkpoint. Alterations in abundance and activity of MCAK, as implicated in aged oocytes, may therefore contribute to the loss of control of cell cycle and chromosome behaviour, thus increasing risk for errors in chromosome segregation and aneuploidy.