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Protein kinases that phosphorylate histones are ideally-placed to influence the behavior of chromosomes during cell division. Indeed, a number of conserved histone phosphorylation events occur prominently during mitosis and meiosis in most eukaryotes, including on histone H3 at threonine-3 (H3T3ph). At least two kinases, Haspin and VRK1 (NHK-1/ballchen in Drosophila), have been proposed to carry out this modification. Phosphorylation of H3 by Haspin has defined roles in mitosis, but the significance of VRK1 activity towards histones in dividing cells has been unclear. Here, using in vitro kinase assays, KiPIK screening, RNA interference, and CRISPR/Cas9 approaches, we were unable to substantiate a direct role for VRK1, or its paralogue VRK2, in the phosphorylation of threonine-3 or serine-10 of Histone H3 in mitosis, although loss of VRK1 did slow cell proliferation. We conclude that the role of VRKs, and their more recently identified association with neuromuscular disease and importance in cancers of the nervous system, are unlikely to involve mitotic histone kinase activity. In contrast, Haspin is required to generate H3T3ph during mitosis., (© 2022. The Author(s).)

Aurora B kinase plays an essential role in chromosome bi-orientation, which is a prerequisite for equal segregation of chromosomes during mitosis. However, it remains largely unclear whether centromere-localized Aurora B is required for faithful chromosome segregation. Here we show that histone H3 Thr-3 phosphorylation (H3pT3) and H2A Thr-120 phosphorylation (H2ApT120) can independently recruit Aurora B. Disrupting H3pT3-mediated localization of Aurora B at the inner centromere impedes the decline in H2ApT120 during metaphase and causes H2ApT120-dependent accumulation of Aurora B at the kinetochore-proximal centromere. Consequently, silencing of the spindle assembly checkpoint (SAC) is delayed, whereas the fidelity of chromosome segregation is negligibly affected. Further eliminating an H2ApT120-dependent pool of Aurora B restores proper timing for SAC silencing but increases chromosome missegregation. Our data indicate that H2ApT120-mediated localization of Aurora B compensates for the loss of an H3pT3-dependent pool of Aurora B to correct improper kinetochore-microtubule attachments. This study provides important insights into how centromeric Aurora B regulates SAC and kinetochore attachment to microtubules to ensure error-free chromosome segregation., (© 2019 Zhejiang University.)

The chromosomal passenger complex (CPC) is a master regulator of mitosis. CPC consists of inner centromere protein (INCENP), Survivin, Borealin, and the kinase Aurora B and plays key roles in regulating kinetochore-microtubule attachments and spindle assembly checkpoint signaling. However, the role of CPC in sister chromatid cohesion, mediated by the cohesin complex, remains incompletely understood. Here, we show that Aurora B kinase activity contributes to centromeric cohesion protection partly through promoting kinetochore localization of the kinase Bub1. Interestingly, disrupting the interaction of INCENP with heterochromatin protein 1 (HP1) in HeLa cells selectively weakens cohesion at mitotic centromeres without detectably reducing the kinase activity of Aurora B. Thus, through this INCENP-HP1 interaction, the CPC also protects centromeric cohesion independently of Aurora B kinase activity. Moreover, the requirement for the INCENP-HP1 interaction in centromeric cohesion protection can be bypassed by tethering HP1 to centromeres or by depleting the cohesin release factor Wapl. We provide further evidence suggesting that the INCENP-HP1 interaction protects centromeric cohesion by promoting the centromere localization of Haspin, a protein kinase that antagonizes Wapl activity at centromeres. Taken together, this study identifies Aurora B kinase activity-dependent and -independent roles for the CPC in regulating centromeric cohesion during mitosis in human cells., (© 2019 Yi et al.)

The inner centromere region of a mitotic chromosome critically regulates sister chromatid cohesion and kinetochore-microtubule attachments. However, the molecular mechanism underlying inner centromere assembly remains elusive. Here, using CRISPR/Cas9-based gene editing in HeLa cells, we disrupted the interaction of Shugoshin 1 (Sgo1) with histone H2A phosphorylated on Thr-120 (H2ApT120) to selectively release Sgo1 from mitotic centromeres. Interestingly, cells expressing the H2ApT120-binding defective mutant of Sgo1 have an elevated rate of chromosome missegregation accompanied by weakened centromeric cohesion and decreased centromere accumulation of the chromosomal passenger complex (CPC), an integral part of the inner centromere and a key player in the correction of erroneous kinetochore-microtubule attachments. When artificially tethered to centromeres, a Sgo1 mutant defective in binding protein phosphatase 2A (PP2A) is not able to support proper centromeric cohesion and CPC accumulation, indicating that the Sgo1-PP2A interaction is essential for the integrity of mitotic centromeres. We further provide evidence indicating that Sgo1 protects centromeric cohesin to create a binding site for the histone H3-associated protein kinase Haspin, which not only inhibits the cohesin release factor Wapl and thereby strengthens centromeric cohesion but also phosphorylates histone H3 at Thr-3 to position CPC at inner centromeres. Taken together, our findings reveal a positive feedback-based mechanism that ensures proper assembly of the functional inner centromere during mitosis. They further suggest a causal link between centromeric cohesion defects and chromosomal instability in cancer cells., (© 2019 Liang et al.)

The key mitotic regulator Polo-like kinase 1 (Plk1) is activated during G2 phase by Aurora A kinase (AurkA)-mediated phosphorylation of its activation loop, which is important for timely mitotic entry. The mechanism for Plk1 activation remains incompletely understood. Here, we report that the activation of Plk1 requires WAC, a WW domain-containing adaptor protein with a coiled-coil region that predominantly localizes to the nucleus in interphase. Cyclin-dependent kinase 1 (Cdk1) phosphorylates WAC, priming its direct interaction with the polo-box domain of Plk1. Knockdown of WAC compromises Plk1 activity and delays mitotic entry. These defects are rescued by exogenous expression of wild-type WAC, but not the Plk1-binding-deficient mutant. WAC also binds AurkA and can enhance Plk1 phosphorylation by AurkA in vitro. Taken together, these results indicate an important role for WAC in promoting Plk1 activation and the timely entry into mitosis., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Sister-chromatid cohesion, mediated by the multi-subunit cohesin complex, must be precisely regulated to prevent chromosome mis-segregation. In prophase and prometaphase, whereas the bulk of cohesin on chromosome arms is removed by its antagonist Wapl, cohesin at centromeres is retained to ensure chromosome biorientation until anaphase onset. It remains incompletely understood how centromeric cohesin is protected against Wapl in mitosis. Here we show that the mitotic histone kinase Haspin binds to the cohesin regulatory subunit Pds5B through a conserved YGA/R motif in its non-catalytic N terminus, which is similar to the recently reported YSR-motif-dependent binding of Wapl to Pds5B. Knockout of Haspin or disruption of Haspin-Pds5B interaction causes weakened centromeric cohesion and premature chromatid separation, which can be reverted by centromeric targeting of a N-terminal short fragment of Haspin containing the Pds5B-binding motif or by prevention of Wapl-dependent cohesin removal. Conversely, excessive Haspin capable of binding Pds5B displaces Wapl from Pds5B and suppresses Wapl activity, and it largely bypasses the Wapl antagonist Sgo1 for cohesion protection. Taken together, these data indicate that the Haspin-Pds5B interaction is required to ensure proper sister-chromatid cohesion, most likely through antagonizing Wapl-mediated cohesin release from mitotic centromeres., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Histone modifications coordinate the chromatin localization of key regulatory factors in mitosis. For example, mitotic phosphorylation of Histone H3 threonine-3 (H3T3ph) by Haspin creates a binding site for the chromosomal passenger complex (CPC). However, how these histone modifications are spatiotemporally controlled during the cell cycle is unclear. Here we show that Plk1 binds to Haspin in a Cdk1-phosphorylation-dependent manner. Reducing Plk1 activity decreases the phosphorylation of Haspin and inhibits H3T3ph, particularly in prophase, suggesting that Plk1 is required for initial activation of Haspin in early mitosis. These studies demonstrate that Plk1 can positively regulate CPC recruitment in mitosis.

The roles of post-translational histone modifications in regulating transcription and DNA damage have been widely studied and discussed. Although mitotic histone marks, particularly phosphorylation, were discovered four decades ago, their roles in mitosis have been outlined only in the past few years. Here we aim to provide an integrated view of how histone modifications act as 'countermarks', 'landmarks', and 'bookmarks' to displace, recruit, and 'remember' the location of regulatory proteins during and shortly after mitosis. These capabilities allow histone marks to help downregulate interphase functions such as transcription during mitosis, to facilitate chromatin events required to accomplish chromosome segregation, and to contribute to the maintenance of epigenetic states through mitosis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Haspin phosphorylates histone H3 at Thr3 (H3T3ph) during mitosis [1, 2], providing a chromatin binding site for the chromosomal passenger complex (CPC) at centromeres to regulate chromosome segregation [3-5]. H3T3ph becomes increasingly focused at inner centromeres during prometaphase [1, 2], but little is known about how its level or location and the consequent chromosomal localization of the CPC are regulated. In addition, CPC binding to shugoshin proteins contributes to centromeric Aurora B localization [5, 6]. Recruitment of the shugoshins to centromeres requires the phosphorylation of histone H2A at Thr120 (H2AT120ph) by the kinetochore kinase Bub1 [7], but the molecular basis for the collaboration of this pathway with H3T3ph has been unclear. Here, we show that Aurora B phosphorylates Haspin to promote generation of H3T3ph and that Aurora B kinase activity is required for normal chromosomal localization of the CPC, indicating an intimate linkage between Aurora B and Haspin functions in mitosis. We propose that Aurora B activity triggers a CPC-Haspin-H3T3ph feedback loop that promotes generation of H3T3ph on chromatin. We also provide evidence that the Bub1-shugoshin-CPC pathway supplies a signal that boosts the CPC-Haspin-H3T3ph feedback loop specifically at centromeres to produce the well-known accumulation of the CPC in these regions., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Histone methylation patterns are correlated with eukaryotic gene transcription. High-affinity binding of the plant homeodomain (PHD) of TFIID subunit TAF3 to trimethylated lysine-4 of histone H3 (H3K4me3) is involved in promoter recruitment of this basal transcription factor. Here, we show that for transcription activation the PHD of TAF3 can be replaced by PHDs of other high-affinity H3K4me3 binders. Interestingly, H3K4me3 binding of TFIID and the TAF3-PHD is decreased by phosphorylation of the adjacent threonine residue (H3T3), which coincides with mitotic inhibition of transcription. Ectopic expression of the H3T3 kinase haspin repressed TAF3-mediated transcription of endogenous and of reporter genes and decreased TFIID association with chromatin. Conversely, immunofluorescence and live-cell microscopy studies showed an increased association of TFIID with mitotic chromosomes upon haspin knockdown. Based on our observations, we propose that a histone H3 phospho-methyl switch regulates TFIID-mediated transcription during mitotic progression of the cell cycle.

Aurora B is a component of the chromosomal passenger complex (CPC) required for correct spindle-kinetochore attachments during chromosome segregation and for cytokinesis. The chromatin factors that recruit the CPC to centromeres are unknown, however. Here we show that phosphorylation of histone H3 threonine 3 (H3T3ph) by Haspin is necessary for CPC accumulation at centromeres and that the CPC subunit Survivin binds directly to H3T3ph. A nonbinding Survivin-D70A/D71A mutant does not support centromeric CPC concentration, and both Haspin depletion and Survivin-D70A/D71A mutation diminish centromere localization of the kinesin MCAK and the mitotic checkpoint response to taxol. Survivin-D70A/D71A mutation and microinjection of H3T3ph-specific antibody both compromise centromeric Aurora B functions but do not prevent cytokinesis. Therefore, H3T3ph generated by Haspin positions the CPC at centromeres to regulate selected targets of Aurora B during mitosis.

Error-free mitosis depends on accurate chromosome attachment to spindle microtubules, which is monitored by the spindle assembly checkpoint (SAC) signaling. As an upstream factor of SAC, the precise and dynamic kinetochore localization of Mps1 kinase is critical for initiating and silencing SAC signaling. However, the underlying molecular mechanism remains elusive. Here, we demonstrated that the multisite interactions between Mps1 and Ndc80 complex (Ndc80C) govern Mps1 kinetochore targeting. Importantly, we identified direct interaction between Mps1 tetratricopeptide repeat domain and Ndc80C. We further identified that Mps1 C-terminal fragment, which contains the protein kinase domain and C-tail, enhances Mps1 kinetochore localization. Mechanistically, Mps1 C-terminal fragment mediates its dimerization. Perturbation of C-tail attenuates the kinetochore targeting and activity of Mps1, leading to aberrant mitosis due to compromised SAC function. Taken together, our study highlights the importance of Mps1 dimerization and multisite interactions with Ndc80C in enabling responsive SAC signaling. [ABSTRACT FROM AUTHOR]

Sister chromatid cohesion plays a key role in ensuring precise chromosome segregation during mitosis, which is mediated by the multisubunit cohesin complex. However, the molecular regulation of cohesin subunits stability remains unclear. Here, we show that NudCL2 (NudC-like protein 2) is essential for the stability of cohesin subunits by regulating Hsp90 ATPase activity in mammalian cells. Depletion of NudCL2 induces mitotic defects and premature sister chromatid separation and destabilizes cohesin subunits that interact with NudCL2. Similar defects are also observed upon inhibition of Hsp90 ATPase activity. Interestingly, ectopic expression of Hsp90 efficiently rescues the protein instability and functional deficiency of cohesin induced by NudCL2 depletion, but not vice versa. Moreover, NudCL2 not only binds to Hsp90, but also significantly modulates Hsp90 ATPase activity and promotes the chaperone function of Hsp90. Taken together, these data suggest that NudCL2 is a previously undescribed Hsp90 cochaperone to modulate sister chromatid cohesion by stabilizing cohesin subunits, providing a hitherto unrecognized mechanism that is crucial for faithful chromosome segregation during mitosis. [ABSTRACT FROM AUTHOR]

Chromosome segregation in mitosis requires the removal of catenation between sister chromatids. Timely decatenation of sister DNAs at mitotic centromeres by topoisomerase IIα (TOP2A) is crucial to maintain genomic stability. The chromatin factors that recruit TOP2A to centromeres during mitosis remain unknown. Here, we show that histone H2A Thr‐120 phosphorylation (H2ApT120), a modification generated by the mitotic kinase Bub1, is necessary and sufficient for the centromeric localization of TOP2A. Phosphorylation at residue‐120 enhances histone H2A binding to TOP2A in vitro. The C‐gate and the extreme C‐terminal region are important for H2ApT120‐dependent localization of TOP2A at centromeres. Preventing H2ApT120‐mediated accumulation of TOP2A at mitotic centromeres interferes with sister chromatid disjunction, as evidenced by increased frequency of anaphase ultra‐fine bridges (UFBs) that contain catenated DNA. Tethering TOP2A to centromeres bypasses the requirement for H2ApT120 in suppressing anaphase UFBs. These results demonstrate that H2ApT120 acts as a landmark that recruits TOP2A to mitotic centromeres to decatenate sister DNAs. Our study reveals a fundamental role for histone phosphorylation in resolving centromere DNA entanglements and safeguarding genomic stability during mitosis. Synopsis: Chromosome segregation requires timely decatenation of sister chromatids at mitotic centromeres by topoisomerase IIα (TOP2A). Histone H2A threonine‐120 phosphorylation by Bub1 recruits TOP2A to mitotic centromeres to decatenate sister DNAs, thereby safeguarding genomic stability. Bub1‐mediated histone H2A threonine‐120 phosphorylation (H2ApT120) during mitosis is necessary and sufficient for the centromeric localization of TOP2A.Phosphorylation at Thr120 enhances histone H2A binding to TOP2A in vitro.The C‐gate and the extreme C‐terminal region of TOP2A are important for its H2ApT120‐dependent localization to mitotic centromeres.H2ApT120‐mediated localization of TOP2A at centromeres facilitates the timely decatenation of sister chromatids. [ABSTRACT FROM AUTHOR]