Publications by authors named "Linda Clijsters"

11 Publications

  • Page 1 of 1

Molecular mechanisms of APC/C release from spindle assembly checkpoint inhibition by APC/C SUMOylation.

Cell Rep 2021 Mar;34(13):108929

MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK. Electronic address:

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that controls cell cycle transitions. Its regulation by the spindle assembly checkpoint (SAC) is coordinated with the attachment of sister chromatids to the mitotic spindle. APC/C SUMOylation on APC4 ensures timely anaphase onset and chromosome segregation. To understand the structural and functional consequences of APC/C SUMOylation, we reconstituted SUMOylated APC/C for electron cryo-microscopy and biochemical analyses. SUMOylation of the APC/C causes a substantial rearrangement of the WHB domain of APC/C's cullin subunit (APC2). Although APC/C SUMOylation results in a modest impact on normal APC/C activity, repositioning APC2 reduces the affinity of APC/C for the mitotic checkpoint complex (MCC), the effector of the SAC. This attenuates MCC-mediated suppression of APC/C activity, allowing for more efficient ubiquitination of APC/C substrates in the presence of the MCC. Thus, SUMOylation stimulates the reactivation of APC/C when the SAC is silenced, contributing to timely anaphase onset.
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http://dx.doi.org/10.1016/j.celrep.2021.108929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8028313PMC
March 2021

Two Distinct E2F Transcriptional Modules Drive Cell Cycles and Differentiation.

Cell Rep 2019 06 23;27(12):3547-3560.e5. Epub 2019 May 23.

Department of Biochemistry and Molecular Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA. Electronic address:

Orchestrating cell-cycle-dependent mRNA oscillations is critical to cell proliferation in multicellular organisms. Even though our understanding of cell-cycle-regulated transcription has improved significantly over the last three decades, the mechanisms remain untested in vivo. Unbiased transcriptomic profiling of G, G-S, and S-G-M sorted cells from FUCCI mouse embryos suggested a central role for E2Fs in the control of cell-cycle-dependent gene expression. The analysis of gene expression and E2F-tagged knockin mice with tissue imaging and deep-learning tools suggested that post-transcriptional mechanisms universally coordinate the nuclear accumulation of E2F activators (E2F3A) and canonical (E2F4) and atypical (E2F8) repressors during the cell cycle in vivo. In summary, we mapped the spatiotemporal expression of sentinel E2F activators and canonical and atypical repressors at the single-cell level in vivo and propose that two distinct E2F modules relay the control of gene expression in cells actively cycling (E2F3A-8-4) and exiting the cycle (E2F3A-4) during mammalian development.
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http://dx.doi.org/10.1016/j.celrep.2019.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6673649PMC
June 2019

Cyclin F Controls Cell-Cycle Transcriptional Outputs by Directing the Degradation of the Three Activator E2Fs.

Mol Cell 2019 06 23;74(6):1264-1277.e7. Epub 2019 May 23.

Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA; Perlmutter NYU Cancer Center, New York University School of Medicine, New York, NY 10016, USA; Howard Hughes Medical Institute, New York University School of Medicine, New York, NY 10016, USA. Electronic address:

E2F1, E2F2, and E2F3A, the three activators of the E2F family of transcription factors, are key regulators of the G1/S transition, promoting transcription of hundreds of genes critical for cell-cycle progression. We found that during late S and in G2, the degradation of all three activator E2Fs is controlled by cyclin F, the substrate receptor of 1 of 69 human SCF ubiquitin ligase complexes. E2F1, E2F2, and E2F3A interact with the cyclin box of cyclin F via their conserved N-terminal cyclin binding motifs. In the short term, E2F mutants unable to bind cyclin F remain stable throughout the cell cycle, induce unscheduled transcription in G2 and mitosis, and promote faster entry into the next S phase. However, in the long term, they impair cell fitness. We propose that by restricting E2F activity to the S phase, cyclin F controls one of the main and most critical transcriptional engines of the cell cycle.
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http://dx.doi.org/10.1016/j.molcel.2019.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588466PMC
June 2019

Cyclin F-Mediated Degradation of SLBP Limits H2A.X Accumulation and Apoptosis upon Genotoxic Stress in G2.

Mol Cell 2016 11 20;64(3):507-519. Epub 2016 Oct 20.

Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 522 First Avenue, SRB 1107, New York, NY 10016, USA; Perlmutter NYU Cancer Center, New York University School of Medicine, 522 First Avenue, SRB 1107, New York, NY 10016, USA; Howard Hughes Medical Institute, 522 First Avenue, SRB 1107, New York, NY 10016, USA. Electronic address:

SLBP (stem-loop binding protein) is a highly conserved factor necessary for the processing, translation, and degradation of H2AFX and canonical histone mRNAs. We identified the F-box protein cyclin F, a substrate recognition subunit of an SCF (Skp1-Cul1-F-box protein) complex, as the G2 ubiquitin ligase for SLBP. SLBP interacts with cyclin F via an atypical CY motif, and mutation of this motif prevents SLBP degradation in G2. Expression of an SLBP stable mutant results in increased loading of H2AFX mRNA onto polyribosomes, resulting in increased expression of H2A.X (encoded by H2AFX). Upon genotoxic stress in G2, high levels of H2A.X lead to persistent γH2A.X signaling, high levels of H2A.X phosphorylated on Tyr142, high levels of p53, and induction of apoptosis. We propose that cyclin F co-evolved with the appearance of stem-loops in vertebrate H2AFX mRNA to mediate SLBP degradation, thereby limiting H2A.X synthesis and cell death upon genotoxic stress.
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http://dx.doi.org/10.1016/j.molcel.2016.09.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5097008PMC
November 2016

Inefficient degradation of cyclin B1 re-activates the spindle checkpoint right after sister chromatid disjunction.

Cell Cycle 2014 ;13(15):2370-8

a Division of Cell Biology I (B5) and Division of Molecular Carcinogenesis (B7); The Netherlands Cancer Institute (NKI-AvL); Amsterdam, The Netherlands.

Sister chromatid separation creates a sudden loss of tension on kinetochores, which could, in principle, re-activate the spindle checkpoint in anaphase. This so-called "anaphase problem" is probably avoided by timely inactivation of cyclin B1-Cdk1, which may prevent the spindle tension sensing Aurora B kinase from destabilizing kinetochore-microtubule interactions as they lose tension in anaphase. However, exactly how spindle checkpoint re-activation is prevented remains unclear. Here, we investigated how different degrees of cyclin B1 stabilization affected the spindle checkpoint in metaphase and anaphase. Cells expressing a strongly stabilized (R42A) mutant of cyclin B1 degraded APC/C(Cdc20) substrates normally, showing that checkpoint release was not inhibited by high cyclin B1-Cdk1 activity. However, after this initial wave of APC/C(Cdc20) activity, the spindle checkpoint returned in cells with uncohesed sister chromatids. Expression of a lysine mutant of cyclin B1 that is degraded only slightly inefficiently allowed a normal metaphase-to-anaphase transition. Strikingly, however, the spindle checkpoint returned in cells that had not degraded the cyclin B1 mutant 10-15 min after anaphase onset. When cyclin B1 remained in late anaphase, cytokinesis stalled, and translocation of INCENP from separated sister chromatids to the spindle midzone was blocked. This late anaphase arrest required the activity of Aurora B and Mps1. In conclusion, our results reveal that complete removal of cyclin B1 is essential to prevent the return of the spindle checkpoint following sister chromatid disjunction. Speculatively, increasing activity of APC/C(Cdc20) in late anaphase helps to keep cyclin B1 levels low.
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http://dx.doi.org/10.4161/cc.29336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4128882PMC
August 2015

PIP-box-mediated degradation prohibits re-accumulation of Cdc6 during S phase.

J Cell Sci 2014 Mar 16;127(Pt 6):1336-45. Epub 2014 Jan 16.

Division of Cell Biology (B5) and Division of Molecular Carcinogenesis (B7), The Netherlands Cancer Institute (NKI-AVL), Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.

Cdc6 and Cdt1 initiate DNA replication licensing when cells exit mitosis. In cycling cells, Cdc6 is efficiently degraded from anaphase onwards as a result of APC/C-Cdh1 activity. When APC/C-Cdh1 is switched off again, at the end of G1 phase, Cdc6 could thus re-accumulate, risking the re-licensing of DNA as long as Cdt1 is present. Here, we carefully investigated the dynamics of Cdt1 and Cdc6 in cycling cells. We reveal a novel APC/C-Cdh1-independent degradation pathway that prevents nuclear Cdc6 re-accumulation at the G1-S transition and during S phase. Similar to Cdt1, nuclear clearance of Cdc6 depends on an N-terminal PIP-box and the Cdt2-containing CRL4 complex. When cells reach G2 phase, Cdc6 rapidly re-accumulates but, at this time, Cdt1 is mostly absent and expression of Cdc6 is limited to the cytoplasm. We propose that Cdk1 contributes to the nuclear export of Cdc6 at the S-to-G2 transition. In summary, our results show that different control mechanisms of Cdc6 restrain erroneous licensing of DNA replication during G1, S and G2 phase.
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http://dx.doi.org/10.1242/jcs.145862DOI Listing
March 2014

The spindle checkpoint, APC/C(Cdc20), and APC/C(Cdh1) play distinct roles in connecting mitosis to S phase.

J Cell Biol 2013 Jun 17;201(7):1013-26. Epub 2013 Jun 17.

Division of Cell Biology I (B5), The Netherlands Cancer Institute (NKI-AvL), 1066 CX Amsterdam, Netherlands.

DNA replication depends on a preceding licensing event by Cdt1 and Cdc6. In animal cells, relicensing after S phase but before mitosis is prevented by the Cdt1 inhibitor geminin and mitotic cyclin activity. Here, we show that geminin, like cyclin B1 and securin, is a bona fide target of the spindle checkpoint and APC/C(Cdc20). Cyclin B1 and geminin are degraded simultaneously during metaphase, which directs Cdt1 accumulation on segregating sister chromatids. Subsequent activation of APC/C(Cdh1) leads to degradation of Cdc6 well before Cdt1 becomes unstable in a replication-coupled manner. In mitosis, the spindle checkpoint supports Cdt1 accumulation, which promotes S phase onset. We conclude that the spindle checkpoint, APC/C(Cdc20), and APC/C(Cdh1) act successively to ensure that the disappearance of licensing inhibitors coincides exactly with a peak of Cdt1 and Cdc6. Whereas cell cycle entry from quiescence requires Cdc6 resynthesis, our results indicate that proliferating cells use a window of time in mitosis, before Cdc6 is degraded, as an earlier opportunity to direct S phase.
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http://dx.doi.org/10.1083/jcb.201211019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3691463PMC
June 2013

The miRNA-192/194 cluster regulates the Period gene family and the circadian clock.

FEBS J 2009 Oct 13;276(19):5447-55. Epub 2009 Aug 13.

Division of Gene Regulation, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Several biological functions in mammals are regulated in a circadian fashion. The molecular mechanisms orchestrating these circadian rhythms have been unravelled. The biological clock, with its core transcriptional unit Bmal1/CLOCK, is composed of several self-sustaining feedback loops. In this study, we describe another mechanism impinging on the core components of the circadian clock. Using a forward genetic screen, we identified the miR-192/194 cluster as a potent inhibitor of the entire Period gene family. In accordance, the exogenous expression of miR-192/194 leads to an altered circadian rhythm. Thus, our results have uncovered a new mechanism for the control of the circadian clock at the post-transcriptional level.
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http://dx.doi.org/10.1111/j.1742-4658.2009.07229.xDOI Listing
October 2009

Distinct phases of cardiomyocyte differentiation regulate growth of the zebrafish heart.

Development 2009 May;136(10):1633-41

Hubrecht Institute and University Medical Centre Utrecht, 3584 CT, Utrecht, The Netherlands.

Amongst animal species, there is enormous variation in the size and complexity of the heart, ranging from the simple one-chambered heart of Ciona intestinalis to the complex four-chambered heart of lunged animals. To address possible mechanisms for the evolutionary adaptation of heart size, we studied how growth of the simple two-chambered heart in zebrafish is regulated. Our data show that the embryonic zebrafish heart tube grows by a substantial increase in cardiomyocyte number. Augmented cardiomyocyte differentiation, as opposed to proliferation, is responsible for the observed growth. By using transgenic assays to monitor developmental timing, we visualized for the first time the dynamics of cardiomyocyte differentiation in a vertebrate embryo. Our data identify two previously unrecognized phases of cardiomyocyte differentiation separated in time, space and regulation. During the initial phase, a continuous wave of cardiomyocyte differentiation begins in the ventricle, ends in the atrium, and requires Islet1 for its completion. In the later phase, new cardiomyocytes are added to the arterial pole, and this process requires Fgf signaling. Thus, two separate processes of cardiomyocyte differentiation independently regulate growth of the zebrafish heart. Together, our data support a model in which modified regulation of these distinct phases of cardiomyocyte differentiation has been responsible for the changes in heart size and morphology among vertebrate species.
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http://dx.doi.org/10.1242/dev.030924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2673760PMC
May 2009

Polo-like kinase-1 controls Aurora A destruction by activating APC/C-Cdh1.

PLoS One 2009 23;4(4):e5282. Epub 2009 Apr 23.

Department of Medical Oncology, University Medical Centre Utrecht, Utrecht, The Netherlands.

Polo-like kinase-1 (Plk1) is activated before mitosis by Aurora A and its cofactor Bora. In mitosis, Bora is degraded in a manner dependent on Plk1 kinase activity and the E3 ubiquitin ligase SCF-betaTrCP. Here, we show that Plk1 is also required for the timely destruction of its activator Aurora A in late anaphase. It has been shown that Aurora A destruction is controlled by the auxiliary subunit Cdh1 of the Anaphase-Promoting Complex/Cyclosome (APC/C). Remarkably, we found that Plk1-depletion prevented the efficient dephosphorylation of Cdh1 during mitotic exit. Plk1 mediated its effect on Cdh1, at least in part, through direct phosphorylation of the human phosphatase Cdc14A, controlling the phosphorylation state of Cdh1. We conclude that Plk1 facilitates efficient Aurora A degradation through APC/C-Cdh1 activation after mitosis, with a potential role for hCdc14A.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0005282PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2668763PMC
July 2009

To cell cycle, swing the APC/C.

Biochim Biophys Acta 2008 Sep 21;1786(1):49-59. Epub 2008 May 21.

Department of Medical Oncology, University Medical Center, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.

For successful mitosis, Cyclin B1 and Securin must be degraded efficiently before anaphase. Destruction of these mitotic regulators by the 26S proteasome is the result of their poly-ubiquitination by a multi-subunit E3 ligase: the Anaphase-Promoting Complex or Cyclosome (APC/C). Clearly, the APC/C is not just important for mitosis. Destruction of APC/C substrates such as Cdc20, Plk1, Aurora A and Skp2 directs events in G1. Strikingly, the APC/C needs to stay active even in quiescent cells to keep them out of the cell cycle and forms an intriguing link with pRb. An inactive APC/C stabilizes Geminin, Cyclin A and Cyclin B1, thereby securing completion of DNA synthesis and progression through G2-phase. In prometaphase the APC/C becomes active again, but is controlled by the spindle assembly checkpoint. Here we discuss how the APC/C is either held in check or released. We argue that shedding more light on the APC/C is also important to understand cancer and could help the design of treatment.
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http://dx.doi.org/10.1016/j.bbcan.2008.05.002DOI Listing
September 2008