Topoisomerase 1 activity during mitotic transcription favors the transition from mitosis to G1.

As cells enter mitosis, chromatin compacts to facilitate chromosome segregation yet remains transcribed. Transcription supercoils DNA to levels that can impede further progression of RNA polymerase II (RNAPII) unless it is removed by DNA topoisomerase 1 (TOP1). Using ChIP-seq on mitotic cells, we found that TOP1 is required for RNAPII translocation along genes. The stimulation of TOP1 activity by RNAPII during elongation allowed RNAPII clearance from genes in prometaphase and enabled chromosomal segregation. Disruption of the TOP1-RNAPII interaction impaired RNAPII spiking at promoters and triggered defects in the post-mitotic transcription program. This program includes factors necessary for cell growth, and cells with impaired TOP1-RNAPII interaction are more sensitive to inhibitors of mTOR signaling. We conclude that TOP1 is necessary for assisting transcription during mitosis with consequences for growth and gene expression long after mitosis is completed. In this sense, TOP1 ensures that cellular memory is preserved in subsequent generations.

Cyclin A2 localises in the cytoplasm at the S/G2 transition to activate PLK1.

Cyclin A2 is a key regulator of the cell cycle, implicated both in DNA replication and mitotic entry. Cyclin A2 participates in feedback loops that activate mitotic kinases in G2 phase, but why active Cyclin A2-CDK2 during the S phase does not trigger mitotic kinase activation remains unclear. Here, we describe a change in localisation of Cyclin A2 from being only nuclear to both nuclear and cytoplasmic at the S/G2 border. We find that Cyclin A2-CDK2 can activate the mitotic kinase PLK1 through phosphorylation of Bora, and that only cytoplasmic Cyclin A2 interacts with Bora and PLK1. Expression of predominately cytoplasmic Cyclin A2 or phospho-mimicking PLK1 T210D can partially rescue a G2 arrest caused by Cyclin A2 depletion. Cytoplasmic presence of Cyclin A2 is restricted by p21, in particular after DNA damage. Cyclin A2 chromatin association during DNA replication and additional mechanisms contribute to Cyclin A2 localisation change in the G2 phase. We find no evidence that such mechanisms involve G2 feedback loops and suggest that cytoplasmic appearance of Cyclin A2 at the S/G2 transition functions as a trigger for mitotic kinase activation.

FRET-Based Sorting of Live Cells Reveals Shifted Balance between PLK1 and CDK1 Activities During Checkpoint Recovery.

Cells recovering from the G2/M DNA damage checkpoint rely more on Aurora A-PLK1 signaling than cells progressing through an unperturbed G2 phase, but the reason for this discrepancy is not known. Here, we devised a method based on a FRET reporter for PLK1 activity to sort cells in distinct populations within G2 phase. We employed mass spectroscopy to characterize changes in protein levels through an unperturbed G2 phase and validated that ATAD2 levels decrease in a proteasome-dependent manner. Comparing unperturbed cells with cells recovering from DNA damage, we note that at similar PLK1 activities, recovering cells contain higher levels of Cyclin B1 and increased phosphorylation of CDK1 targets. The increased Cyclin B1 levels are due to continuous Cyclin B1 production during a DNA damage response and are sustained until mitosis. Whereas partial inhibition of PLK1 suppresses mitotic entry more efficiently when cells recover from a checkpoint, partial inhibition of CDK1 suppresses mitotic entry more efficiently in unperturbed cells. Our findings provide a resource for proteome changes during G2 phase, show that the mitotic entry network is rewired during a DNA damage response, and suggest that the bottleneck for mitotic entry shifts from CDK1 to PLK1 after DNA damage.

Spatial organization-dependent EphA2 transcriptional responses revealed by ligand nanocalipers.

Ligand binding induces extensive spatial reorganization and clustering of the EphA2 receptor at the cell membrane. It has previously been shown that the nanoscale spatial distribution of ligands modulates EphA2 receptor reorganization, activation and the invasive properties of cancer cells. However, intracellular signaling downstream of EphA2 receptor activation by nanoscale spatially distributed ligands has not been elucidated. Here, we used DNA origami nanostructures to control the positions of ephrin-A5 ligands at the nanoscale and investigated EphA2 activation and transcriptional responses following ligand binding. Using RNA-seq, we determined the transcriptional profiles of human glioblastoma cells treated with DNA nanocalipers presenting a single ephrin-A5 dimer or two dimers spaced 14, 40 or 100 nm apart. These cells displayed divergent transcriptional responses to the differing ephrin-A5 nano-organization. Specifically, ephrin-A5 dimers spaced 40 or 100 nm apart showed the highest levels of differential expressed genes compared to treatment with nanocalipers that do not present ephrin-A5. These findings show that the nanoscale organization of ephrin-A5 modulates transcriptional responses to EphA2 activation.

The human long non-coding RNA gene RMRP has pleiotropic effects and regulates cell-cycle progression at G2.

RMRP was the first non-coding nuclear RNA gene implicated in a disease. Its mutations cause cartilage-hair hypoplasia (CHH), an autosomal recessive skeletal dysplasia with growth failure, immunodeficiency, and a high risk for malignancies. This study aimed to gain further insight into the role of RNA Component of Mitochondrial RNA Processing Endoribonuclease (RMRP) in cellular physiology and disease pathogenesis. We combined transcriptome analysis with single-cell analysis using fibroblasts from CHH patients and healthy controls. To directly assess cell cycle progression, we followed CHH fibroblasts by pulse-labeling and time-lapse microscopy. Transcriptome analysis identified 35 significantly upregulated and 130 downregulated genes in CHH fibroblasts. The downregulated genes were significantly connected to the cell cycle. Multiple other pathways, involving regulation of apoptosis, bone and cartilage formation, and lymphocyte function, were also affected, as well as PI3K-Akt signaling. Cell-cycle studies indicated that the CHH cells were delayed specifically in the passage from G2 phase to mitosis. Our findings expand the mechanistic understanding of CHH, indicate possible pathways for therapeutic intervention and add to the limited understanding of the functions of RMRP.

Neutralization of the Positive Charges on Histone Tails by RNA Promotes an Open Chromatin Structure.

RNA associates extensively with chromatin and can influence its structure; however, the potential role of the negative charges of RNA on chromatin structure remains unknown. Here, we demonstrate that RNA prevents precipitation of histones and can attenuate electrostatic interactions between histones and DNA, thereby loosening up the chromatin structure. This effect is independent of the sequence of RNA but dependent on its single-stranded nature, length, concentration, and negative charge. Opening and closure of chromatin by RNA occurs rapidly (within minutes) and passively (in permeabilized cells), in agreement with electrostatics. Accordingly, chromatin compaction following removal of RNA can be prevented by high ionic strength or neutralization of the positively charged histone tails by hyperacetylation. Finally, LINE1 repeat RNAs bind histone H2B and can decondense chromatin. We propose that RNA regulates chromatin opening and closure by neutralizing the positively charged tails of histones, reducing their electrostatic interactions with DNA.

Mapping Metabolic Events in the Cancer Cell Cycle Reveals Arginine Catabolism in the Committed SG2M Phase.

Alterations in cell-cycle regulation and cellular metabolism are associated with cancer transformation, and enzymes active in the committed cell-cycle phase may represent vulnerabilities of cancer cells. Here, we map metabolic events in the G1 and SG2M phases by combining cell sorting with mass spectrometry-based isotope tracing, revealing hundreds of cell-cycle-associated metabolites. In particular, arginine uptake and ornithine synthesis are active during SG2M in transformed but not in normal cells, with the mitochondrial arginase 2 (ARG2) enzyme as a potential mechanism. While cancer cells exclusively use ARG2, normal epithelial cells synthesize ornithine via ornithine aminotransferase (OAT). Knockdown of ARG2 markedly reduces cancer cell growth and causes G2M arrest, while not inducing compensation via OAT. In human tumors, ARG2 is highly expressed in specific tumor types, including basal-like breast tumors. This study sheds light on the interplay between metabolism and cell cycle and identifies ARG2 as a potential metabolic target.

DNA Replication Determines Timing of Mitosis by Restricting CDK1 and PLK1 Activation.

To maintain genome stability, cells need to replicate their DNA before dividing. Upon completion of bulk DNA synthesis, the mitotic kinases CDK1 and PLK1 become active and drive entry into mitosis. Here, we have tested the hypothesis that DNA replication determines the timing of mitotic kinase activation. Using an optimized double-degron system, together with kinase inhibitors to enforce tight inhibition of key proteins, we find that human cells unable to initiate DNA replication prematurely enter mitosis. Preventing DNA replication licensing and/or firing causes prompt activation of CDK1 and PLK1 in S phase. In the presence of DNA replication, inhibition of CHK1 and p38 leads to premature activation of mitotic kinases, which induces severe replication stress. Our results demonstrate that, rather than merely a cell cycle output, DNA replication is an integral signaling component that restricts activation of mitotic kinases. DNA replication thus functions as a brake that determines cell cycle duration.

ATM/Wip1 activities at chromatin control Plk1 re-activation to determine G2 checkpoint duration.

After DNA damage, the cell cycle is arrested to avoid propagation of mutations. Arrest in G2 phase is initiated by ATM-/ATR-dependent signaling that inhibits mitosis-promoting kinases such as Plk1. At the same time, Plk1 can counteract ATR-dependent signaling and is required for eventual resumption of the cell cycle. However, what determines when Plk1 activity can resume remains unclear. Here, we use FRET-based reporters to show that a global spread of ATM activity on chromatin and phosphorylation of ATM targets including KAP1 control Plk1 re-activation. These phosphorylations are rapidly counteracted by the chromatin-bound phosphatase Wip1, allowing cell cycle restart despite persistent ATM activity present at DNA lesions. Combining experimental data and mathematical modeling, we propose a model for how the minimal duration of cell cycle arrest is controlled. Our model shows how cell cycle restart can occur before completion of DNA repair and suggests a mechanism for checkpoint adaptation in human cells.

Residual Cdk1/2 activity after DNA damage promotes senescence.

In response to DNA damage, a cell can be forced to permanently exit the cell cycle and become senescent. Senescence provides an early barrier against tumor development by preventing proliferation of cells with damaged DNA. By studying single cells, we show that Cdk activity persists after DNA damage until terminal cell cycle exit. This low level of Cdk activity not only allows cell cycle progression, but also promotes cell cycle exit at a decision point in G2 phase. We find that residual Cdk1/2 activity is required for efficient p21 production, allowing for nuclear sequestration of Cyclin B1, subsequent APC/CCdh1 -dependent degradation of mitotic inducers and induction of senescence. We suggest that the same activity that triggers mitosis in an unperturbed cell cycle enforces senescence in the presence of DNA damage, ensuring a robust response when most needed.

Loss of CSL Unlocks a Hypoxic Response and Enhanced Tumor Growth Potential in Breast Cancer Cells.

Notch signaling is an important regulator of stem cell differentiation. All canonical Notch signaling is transmitted through the DNA-binding protein CSL, and hyperactivated Notch signaling is associated with tumor development; thus it may be anticipated that CSL deficiency should reduce tumor growth. In contrast, we report that genetic removal of CSL in breast tumor cells caused accelerated growth of xenografted tumors. Loss of CSL unleashed a hypoxic response during normoxic conditions, manifested by stabilization of the HIF1α protein and acquisition of a polyploid giant-cell, cancer stem cell-like, phenotype. At the transcriptome level, loss of CSL upregulated more than 1,750 genes and less than 3% of those genes were part of the Notch transcriptional signature. Collectively, this suggests that CSL exerts functions beyond serving as the central node in the Notch signaling cascade and reveals a role for CSL in tumorigenesis and regulation of the cellular hypoxic response.

Spatial Separation of Plk1 Phosphorylation and Activity.

Polo-like kinase 1 (Plk1) is one of the major kinases controlling mitosis and cell division. Plk1 is first recruited to the centrosome in S phase, then appears on the kinetochores in late G2, and at the end of mitosis, it translocates to the central spindle. Activation of Plk1 requires phosphorylation of T210 by Aurora A, an event that critically depends on the co-factor Bora. However, conflicting reports exist as to where Plk1 is first activated. Phosphorylation of T210 is first observed at the centrosomes, but kinase activity seems to be restricted to the nucleus in the earlier phases of G2. Here, we demonstrate that Plk1 activity manifests itself first in the nucleus using a nuclear FRET-based biosensor for Plk1 activity. However, we find that Bora is restricted to the cytoplasm and that Plk1 is phosphorylated on T210 at the centrosomes. Our data demonstrate that while Plk1 activation occurs on centrosomes, downstream target phosphorylation by Plk1 first occurs in the nucleus. We discuss several explanations for this surprising separation of activation and function.

Bystander communication and cell cycle decisions after DNA damage.

The DNA damage response (DDR) has two main goals, to repair the damaged DNA and to communicate the presence of damaged DNA. This communication allows the adaptation of cellular behavior to minimize the risk associated with DNA damage. In particular, cell cycle progression must be adapted after a DNA-damaging insult, and cells either pause or terminally exit the cell cycle during a DDR. As cells can accumulate mutations after a DDR due to error-prone DNA repair, terminal cell cycle exit may prevent malignant transformation. The tumor suppressor p53 plays a key role in promoting terminal cell cycle exit. Interestingly, p53 has been implicated in communication of a stress response to surrounding cells, known as the bystander response. Recently, surrounding cells have also been shown to affect the damaged cell, suggesting the presence of intercellular feedback loops. How such feedback may affect terminal cell cycle exit remains unclear, but its presence calls for caution in evaluating cellular outcome without controlling the cellular surrounding. In addition, such feedback may contribute to how the cellular environment affects malignant transformation after DNA damage.

Nuclear translocation of Cyclin B1 marks the restriction point for terminal cell cycle exit in G2 phase.

Upon DNA damage, cell cycle progression is temporally blocked to avoid propagation of mutations. While transformed cells largely maintain the competence to recover from a cell cycle arrest, untransformed cells past the G1/S transition lose mitotic inducers, and thus the ability to resume cell division. This permanent cell cycle exit depends on p21, p53, and APC/C(Cdh1). However, when and how permanent cell cycle exit occurs remains unclear. Here, we have investigated the cell cycle response to DNA damage in single cells that express Cyclin B1 fused to eYFP at the endogenous locus. We find that upon DNA damage Cyclin B1-eYFP continues to accumulate up to a threshold level, which is reached only in G2 phase. Above this threshold, a p21 and p53-dependent nuclear translocation required for APC/C(Cdh1)-mediated Cyclin B1-eYFP degradation is initiated. Thus, cell cycle exit is decoupled from activation of the DNA damage response in a manner that correlates to Cyclin B1 levels, suggesting that G2 activities directly feed into the decision for cell cycle exit. Once Cyclin B1-eYFP nuclear translocation occurs, checkpoint inhibition can no longer promote mitotic entry or re-expression of mitotic inducers, suggesting that nuclear translocation of Cyclin B1 marks the restriction point for permanent cell cycle exit in G2 phase.

Picropodophyllin causes mitotic arrest and catastrophe by depolymerizing microtubules via insulin-like growth factor-1 receptor-independent mechanism.

Picropodophyllin (PPP) is an anticancer drug undergoing clinical development in NSCLC. PPP has been shown to suppress IGF-1R signaling and to induce a G2/M cell cycle phase arrest but the exact mechanisms remain to be elucidated. The present study identified an IGF-1-independent mechanism of PPP leading to pro-metaphase arrest. The mitotic block was induced in human cancer cell lines and in an A549 xenograft mouse but did not occur in normal hepatocytes/mouse tissues. Cell cycle arrest by PPP occurred in vitro and in vivo accompanied by prominent CDK1 activation, and was IGF-1R-independent since it occurred also in IGF-1R-depleted and null cells. The tumor cells were not arrested in G2/M but in mitosis. Centrosome separation was prevented during mitotic entry, resulting in a monopolar mitotic spindle with subsequent prometaphase-arrest, independent of Plk1/Aurora A or Eg5, and leading to cell features of mitotic catastrophe. PPP also increased soluble tubulin and decreased spindle-associated tubulin within minutes, indicating that it interfered with microtubule dynamics. These results provide a novel IGF-1R-independent mechanism of antitumor effects of PPP.

Centmitor-1, a novel acridinyl-acetohydrazide, possesses similar molecular interaction field and antimitotic cellular phenotype as rigosertib, on 01910.Na.

Mitosis is an attractive target for the development of new anticancer drugs. In a search for novel mitotic inhibitors, we virtually screened for low molecular weight compounds that would possess similar steric and electrostatic features, but different chemical structure than rigosertib (ON 01910.Na), a putative inhibitor of phosphoinositide 3-kinase (PI3K) and polo-like kinase 1 (Plk1) pathways. Highest scoring hit compounds were tested in cell-based assays for their ability to induce mitotic arrest. We identified a novel acridinyl-acetohydrazide, here named as Centmitor-1 (Cent-1), that possesses highly similar molecular interaction field as rigosertib. In cells, Cent-1 phenocopied the cellular effects of rigosertib and caused mitotic arrest characterized by chromosome alignment defects, multipolar spindles, centrosome fragmentation, and activated spindle assembly checkpoint. We compared the effects of Cent-1 and rigosertib on microtubules and found that both compounds modulated microtubule plus-ends and reduced microtubule dynamics. Also, mitotic spindle forces were affected by the compounds as tension across sister kinetochores was reduced in mitotic cells. Our results showed that both Cent-1 and rigosertib target processes that occur during mitosis as they had immediate antimitotic effects when added to cells during mitosis. Analysis of Plk1 activity in cells using a Förster resonance energy transfer (FRET)-based assay indicated that neither compound affected the activity of the kinase. Taken together, these findings suggest that Cent-1 and rigosertib elicit their antimitotic effects by targeting mitotic processes without impairment of Plk1 kinase activity.

Assessing kinetics from fixed cells reveals activation of the mitotic entry network at the S/G2 transition.

During the cell cycle, DNA duplication in S phase must occur before a cell divides in mitosis. In the intervening G2 phase, mitotic inducers accumulate, which eventually leads to a switch-like rise in mitotic kinase activity that triggers mitotic entry. However, when and how activation of the signaling network that promotes the transition to mitosis occurs remains unclear. We have developed a system to reduce cell-cell variation and increase accuracy of fluorescence quantification in single cells. This allows us to use immunofluorescence of endogenous marker proteins to assess kinetics from fixed cells. We find that mitotic phosphorylations initially occur at the completion of S phase, showing that activation of the mitotic entry network does not depend on protein accumulation through G2. Our data show insights into how mitotic entry is linked to the completion of S phase and forms a quantitative resource for mathematical models of the human cell cycle.

Phosphorylation-mediated stabilization of Bora in mitosis coordinates Plx1/Plk1 and Cdk1 oscillations.

Cdk1 and Plk1/Plx1 activation leads to their inactivation through negative feedback loops. Cdk1 deactivates itself by activating the APC/C, consequently generating embryonic cell cycle oscillations. APC/C inhibition by the mitotic checkpoint in somatic cells and the cytostatic factor (CSF) in oocytes sustain the mitotic state. Plk1/Plx1 targets its co-activator Bora for degradation, but it remains unclear how embryonic oscillations in Plx1 activity are generated, and how Plk1/Plx1 activity is sustained during mitosis. We show that Plx1-mediated degradation of Bora in interphase generates oscillations in Plx1 activity and is essential for development. In CSF extracts, phosphorylation of Bora on the Cdk consensus site T52 blocks Bora degradation. Upon fertilization, Calcineurin dephosphorylates T52, triggering Plx1 oscillations. Similarly, we find that GFP-Bora is degraded when Plk1 activity spreads to somatic cell cytoplasm before mitosis. Interestingly, GFP-Bora degradation stops upon mitotic entry when Cdk1 activity is high. We hypothesize that Cdk1 controls Bora through an incoherent feedforward loop synchronizing the activities of mitotic kinases.

Astral microtubules control redistribution of dynein at the cell cortex to facilitate spindle positioning.

Cytoplasmic dynein is recruited to the cell cortex in early mitosis, where it can generate pulling forces on astral microtubules to position the mitotic spindle. Recent work has shown that dynein displays a dynamic asymmetric cortical localization, and that dynein recruitment is negatively regulated by spindle pole-proximity. This results in oscillating dynein recruitment to opposite sides of the cortex to center the mitotic spindle. However, although the centrosome-derived signal that promotes displacement of dynein has been identified, it is currently unknown how dynein is re-recruited to the cortex once it has been displaced. Here we show that re-recruitment of cortical dynein requires astral microtubules. We find that microtubules are necessary for the sustained localized enrichment of dynein at the cortex. Furthermore, we show that stabilization of astral microtubules causes spindle misorientation, followed by mispositioning of dynein at the cortex. Thus, our results demonstrate the importance of astral microtubules in the dynamic regulation of cortical dynein recruitment in mitosis.

Bora and Aurora-A continue to activate Plk1 in mitosis.

Polo-like kinase-1 (Plk1) is required for proper cell division. Activation of Plk1 requires phosphorylation on a conserved threonine in the T-loop of the kinase domain (T210). Plk1 is first phosphorylated on T210 in G2 phase by the kinase Aurora-A, in concert with its cofactor Bora. However, Bora was shown to be degraded prior to entry into mitosis, and it is currently unclear how Plk1 activity is sustained in mitosis. Here we show that the Bora-Aurora-A complex remains the major activator of Plk1 in mitosis. We show that a small amount of Aurora-A activity is sufficient to phosphorylate and activate Plk1 in mitosis. In addition, a fraction of Bora is retained in mitosis, which is essential for continued Aurora-A-dependent T210 phosphorylation of Plk1. We find that once Plk1 is activated, minimal amounts of the Bora-Aurora-A complex are sufficient to sustain Plk1 activity. Thus, the activation of Plk1 by Aurora-A may function as a bistable switch; highly sensitive to inhibition of Aurora-A in its initial activation, but refractory to fluctuations in Aurora-A activity once Plk1 is fully activated. This provides a cell with robust Plk1 activity once it has committed to mitosis.