Bulk mitotic ATACseq for Polo-like kinase 1 maintains transcription and chromosomal accessibility during mitosis
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE282486 | GEO | 2025/11/18
REPOSITORIES: GEO
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Project description:Polo-like kinase 1 (PLK1) protects against genome instability by ensuring timely and accurate mitotic cell division, and its activity is tightly regulated throughout the cell cycle. Although the pathways that initially activate PLK1 in G2 are well-characterized, the factors that directly regulate PLK1 in mitosis remain poorly understood. Here, we identify that human PLK1 activity is sustained by the DNA damage response kinase Checkpoint kinase 2 (Chk2) in mitosis. Chk2 directly phosphorylates PLK1 T210, a residue on its T-loop whose phosphorylation is essential for full PLK1 kinase activity. Loss of Chk2-dependent PLK1 activity causes increased mitotic errors, including chromosome misalignment, chromosome missegregation, and cytokinetic defects. Moreover, Chk2 deficiency increases sensitivity to PLK1 inhibitors, suggesting that Chk2 status may be an informative biomarker for PLK1 inhibitor efficacy. This work demonstrates that Chk2 sustains mitotic PLK1 activity and protects genome stability through discrete functions in interphase DNA damage repair and mitotic chromosome segregation.
2024-11-01 | PXD053933 | Pride
Project description:Plasticity delineates cancer subtypes with more or less favourable outcomes. In breast cancer, triple-negative is the subtype that lacks the expression of major differentiation markers (i.e. estrogen receptor [ER]), ant its high cellular plasticity results in higher aggressiveness and poor prognosis compared to other subtypes. Whether plasticity poses a vulnerability to cancer cells remains elusive. Here, we show that cancer cell plasticity can be exploited to differentiate triple-negative breast cancer. Using a high-throughput reporter drug screen with 9,501 compounds, we identify three polo-like kinase 1 (PLK1) inhibitors as major inducers of ER protein expression and downstream activity in triple-negative breast cancer cells via the transcription factor BATF. PLK1 inhibition upregulates a cell differentiation program characterized by increased DNA damage, mitotic arrest and ultimately cell death. Notably, cells surviving PLK1 inhibition have decreased tumorigenic potential, and targeting PLK1 in already established tumours reduces tumour growth both in cell line and patient-derived xenograft models. In addition, genes upregulated upon PLK1 inhibition are correlated with expression in normal breast tissue and confer better overall survival in breast cancer patients. Our results indicate that differentiation therapy based on PLK1 inhibition might be an alternative strategy to treat triple-negative breast cancer.
2022-08-23 | PXD028495 | Pride
Project description:The Eyes Absent family of proteins (EYA1-4) are a biochemically unique group of tyrosine phosphatases known to be tumour promoting across a range of cancer types. To date, the molecular targets of EYA phosphatase activity remain largely uncharacterised. Here, we identify Polo-like kinase 1 (PLK1) as a direct interactor and phosphatase substrate of both EYA4 and EYA1, with pY445 on PLK1 being the primary target site. EYA-mediated dephosphorylation of PLK1 in the G2 phase of the cell cycle is required for centrosome maturation, PLK1 localization to centrosomes, and polo-box domain (PBD) dependent interactions between PLK1 and the PLK1-activating proteins BORA and CEP192. Molecular dynamics simulations support the rationale that pY445 confers a structural impairment to PBD-substrate interactions that is relieved by EYA-mediated dephosphorylation. Depletion of EYA4 or EYA1, or chemical inhibition of EYA phosphatase activity, dramatically reduces PLK1 activation, causing mitotic defects and cell death. Overall, we have characterized a novel phosphotyrosine signalling network governing PLK1 and mitosis. This work provides a mechanism of cell killing for EYA phosphatase inhibitors with important therapeutic implications.
2024-02-26 | PXD036430 | Pride
Project description:Polo-like kinase 1 (PLK1) is a serine/threonine kinase required for mitosis and cytokinesis. As cancer cells are often hypersensitive to partial PLK1 inactivation, chemical inhibitors of PLK1 have been developed and tested in clinical trials. However, these molecules alone were not completely effective. PLK1 promotes numerous molecular and cellular events in the cell division cycle. To date, it is unclear which of these events most crucially depend on PLK1 activity. We used a CRISPR-based genome-wide screening strategy to identify genes whose inactivation enhances cell proliferation defects upon partial chemical inhibition of PLK1. Genes identified encode proteins that are functionally linked to PLK1 in multiple ways. Among them, factors that promote centromere and kinetochore function were clearly enriched. In particular, inactivation of the kinesin KIF18A or SKA1 in PLK1-compromised cells results in mitotic defects, activation of the spindle assembly checkpoint and nuclear reassembly defects. Our results suggest that functions of PLK1 at kinetochores are most sensitive to its inhibition, and point at KIF18A as a possible target for combinatorial therapies using existing PLK1 inhibitors.
2023-10-02 | GSE228155 | GEO
Project description:Plk1 is a key mitotic kinase that localizes to distinct subcellular structures to promote accurate mitotic progression. Plk1 recruitment depends on direct interaction between polo-box domain (PBD) on Plk1 and PBD binding motif (PBD BM) on the interactors. However, recent study showed that PBD BM alone is not enough for stable binding between CENP-U and Plk1 highlighting the complexity of the interaction which warrants further investigation. An important interactor for Plk1 during mitosis is the checkpoint protein BubR1. Plk1 bound to BubR1 via PBD interaction with pT620 phosphorylates BubR1 S676/T680 to promote BubR1-PP2A/B56 interaction. The BubR1-PP2A/B56 complex counteracts the destablizing effect on kinetochore-microtubule attachments by mitotic kinases to promote mitotic progression.
Here we show that Plk1 phosphorylates T600/T608 on BubR1 and the double phosphorylation is critical for BubR1-Plk1 interaction. A similar mechanism for Plk1-Bub1 interaction also exists indicating a general principle for Plk1 kinetochore recruitment through self-priming. Mechanistically preventing BubR1 T600/T608 phosphorylation impairs chromosome congression and checkpoint silencing by reducing Plk1 and PP2A/B56 binding to BubR1. Increasing the binding affinity towards Plk1 and PP2A/B56 in BubR1 through protein engineering bypasses the requirement of T600/T608 phosphorylation for mitotic progression. These results reveal a new layer of regulation for accurate mitotic progression.
2024-10-31 | MSV000096264 | MassIVE
Project description:Polo-like kinase 1 (Plk1) is an essential protein kinase that promotes faithful mitotic progression in eukaryotes. Plk1 subcellular localization and substrate interactions are tightly controlled and require binding of Plk1 to phosphorylated sequences. Here, we use quantitative proteomics to identify phosphorylation-dependent interactions within the Plk1 network in human mitotic HeLa cells using kinase inhibitors and a Plk1 mutant deficient in phosphorylation-dependent substrate binding. We find that many interactions are abolished upon kinase inhibition; however, a subset are protected from phosphatase opposition or are unopposed, resulting in persistent Plk1-substrate interactions. This subset includes Phosphoprotein Phosphatase 6 (PP6), whose activity towards Aurora kinase A (AURKA) is inhibited by Plk1. This Plk1-PP6 interaction creates a feedback loop that coordinates and reinforces the activities of Plk1 and AURKA during mitotic entry and is terminated by Plk1 degradation during mitotic exit. Collectively, this work provides a cellular mechanism for the observed regulation of AURKA by Plk1.
2018-03-09 | MSV000082147 | MassIVE
Project description:Mitosis entails global alterations to chromosome structure and nuclear architecture, concomitant with transient silencing of transcription. How cells transmit transcriptional states through mitosis remains incompletely understood. While many nuclear factors dissociate from mitotic chromosomes, the observation that certain nuclear factors and chromatin features remain associated with individual loci during mitosis originated the hypothesis that they could provide transcriptional memory through mitosis. To obtain the first genome-wide view of the dynamics of chromatin structure during mitosis, we compared the DNase sensitivity of interphase and mitotic chromatin at two stages of cellular maturation in a rapidly dividingmurine erythroblastmodel. Despite global chromosome condensation visible during mitosis at the microscopic level, the chromatin accessibility landscape is largely unaltered. However, mitotic chromatin accessibility is locally dynamic, with individual loci maintaining none, some, or all of their interphase accessibility. Mitotic reduction in accessibility occurs primarily within narrow, highly hypersensitive sites that frequently coincide with transcription factor binding sites, whereas broader domains of moderate accessibility tend to be more stable. In mitosis, proximal promoters generally maintain their accessibility, whereas distal regulatory elements preferentially lose accessibility. Promoters with the highest degree of accessibility preservation in mitosis tend to also be accessible across many murine tissues in interphase. Transcription factor GATA1 exerts site-specific changes in interphase accessibility that are most pronounced at distal regulatory elements, but does not visibly influence mitotic accessibility. We conclude that features of open chromatin are remarkably stable through mitosis and are modulated at the level of individual genes and regulatory elements. Dnase-Seq data is integrated with Chip-seq [GSE36589, GSE30142] and RNA-seq to examine epigentic changes in mitosis. We performed DNase-seq on two cell lines, G1E and G1E-ER4, both on an asynchronus population, and on a sample of cells in mitosis; each of the 4 experiments in triplicate.
2014-10-01 | E-GEOD-61885 | biostudies-arrayexpress
Project description:Phosphorylation of Ser10 of histone H3 (H3S10p), together with the adjacent dimethylation of Lys9 (H3K9me2), has been proposed to function as a ‘phospho-methyl switch’ to regulate mitotic chromosome dynamics and control peripheral heterochromatin localization (1, 2). Despite of immensely understanding the regulation of H3S10 phosphorylation, how this modification temporarily masks the H3K9me2 mark during mitosis is poorly understood. Herein, we report that the master mitotic kinase Plk1 phosphorylates G9a at Thr1045 (pT1045) in vitro and in vivo at early mitosis. T1045p inhibits G9a catalytic activity, leading to decrease H3K9me2 levels. RNA-sequencing analysis showed that the phospho-mimic T1045 mutant of G9a (T1045E) compromised its repressive activity on several targeted genes, which was confirmed by luciferase reporter assays. Using genome-wide epigenomic technologies, we found that T1045E mutant displayed reduced H3K9me2 level, accompanied with increased chromatin accessibility, compared to the wild-type G9a cells. Finally, we found that T1045 phosphorylation could be removed by the phosphatase PP2A at late mitosis, which inversely reactivated G9a activity on H3K9me2 level, correlated with lower levels of H3S10p. Therefore, our result may provide a mechanistic explanation for ‘phospho-methyl switch’ and highlight the importance of Plk1 and PP2A-mediated dynamic regulation of G9a during mitosis.
2023-07-17 | GSE223359 | GEO
Project description:Mutations in the Fused in Sarcoma (FUS) gene cause familial amyotrophic lateral sclerosis (ALS), characterized by selective degeneration of spinal motor neurons (sMNs) with relative sparing of cortical neurons (CNs). The mechanisms underlying this cell-type vulnerability remain unclear. Here, we compare CNs and sMNs derived from FUS-ALS models to assess differential responses to FUS mutations. We find that CNs are less affected than sMNs in DNA damage repair, axonal organelle trafficking, and stress granule dynamics. RNA sequencing (RNA-seq) reveals distinct transcriptomic signatures, with sMNs uniquely activating DNA damage responses involving cell cycle regulators, particularly polo-like kinase 1 (PLK1). PLK1 is highly expressed in sMNs but not CNs, correlating with greater nuclear FUS loss and splicing defects in sMNs. Cross-comparison with other familial ALS RNA-seq datasets highlights PLK1 upregulation as a shared molecular feature. These findings identify intrinsic differences between CNs and sMNs in FUS-ALS and suggest PLK1 as a potential driver of sMN vulnerability.
2024-08-27 | GSE272827 | GEO
Project description:We show that Polθ is recruited to mitotic Double-strand breaks (DSBs) to slow down cell cycle progression and allow DNA repair. Because Polθ is one of the only repair protein to forms repair foci during mitosis, we investigated its regulation during mitosis. We performed immunoprecipitation (IP) of Polθ and assessed phosphorylation by immunoblot analysis (using pan phospho antibodies). We observed a phosphorylation signal corresponding to the size of Polθ when IP was performed from mitotic cell extracts. This phosphorylation signal was abolished when cells where treated with two different PLK1 inhibitors (PLK1i), indicating that PLK1 is responsible for Polθ phosphorylation in mitosis. In order to elucidate the regulation of mitotic Polθ activity, we performed mass spectrometry (MS)-based phosphorylation analysis of Polθ in mitosis with or without the PLK1 inhibitor Volasertib. We found 5 phosphorylated residues. To assess the functional consequences of Polθ phosphorylation by PLK1, we mutated some of the identified residues and found that the phospho dead mutant of Polθ fails be recruited to DSBs in mitosis. This indicates that PLK1-mediated regulation of mitotic Polθ repair is essential for its proper functioning
2023-09-15 | PXD036486 | Pride