Identifying the mitotic interactome of the human PLK1 protein kinase
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Cell Culture
SUBMITTER: 
Renata Soares
LAB HEAD: John Rouse
PROVIDER: PXD080479 | Pride | 2026-07-03
REPOSITORIES: Pride
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Project description:Accurate chromosome segregation requires the attachment of spindle microtubules to centromeres, which are epigenetically defined by the enrichment of CENP-A nucleosomes. During DNA replication, existing CENP-A nucleosomes undergo dilution as they get redistributed among the two DNA strands. To preserve centromere identity, CENP-A levels must be restored in a cell-cycle controlled manner orchestrated by the Mis18 complex. Here we provide a comprehensive mechanistic basis for PLK1-mediated licensing of CENP-A loading. We demonstrate that PLK1 interacts with Mis18α and Mis18BP1 subunits of the Mis18 complex by recognising self-primed phosphorylations of Mis18α (S54) and Mis18BP1 (T78 and S93) through its Polo-box binding domain. Disrupting these PLK1 phosphorylations perturbed the centromere recruitment of HJURP and new CENP-A loading. Biochemical and functional analyses show that phosphorylation of Mis18α and subsequent PLK1 binding is required to activate the Mis18α/β complex for robust Mis18α/β-HJURP interaction. Thus, our study reveals key molecular events underpinning the licensing role of PLK1 in ensuring accurate centromere inheritance.
2025-02-15 | PXD053197 | Pride
Project description:The fidelity of chromosome segregation in mitosis is safeguarded by the precise regulation of kinetochore microtubule (k-MT) attachment stability. Previously, we demonstrated that Cyclin A/Cdk1 destabilizes k-MT attachments to promote faithful chromosome segregation. Here, we use quantitative phosphoproteomics to identify 156 Cyclin A/Cdk1 substrates in prometaphase. One Cyclin A/Cdk1 substrate is myosin phosphatase targeting subunit 1 (MYPT1), and we show that MYPT1 localization to kinetochores depends on Cyclin A/Cdk1 activity and that MYPT1 destabilizes k-MT attachments by negatively regulating Plk1 at kinetochores. Thus, Cyclin A/Cdk1 phosphorylation primes MYPT1 for Plk1 binding. Interestingly, priming of PBIP1 by Plk1 itself (self-priming) increased in MYPT1-depleted cells showing that MYPT1 provides a molecular link between the processes of Cdk1-dependent priming and self-priming of Plk1 substrates. These data demonstrate cross-regulation between Cyclin A/Cdk1-dependent and Plk1-dependent phosphorylation of substrates during mitosis to ensure efficient correction of k-MT attachment errors necessary for high mitotic fidelity.
2017-12-01 | PXD008316 | Pride
Project description:Anterior foregut endoderm (AFE) gives rise to many tissue types of interest for therapeutic research including the esophagus, salivary glands, lung, thymus, parathyroid and thyroid. Despite its importance, only few reports describe the generation of AFE from pluripotent stem cells (PSCs) by directed differentiation. Here, we describe a novel protocol to derive a subdomain of AFE, identified by expression of Pax9, from PSCs using small molecules and chemically defined conditions. Generation of a reporter PSC line allows isolation and characterization of Pax9+ AFE cells. When transplanted in vivo, Pax9+ AFE can form several distinct types of complex anterior foregut epithelia including mucosal glands and stratified squamous epithelium. Finally, we show that the directed differentiation protocol can be used to generate AFE from DiGeorge Syndrome patient-specific human induced PSCs, thus creating a platform to produce anterior foregut derivatives for therapy and to enable the study of disorders of the AFE. Total RNA obtained from FACS purified from in vitro dervied mouse definitive endoderm, anterior foregut and ES cells. AFE cells were derived from a 129X1/SvJ background, DE cells from 129X1/SvJ x 129S1/SV-+p+Tyr- cKitlSl-J/+ (R1 ES cells) and non reporter ES cells from a 129P2/OlaHsd background.
2013-04-15 | E-GEOD-42139 | biostudies-arrayexpress
Project description:Two inbred mouse strains, C57BL/6J and CAST/EiJ, were crossed to generate both initial and reciprocal F1 crosses. For each genetically distinct class of mice (F0 C57BL/6J, F0 CAST/EiJ, F1i - C57BL/6J x CAST/EiJ, F1r - CAST/EiJ x C57BL/6J, where the male parent is listed first), samples were collected from a single lobe of the liver from 6 male mice between the ages of 4 and 6 months. The 24 samples were then processed to generate strand-specific RNA-seq libraries, which were sequenced on the Illumina GAII platform using 72bp paired-end reads.
2012-09-07 | E-MTAB-1091 | biostudies-arrayexpress
Project description:The high level of 5-hydroxymethylcytosine (5hmC) present in neuronal genomes suggests that mechanisms interpreting 5hmC in the central nervous system (CNS) may differ from those present in embryonic stem cells. Here we present quantitative, genome-wide analysis of 5hmC, 5- methylcytosine (5mC) and gene expression in differentiated CNS cell types in vivo. We report that 5hmC is enriched in active genes, and that surprisingly strong depletion of 5mC is observed over these regions. The contribution of these epigenetic marks to gene expression depends critically on cell type. We identify methyl-CpG binding protein 2 (MeCP2) as the major 5hmC binding protein in the brain, and demonstrate that MeCP2 binds 5hmC and 5mC containing DNA with similar high affinities. The Rett Syndromecausing mutation R133C preferentially inhibits 5hmC binding. These findings support a model in which 5hmC and MeCP2 constitute a cell specific epigenetic mechanism for regulation of chromatin structure and gene expression. Isolation and sorting was performed as described in (Kriaucionis and Heintz, 2009). Briefly, cerebella were dissected as described above and homogenized in homogenization medium (0.25 M sucrose, 150 mM KCl, 5 mM MgCl2, 20 mM Tricine pH 7.8, 0.15 mM spermine, 0.5 mM spermidine, EDTA-free protease inhibitor cocktail (Roche) using loose (A) and tight (B) glassglass dounce.Homogenate was supplemented with 50% iodixanol (OptiPrep, Axis-Shield, Scotland), 150 mM KCl, 5 mM MgCl2, 20 mM Tricine pH 7.8; and laid on a 29% iodixanol cushion. GC nuclei from WT and Mecp2-null mice were sorted by selecting a specific FSC/SSC population identified from GC EGFP+ mice. We observed that this FSC/SSC gated population in GC EGFP+ animals resulted >92% of the sorted nuclei being EGFP positive. Thus, the gate selected allows us to enrich the sample from 65-70% to 92-96% on GC cells. 5hmC was pulled down as described (Song et al., 2010). After purification DNA was amplified as described in TruSeq DNA Sample kit. 1-0.5 M-NM-<g of DNA was used for each experiment. Sonicated DNA was end-repaired following by ligation to Illumina paired end sequencing adapters (Illumina, PEM-bM-^@M-^P 102M-bM-^@M-^P 1003).), following by amplification with Illumina primers. Input samples were produced for each cell types in both procedures 5hmC enriched were then sequenced using Illumina platform obtaining more than 50 x10-6, 36- bp single-end reads per sample. Reads were aligned to mm9 mouse genome assembly using Bowtie v0.12.7 (-m1 --best). Two biological replicas were done for each of the cell types Single animals per replica were euthanized with CO2 and cerebella were dissected. 4 cerebella was immediately homogenized in ice-cold homogenization buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 100 M-NM-<g/ml cycloheximide, Complete-EDTA-free protease inhibitors (Roche) and RNasin RNase inhibitors (Promega, Madison, WI) using a Teflon-glass homogenizer. Homogenates were centrifuged for 10 minutes at 2,000 M-CM-^W g, 4 M-BM-0C, to pellet cell debris, and incubated with 1% IGEPAL CA-630 (NP-40, Sigma) and 30 mM DHPC (Avanti Polar Lipids, Alabaster, AL) for 5 min. Lysates were centrifuged for 15 minutes at 20,000 M-CM-^W g to pellet insoluble material. At this point, 30 M-NM-<L of supernatant was saved as input sample. Two custom made mouse anti-GFP (clones 19C8 and 19F7; see Heiman et al., 2008) antibodies were captured on Dynal magnetic beads (Invitrogen Corporation, Carlsbad, CA) coupled with protein L (Pierce, Rockford, IL) . The homogenate was incubated with antibody coupled beads at 4M-BM-0C with end-over-end rotation for approximately 16 hours. Beads were subsequently collected on a magnetic rack, washed three times with high-salt wash buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5mM DTT, 100 M-NM-<g/ml cycloheximide) and RNA was released and purified using Rneasy Micro Kit (Qiagen, Valencia, CA) with in-column DNase digestion. RNA quantity and quality were determined with a Nanodrop 1000 spectrophotometer (Wilmington, DE) and Agilent 2100 Bioanalyzer using RNA 6000 Pico Chip. 4 biological replicas was produced per each cell type. 10 ng of total RNA from cerebellum were converted to cDNA using the NuGEN Ovation RNA-Seq (NuGEN,San Carlos,CA,USA) following manufacturersM-bM-^@M-^Y instructions. The Single Primer Isothermal Amplification (SPIA) method used in this protocol allows the amplification of RNA target in double stranded cDNA under standardized conditions that markedly deplete rRNA without preselecting mRNA. cDNA obtained was quality scored by RNA 6000 PicoChip for Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). 1 M-NM-<g of cDNA per sample was sonicated using Covaris-S2 system (Covaris Inc., Woburn, MA, USA), cDNA fragments of 200 bp were end-repaired and adapters were ligated for HiSeq 2000 (Ilumina Inc., San Diego, CA, USA) technology using TruSeq DNA Sample kit (Illumina) and following manufacturer`s instructions. Quality of libraries was assesed using HT DNA High Sensitivity Chip (Agilent) for 2100 Bioanalyzer. Single animals per replica were euthanized with CO2 and cerebella were dissected. 4 cerebella was immediately homogenized in ice-cold homogenization buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 100 M-NM-<g/ml cycloheximide, Complete-EDTA-free protease inhibitors (Roche) and RNasin RNase inhibitors (Promega, Madison, WI) using a Teflon-glass homogenizer. Homogenates were centrifuged for 10 minutes at 2,000 M-CM-^W g, 4 M-BM-0C, to pellet cell debris, and incubated with 1% IGEPAL CA-630 (NP-40, Sigma) and 30 mM DHPC (Avanti Polar Lipids, Alabaster, AL) for 5 min. Lysates were centrifuged for 15 minutes at 20,000 M-CM-^W g to pellet insoluble material. At this point, 30 M-NM-<L of supernatant was saved as input sample. Two custom made mouse anti-GFP (clones 19C8 and 19F7; see Heiman et al., 2008) antibodies were captured on Dynal magnetic beads (Invitrogen Corporation, Carlsbad, CA) coupled with protein L (Pierce, Rockford, IL) . The homogenate was incubated with antibody coupled beads at 4M-BM-0C with end-over-end rotation for approximately 16 hours. Beads were subsequently collected on a magnetic rack, washed three times with high-salt wash buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5mM DTT, 100 M-NM-<g/ml cycloheximide) and RNA was released and purified using Rneasy Micro Kit (Qiagen, Valencia, CA) with in-column DNase digestion. RNA quantity and quality were determined with a Nanodrop 1000 spectrophotometer (Wilmington, DE) and Agilent 2100 Bioanalyzer using RNA 6000 Pico Chip. 4 biological replicas was produced per each cell type. 10 ng of total RNA per IP or input sample were converted to cDNA using the NuGEN Ovation RNA-Seq (NuGEN,San Carlos,CA,USA) following manufacturersM-bM-^@M-^Y instructions. The Single Primer Isothermal Amplification (SPIA) method used in this protocol allows the amplification of RNA target in double stranded cDNA under standardized conditions that markedly deplete rRNA without preselecting mRNA. cDNA obtained was quality scored by RNA 6000 PicoChip for Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). 1 M-NM-<g of cDNA per sample was sonicated using Covaris-S2 system (Covaris Inc., Woburn, MA, USA), cDNA fragments of 200 bp were end-repaired and adapters were ligated for HiSeq 2000 (Ilumina Inc., San Diego, CA, USA) technology using TruSeq DNA Sample kit (Illumina) and following manufacturer`s instructions. Quality of libraries was assesed using HT DNA High Sensitivity Chip (Agilent) for 2100 Bioanalyzer. Single animals per replica were euthanized with CO2 and cerebella were dissected. 4 cerebella was immediately homogenized in ice-cold homogenization buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 100 M-NM-<g/ml cycloheximide, Complete-EDTA-free protease inhibitors (Roche) and RNasin RNase inhibitors (Promega, Madison, WI) using a Teflon-glass homogenizer. Homogenates were centrifuged for 10 minutes at 2,000 M-CM-^W g, 4 M-BM-0C, to pellet cell debris, and incubated with 1% IGEPAL CA-630 (NP-40, Sigma) and 30 mM DHPC (Avanti Polar Lipids, Alabaster, AL) for 5 min. Lysates were centrifuged for 15 minutes at 20,000 M-CM-^W g to pellet insoluble material. At this point, 30 M-NM-<L of supernatant was saved as input sample. Two custom made mouse anti-GFP (clones 19C8 and 19F7; see Heiman et al., 2008) antibodies were captured on Dynal magnetic beads (Invitrogen Corporation, Carlsbad, CA) coupled with protein L (Pierce, Rockford, IL) . The homogenate was incubated with antibody coupled beads at 4M-BM-0C with end-over-end rotation for approximately 16 hours. Beads were subsequently collected on a magnetic rack, washed three times with high-salt wash buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5mM DTT, 100 M-NM-<g/ml cycloheximide) and RNA was released and purified using Rneasy Micro Kit (Qiagen, Valencia, CA) wit
2013-01-14 | E-GEOD-42880 | biostudies-arrayexpress
Project description:Polo-like kinase 1 (plk1) is a conserved regulator of cell division. During prophase, plk1 phosphorylates direct substrates and is involved in activation of the cyclin-dependent kinase 1 (cdk1). However, the exact functions of plk1 in prophase remain incompletely understood. Here, we inhibited plk1 in synchronous G2 populations of multiple mammalian cell lines. This led to delay or prevention of mitotic entry showing high variability between individual cells. Using a mathematical model, we recapitulate and provide an explanation for this seemingly variable phenotype. We show that plk1-inhibited cells are delayed in a prophase-like state with low cdk1 activity that increases slowly and gradually over hours. These cells display progressively condensing chromosomes, increased microtubule dynamics and reorganization of the actin cortex, while the nuclear envelope remains intact. We then characterize this state by phosphoproteomics, revealing phosphorylation of regulators of chromatin organization and the cytoskeleton consistent with cellular phenotypes. Together, we show that plk1 inhibition stabilizes cells in a prophase-like state with low cdk1 activity displaying a specific set of early mitotic phosphorylations.
2025-03-17 | PXD060406 | Pride
Project description:This dataset has been designed to test whether codons in mRNAs and anticodons in tRNAs vary in order to maximize translation in specific cellular conditions in mammals. Prokaryotes and simple unicellular eukaryotes optimize their translational rates by adjusting the codons in the protein-coding transcriptome to the available pool of anticodons in the tRNA transcriptome. We found no evidence supporting this mechanism in mammals, even when subsets of genes were considered, such as those found in Gene Ontology functional categories or in tissue-specific transcriptional signatures. The simplest explanation accounting for the observed codon distributions in mammals is the variation in GC content of gene categories. GC variation across the mammalian genome is most likely to result from the interplay of genome repair and gene duplication mechanisms, rather than selective pressures caused by codon-driven translational rates. This work is part of experiment series: ChIP-Seq E-MTAB-23282326.
2016-04-18 | E-MTAB-4052 | biostudies-arrayexpress
Project description:The pathogenesis of diffuse large B cell lymphomas (DLBCL) is only partly understood. We analyzed 148 DLBCL by high resolution single nucleotide polymorphism (SNP)-chips to characterize genomic imbalances. Seventy-nine cases were of the germinal center B-cell like (GCB) type of DLBCL, 49 of the activated B-cell like (ABC) subtype and 20 were type 3 DLBCL. Twenty-four regions of recurrent genomic gains and 38 regions of recurrent genomic losses were identified over the whole cohort, with a median of 25 imbalances per case for ABC-DLBCL and 19 per case for GCB-DLBCL. Several recurrent copy number changes showed differential frequencies in the GCB- and ABC-DLBCL subgroups, including gains of HDAC7A predominantly in GCB-DLBCL (38% of cases) and losses of BACH2 and CASP8AP2 predominantly in ABC-DLBCL (35%), hinting at disparate pathogenetic mechanisms in these entities. Correlating gene expression and copy number revealed a strong gene dosage effect in all tumors, with 34% of probesets showing a concordant expression change in affected regions. Two new potential tumor suppressor genes emerging from the analysis, CASP3 and IL5RA, were sequenced in 10 and 16 candidate cases, respectively. However, no mutations were found, pointing to a potential haploinsufficiency effect of these genes, considering their reduced expression in cases with deletions. This work thus describes differences and similarities in the landscape of genomic aberrations in the DLBCL subgroups in a large collection of cases, confirming already known targets, but also discovering novel copy number changes with possible pathogenetic relevance. Affymetrix HGU133A arrays were performed according to the manufacturer's directions on RNA extracted from cryopreserved lymph node biopsies of tumor samples.
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2019-11-18 | PXD015513 | Pride
Project description:Cereblon (CRBN), a substrate receptor of the E3 ubiquitin ligase complex CRL4CRBN, is the target of the immunomodulatory drugs lenalidomide and pomalidomide. Recently, it was demonstrated that binding of these drugs to CRBN promotes the ubiquitination and subsequent degradation of two common substrates, transcription factors Aiolos and Ikaros. Here we report that the pleiotropic pathway modifier CC-122, a new chemical entity termed pleiotropic pathway modifier binds CRBN and promotes degradation of Aiolos and Ikaros in diffuse large B-cell lymphoma (DLBCL) and T cells in vitro, in vivo and in patients, resulting in both cell autonomous as well as immunostimulatory effects. In DLBCL cell lines, CC-122-induced degradation or shRNA mediated knockdown of Aiolos and Ikaros correlates with increased transcription of interferon stimulated genes (ISGs) independent of interferon α, β, γ production and/or secretion and results in apoptosis in both ABC and GCB-DLBCL cell lines. Our results provide mechanistic insight into the cell of origin independent anti-lymphoma activity of CC-122, in contrast to the ABC subtype selective activity of lenalidomide. Microarray analysis of the OCI-LY10 activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) cell line treated with the compound CC-122 for 18 hours
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