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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Differential contribution of the chromatin-associated PP1 binding proteins PNUTS, Repo-Man and Ki-67 to mitotic exit and G1 re-establishment.

ABSTRACT: Mitotic exit is an important part of the cell cycle that requires coordination of many chromatin and cytoskeleton remodelling events to successfully complete cell division and maintain cell identity. Protein de-phosphorylation is a key step in directing mitotic exit and protein phosphatase 1 (PP1) is essential to this process. However, the specific contribution of its numerous targeting subunits is still unknown. Here we have investigated the function of three chromatin-associated PP1 targeting subunits in exiting mitosis: Repo-Man, Ki-67 and PNUTS. We have generated endogenously tagged, auxin-degradable alleles for each subunit and used a multi-omic approach to address their specific contribution to transcription resumption, chromatin accessibility and protein phosphorylation at the transition from mitosis to G1. This approach has identified their distinct role in mitotic exit, provided unique datasets for the cell cycle community, and highlighted specific and novel functions for Ki-67 and Repo-Man in genome stability and organisation.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER: Paola Vagnarelli

PROVIDER: E-MTAB-13827 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Differential contribution of the chromatin-associated PP1 binding proteins PNUTS, Repo-Man and Ki-67 to mitotic exit and G1 re-establishment.

Project description:Mitotic exit is an important part of the cell cycle that requires coordination of many chromatin and cytoskeleton remodelling events to successfully complete cell division and maintain cell identity. Protein de-phosphorylation is a key step in directing mitotic exit and protein phosphatase 1 (PP1) is essential to this process. However, the specific contribution of its numerous targeting subunits is still unknown. Here we have investigated the function of three chromatin-associated PP1 targeting subunits in exiting mitosis: Repo-Man, Ki-67 and PNUTS. We have generated endogenously tagged, auxin-degradable alleles for each subunit and used a multi-omic approach to address their specific contribution to transcription resumption, chromatin accessibility and protein phosphorylation at the transition from mitosis to G1. This approach has identified their distinct role in mitotic exit, provided unique datasets for the cell cycle community, and highlighted specific and novel functions for Ki-67 and Repo-Man in genome stability and organisation.

2025-01-01 | E-MTAB-13826 | biostudies-arrayexpress

Differential contribution of the chromatin-associated PP1 binding proteins PNUTS, Repo-Man and Ki-67 to mitotic exit and G1 re-establishment.

Project description:Differential contribution of the chromatin-associated PP1 binding proteins PNUTS, Repo-Man and Ki-67 to mitotic exit and G1 re-establishment.

| PRJEB72791 | ENA

Differential contribution of the chromatin-associated PP1 binding proteins PNUTS, Repo-Man and Ki-67 to mitotic exit and G1 re-establishment.

Project description:Differential contribution of the chromatin-associated PP1 binding proteins PNUTS, Repo-Man and Ki-67 to mitotic exit and G1 re-establishment.

| PRJEB72792 | ENA

Homo sapiens

Project description:Repo-Man/PP1 regulates heterochromatin formation in interphase

| PRJNA327850 | ENA

Ki-67 is necessary during DNA replication for forks protection and genome stability

Project description:Ki-67 molecular functions and properties have remained quite obscure for a long time, despite its importance as a major proliferation marker in clinic. Only recently it has been shown that Ki-67 has a major role in the formation of the mitotic chromosome periphery compartment, and it is a protein phosphatase 1 (PP1) binding protein. Understanding Ki-67 functions at different stages of the cell cycle has been so far hindered by the fact that the different phenotypes observed in cells upon Ki-67 depletion could be the outcome of secondary effects. Here, by using Auxin-inducible-degron (AID) system, we show that Ki-67 degradation at the G1/S boundary causes the disassembly of the replication machinery and leads to DNA damage. This triggers the interferon response and causes replication delay. Our results support the model whereby Ki-67 contributes to replication forks protection, and it is important for timely DNA replication and genome maintenance.

2024-04-13 | PXD037513 | Pride

RNA sequencing from publication: Ki-67 is necessary during DNA replication for forks protection and genome stability

Project description:Ki-67 is highly expressed in proliferating cells, characteristic that made the protein a very important marker widely used in the clinic. However, its molecular functions and properties have remained quite obscure for a long time. Only recently important discoveries have shed some light on Ki-67 function and shown that Ki-67 has a major role in the formation of mitotic chromosome periphery compartment, it is associated with protein phosphatase one (PP1) and regulates chromatin structure in both interphase and mitosis. However, it is still difficult to understand the specific molecular function of this protein since the different phenotypes could be the outcome of secondary effects. In fact, the studies so far conducted have used either RNAi or knock-out cells: the former could lead to phenotypes which are the sum of a cascade of complex events while the latter could be the outcome of compensatory mechanisms taking place. To address these problems, we have generated a cell line system for the rapid manipulation of Ki-67 by introducing an Auxin-inducible-degron (AID) tag in both alleles of the endogenous Ki-67 gene in HCT116 cells. This AID system allows rapid protein depletion by the addition of Auxin.

2024-04-07 | E-MTAB-12279 | biostudies-arrayexpress

Repo-Man/PP1 regulates heterochromatin formation in interphase

Project description:Repo-Man chromatin binding sites were obtained by expression of GST:Repo-Man and incubation with nucleosomes extracted from HeLa cells (GST alone signal was used as a negative control and subtracted from the GST:Repo-Man chromatin bound fraction)

2016-11-23 | GSE84035 | GEO

Distinct dephosphorylation site signatures of PP1 and PP2A-B55 coordinate mitotic exit.

Project description:Mitotic exit requires extensive dephosphorylation of Ser/Thr residues by the PP1 and PP2A-B55 protein phosphatases in human cells. Several aspects of this are poorly understood including specific substrates and determinants of phosphatase specificity. Here we develop a novel in vitro assay, MRBLE-dephos, that allows multiplexing of dephosphorylation reactions to determine phosphatase specificity. Using MRBLE-dephos, we establish amino acid preferences of the residues surrounding the phosphorylation site for PP1 and PP2A-B55, which reveals common and unique preferences for the two phosphatases. We use specific inhibition of PP1 and PP2A-B55 in mitotic exit lysates coupled with quantitative phosphoproteomics to identify more than 2000 regulated phosphorylation sites and integrate this with mitotic interactomes to obtain a comprehensive map of mitotic dephosphorylation events. Importantly, the sites dephosphorylated during mitotic exit reveal key signatures that are consistent with the MRBLE-dephos results. We use these insights to specifically alter INCENP dephosphorylation kinetics at mitotic exit resulting in defective cytokinesis underscoring the biological relevance of our determined specificity principles. Finally, we provide a comprehensive characterization of PP1 binding motifs and show how these can shape dephosphorylation and how PP1 primes its own association with these motifs at mitotic exit. Collectively we provide a framework for understanding mitotic exit regulation by dephosphorylation and novel approaches to dissect protein phosphatase specificity.

2023-05-12 | MSV000091928 | MassIVE

Ki-67 organises heterochromatin during cell proliferation

Project description:Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not inhibit cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 at heterochromatin was strongly reduced. Overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression.

2018-10-26 | PXD003551 | Pride

Repo-Man/PP1 regulates heterochromatin formation in interphase

Project description:GST:Repo-Man(C-terminus) or GST:alone were expressed as in Vagnarelli et al. 2011. Before elution, beads were first incubated with nucleosomes extracted from HeLa. HeLa cells were lysed and nucleosomes were digested with MNase (20min, 37C). Nucleosomes were firstly incubated with GST alone for 2hrs. This cleared lysate was then incubated with GST:Repo-Man and GST alone. Beads were then eluted and mix with 3x Laemli Buffer. Histone bands were sent to Mass Spec.

2016-12-07 | PXD004613 | Pride

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