Repo-Man/PP1 regulates heterochromatin formation in interphase
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE84035 | GEO | 2016/11/23
SECONDARY ACCESSION(S): PRJNA327850
REPOSITORIES: GEO
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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.
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Project description:Repo-Man is a protein phosphatase 1 (PP1) targeting subunit that regulates mitotic progression and chromatin remodelling. After mitosis, Repo-Man/PP1 remains associated with chromatin but its function in interphase is not known. Here we show that Repo-Man, via Nup153, is enriched on condensed chromatin at the nuclear periphery and at the edge of the nucleopore basket. Repo-Man/PP1 regulates the formation of heterochromatin, dephosphorylates H3S28 and it is necessary and sufficient for heterochromatin protein 1 binding and H3K27me3 recruitment. Using a novel proteogenomic approach, we show that Repo-Man is enriched at subtelomeric regions together with H2AZ and H3.3 and that depletion of Repo-Man alters the peripheral localization of a subset of these regions and alleviates repression of some polycomb telomeric genes. This study shows a role for a mitotic phosphatase in the regulation of the epigenetic landscape and gene expression in interphase.
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Project description:Simulium sp. D3-Man isolate:D3-Man Genome sequencing
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Project description:Repo-Man/PP1 regulates heterochromatin formation in interphase
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Project description:Dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine (Ser) and threonine (Thr) residues (O-GlcNAcylation) is an essential process in all eukaryotic cells except yeast, e.g Saccharomyces cerevisiae. O-GlcNAcylation modulates signaling and cellular processes in an intricate interplay with protein phosphorylation, and serves as a key sensor of nutrients by linking the hexosamine biosynthetic pathway (HBP) to cellular signaling. A longstanding conundrum has been how yeast survives without O-GlcNAcylation in light of its similar phosphorylation signaling system. We previously developed a sensitive lectin enrichment and mass spectrometry workflow for identification of the human O-Mannose (O-Man) glycoproteome, and used this to identify a pleothora of O-linked mannose glycoproteins in human cell lines including the large family of cadherins and protocadherins. Here, we applied the workflow to yeast with the aim to characterize the yeast O-Man glycoproteome, and doing so we discovered hitherto unknown O-Man glycosites on nuclear, cytoplasmic and mitochondrial proteins in Saccharomyces cerevisiae. The type of nucleocytoplasmic proteins and the localization of identified O-Man residues mirrors that of the O-GlcNAc glycoproteome in other eukaryotic cells, indicating that the two different types of O-glycosylations serve the same important biological functions. The discovery opens for exploration of the enzyme machinery that is predicted to regulate the discovered nucleocytoplasmic O-Man glycosylation. It is likely that manipulation of this type of O-Man glycosylation will have wide applications for yeast bioprocessing.
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Project description:Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase regulating many cellular processes. PP1alpha and -gamma are closely related isoforms with distinct localization patterns, shown here by time-lapse microscopy of stably expressed fluorescent protein fusions. A pool of PP1gamma is selectively loaded onto chromatin at anaphase. Using stable isotope labeling and proteomics, we identified a novel PP1 binding protein, Repo-Man, which selectively recruits PP1gamma onto mitotic chromatin at anaphase and into the following interphase. This approach revealed both novel and known PP1 binding proteins, quantitating their relative distribution between PP1alpha and -gamma in vivo. When overexpressed, Repo-Man can also recruit PP1alpha to chromatin. Mutating Repo-Man's PP1 binding domain does not disrupt chromatin binding but abolishes recruitment of PP1 onto chromatin. RNA interference-induced knockdown of Repo-Man caused large-scale cell death by apoptosis, as did overexpression of this dominant-negative mutant. The data indicate that Repo-Man forms an essential complex with PP1gamma and is required for the recruitment of PP1 to chromatin.
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Project description:The androgen receptor (AR) is the major transcriptional driver of prostate cell growth in man. For the first time, we define AR targets in prostate cancer (PC) tissue representing progression from treatment-naive to castrate-resistant disease (CRPC). We employed chromatin immunoprecipitation with high through-put sequencing (ChIP-seq) in human tissue, with cell-line and xenograft studies. We uncovered an AR transcriptional network not observed in cultured cells, with significant over-representation of MYC, E2F and STAT binding sites, progenitor cell gene signatures and targets which regulate metabolism, cell cycle and steroid biosynthesis. We identified AR targets unique to CRPC tissue, with a subset over-expressed in CRPC and predictive of clinical outcome, highlighting persistence of AR signalling. Our data support a model whereby endocrine and paracrine signals converge on the AR in PC tissue to drive oncogenic transcriptional programs, highlighting the critical role of cellular context in the regulation of target selection and gene expression. chromatin immunoprecipitation with high through-put sequencing (ChIP-seq) in human tissue, with cell-line and xenograft studies
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Project description:Spindle orientation determines the axis of division and is crucial for cell fate, tissue morphogenesis, and the development of an organism. In animal cells, spindle orientation is regulated by the conserved Gαi-LGN-NuMA complex, which targets the force generator dynein-dynactin to the cortex. In this study, we show that p37/UBXN2B, a cofactor of the p97 AAA ATPase, regulates spindle orientation in mammalian cells by limiting the levels of cortical NuMA. p37 controls cortical NuMA levels via the phosphatase PP1 and its regulatory subunit Repo-Man, but it acts independently of Gαi, the kinase Aurora A, and the phosphatase PP2A. Our data show that in anaphase, when the spindle elongates, PP1/Repo-Man promotes the accumulation of NuMA at the cortex. In metaphase, p37 negatively regulates this function of PP1, resulting in lower cortical NuMA levels and correct spindle orientation.
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Project description:The reversible condensation of chromosomes during cell division remains a classic problem in cell biology. Condensation requires the condensin complex in certain experimental systems, but not in many others. Anaphase chromosome segregation almost always fails in condensin-depleted cells, leading to the formation of prominent chromatin bridges and cytokinesis failure. Here, live-cell analysis of chicken DT40 cells bearing a conditional knockout of condensin subunit SMC2 revealed that condensin-depleted chromosomes abruptly lose their compact architecture during anaphase and form massive chromatin bridges. The compact chromosome structure can be preserved and anaphase chromosome segregation rescued by preventing the targeting subunit Repo-Man from recruiting protein phosphatase 1 (PP1) to chromatin at anaphase onset. This study identifies an activity critical for mitotic chromosome structure that is inactivated by Repo-Man-PP1 during anaphase. This activity, provisionally termed 'regulator of chromosome architecture' (RCA), cooperates with condensin to preserve the characteristic chromosome architecture during mitosis.
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