Mapping and characterization of promoter-binding sites of protein phosphatase 1
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE54170 | GEO | 2015/06/17
SECONDARY ACCESSION(S): PRJNA235324
REPOSITORIES: GEO
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Project description:Protein phosphatase 1 (PP1) is a Ser/Thr phosphatase that has been implicated in many key cellular functions including transcriptional regulation. Due to its involvement these many processes, it becomes difficult to directly link PP1 to transcriptional regulation on the chromatin level as no direct genomic binding sites have been identified. Previous work has failed to address this as the most common method used, namely chromatin immunoprecipitation (ChIP), is an antibody-dependent technique and currently no ChIP-grade PP1 antibodies have been developed. Using DamID, an alternative to ChIP, we have identified PP1 isoform-specific binding sites on the promoter regions of genes. We also identified the binding sites of three main PP1 regulatory subunits (R-subunits) in order to identify potential PP1 holo-enzymes binding sites. Our study revealed the full extent of PP1 isoform specific binding an allowed us to investigate the dependency of the R-subunits on PP1 for chromatin targeting. This data establishes PP1 as a chromatin interactor and allow for the identification of direct effects PP1 can have on the regulation of the genes on whose promoter it is bound. HeLa stable cell lines were created using constructs derived from pIND-(V5)-EcoDam. These constructs express trace amounts of Dam or C-terminal fusions with PP1α, PP1β, PP1γ and three R-subunits, PNUTS, NIPP1 and RepoMan, both wildtype (WT) and their PP1-binding mutants (RATA). Two independent stable cell lines were set up for each of the constructs. DamID-DNA was labeled and hybridized to a GeneChip Human Promoter 1.0R Array (Affymetrix, Santa Clara, CA, USA). The tiling array readouts were analyzed with the “model-based analysis of tiling arrays” (MAT) algorithm (version 1.0.0) against the hg19 reference genome. We normalized two biological replicates of each Dam-fusion over two Dam-only biological replicates. Each biological repeat consisted of 2 technical repeats pooled together prior to hybridization to the tiling array.
2015-06-17 | E-GEOD-54170 | biostudies-arrayexpress
Project description:To gain unbiased insight into the association of NIPP1 with regulatory, coding and intergenic regions of the human genome, DamID experiments and subsequent analysis by ENCODE arrays were performed. The DamID method uses fusions of the bacterial Dam DNA methylase and the protein of interest, to direct the enzymatic activity to the protein’s genomic binding sites, where the DNA is methylated. Methylated DNA is then extracted, enriched and further analysed by microarray. The PP1 (Protein Phosphatase 1) interacting protein NIPP1 has been implicated to play a role in regulation of gene expression through PRC2 function, but also has a function in splicing. The array analysis was performed to obtain a general picture of the sites of chromatin association of NIPP1 and specifically their spatial relation to coding regions as well as to identify target regions to be analysed in more detail. In addition, a PP1 binding deficient mutant (NIPP1-RATA) was analysed, thus allowing to distinguish PP1 dependent and independent sites of association with the genome by microarray analysis. NIPP1-WT is referred to as FDN and the mutant referred to as RATA in the following descriptions.
2010-07-19 | GSE22123 | GEO
Project description:The p52 isoform of Psip1/Ledgf links histone H3K36 methylation and the regulation of alternative splicing. Chromatin immunoprecipitation (ChIP) of Psip1 together with H3K36me3 and H3K4me3 and by ChIP-on-chip analysis demonstrated that, Like H3K36me3, Psip1 is enriched on exons of highly expressed genes, Comparision of H3K36me3 and Psip1 binding sites on the expressed and non expressed genes.
2012-04-11 | E-GEOD-28153 | biostudies-arrayexpress
Project description:The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to ensure proper mRNA maturation and prevent interference between neighboring genes. Pol II slowing downstream of the cleavage and polyadenylation signal (CPS) leads to recruitment of cleavage and polyadenylation factors and termination, but how this chain of events is initiated remains unclear. In a chemical-genetic screen we identified protein phosphatase 1 (PP1) isoforms as substrates of human positive transcription elongation factor b (P-TEFb), the cyclin-dependent kinase 9 (Cdk9)-cyclin T1 complex. Here we show that Cdk9 and PP1 govern phosphorylation of the conserved transcription factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis2. Sites phosphorylated by Cdk9 in the Spt5 carboxy-terminal domain (CTD) are dephosphorylated by Dis2 in vitro, and Cdk9 inhibition in vivo leads to rapid Spt5 dephosphorylation that is retarded by concurrent Dis2 inactivation. Chromatin immunoprecipitation and sequencing (ChIP-seq) analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the CPS, prior to or concomitant with Pol II pausing. A Dis2-inactivating mutation stabilizes Spt5 phosphorylation (pSpt5) on chromatin, promotes transcription beyond the normal termination zone detected by precision run-on transcription and sequencing (PRO-seq), and is suppressed by ablation of Cdk9 target sites in Spt5. These results support a model whereby the transition of Pol II from elongation to termination is regulated by opposing activities of Cdk9 and Dis2 towards their common substrate Spt5—a bistable switch analogous to a Cdk1-PP1 module that controls exit from mitosis.
2018-06-07 | GSE102590 | GEO
Project description:SDS22 forms an inactive complex with nascent protein phosphatase-1 (PP1) and Inhibitor-3 (I3). SDS22:PP1:I3 is a substrate for the ATPase p97/VCP, which liberates PP1 for binding to canonical regulatory subunits. The exact role of SDS22 in PP1-holoenzyme assembly remains elusive. Here, we show that SDS22 prevents the aggregation of nascent PP1. In the absence of SDS22, PP1 was gradually lost, resulting in substrate hyperphosphorylation and a proliferation arrest. A human patient with an unstable SDS22 mutant also expressed reduced levels of PP1 and suffered from neurodevelopmental retardation. We furthermore found that SDS22 directly binds to I3 and that this is essential for the stable assembly of SDS22:PP1:I3, the recruitment of p97/VCP, and the extraction of SDS22 during holoenzyme assembly. SDS22 with a disabled I3-binding site co-transfered with PP1 to canonical regulatory subunits, thereby forming non-functional holoenzymes. Our data show that SDS22, by its ability to bind to both PP1 and I3, integrates the major steps of PP1 holoenzyme assembly.
2024-03-18 | MSV000094347 | MassIVE
Project description:Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects the majority of the world’s population. Lytic HCMV replication in immunocompromised individuals or neonates can lead to severe disease in multiple organ systems and even death. The establishment of lytic replication is driven by the first viral proteins expressed upon infection, the immediate early proteins, which play a key role in creating an intracellular environment conducive to virus replication. Two immediate early proteins, the functional orthologs pTRS1 and pIRS1, stimulate immediate early gene expression by suppressing antiviral PKR/eIF2a signaling and enhance the translation of viral mRNAs independent of PKR antagonism. To better understand the molecular functions of pTRS1, we used proximity labeling proteomics to identify proteins that interact with pTRS1 in infected cells. Multiple novel host and viral interactors were identified, including the catalytic subunits of the protein phosphatase 1 (PP1) holoenzyme. Mutations to a PP1 catalytic subunit known to disrupt binding to PP1 regulatory subunits decreased binding to pTRS1. pTRS1 immune complexes contained phosphatase activity, and inhibition of phosphatase activity in transfected or infected cells reversed the ability of pTRS1 to inhibit the antiviral kinase PKR. Depletion of individual PP1 catalytic subunits decreased virus replication and increased the phosphorylation of the PKR substrate eIF2a. Together our data suggest potential novel functions for pTRS1, and define a novel role for PP1 as an antagonist of the antiviral PKR/eIF2 signaling axis during HCMV infection.
2024-10-11 | PXD053924 | Pride
Project description:The essential process of dosage compensation equalizes X-chromosome gene expression between C. elegans XO males and XX hermaphrodites through a dosage compensation complex (DCC) that resembles condensin. The DCC binds to both X chromosomes of hermaphrodites to repress transcription by half. Here we show that post-translational modification by the SUMO conjugation pathway is essential for sex-specific assembly of the DCC onto X. Depletion of the SUMO peptide in vivo severely disrupts binding of particular DCC subunits and causes changes in X-linked gene expression similar to those caused by disrupting genes encoding DCC subunits. Three DCC subunits are themselves SUMOylated, and depletion of SUMO preferentially reduces their binding to X, suggesting that SUMOylation of DCC subunits is essential for robust association with X. DCC SUMOylation is triggered by the signal that initiates DCC assembly onto X. The initial step of assembly--binding of X-targeting factors to recruitment sites on X (rex sites)--is independent of SUMOylation, but robust binding of the complete complex requires SUMOylation. SUMOylated DCC subunits are enriched at rex sites, and SUMOylation enhances interactions between X-targeting factors and condensin subunits that facilitate DCC binding beyond the low level achieved without SUMOylation. DCC subunits also participate in condensin complexes essential for chromosome segregation, but their SUMOylation occurs only in the context of the DCC. Our results reinforce a newly emerging theme in which multiple proteins of a complex are SUMOylated in response to a specific stimulus, leading to accelerated complex formation and enhanced function. ChIP-chip experiments using antibodies against DPY-27, SDC-3, DPY-30, DPY-26, DPY-28 and FLAG-tagged SDC-2 in wild-type and smo-1 RNAi treated mixed embryos, with IGG controls. Also, sequential ChIP-chip experiments: (1) ChIP using FLAG antibodies to determine the genome-wide binding sites for SUMOylated proteins, (2) ChIP using FLAG antibodies followed by re-ChIP of eluted protein-chromatin complexes with DPY-27 antibodies to determine genome-wide binding sites for SUMOylated DPY-27 (referred to as DPY-27 re-ChIP experiments), (3) ChIP using FLAG antibodies followed by re-ChIP of eluted protein-chromatin with IGG antibodies to determine background binding (referred to as IGG re-ChIP experiments, and (4) ChIP using DPY-27 antibodies as a control to assess the efficiency of DPY-27 binding and detection in control vs. FLAG-tagged strains.
2013-08-31 | E-GEOD-48413 | biostudies-arrayexpress
Project description:It was previously shown that expression of an activated Phactr1 mutant (Phactr1XXX) that constitutively forms the Phactr1/PP1 phosphatase holoenzyme, induces F-actin rearrangements in NIH3T3 fibroblasts. Expression of the Phactr1 PP1-binding domain (C-terminal) alone is also sufficient to induce such cytoskeletal changes. In contrast, expression of Phactr1XXXC derivative, which lacks the PP1 binding sequences does not result in alteration of cytoskeletal morphology (Wiezlak et al., 2012). These observations suggest that Phactr1/PP1 dephosphorylates target proteins involved in cytoskeletal dynamics. To identify potential Phactr1/PP1 substrates, we used differential SILAC phosphoproteomics in NIH3T3 cells inducibly expressing Phactr1XXX, Phactr1XXXC constructs or vector alone. Over 3000 phosphorylation sites were quantified, among which we determined Phactr1/PP1 target dephosphorylation sites by comparative analysis.
2020-10-28 | PXD019977 | Pride
Project description:Objectives: To test the hypothesis that serine/threonine protein phosphatase type-1 (PP1) is dysregulated in paroxysmal atrial fibrillation (pAF) at the level of its regulatory subunits (R-subunits). Background: AF is the most common sustained cardiac arrhythmia yet current pharmacologic treatment is ineffective. PP1, a major phosphatase in the heart, consists of a catalytic subunit (PP1c) and a large set of R-subunits that confer localization and substrate specificity to the holoenzyme. Previous studies suggest that PP1 is dysregulated in AF but the mechanisms are unknown. Methods: Cardiac lysates were co-immunoprecipitated with anti-PP1c antibody followed by mass spectrometry-based (quantitative) profiling of associated R-subunits. Subsequently, label-free quantification was used to evaluate altered R-subunit-PP1c interactions in pAF patients. R-subunits with altered binding to PP1c in pAF were further validated using qRT-PCR, Western blotting (WB), immunocytochemistry, and co-immunoprecipitation. Results: 135 and 78 putative PP1c-interactors were captured respectively from mouse ventricles and human atria, with many previously unreported interactors with conserved PP1c-docking motifs. Increases in binding were found between PP1c and PPP1R7, CSDA, and PDE5A in pAF patients, with CSDA and PDE5A being novel interactors validated by bioinformatics, immunocytochemistry and co-immunoprecipitation. WB confirmed that these upregulated associations cannot be ascribed to changes in global protein expression alone. Conclusion: Subcellular heterogeneity in PP1 activity and downstream protein phosphorylation in AF may be attributed to alterations in PP1c-R-subunits interactions, which impair PP1 targeting to proteins involved in electrical and Ca2+-remodeling. This represents a novel concept in AF pathogenesis and provides highly-specific drug targets for treating AF.
2015-03-30 | PXD000697 | Pride
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