Homo sapiens
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ABSTRACT:
PROVIDER: PRJNA122533 | ENA |
REPOSITORIES: ENA
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Project description:NIPP1, an established interactor of protein phosphatase 1 (PP1), is implicated in PRC2-mediated regulation of gene expression. Here, we explore whether PP1 associated with NIPP1 is involved in NIPP1-mediated regulation of genes. Therefore, we generated Hela Tet-off (HTO) cell lines that stably and inducibly express a Flag-tagged version of wild-type NIPP1 (HTO-NIPP1wt) or mutant NIPP1 (HTO_NIPP1m). The latter mutant lacks the major PP1 binding site by two point mutations in the RVXF-motif, an established PP1 - binding motif. All cell lines were derived from the same parental HeLa Tet-Off (HTO-PT) cell line which expresses only tTA transactivator and is used as a control. The Flag fusions were only expressed in the absence of doxycyline and at levels that were up to twofold higher than that of endogenous NIPP1. We performed a gene expression profiling of the HTO cell lines, using Whole Human Genome Oligo microarrays from Agilent. A Paired SAM analysis identified 1365 genes with an altered expression (P < 0.01) between the Flag-NIPP1 and parental cell lines. Importantly, only 185 genes were differentially expressed (P<0.01) between the Flag-NIPP1m and parental cell lines. Even more strikingly, only 5% of the genes that were affected by the expression of Flag-NIPP1 also showed a significantly different expression in the Flag-NIPP1m cells. A similar small overlap was noted when the analysis was restricted to the 50 genes that were most upregulated or downregulated by the expression of Flag-NIPP1. Finally, scatter plot analysis revealed no significant correlation between the genes that were affected by the expression of Flag-NIPP1 or Flag-NIPP1m. Collectively, these data demonstrate that a moderate increase in the concentration of NIPP1 affects the expression of numerous genes by a mechanism that depends on associated PP1.
2010-07-19 | GSE19642 | GEO
Project description:NIPP1, an established interactor of protein phosphatase 1 (PP1), is implicated in PRC2-mediated regulation of gene expression. Here, we explore whether PP1 associated with NIPP1 is involved in NIPP1-mediated regulation of genes. Therefore, we generated Hela Tet-off (HTO) cell lines that stably and inducibly express a Flag-tagged version of wild-type NIPP1 (HTO-NIPP1wt) or mutant NIPP1 (HTO_NIPP1m). The latter mutant lacks the major PP1 binding site by two point mutations in the RVXF-motif, an established PP1 - binding motif. All cell lines were derived from the same parental HeLa Tet-Off (HTO-PT) cell line which expresses only tTA transactivator and is used as a control. The Flag fusions were only expressed in the absence of doxycyline and at levels that were up to twofold higher than that of endogenous NIPP1. We performed a gene expression profiling of the HTO cell lines, using Whole Human Genome Oligo microarrays from Agilent. A Paired SAM analysis identified 1365 genes with an altered expression (P < 0.01) between the Flag-NIPP1 and parental cell lines. Importantly, only 185 genes were differentially expressed (P<0.01) between the Flag-NIPP1m and parental cell lines. Even more strikingly, only 5% of the genes that were affected by the expression of Flag-NIPP1 also showed a significantly different expression in the Flag-NIPP1m cells. A similar small overlap was noted when the analysis was restricted to the 50 genes that were most upregulated or downregulated by the expression of Flag-NIPP1. Finally, scatter plot analysis revealed no significant correlation between the genes that were affected by the expression of Flag-NIPP1 or Flag-NIPP1m. Collectively, these data demonstrate that a moderate increase in the concentration of NIPP1 affects the expression of numerous genes by a mechanism that depends on associated PP1. In total 12 samples were processed. Three different cell lines were analysed: HTO_parental, HTO_NIPP1wt and HTO_NIPP1m. For each cell line 4 replicates were obtained. The HTO_parental cell line is the control cell line. For the HTO_NIPP1wt the 4 replicates were obtained from two replicates of two different transgenic cell lines expressing FlagNIPP1wt (cell line wt n°1 and 2). Each cell line derived from the same parental control cell line.
2010-07-19 | E-GEOD-19642 | biostudies-arrayexpress
Project description:The deletion of the protein phosphatase-1 (PP1) regulator NIPP1 is embryonic lethal during gastrulation, hinting at a key role of PP1-NIPP1 in lineage specification. Consistent with this notion we show here that a mild, stable overexpression of NIPP1 in HeLa cells caused a massive induction of genes of the mesenchymal lineage, in particular smooth/cardiac-muscle and matrix markers. This reprogramming was associated with the formation of actin-based stress fibers and retracting filopodia, and a reduced proliferation potential. The NIPP1-induced mesenchymal transition required functional substrate and PP1-binding domains, suggesting that it involves the selective dephosphorylation of substrates of PP1-NIPP1. In total 16 samples were processed. Four different cell lines were analysed: HTO_parental, HTO_NIPP1wt, HTO_NIPP1m (= alias NIPP1-Pm) and HTO_NIPP1-Pa. For each cell line 4 replicates were obtained. The HTO_parental cell line is the control cell line. For the HTO_NIPP1wt and the HTO_NIPP1-Pa the 4 replicates were obtained from two replicates of two different transgenic cell lines expressing FlagNIPP1wt (cell line wt n°1 and 2) and FlagNIPP1-Pa (cell line n°1 and 2), respectively. Each cell line was derived from the same parental control cell line.
2015-04-04 | E-GEOD-67558 | biostudies-arrayexpress
Project description:The deletion of the protein phosphatase-1 (PP1) regulator NIPP1 is embryonic lethal during gastrulation, hinting at a key role of PP1-NIPP1 in lineage specification. Consistent with this notion we show here that a mild, stable overexpression of NIPP1 in HeLa cells caused a massive induction of genes of the mesenchymal lineage, in particular smooth/cardiac-muscle and matrix markers. This reprogramming was associated with the formation of actin-based stress fibers and retracting filopodia, and a reduced proliferation potential. The NIPP1-induced mesenchymal transition required functional substrate and PP1-binding domains, suggesting that it involves the selective dephosphorylation of substrates of PP1-NIPP1.
2015-04-04 | GSE67558 | GEO
Project description:RNAi-seq of stable Flp-In™ T-Rex Hela cells that inducibly expressed PP1-NIPP1 and mutants.
2018-06-28 | GSE104320 | GEO
Project description:This study aims at confidently identifying endogenous nonsense mediated decay (NMD) targets. To achieve this purpose, we performed KD of a few NMD factors in HeLa cells. Additionally, we performed rescue experiments for each factor, expressing an RNAi-resistant version of the gene from a plasmid. To determine transcripts bound by UPF1 in HeLa cells, A construct with a C-terminally flag tagged version of UPF1 was expressed. In order to avoid competition with endogenous UPF1, a KD was performed.
2016-12-19 | GSE86148 | GEO
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 purpose of this experiment was to show that the iron-sulphur cluster ([4Fe-4S]) of whiB is essential for the DNA binding by WhiB and WhiA. The \\"serine version of whiB\\" mentioned below is a whiB in which the 4 cysteine residues which coordinate the [4Fe-4S] cluster were mutated to serines. ChIP-Seq using anti-FLAG anitbodies was performed in the following: 1. A whiB deletion strain expressing the FLAG-tagged serine version of WhiB (WhiBFLAG-Ser). 2. A whiB and whiA deletion strain expressing the FLAG-tagged serine version of WhiB (delAB_WhiBFLAG-Ser). 3. A whiB and whiA deletion strain expressing the serine version of WhiB and FLAG-tagged whiA (delAB_WhiAFLAG_WhiBcomp-Ser).
2021-08-16 | E-MTAB-10818 | biostudies-arrayexpress
Project description:NPAC ChIP were performed by anti-Flag and anti-HA tandem affinity purification from HeLa stably expressing Flag-HA tagged NPAC from pOZ-N vector, and enrichement on chromosome 3, 21, and 22 were determined by chip microarray analysis using Affymatrix HumanTiling 2.0 arrays
2021-10-31 | GSE160129 | GEO