Protein phosphatase PP1-NIPP1 activates mesenchymal genes in HeLa cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Wouter Van Delm
PROVIDER: E-GEOD-67558 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
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: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: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: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:NIPP1 and EZH2 contribute to the silencing of a common set of genes. Since both NIPP1 and EZH2 turned out to be essential for the initiation and maintenance of global H3K27 trimethylation, a key step in the PcG-mediated transcriptional regulation of genes, we have subsequently examined by DNA-microarray analyses whether the loss of NIPP1 or EZH2 results in the expected regulation of a common set of genes. <br> <br> PC-3 cells were transfected in four independent experiments with a control siRNA or siRNA duplexes for the knockdown of EZH2 or NIPP1. To minimize indirect effects, RNA was already isolated 48h after transfection. At this time, the knockdown of NIPP1 or EZH2 was verified by quantitative qRT-PCR and amounted to 73 ± 7% and 82 ± 3% (n = 4), respectively. The isolated RNA pools were labeled with a fluorescent dye and were hybridized onto whole human genome Agilent gene chips. These chips contained 44,000 60-mer oligonucleotides, corresponding to ca 27,000 unambiguously annotated genes. Using SAM (significance analysis of microarray) analysis (P < 0.05), performed by comparing the NIPP1 or EZH2 knockdown against RNAi control, we selected 1581 and 2024 genes as significant in the NIPP1 or EZH2 knockdown, respectively. Using p<0.01, the loss of NIPP1 or EZH2 resulted in a significant upregulation of 281 genes and 319 genes, respectively, and downregulation of 409 and 175 genes respectively ). The knockdown of NIPP1 or EZH2 was confirmed in the microarray and the results of the expression array data were confirmed by qRT-PCR on a selection of affected genes, representing the entire range of fold-changes and using samples independent of those used for the microarray analysis.<br> <br> Strikingly, the list of the 50 genes that were most upregulated (SAM test, p<0.01) by the knockdown of NIPP1 were, in most cases, also upregulated following the knockdown of EZH2. In contrast, there was no obvious correlation between the 50 genes that were most downregulated after the knockdown of NIPP1 or EZH2. More detailed analysis revealed that 43% (121 genes) of the genes that were upregulated after the knockdown of NIPP1 were also upregulated by the loss of EZH2. In contrast, only 6% (25 genes) of the genes that were downregulated by the knockdown of NIPP1 were also downregulated by the loss of EZH2. Using another approach and using the list of significant genes based upon SAM test and p<0.05, we noted a significant linear correlation (R= 0.59 ± 0.01) between the extent to which genes were significantly upregulated by the loss of NIPP1 and the changes in the expression of these same genes following the knockdown of EZH2. No such correlation (R= 0.01 ± 0.01) was obtained for genes that were significantly downregulated. In the residual plot of these regressions, an obvious bend became apparent between the up and downregulated genes of the NIPP1 knockdown, confirming that we are in fact dealing with two differently regulated subpopulations. To validate this subdivision, the microarray data for a knockdown of EZH2, EED or SUZ12 which represent the core of the PRC2 complex, recently published by Bracken et al. ( 2006 ), were identically analyzed. In the residual plots, a bend between the up and downregulated genes was consistently present. We can conclude from our microarray data that NIPP1 silence a same set of genes as EZH2 and that NIPP1 also act as a transcriptional activator independent of EZH2.
2007-08-21 | E-TABM-128 | biostudies-arrayexpress
Project description:Background: Pachymic acid (PA) is a purified triterpene extracted from medicinal fungus Poria cocos. In this paper, we investigated the anticancer effects of PA on human chemotherapy resistant pancreatic cancer cells. Methods: Gemcitabine-resistant pancreatic cancer cells PANC-1 and MIA PaCa-2 were used, along with a xenograft model of MIA PaCa-2 cells implanted in mice. Apoptosis was assessed by quantitation of cytoplasmic histone-associated DNA fragments and expression of cleaved PARP. Differential expression of genes was identified using comparative DNA microarray analysis. Protein levels were determined by immunoblotting. Toxicology studies in vivo were assessed by detecting pathological changes in organs and liver enzyme profiles in plasma. Tumor tissues were analyzed by quantification of apoptotic bodies, qRT-PCR and immunoblotting. Principal Findings: PA induced endoplasmic reticulum (ER) stress in chemotherapy resistant pancreatic cancer cells through activation of heat shock response and unfolded protein response related genes, which further triggered apoptosis. The involvement of ER stress was confirmed by increasing expression of XBP-1s, ATF4, Hsp70, CHOP and phospho-eIF2M-NM-1. Moreover, 25 mg kgM-bM-^HM-^R1 of PA significantly suppressed MIA PaCa-2 tumor growth in vivo without toxicity, which correlated with induction of apoptosis, ER stress related genes and proteins expression. Conclusions: Growth inhibition and induction of apoptosis by PA in chemotherapy resistant pancreatic cancer cells were associated with ER stress activation both in vitro and in vivo. Pancreatic cancer cell line treated with pachymic acid vs. control (untreated)
2014-12-12 | E-GEOD-64111 | biostudies-arrayexpress
Project description:Upon recruitment to active enhancers and promoters, RNA polymerase II (Pol_II) generates short non-coding transcripts of unclear function. The mechanisms that control the length and the amount of ncRNAs generated by cis-regulatory elements are largely unknown. Here, we show that the adapter protein WDR82 and its associated complexes actively limit such non-coding transcription. WDR82 targets the SET1/COMPASS H3K4 methyltransferase and the nuclear Protein Phosphatase 1 (PP1) complexes to the initiating Pol_II. WDR82 and PP1 also interact with components of the transcriptional termination and RNA processing machineries. Depletion of WDR82, SET1 or the PP1 subunit required for its nuclear import caused distinct but overlapping transcription termination defects at highly expressed genes, active enhancers and promoters, thus enabling the increased synthesis of unusually long ncRNAs. These data indicate that transcription initiated from cis-regulatory elements is tightly coordinated with termination mechanisms that impose the synthesis of short RNAs. 4C-seq was performed on two different viewpoints (TSSs of the genes Ccl2 and Jun) in BMDMs.
2015-11-09 | E-GEOD-66950 | biostudies-arrayexpress
Project description:Upon recruitment to active enhancers and promoters, RNA polymerase II (Pol_II) generates short non-coding transcripts of unclear function. The mechanisms that control the length and the amount of ncRNAs generated by cis-regulatory elements are largely unknown. Here, we show that the adapter protein WDR82 and its associated complexes actively limit such non-coding transcription. WDR82 targets the SET1/COMPASS H3K4 methyltransferase and the nuclear Protein Phosphatase 1 (PP1) complexes to the initiating Pol_II. WDR82 and PP1 also interact with components of the transcriptional termination and RNA processing machineries. Depletion of WDR82, SET1 or the PP1 subunit required for its nuclear import caused distinct but overlapping transcription termination defects at highly expressed genes, active enhancers and promoters, thus enabling the increased synthesis of unusually long ncRNAs. These data indicate that transcription initiated from cis-regulatory elements is tightly coordinated with termination mechanisms that impose the synthesis of short RNAs. 4C-seq was performed on four different viewpoints (either at the 5' or the 3' of the Mpeg1 and Lgals3 genes, indicated as sx or dx, respectively) in BMDMs. Experiments were carried out in cells containing either a short hairpin targeting Wdr82 or a scrambled as a control.
2015-11-09 | E-GEOD-72958 | biostudies-arrayexpress
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