Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Expression profiling of HeLa Tet-off cells expressing Flag-tagged version of WT NIPP1 or PP1 binding mutant of NIPP1.

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Joke Allemeersch

PROVIDER: E-GEOD-19642 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The phosphatase interactor NIPP1 regulates the occupancy of the histone methyltransferase EZH2 at Polycomb targets.

Van Dessel Nele N Beke Lijs L Görnemann Janina J Minnebo Nikki N Beullens Monique M Tanuma Nobuhiro N Shima Hiroshi H Van Eynde Aleyde A Bollen Mathieu M

Nucleic acids research 20100729 21

Polycomb group (PcG) proteins are key regulators of stem-cell and cancer biology. They mainly act as repressors of differentiation and tumor-suppressor genes. One key silencing step involves the trimethylation of histone H3 on Lys27 (H3K27) by EZH2, a core component of the Polycomb Repressive Complex 2 (PRC2). The mechanism underlying the initial recruitment of mammalian PRC2 complexes is not well understood. Here, we show that NIPP1, a regulator of protein Ser/Thr phosphatase-1 (PP1), forms a c ...[more]

PMID: 20671031

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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.

Dataset Information

Expression profiling of HeLa Tet-off cells expressing Flag-tagged version of WT NIPP1 or PP1 binding mutant of NIPP1.

Publications

The phosphatase interactor NIPP1 regulates the occupancy of the histone methyltransferase EZH2 at Polycomb targets.

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