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Genome-wide maps of chromatin state in HCT116 cells expressing Flag-tagged VCP

ABSTRACT: Genome-wide maps of chromatin state in HCT116 cells expressing Flag-tagged VCP

PROVIDER: PRJNA830344 | ENA |

REPOSITORIES: ENA

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Genome-wide maps of chromatin state in HCT116 cells expressing Flag-tagged VCP

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Project description:RNA-sequencing of H3.3-G34R and H3.3-WT HGG cells was performed to uncover transcriptomic differences related to the presence of H3.3-G34R mutation, using human cells obtained from a patient harboring pHGG and stably tranfected with 3X-FLAG-tagged wild type H3.3 or a 3X-FLAG-tagged H3.3 harboring the G34R mutation.

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

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.

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