Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide map of Six4 in C2C12 cells 24 hours post-differentiation

ABSTRACT: In this study, we used ChIP-seq to map Six4 binding profile in different C2C12 cell lines 24 hours after differentiation (T24). We performed ChIP-seq using two different antibodies: anti-Flag antibody in Flag-Six4 C2C12 cell line or in parental C2C12 cells; a custom-made anti-Six4 antibody in shNS C2C12 cell line (a control cell line) or shSix4 C2C12 (C2C12 with stable Six4 knockdown using short hairpin RNA). We also performed ChIP-seq in parental C2C12 cells using normal rabbit IgG. We were able to identify Six4-bound loci in C2C12 T24 that were recognized by two different antibodies and showed a decrease in peak intensity in shSix4 C2C12 compared to shNS C2C12 cells. We established a C2C12 cell line with stable Six4 knockdown by short hairpin RNA (shSix4) vs. a control cell line (shNS). We also established a C2C12 cell line with stable expression of Flag-Six4-myc by infection of retroviruses expressing pBABE-Flag-Six4-myc (Flag-Six4 C2C12) vs. parental C2C12. We differentiate these cells for 24 hours before using them for ChIP-seq.

ORGANISM(S): Mus musculus

SUBMITTER: Alexandre Blais

PROVIDER: E-GEOD-66901 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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. 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.

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Genome-wide map of Six4 in C2C12 cells 24 hours post-differentiation

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