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Gene expression and ChIP-chip analysis of U251 human glioblastoma cell line

ABSTRACT: Using human U251 glioblastoma cells with endogenous mutp53 expression as a model, we performed a ChIP-chip analysis of mutp53 binding sites on a custom tiling array, coupled with global expression profiling and an analysis of the epigenetic status of mutp53 regulated promoters. Mutp53 binds preferentially, and independent of other transcription factors (e.g. ETS1 and SP1), to G/C-rich DNA stretches around transcriptional start sites (TSS) of many genes. Mutp53-bound regions are frequently located in CpG islands and are highly prone to adopt non-B DNA conformation(s). Analysis of the transcriptional status of mutp53-regulated genes demonstrated that mutp53 generally modulates transcription from active promoters marked by H3K4me3. Based on our data we propose a dual mode model of mutp53 GOF, which includes both stochastic and deterministic components. On a local scale, mutp53 acts as a basal transcriptional co-factor that has the potential to bind autonomously and selectively to non-B DNA structures around TSSs of active genes and to modulate transcription rates of many genes in a context and stimulus-dependent fashion. Resulting stochastic alterations generate transcriptional plasticity and enhance transcriptional competence on a global scale. This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Homo sapiens

PROVIDER: GSE35500 | GEO | 2012/12/10

SECONDARY ACCESSION(S): PRJNA152303

REPOSITORIES: GEO

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Project description:Missense mutations in the TP53 gene are frequent genetic alterations in human tumor tissue and cell lines. In contrast to wild-type p53, the mutant p53 (mutp53) protein has lost the transcriptional activity towards pro-apoptotic and growth arrest genes, but retained the property to interact with DNA in a structure-specific fashion. Expression of mutp53 is advantageous for tumor cells, however the molecular mechanism of mutp53 action is still not known. We used the glioblastoma-derived U-251 MG human cell line to analyze DNA binding of mutant p53 (R273H mutation) on a Nimblegen custom 135k tiling array and to correlate mutp53 binding regions with the epigenetic state and occupation by other transcription factors (ETS1 and SP1). We found that mutp53-binding regions are G/C-rich and are located around transcriptional start sites (TSS) of many protein-coding genes, which in most cases are active, but are not always regulated upon transient mutp53 depletion. We propose a model which does not only rely on interactions of mutp53 with diverse transcriptional regulators at active promoters, but primarily is based on a DNA binding activity of mutp53. We designed a Nimblegen custom 135k tiling array that covers a large set of putative and known p53 (wild-type and mutant) target genes in the human genome. For analysis of the epigenetic state of genes covered by the tiling array in control and mutant p53-depleted U251 cells we focused on changes in active histone marks, H3K4me3 and H3K9Ac, and RNA polymerase II recruitment and processivity. H3K4me3 and H3K9Ac marks are enriched in active promoter regions and the phosphorylation of serine 5 (S5-P) and serine 2 (S2-P) in the CTD of RNA polymerase II have been described to define initiated and elongating complexes, respectively. We performed the ChIP-chip experiments for H3K4me3, H3K9Ac, RNA polymerase II (S5-P) and RNA polymerase II (S2-P) from U251 cells transfected with p53-specific siRNA or control siRNA (2 biological replicates each). To analyze binding of mutant p53 to the genes covered by the tiling array we performed mutant p53 ChIP-chip experiments in 4 biological replicates of. In addition, we analyzed the distribution of SP1 and ETS1 binding sites in 3 biological replicates.

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Gene expression and ChIP-chip analysis of U251 human glioblastoma cell line

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