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Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells.

Menendez D, Nguyen TA, Freudenberg JM, Mathew VJ, Anderson CW, Jothi R, Resnick MA - Nucleic Acids Res. (2013)

Bottom Line: Although the number of sites bound by p53 was six times greater for Nutlin than DXR, expression changes induced by Nutlin were much less dramatic compared with DXR.Unexpectedly, the solvent dimethylsulphoxide (DMSO) alone induced p53 binding to many sites common to DXR; however, this binding had no effect on target gene expression.Furthermore, both p53 binding and transactivation were associated with increased active histone modification histone H3 lysine 4 trimethylation.

View Article: PubMed Central - PubMed

Affiliation: Chromosome Stability Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), Research Triangle Park, NC 27709, USA, Systems Biology Group, Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), Research Triangle Park, NC 27709, USA, William G. Enloe High School, Raleigh, NC 27610, USA and Department of Biology, Brookhaven National Laboratory, Upton, NY 11973, USA.

ABSTRACT
The effects of diverse stresses on promoter selectivity and transcription regulation by the tumor suppressor p53 are poorly understood. We have taken a comprehensive approach to characterizing the human p53 network that includes p53 levels, binding, expression and chromatin changes under diverse stresses. Human osteosarcoma U2OS cells treated with anti-cancer drugs Doxorubicin (DXR) or Nutlin-3 (Nutlin) led to strikingly different p53 gene binding patterns based on chromatin immunoprecipitation with high-throughput sequencing experiments. Although two contiguous RRRCWWGYYY decamers is the consensus binding motif, p53 can bind a single decamer and function in vivo. Although the number of sites bound by p53 was six times greater for Nutlin than DXR, expression changes induced by Nutlin were much less dramatic compared with DXR. Unexpectedly, the solvent dimethylsulphoxide (DMSO) alone induced p53 binding to many sites common to DXR; however, this binding had no effect on target gene expression. Together, these data imply a two-stage mechanism for p53 transactivation where p53 binding only constitutes the first stage. Furthermore, both p53 binding and transactivation were associated with increased active histone modification histone H3 lysine 4 trimethylation. We discovered 149 putative new p53 target genes including several that are relevant to tumor suppression, revealing potential new targets for cancer therapy and expanding our understanding of the p53 regulatory network.

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Genome-wide p53 binding after various treatments. (A) Representative western blot of p53 levels in U2OS cytoplasmic, nuclear or whole-cell extracts from untreated (NT) cells, or after 24 h treatment with 0.1% DMSO, 0.6 µg/ml DXR or 10 µM Nutlin. Lamin B and actin Hsp90, lamin B and actin are loading controls. (B) Number of p53 binding sites identified in U2OS 24 h after treatment with DMSO, DXR or Nutlin. (C) UCSC Genome Browser shots summarizing p53 ChIP-Seq and Input control data for known p53 target genes after various treatments. The Y axis corresponds to the number of reads. The same scale is used for all examples presented. (D) Genomic distribution of p53 binding sites relative to genic regions after the various treatments: TSS, 5 kb upstream through the first intron; Intragenic, second exon through 3′UTR; and Intergenic, everything else.
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gkt504-F1: Genome-wide p53 binding after various treatments. (A) Representative western blot of p53 levels in U2OS cytoplasmic, nuclear or whole-cell extracts from untreated (NT) cells, or after 24 h treatment with 0.1% DMSO, 0.6 µg/ml DXR or 10 µM Nutlin. Lamin B and actin Hsp90, lamin B and actin are loading controls. (B) Number of p53 binding sites identified in U2OS 24 h after treatment with DMSO, DXR or Nutlin. (C) UCSC Genome Browser shots summarizing p53 ChIP-Seq and Input control data for known p53 target genes after various treatments. The Y axis corresponds to the number of reads. The same scale is used for all examples presented. (D) Genomic distribution of p53 binding sites relative to genic regions after the various treatments: TSS, 5 kb upstream through the first intron; Intragenic, second exon through 3′UTR; and Intergenic, everything else.

Mentions: Treatment of U2OS cells with DXR (0.6 µg/ml) or Nutlin (10 µM) resulted in the stabilization and increase of nuclear p53 protein levels (Figure 1A and Supplementary Figure 1A) after 24 h (a time point commonly used in studies of p53 activating drugs; see ‘Discussion’ section). Nutlin treatment induced a clear G1 and G2 cell cycle arrest, whereas DXR-treated cells were arrested at G2. DMSO treatment had no effect on cell cycle progression when compared with the No Treatment (NT) control. There was no substantial induction of apoptosis (measured as a sub-G1 fraction) for any of the treatment conditions at 24 h (Supplementary Figure S1B). ChIP-seq analysis using the p53 specific antibody DO-1 revealed considerable difference in the number and pattern of binding sites across the genome after DXR or Nutlin treatment. Genome-wide analysis of the ChIP-seq tags using the SISSRs peak-finding tool (16) identified 496 binding sites (peaks) for the NT control, 3087 sites after DXR treatment and nearly 6-fold more sites (18 159) in cells treated with Nutlin (Figure 1B, Supplementary Figure S1C and Supplementary Table S1). Surprisingly, the solvent DMSO (0.1%) alone caused substantial p53 binding (2793 sites). No relationship between the number of binding sites and the level of p53 expression was seen: DXR led to the largest induction of p53; Nutlin treatment induced less p53; there was little, if any, p53 induction after DMSO incubation (Figure 1A and Supplementary Figure S1A). Specific binding to the REs of the well-characterized p53 target genes BAX, CDKN1A (p21) and MDM2 attest to the quality of ChIP-Seq data (Figure 1C and Supplementary Figure S1D). To ensure the reliability of our ChIP-Seq data, we validated 36 of the identified p53 sites by ChIP-qPCR (described in Supplementary Table S2 and Supplementary Figure S1E and S1F). Nearly half of the sites bound by p53 after DXR treatment were also bound by p53 under DMSO and Nutlin treatments, suggesting common features to these presumably different modes of induced binding. However, nearly 80% of the Nutlin-induced p53-binding sites were unique (Figure 1B and Supplementary Figure S1C), with only seven sites in common between all three treatments and the NT control (Supplementary Figure S1C), suggesting a different mechanism of p53 binding in the basal condition without any treatment.Figure 1.


Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells.

Menendez D, Nguyen TA, Freudenberg JM, Mathew VJ, Anderson CW, Jothi R, Resnick MA - Nucleic Acids Res. (2013)

Genome-wide p53 binding after various treatments. (A) Representative western blot of p53 levels in U2OS cytoplasmic, nuclear or whole-cell extracts from untreated (NT) cells, or after 24 h treatment with 0.1% DMSO, 0.6 µg/ml DXR or 10 µM Nutlin. Lamin B and actin Hsp90, lamin B and actin are loading controls. (B) Number of p53 binding sites identified in U2OS 24 h after treatment with DMSO, DXR or Nutlin. (C) UCSC Genome Browser shots summarizing p53 ChIP-Seq and Input control data for known p53 target genes after various treatments. The Y axis corresponds to the number of reads. The same scale is used for all examples presented. (D) Genomic distribution of p53 binding sites relative to genic regions after the various treatments: TSS, 5 kb upstream through the first intron; Intragenic, second exon through 3′UTR; and Intergenic, everything else.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3753631&req=5

gkt504-F1: Genome-wide p53 binding after various treatments. (A) Representative western blot of p53 levels in U2OS cytoplasmic, nuclear or whole-cell extracts from untreated (NT) cells, or after 24 h treatment with 0.1% DMSO, 0.6 µg/ml DXR or 10 µM Nutlin. Lamin B and actin Hsp90, lamin B and actin are loading controls. (B) Number of p53 binding sites identified in U2OS 24 h after treatment with DMSO, DXR or Nutlin. (C) UCSC Genome Browser shots summarizing p53 ChIP-Seq and Input control data for known p53 target genes after various treatments. The Y axis corresponds to the number of reads. The same scale is used for all examples presented. (D) Genomic distribution of p53 binding sites relative to genic regions after the various treatments: TSS, 5 kb upstream through the first intron; Intragenic, second exon through 3′UTR; and Intergenic, everything else.
Mentions: Treatment of U2OS cells with DXR (0.6 µg/ml) or Nutlin (10 µM) resulted in the stabilization and increase of nuclear p53 protein levels (Figure 1A and Supplementary Figure 1A) after 24 h (a time point commonly used in studies of p53 activating drugs; see ‘Discussion’ section). Nutlin treatment induced a clear G1 and G2 cell cycle arrest, whereas DXR-treated cells were arrested at G2. DMSO treatment had no effect on cell cycle progression when compared with the No Treatment (NT) control. There was no substantial induction of apoptosis (measured as a sub-G1 fraction) for any of the treatment conditions at 24 h (Supplementary Figure S1B). ChIP-seq analysis using the p53 specific antibody DO-1 revealed considerable difference in the number and pattern of binding sites across the genome after DXR or Nutlin treatment. Genome-wide analysis of the ChIP-seq tags using the SISSRs peak-finding tool (16) identified 496 binding sites (peaks) for the NT control, 3087 sites after DXR treatment and nearly 6-fold more sites (18 159) in cells treated with Nutlin (Figure 1B, Supplementary Figure S1C and Supplementary Table S1). Surprisingly, the solvent DMSO (0.1%) alone caused substantial p53 binding (2793 sites). No relationship between the number of binding sites and the level of p53 expression was seen: DXR led to the largest induction of p53; Nutlin treatment induced less p53; there was little, if any, p53 induction after DMSO incubation (Figure 1A and Supplementary Figure S1A). Specific binding to the REs of the well-characterized p53 target genes BAX, CDKN1A (p21) and MDM2 attest to the quality of ChIP-Seq data (Figure 1C and Supplementary Figure S1D). To ensure the reliability of our ChIP-Seq data, we validated 36 of the identified p53 sites by ChIP-qPCR (described in Supplementary Table S2 and Supplementary Figure S1E and S1F). Nearly half of the sites bound by p53 after DXR treatment were also bound by p53 under DMSO and Nutlin treatments, suggesting common features to these presumably different modes of induced binding. However, nearly 80% of the Nutlin-induced p53-binding sites were unique (Figure 1B and Supplementary Figure S1C), with only seven sites in common between all three treatments and the NT control (Supplementary Figure S1C), suggesting a different mechanism of p53 binding in the basal condition without any treatment.Figure 1.

Bottom Line: Although the number of sites bound by p53 was six times greater for Nutlin than DXR, expression changes induced by Nutlin were much less dramatic compared with DXR.Unexpectedly, the solvent dimethylsulphoxide (DMSO) alone induced p53 binding to many sites common to DXR; however, this binding had no effect on target gene expression.Furthermore, both p53 binding and transactivation were associated with increased active histone modification histone H3 lysine 4 trimethylation.

View Article: PubMed Central - PubMed

Affiliation: Chromosome Stability Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), Research Triangle Park, NC 27709, USA, Systems Biology Group, Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), Research Triangle Park, NC 27709, USA, William G. Enloe High School, Raleigh, NC 27610, USA and Department of Biology, Brookhaven National Laboratory, Upton, NY 11973, USA.

ABSTRACT
The effects of diverse stresses on promoter selectivity and transcription regulation by the tumor suppressor p53 are poorly understood. We have taken a comprehensive approach to characterizing the human p53 network that includes p53 levels, binding, expression and chromatin changes under diverse stresses. Human osteosarcoma U2OS cells treated with anti-cancer drugs Doxorubicin (DXR) or Nutlin-3 (Nutlin) led to strikingly different p53 gene binding patterns based on chromatin immunoprecipitation with high-throughput sequencing experiments. Although two contiguous RRRCWWGYYY decamers is the consensus binding motif, p53 can bind a single decamer and function in vivo. Although the number of sites bound by p53 was six times greater for Nutlin than DXR, expression changes induced by Nutlin were much less dramatic compared with DXR. Unexpectedly, the solvent dimethylsulphoxide (DMSO) alone induced p53 binding to many sites common to DXR; however, this binding had no effect on target gene expression. Together, these data imply a two-stage mechanism for p53 transactivation where p53 binding only constitutes the first stage. Furthermore, both p53 binding and transactivation were associated with increased active histone modification histone H3 lysine 4 trimethylation. We discovered 149 putative new p53 target genes including several that are relevant to tumor suppression, revealing potential new targets for cancer therapy and expanding our understanding of the p53 regulatory network.

Show MeSH
Related in: MedlinePlus