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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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Gene expression analysis following various treatments. (A) Number of genes with altered expression following DXR or Nutlin treatment as compared with the control NT or DMSO controls, respectively. (B) Heat map showing gene expression changes based on microarray analysis of cells after 24 h of DXR, DMSO or Nutlin treatment. DXR and DMSO changes were assessed in relation to NT, and Nutlin changes were assessed in relation to DMSO. The KEGG/GO terms shown are non-redundant terms manually selected among the most significantly enriched categories for each of the bracketed clusters. The full lists of terms are available in Supplementary Data Set 5. (C) KEGG pathways and GO biological processes enriched among all the differentially expressed genes in (A) after various treatments. (D) Breakdown of expression changes of genes with a p53-binding site near the TSS or (E) in the intragenic region. (F) Number of genes that were bound by p53 near a TSS and were differentially expressed after DXR or Nutlin treatment.
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gkt504-F3: Gene expression analysis following various treatments. (A) Number of genes with altered expression following DXR or Nutlin treatment as compared with the control NT or DMSO controls, respectively. (B) Heat map showing gene expression changes based on microarray analysis of cells after 24 h of DXR, DMSO or Nutlin treatment. DXR and DMSO changes were assessed in relation to NT, and Nutlin changes were assessed in relation to DMSO. The KEGG/GO terms shown are non-redundant terms manually selected among the most significantly enriched categories for each of the bracketed clusters. The full lists of terms are available in Supplementary Data Set 5. (C) KEGG pathways and GO biological processes enriched among all the differentially expressed genes in (A) after various treatments. (D) Breakdown of expression changes of genes with a p53-binding site near the TSS or (E) in the intragenic region. (F) Number of genes that were bound by p53 near a TSS and were differentially expressed after DXR or Nutlin treatment.

Mentions: Global gene expression was examined using microarray analysis of the NT and the 24 h DMSO, Nutlin or DXR-treated cells. To establish clear differences in expression, we identified genes for which changes in mRNA levels were ≥2-fold and had an FDR of ≤0.1. As shown in Figure 3A, 1717 genes were differentially expressed between DXR and NT, whereas for Nutlin versus DMSO, only 416 genes were differentially expressed; only 220 genes had altered gene expression in both DXR and Nutlin treatments. In Figure 3B, we present a heat map of all the differentially expressed genes and a cluster functional analysis. Cell cycle related, p53 signaling and apoptosis categories were enriched based on a combination of GO and KEGG analyses (the complete cluster functional enrichment analysis is available as supplemental information (Supplementary Data Set 4). Further KEGG and GO analysis of all the differentially expressed genes revealed that the global gene expression pathways activated by DXR and Nutlin includes processes commonly associated with p53 functions (Figure 3C and Supplementary Data Set 4) such as cell cycle checkpoints and regulation of cell proliferation, consistent with the cell cycle arrest induced by these two drugs (Supplementary Figure S1B). Remarkably, little change in gene expression was observed following incubation of cells with DMSO versus NT, even though DMSO induces nearly 3000 binding sites, about half of which also are induced during DXR treatments (Figures 3B and 1B). Additionally, although the number of sites bound by p53 were six times greater for Nutlin than for DXR (Figure 1B), expression changes induced by Nutlin were much less dramatic compared with those induced by DXR (Figure 3B). Taken together, these data support the view that p53 binding does not predict transactivation.Figure 3.


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)

Gene expression analysis following various treatments. (A) Number of genes with altered expression following DXR or Nutlin treatment as compared with the control NT or DMSO controls, respectively. (B) Heat map showing gene expression changes based on microarray analysis of cells after 24 h of DXR, DMSO or Nutlin treatment. DXR and DMSO changes were assessed in relation to NT, and Nutlin changes were assessed in relation to DMSO. The KEGG/GO terms shown are non-redundant terms manually selected among the most significantly enriched categories for each of the bracketed clusters. The full lists of terms are available in Supplementary Data Set 5. (C) KEGG pathways and GO biological processes enriched among all the differentially expressed genes in (A) after various treatments. (D) Breakdown of expression changes of genes with a p53-binding site near the TSS or (E) in the intragenic region. (F) Number of genes that were bound by p53 near a TSS and were differentially expressed after DXR or Nutlin treatment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3753631&req=5

gkt504-F3: Gene expression analysis following various treatments. (A) Number of genes with altered expression following DXR or Nutlin treatment as compared with the control NT or DMSO controls, respectively. (B) Heat map showing gene expression changes based on microarray analysis of cells after 24 h of DXR, DMSO or Nutlin treatment. DXR and DMSO changes were assessed in relation to NT, and Nutlin changes were assessed in relation to DMSO. The KEGG/GO terms shown are non-redundant terms manually selected among the most significantly enriched categories for each of the bracketed clusters. The full lists of terms are available in Supplementary Data Set 5. (C) KEGG pathways and GO biological processes enriched among all the differentially expressed genes in (A) after various treatments. (D) Breakdown of expression changes of genes with a p53-binding site near the TSS or (E) in the intragenic region. (F) Number of genes that were bound by p53 near a TSS and were differentially expressed after DXR or Nutlin treatment.
Mentions: Global gene expression was examined using microarray analysis of the NT and the 24 h DMSO, Nutlin or DXR-treated cells. To establish clear differences in expression, we identified genes for which changes in mRNA levels were ≥2-fold and had an FDR of ≤0.1. As shown in Figure 3A, 1717 genes were differentially expressed between DXR and NT, whereas for Nutlin versus DMSO, only 416 genes were differentially expressed; only 220 genes had altered gene expression in both DXR and Nutlin treatments. In Figure 3B, we present a heat map of all the differentially expressed genes and a cluster functional analysis. Cell cycle related, p53 signaling and apoptosis categories were enriched based on a combination of GO and KEGG analyses (the complete cluster functional enrichment analysis is available as supplemental information (Supplementary Data Set 4). Further KEGG and GO analysis of all the differentially expressed genes revealed that the global gene expression pathways activated by DXR and Nutlin includes processes commonly associated with p53 functions (Figure 3C and Supplementary Data Set 4) such as cell cycle checkpoints and regulation of cell proliferation, consistent with the cell cycle arrest induced by these two drugs (Supplementary Figure S1B). Remarkably, little change in gene expression was observed following incubation of cells with DMSO versus NT, even though DMSO induces nearly 3000 binding sites, about half of which also are induced during DXR treatments (Figures 3B and 1B). Additionally, although the number of sites bound by p53 were six times greater for Nutlin than for DXR (Figure 1B), expression changes induced by Nutlin were much less dramatic compared with those induced by DXR (Figure 3B). Taken together, these data support the view that p53 binding does not predict transactivation.Figure 3.

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