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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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Validation of new p53 direct target genes. Real-time PCR validation of newly identified putative p53 targets in U2OS cells using shRNA-mediated silencing of p53 expression. Parental U2OS cells treated with (A) no shRNA, (B) scramble (control) shRNA, (C) p53shRNA 3755 or (D) p53shRNA 3756. mRNA levels were quantified in U2OS cells 24 h after NT, DMSO, DXR or Nutlin treatment using qRT-PCR and normalized to GAPDH. mRNA levels are shown as fold change after treatment relative to the NT control. Expression of the CDKN1A gene was used as a positive control. Shown are averages of three independent experiments. The dashed line corresponds to a 2-fold change in expression. Error bars represent standard deviation (SD).
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gkt504-F5: Validation of new p53 direct target genes. Real-time PCR validation of newly identified putative p53 targets in U2OS cells using shRNA-mediated silencing of p53 expression. Parental U2OS cells treated with (A) no shRNA, (B) scramble (control) shRNA, (C) p53shRNA 3755 or (D) p53shRNA 3756. mRNA levels were quantified in U2OS cells 24 h after NT, DMSO, DXR or Nutlin treatment using qRT-PCR and normalized to GAPDH. mRNA levels are shown as fold change after treatment relative to the NT control. Expression of the CDKN1A gene was used as a positive control. Shown are averages of three independent experiments. The dashed line corresponds to a 2-fold change in expression. Error bars represent standard deviation (SD).

Mentions: Of the 149 potential new p53 targets, 11 candidate genes were characterized further for responses to DMSO, DXR or Nutlin (Figure 5). Included were genes with p53 REs having zero spacer (CPEB4, APOBEC3C, ARHGEF3, ACYP2, SYTL1, DYRK3, NF2), a 9 nt spacer (LASS5) or no p53 motif in the region near the TSS (INPP1, EBI3, CDC27). We also examined three candidate genes that were bound by p53 but showed no expression changes: APOBEC3H, zero spacer; CYP3A7 and EML2, half sites. Quantitative RT-PCR analysis confirmed that among the 14 genes examined, 12 were upregulated while 2 were downregulated by DXR or Nutlin treatment (Figure 5A). As previously observed in the microarray analysis, no significant changes were observed in DMSO-treated cells. Although APOBEC3H, CYP3A7 and EML2 showed no differential expression using microarray analysis, expression changes could be detected with qRT-PCR.Figure 5.


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)

Validation of new p53 direct target genes. Real-time PCR validation of newly identified putative p53 targets in U2OS cells using shRNA-mediated silencing of p53 expression. Parental U2OS cells treated with (A) no shRNA, (B) scramble (control) shRNA, (C) p53shRNA 3755 or (D) p53shRNA 3756. mRNA levels were quantified in U2OS cells 24 h after NT, DMSO, DXR or Nutlin treatment using qRT-PCR and normalized to GAPDH. mRNA levels are shown as fold change after treatment relative to the NT control. Expression of the CDKN1A gene was used as a positive control. Shown are averages of three independent experiments. The dashed line corresponds to a 2-fold change in expression. Error bars represent standard deviation (SD).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3753631&req=5

gkt504-F5: Validation of new p53 direct target genes. Real-time PCR validation of newly identified putative p53 targets in U2OS cells using shRNA-mediated silencing of p53 expression. Parental U2OS cells treated with (A) no shRNA, (B) scramble (control) shRNA, (C) p53shRNA 3755 or (D) p53shRNA 3756. mRNA levels were quantified in U2OS cells 24 h after NT, DMSO, DXR or Nutlin treatment using qRT-PCR and normalized to GAPDH. mRNA levels are shown as fold change after treatment relative to the NT control. Expression of the CDKN1A gene was used as a positive control. Shown are averages of three independent experiments. The dashed line corresponds to a 2-fold change in expression. Error bars represent standard deviation (SD).
Mentions: Of the 149 potential new p53 targets, 11 candidate genes were characterized further for responses to DMSO, DXR or Nutlin (Figure 5). Included were genes with p53 REs having zero spacer (CPEB4, APOBEC3C, ARHGEF3, ACYP2, SYTL1, DYRK3, NF2), a 9 nt spacer (LASS5) or no p53 motif in the region near the TSS (INPP1, EBI3, CDC27). We also examined three candidate genes that were bound by p53 but showed no expression changes: APOBEC3H, zero spacer; CYP3A7 and EML2, half sites. Quantitative RT-PCR analysis confirmed that among the 14 genes examined, 12 were upregulated while 2 were downregulated by DXR or Nutlin treatment (Figure 5A). As previously observed in the microarray analysis, no significant changes were observed in DMSO-treated cells. Although APOBEC3H, CYP3A7 and EML2 showed no differential expression using microarray analysis, expression changes could be detected with qRT-PCR.Figure 5.

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