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Induction of PPM1D following DNA-damaging treatments through a conserved p53 response element coincides with a shift in the use of transcription initiation sites.

Rossi M, Demidov ON, Anderson CW, Appella E, Mazur SJ - Nucleic Acids Res. (2008)

Bottom Line: PPM1D also has been identified as a CREB-regulated gene due to the presence of a cyclic AMP response element (CRE) in the promoter.CREB binding to the CRE contributes to the regulation of basal expression of PPM1D and directs transcription initiation at upstream sites.In cells containing wild-type p53, exposure to UV resulted in increased PPM1D protein levels even when PPM1D mRNA levels remained constant, indicating post-transcriptional regulation of PPM1D protein levels.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
PPM1D (Wip1), a type PP2C phosphatase, is expressed at low levels in most normal tissues but is overexpressed in several types of cancers. In cells containing wild-type p53, the levels of PPM1D mRNA and protein increase following exposure to genotoxic stress, but the mechanism of regulation by p53 was unknown. PPM1D also has been identified as a CREB-regulated gene due to the presence of a cyclic AMP response element (CRE) in the promoter. Transient transfection and chromatin immunoprecipitation experiments in HCT116 cells were used to characterize a conserved p53 response element located in the 5' untranslated region (UTR) of the PPM1D gene that is required for the p53-dependent induction of transcription from the human PPM1D promoter. CREB binding to the CRE contributes to the regulation of basal expression of PPM1D and directs transcription initiation at upstream sites. Following exposure to ultraviolet (UV) or ionizing radiation, the abundance of transcripts with short 5' UTRs increased in cells containing wild-type p53, indicating increased utilization of downstream transcription initiation sites. In cells containing wild-type p53, exposure to UV resulted in increased PPM1D protein levels even when PPM1D mRNA levels remained constant, indicating post-transcriptional regulation of PPM1D protein levels.

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(A) Northern blot analysis of PPM1D expression in HCT116 p53+/+ and p53–/– cells following exposure to 6-Gy IR. (B) Immunoblot analysis of PPM1D protein levels in extracts from HCT116 p53+/+ and p53–/– cells at the indicated times following exposure to 10-Gy IR.
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Figure 1: (A) Northern blot analysis of PPM1D expression in HCT116 p53+/+ and p53–/– cells following exposure to 6-Gy IR. (B) Immunoblot analysis of PPM1D protein levels in extracts from HCT116 p53+/+ and p53–/– cells at the indicated times following exposure to 10-Gy IR.

Mentions: To investigate the mechanism of the p53-dependent induction of PPM1D, we used the human colorectal cell line HCT116, which contains wild-type p53 protein and exhibits a normal p53 response following treatments with DNA damage-inducing agents, and an HCT116 p53–/– derivative that lacks p53. Northern blot analysis of PPM1D expression in HCT116 p53+/+ cells showed that PPM1D mRNA levels increased after exposure to 10 Gy IR in p53+/+ cells with the maximum induction occurring 3 h post-irradiation (Figure 1A). Although the levels of PPM1D mRNA were comparable in HCT116 p53+/+ and p53–/– cells prior to irradiation, PPM1D mRNA levels decreased after IR in p53–/– cells. As shown in the immunoblot depicted in Figure 1B, the levels of PPM1D protein also increase markedly following exposure of cells containing wild-type p53 to IR. These results are in accord with previous work establishing that the induction of PPM1D mRNA and protein following IR in human or mouse cells is dependent on wild-type p53 (1,2).Figure 1.


Induction of PPM1D following DNA-damaging treatments through a conserved p53 response element coincides with a shift in the use of transcription initiation sites.

Rossi M, Demidov ON, Anderson CW, Appella E, Mazur SJ - Nucleic Acids Res. (2008)

(A) Northern blot analysis of PPM1D expression in HCT116 p53+/+ and p53–/– cells following exposure to 6-Gy IR. (B) Immunoblot analysis of PPM1D protein levels in extracts from HCT116 p53+/+ and p53–/– cells at the indicated times following exposure to 10-Gy IR.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602757&req=5

Figure 1: (A) Northern blot analysis of PPM1D expression in HCT116 p53+/+ and p53–/– cells following exposure to 6-Gy IR. (B) Immunoblot analysis of PPM1D protein levels in extracts from HCT116 p53+/+ and p53–/– cells at the indicated times following exposure to 10-Gy IR.
Mentions: To investigate the mechanism of the p53-dependent induction of PPM1D, we used the human colorectal cell line HCT116, which contains wild-type p53 protein and exhibits a normal p53 response following treatments with DNA damage-inducing agents, and an HCT116 p53–/– derivative that lacks p53. Northern blot analysis of PPM1D expression in HCT116 p53+/+ cells showed that PPM1D mRNA levels increased after exposure to 10 Gy IR in p53+/+ cells with the maximum induction occurring 3 h post-irradiation (Figure 1A). Although the levels of PPM1D mRNA were comparable in HCT116 p53+/+ and p53–/– cells prior to irradiation, PPM1D mRNA levels decreased after IR in p53–/– cells. As shown in the immunoblot depicted in Figure 1B, the levels of PPM1D protein also increase markedly following exposure of cells containing wild-type p53 to IR. These results are in accord with previous work establishing that the induction of PPM1D mRNA and protein following IR in human or mouse cells is dependent on wild-type p53 (1,2).Figure 1.

Bottom Line: PPM1D also has been identified as a CREB-regulated gene due to the presence of a cyclic AMP response element (CRE) in the promoter.CREB binding to the CRE contributes to the regulation of basal expression of PPM1D and directs transcription initiation at upstream sites.In cells containing wild-type p53, exposure to UV resulted in increased PPM1D protein levels even when PPM1D mRNA levels remained constant, indicating post-transcriptional regulation of PPM1D protein levels.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
PPM1D (Wip1), a type PP2C phosphatase, is expressed at low levels in most normal tissues but is overexpressed in several types of cancers. In cells containing wild-type p53, the levels of PPM1D mRNA and protein increase following exposure to genotoxic stress, but the mechanism of regulation by p53 was unknown. PPM1D also has been identified as a CREB-regulated gene due to the presence of a cyclic AMP response element (CRE) in the promoter. Transient transfection and chromatin immunoprecipitation experiments in HCT116 cells were used to characterize a conserved p53 response element located in the 5' untranslated region (UTR) of the PPM1D gene that is required for the p53-dependent induction of transcription from the human PPM1D promoter. CREB binding to the CRE contributes to the regulation of basal expression of PPM1D and directs transcription initiation at upstream sites. Following exposure to ultraviolet (UV) or ionizing radiation, the abundance of transcripts with short 5' UTRs increased in cells containing wild-type p53, indicating increased utilization of downstream transcription initiation sites. In cells containing wild-type p53, exposure to UV resulted in increased PPM1D protein levels even when PPM1D mRNA levels remained constant, indicating post-transcriptional regulation of PPM1D protein levels.

Show MeSH
Related in: MedlinePlus