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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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Involvement of the CRE and CREB in the control of PPM1D transcription following exposure to IR. (A) The CRE and p53RE sites independently regulate PPM1D promoter activity. HCT116 p53–/– cells were transfected with constructs containing a 600-bp PPM1D promoter fragment or with mutations in the p53RE site, the CRE site or both sites, along with pRL-TK and the empty vector or wild-type p53 expression vector. Cellular extracts were prepared 4 h after exposure to 10 Gy IR. (B) CREB dissociates from the PPM1D promoter region following exposure to IR. Chromatin fragments immunoprecipitated by anti-CREB antibody or nonspecific IgG from HCT116 p53+/+ or HCT116 p53–/– cell extracts at the indicated times following exposure to 5-Gy IR were detected by PCR using primers specific for the PPM1D promoter. Fragments amplified by PCR from 0.62% of the input material are shown.
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Figure 5: Involvement of the CRE and CREB in the control of PPM1D transcription following exposure to IR. (A) The CRE and p53RE sites independently regulate PPM1D promoter activity. HCT116 p53–/– cells were transfected with constructs containing a 600-bp PPM1D promoter fragment or with mutations in the p53RE site, the CRE site or both sites, along with pRL-TK and the empty vector or wild-type p53 expression vector. Cellular extracts were prepared 4 h after exposure to 10 Gy IR. (B) CREB dissociates from the PPM1D promoter region following exposure to IR. Chromatin fragments immunoprecipitated by anti-CREB antibody or nonspecific IgG from HCT116 p53+/+ or HCT116 p53–/– cell extracts at the indicated times following exposure to 5-Gy IR were detected by PCR using primers specific for the PPM1D promoter. Fragments amplified by PCR from 0.62% of the input material are shown.

Mentions: The presence of a conserved CRE within the promoter of the PPM1D gene (16), reported protein–protein interactions between p53 and CREB (19) and reported cooperation in the control of the Bradykinin B2 receptor gene by p53 and CREB (20) suggested a possible involvement of the CRE site and CREB/ATF family transcription factors in the IR-responsiveness of the PPM1D promoter. To investigate whether the CRE site was necessary for the p53-dependent induction of the PPM1D promoter, we transiently transfected expression vectors and luciferase reporter constructs into HCT116 p53–/– cells. In agreement with results described above, the relative luciferase activity resulting from the wild-type PPM1D promoter was robust in the absence of p53 and increased ∼1.5-fold in the presence of p53; mutation of p53RE [m(123)] did not affect the promoter activity in the absence of p53 but reduced promoter activity in the presence of p53 to 44% (Figure 5A). In the absence of p53, mutation of the CRE reduced the promoter activity to 63% of the wild-type promoter, but in the presence of p53, the CRE-mutated promoter exhibited 1.7-fold increased expression. The construct in which both the CRE and p53RE sites (mm) were mutated produced an intermediate level of expression in the absence of p53 and a reduced expression in the presence of p53.Figure 5.


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)

Involvement of the CRE and CREB in the control of PPM1D transcription following exposure to IR. (A) The CRE and p53RE sites independently regulate PPM1D promoter activity. HCT116 p53–/– cells were transfected with constructs containing a 600-bp PPM1D promoter fragment or with mutations in the p53RE site, the CRE site or both sites, along with pRL-TK and the empty vector or wild-type p53 expression vector. Cellular extracts were prepared 4 h after exposure to 10 Gy IR. (B) CREB dissociates from the PPM1D promoter region following exposure to IR. Chromatin fragments immunoprecipitated by anti-CREB antibody or nonspecific IgG from HCT116 p53+/+ or HCT116 p53–/– cell extracts at the indicated times following exposure to 5-Gy IR were detected by PCR using primers specific for the PPM1D promoter. Fragments amplified by PCR from 0.62% of the input material are shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Involvement of the CRE and CREB in the control of PPM1D transcription following exposure to IR. (A) The CRE and p53RE sites independently regulate PPM1D promoter activity. HCT116 p53–/– cells were transfected with constructs containing a 600-bp PPM1D promoter fragment or with mutations in the p53RE site, the CRE site or both sites, along with pRL-TK and the empty vector or wild-type p53 expression vector. Cellular extracts were prepared 4 h after exposure to 10 Gy IR. (B) CREB dissociates from the PPM1D promoter region following exposure to IR. Chromatin fragments immunoprecipitated by anti-CREB antibody or nonspecific IgG from HCT116 p53+/+ or HCT116 p53–/– cell extracts at the indicated times following exposure to 5-Gy IR were detected by PCR using primers specific for the PPM1D promoter. Fragments amplified by PCR from 0.62% of the input material are shown.
Mentions: The presence of a conserved CRE within the promoter of the PPM1D gene (16), reported protein–protein interactions between p53 and CREB (19) and reported cooperation in the control of the Bradykinin B2 receptor gene by p53 and CREB (20) suggested a possible involvement of the CRE site and CREB/ATF family transcription factors in the IR-responsiveness of the PPM1D promoter. To investigate whether the CRE site was necessary for the p53-dependent induction of the PPM1D promoter, we transiently transfected expression vectors and luciferase reporter constructs into HCT116 p53–/– cells. In agreement with results described above, the relative luciferase activity resulting from the wild-type PPM1D promoter was robust in the absence of p53 and increased ∼1.5-fold in the presence of p53; mutation of p53RE [m(123)] did not affect the promoter activity in the absence of p53 but reduced promoter activity in the presence of p53 to 44% (Figure 5A). In the absence of p53, mutation of the CRE reduced the promoter activity to 63% of the wild-type promoter, but in the presence of p53, the CRE-mutated promoter exhibited 1.7-fold increased expression. The construct in which both the CRE and p53RE sites (mm) were mutated produced an intermediate level of expression in the absence of p53 and a reduced expression in the presence of p53.Figure 5.

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