Limits...
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 (Wip1), a type PP2C phosphatase, is expressed at low levels in most normal tissues but is overexpressed in several types of cancers.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.

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

Effects of UV exposure on the PPM1D gene expression. (A) Effects of UV exposure on PPM1D mRNA levels. HCT116 p53+/+ or p53–/– cells were untreated (NT) or exposed to the indicated doses of UV and total RNA was extracted 4 h later. The relative levels of PPM1D mRNAs were determined by quantitative RT-PCR, normalized to β-actin mRNA levels and expressed as a ratio of the level in untreated p53+/+ cells. (B) Effects of UV exposure on PPM1D and p53 protein levels. HCT116 p53+/+ or p53–/– cells were untreated (NT) or exposed to the indicated doses of UV, cellular proteins were extracted 6 h later and specific proteins were detected by immunoblot. (C) Patterns of transcription initiation in the PPM1D promoter following exposure to UV. Total RNA was isolated from untreated cells or 4 h after exposure to 20 J/m2 UV. A quantitative PCR-based method was used to determine the relative abundances of PPM1D mRNAs with long 5′ UTRs compared to all PPM1D transcripts. The amount of PPM1D transcripts with short 5′ UTRs was determined by difference.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2602757&req=5

Figure 7: Effects of UV exposure on the PPM1D gene expression. (A) Effects of UV exposure on PPM1D mRNA levels. HCT116 p53+/+ or p53–/– cells were untreated (NT) or exposed to the indicated doses of UV and total RNA was extracted 4 h later. The relative levels of PPM1D mRNAs were determined by quantitative RT-PCR, normalized to β-actin mRNA levels and expressed as a ratio of the level in untreated p53+/+ cells. (B) Effects of UV exposure on PPM1D and p53 protein levels. HCT116 p53+/+ or p53–/– cells were untreated (NT) or exposed to the indicated doses of UV, cellular proteins were extracted 6 h later and specific proteins were detected by immunoblot. (C) Patterns of transcription initiation in the PPM1D promoter following exposure to UV. Total RNA was isolated from untreated cells or 4 h after exposure to 20 J/m2 UV. A quantitative PCR-based method was used to determine the relative abundances of PPM1D mRNAs with long 5′ UTRs compared to all PPM1D transcripts. The amount of PPM1D transcripts with short 5′ UTRs was determined by difference.

Mentions: The results described above demonstrated that the total amount of PPM1D mRNA, the fraction of PPM1D transcripts with short 5′ UTRs and PPM1D protein levels all increase following exposure to IR in cells with wild-type p53. To discriminate between the effects of the total PPM1D mRNA level and the abundance of transcripts with short 5′ UTRs, we examined the effects of exposure to UV on PPM1D mRNA and protein levels. The increased expression of PPM1D mRNA has been shown to be dependent on both p53 and p38 MAPK (12). However, due to the formation of transcription-blocking photoproducts within the 60-kb PPM1D gene, the production of full-length transcripts is constrained at higher levels of UV (50). As shown in Figure 7A, PPM1D mRNA levels increased in HCT116 p53+/+ cells following exposure to 10 or 20 J/m2 UV but was essentially unchanged following exposure to 30 J/m2. The PPM1D mRNA level was slightly higher in untreated HT116 p53–/– cells than in p53+/+ cells, as observed above (Figure 5B), and decreased with increasing UV dose. This pattern is consistent with a p53-dependent increase in transcription of the PPM1D gene following exposure to UV combined with a UV dose-dependent inhibition of production of full length transcripts (50).Figure 7.


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)

Effects of UV exposure on the PPM1D gene expression. (A) Effects of UV exposure on PPM1D mRNA levels. HCT116 p53+/+ or p53–/– cells were untreated (NT) or exposed to the indicated doses of UV and total RNA was extracted 4 h later. The relative levels of PPM1D mRNAs were determined by quantitative RT-PCR, normalized to β-actin mRNA levels and expressed as a ratio of the level in untreated p53+/+ cells. (B) Effects of UV exposure on PPM1D and p53 protein levels. HCT116 p53+/+ or p53–/– cells were untreated (NT) or exposed to the indicated doses of UV, cellular proteins were extracted 6 h later and specific proteins were detected by immunoblot. (C) Patterns of transcription initiation in the PPM1D promoter following exposure to UV. Total RNA was isolated from untreated cells or 4 h after exposure to 20 J/m2 UV. A quantitative PCR-based method was used to determine the relative abundances of PPM1D mRNAs with long 5′ UTRs compared to all PPM1D transcripts. The amount of PPM1D transcripts with short 5′ UTRs was determined by difference.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 7: Effects of UV exposure on the PPM1D gene expression. (A) Effects of UV exposure on PPM1D mRNA levels. HCT116 p53+/+ or p53–/– cells were untreated (NT) or exposed to the indicated doses of UV and total RNA was extracted 4 h later. The relative levels of PPM1D mRNAs were determined by quantitative RT-PCR, normalized to β-actin mRNA levels and expressed as a ratio of the level in untreated p53+/+ cells. (B) Effects of UV exposure on PPM1D and p53 protein levels. HCT116 p53+/+ or p53–/– cells were untreated (NT) or exposed to the indicated doses of UV, cellular proteins were extracted 6 h later and specific proteins were detected by immunoblot. (C) Patterns of transcription initiation in the PPM1D promoter following exposure to UV. Total RNA was isolated from untreated cells or 4 h after exposure to 20 J/m2 UV. A quantitative PCR-based method was used to determine the relative abundances of PPM1D mRNAs with long 5′ UTRs compared to all PPM1D transcripts. The amount of PPM1D transcripts with short 5′ UTRs was determined by difference.
Mentions: The results described above demonstrated that the total amount of PPM1D mRNA, the fraction of PPM1D transcripts with short 5′ UTRs and PPM1D protein levels all increase following exposure to IR in cells with wild-type p53. To discriminate between the effects of the total PPM1D mRNA level and the abundance of transcripts with short 5′ UTRs, we examined the effects of exposure to UV on PPM1D mRNA and protein levels. The increased expression of PPM1D mRNA has been shown to be dependent on both p53 and p38 MAPK (12). However, due to the formation of transcription-blocking photoproducts within the 60-kb PPM1D gene, the production of full-length transcripts is constrained at higher levels of UV (50). As shown in Figure 7A, PPM1D mRNA levels increased in HCT116 p53+/+ cells following exposure to 10 or 20 J/m2 UV but was essentially unchanged following exposure to 30 J/m2. The PPM1D mRNA level was slightly higher in untreated HT116 p53–/– cells than in p53+/+ cells, as observed above (Figure 5B), and decreased with increasing UV dose. This pattern is consistent with a p53-dependent increase in transcription of the PPM1D gene following exposure to UV combined with a UV dose-dependent inhibition of production of full length transcripts (50).Figure 7.

Bottom Line: PPM1D (Wip1), a type PP2C phosphatase, is expressed at low levels in most normal tissues but is overexpressed in several types of cancers.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.

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