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p53-dependent stimulation of redox-related genes in the lymphoid organs of gamma-irradiated--mice identification of Haeme-oxygenase 1 as a direct p53 target gene.

Meiller A, Alvarez S, Drané P, Lallemand C, Blanchard B, Tovey M, May E - Nucleic Acids Res. (2007)

Bottom Line: Results presented in this article illustrate an additional degree of complexity.Moreover, induction of HO-1 occurs later than that of Waf1/p21.Finally, the higher stimulation of HO-1 is reached when Waf1/p21 stimulation starts to decrease.

View Article: PubMed Central - PubMed

Affiliation: Commissariat à l'Energie Atomique (CEA), Centre National de la Recherche Scientifique (CNRS), UMR217, route du Panorama BP6, 92265 Fontenay-aux-Roses Cedex and CNRS FRE2937, Institut André Lwoff, 7, rue Guy Moquet, BP8, 94801 Villejuif Cedex, France.

ABSTRACT
Recent data showed that p53 stimulates the expression of genes encoding not only pro- but also antioxidant enzymes. It was suggested that antioxidant genes could be induced under physiologic levels of stress while the prooxidant ones respond to higher level of stress. Results presented in this article illustrate an additional degree of complexity. We show that the expression of Haeme-oxygenase 1 (HO-1), a stress-inducible gene that codes for an enzyme having antioxidant properties, is stimulated in a p53-dependent manner in the thymus and spleen of irradiated mice. We prove that HO-1 is a direct p53 target gene by showing that the p53RE identified within human and mouse genes is specifically bound by p53. The threshold of irradiation dose required to induce a significant response of HO-1 in the lymphoid organs of the irradiated mice is higher than that for Waf1/p21 that encodes an universal inhibitor of cell cycle. Moreover, induction of HO-1 occurs later than that of Waf1/p21. Finally, the higher stimulation of HO-1 is reached when Waf1/p21 stimulation starts to decrease.

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The mouse HO-1 gene is directly transactivated by p53. (A-a) Sequence of the 5′ non-coding region of the mouse HO-1 gene with, in the open boxes, the putative p53RE that consists of two adjacent decamers that respond to the definition of a p53-binding site according to El Deiry et al. (41). Nucleotide numbering starts at the A of the translation initiation codon. The lower case letter within the first decamer indicates the variant nucleotide as compared to the consensus decamer: PuPuPuC(A/T)(A/T)GPyPyPy). The two HindIII and NcoI restriction sites were added by PCR amplification in order to clone this sequence within the pluc.min vector to obtain the pGL3-HO1 reporter gene plasmid (see Materials and Methods section). (b) Three mutations (underlined lower case letters) have been introduced within the second decamer by PCR-directed mutagenesis. (B) EMSA shows a specific binding of baculovirus-produced human wt-p53 to the two decamers identified in the mouse HO-1 gene. 32P-labelled DNA probe corresponding to the two decamers was incubated with p53 and the antibody anti-p53 PAb421 (lane2). Competitions were performed by using 100- or 500-fold molar excess of unlabelled double-stranded oligonucleotides either non-specific (ns, lanes 3 and 4); or homologous (s, lanes 5 and 6), as described in Materials and Methods section. (C) Luciferase assay showed that the transcriptional activity of the 5′ non-coding sequence of the mouse HO-1 gene is stimulated by wt-p53. U-2 OS and MCF-7, two cell lines that express wt-p53, were transfected with either pluc.min (control) or pGL3-HO1 (HO-1 sequence) and cotransfected with (+DD) or without (−DD) the plasmid pDDm-TO that encodes a dominant negative mutant of p53. Luciferase activities of cells transfected with pGL3-HO1 were expressed relative to the luciferase activity of cells transfected with pluc.min. The averaged results are of three independent experiments performed in duplicate. Standard deviations are indicated. (D) mHO-1 p53RE confers the ability to be transactivated by wt-p53. U-2OS were transfected with either pGL3-HO1 (wt p53RE) or the plasmid pGL3-HO1mut that is mutated within the p53RE as indicated above (mutant p53RE). Luciferase activities of cells transfected with these two plasmids were expressed relative to the luciferase activity of cells transfected with pluc.min (control). The averaged results are of four independent experiments performed in duplicate.
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Figure 2: The mouse HO-1 gene is directly transactivated by p53. (A-a) Sequence of the 5′ non-coding region of the mouse HO-1 gene with, in the open boxes, the putative p53RE that consists of two adjacent decamers that respond to the definition of a p53-binding site according to El Deiry et al. (41). Nucleotide numbering starts at the A of the translation initiation codon. The lower case letter within the first decamer indicates the variant nucleotide as compared to the consensus decamer: PuPuPuC(A/T)(A/T)GPyPyPy). The two HindIII and NcoI restriction sites were added by PCR amplification in order to clone this sequence within the pluc.min vector to obtain the pGL3-HO1 reporter gene plasmid (see Materials and Methods section). (b) Three mutations (underlined lower case letters) have been introduced within the second decamer by PCR-directed mutagenesis. (B) EMSA shows a specific binding of baculovirus-produced human wt-p53 to the two decamers identified in the mouse HO-1 gene. 32P-labelled DNA probe corresponding to the two decamers was incubated with p53 and the antibody anti-p53 PAb421 (lane2). Competitions were performed by using 100- or 500-fold molar excess of unlabelled double-stranded oligonucleotides either non-specific (ns, lanes 3 and 4); or homologous (s, lanes 5 and 6), as described in Materials and Methods section. (C) Luciferase assay showed that the transcriptional activity of the 5′ non-coding sequence of the mouse HO-1 gene is stimulated by wt-p53. U-2 OS and MCF-7, two cell lines that express wt-p53, were transfected with either pluc.min (control) or pGL3-HO1 (HO-1 sequence) and cotransfected with (+DD) or without (−DD) the plasmid pDDm-TO that encodes a dominant negative mutant of p53. Luciferase activities of cells transfected with pGL3-HO1 were expressed relative to the luciferase activity of cells transfected with pluc.min. The averaged results are of three independent experiments performed in duplicate. Standard deviations are indicated. (D) mHO-1 p53RE confers the ability to be transactivated by wt-p53. U-2OS were transfected with either pGL3-HO1 (wt p53RE) or the plasmid pGL3-HO1mut that is mutated within the p53RE as indicated above (mutant p53RE). Luciferase activities of cells transfected with these two plasmids were expressed relative to the luciferase activity of cells transfected with pluc.min (control). The averaged results are of four independent experiments performed in duplicate.

Mentions: The human HO-1 gene is directly transactivated by p53. (A) Alignment of the 5′ non-coding sequence of mouse, rat and human HO-1 gene. In open boxes, the two adjacent decamers that respond to the definition of El Deiry et al. (41) are shown. The nucleotide variants as compared to the consensus sequence (see legend of Figure 2) were indicated by lower case letters and nucleotide variations in rat and human p53 decamers as compared to mouse are underlined. (B) Stimulation of HO-1 gene expression in irradiated human transformed cells expressing wt-p53 (MCF-7 and SKNSH Neo) but not in cells expressing mutant-p53 (MCF-7/R-A1 and SKNSH DD). Cells were non-irradiated (NI) or irradiated (IR) at a dose of 10 Gy. Total RNA was extracted 3 h post-irradiation. Expression of HO-1 was analysed by real-time quantitative RT-PCR, normalized relative to GAPDH mRNA levels and expressed taking as 1 the HO-1 mRNA level of the non-irradiated cells. (C) ChIP assay of HO-1 and Waf1/p21 in MCF-7 cells. Cross-linked p53 protein/DNA complexes were immunoprecipitated with either anti-p53 or anti-mSim3A (negative control) before PCR amplification as described in Materials and Methods section. Input (positive control) indicates a portion of the sonicated chromatin that has been amplified after immunoprecipitation.


p53-dependent stimulation of redox-related genes in the lymphoid organs of gamma-irradiated--mice identification of Haeme-oxygenase 1 as a direct p53 target gene.

Meiller A, Alvarez S, Drané P, Lallemand C, Blanchard B, Tovey M, May E - Nucleic Acids Res. (2007)

The mouse HO-1 gene is directly transactivated by p53. (A-a) Sequence of the 5′ non-coding region of the mouse HO-1 gene with, in the open boxes, the putative p53RE that consists of two adjacent decamers that respond to the definition of a p53-binding site according to El Deiry et al. (41). Nucleotide numbering starts at the A of the translation initiation codon. The lower case letter within the first decamer indicates the variant nucleotide as compared to the consensus decamer: PuPuPuC(A/T)(A/T)GPyPyPy). The two HindIII and NcoI restriction sites were added by PCR amplification in order to clone this sequence within the pluc.min vector to obtain the pGL3-HO1 reporter gene plasmid (see Materials and Methods section). (b) Three mutations (underlined lower case letters) have been introduced within the second decamer by PCR-directed mutagenesis. (B) EMSA shows a specific binding of baculovirus-produced human wt-p53 to the two decamers identified in the mouse HO-1 gene. 32P-labelled DNA probe corresponding to the two decamers was incubated with p53 and the antibody anti-p53 PAb421 (lane2). Competitions were performed by using 100- or 500-fold molar excess of unlabelled double-stranded oligonucleotides either non-specific (ns, lanes 3 and 4); or homologous (s, lanes 5 and 6), as described in Materials and Methods section. (C) Luciferase assay showed that the transcriptional activity of the 5′ non-coding sequence of the mouse HO-1 gene is stimulated by wt-p53. U-2 OS and MCF-7, two cell lines that express wt-p53, were transfected with either pluc.min (control) or pGL3-HO1 (HO-1 sequence) and cotransfected with (+DD) or without (−DD) the plasmid pDDm-TO that encodes a dominant negative mutant of p53. Luciferase activities of cells transfected with pGL3-HO1 were expressed relative to the luciferase activity of cells transfected with pluc.min. The averaged results are of three independent experiments performed in duplicate. Standard deviations are indicated. (D) mHO-1 p53RE confers the ability to be transactivated by wt-p53. U-2OS were transfected with either pGL3-HO1 (wt p53RE) or the plasmid pGL3-HO1mut that is mutated within the p53RE as indicated above (mutant p53RE). Luciferase activities of cells transfected with these two plasmids were expressed relative to the luciferase activity of cells transfected with pluc.min (control). The averaged results are of four independent experiments performed in duplicate.
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Related In: Results  -  Collection

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Figure 2: The mouse HO-1 gene is directly transactivated by p53. (A-a) Sequence of the 5′ non-coding region of the mouse HO-1 gene with, in the open boxes, the putative p53RE that consists of two adjacent decamers that respond to the definition of a p53-binding site according to El Deiry et al. (41). Nucleotide numbering starts at the A of the translation initiation codon. The lower case letter within the first decamer indicates the variant nucleotide as compared to the consensus decamer: PuPuPuC(A/T)(A/T)GPyPyPy). The two HindIII and NcoI restriction sites were added by PCR amplification in order to clone this sequence within the pluc.min vector to obtain the pGL3-HO1 reporter gene plasmid (see Materials and Methods section). (b) Three mutations (underlined lower case letters) have been introduced within the second decamer by PCR-directed mutagenesis. (B) EMSA shows a specific binding of baculovirus-produced human wt-p53 to the two decamers identified in the mouse HO-1 gene. 32P-labelled DNA probe corresponding to the two decamers was incubated with p53 and the antibody anti-p53 PAb421 (lane2). Competitions were performed by using 100- or 500-fold molar excess of unlabelled double-stranded oligonucleotides either non-specific (ns, lanes 3 and 4); or homologous (s, lanes 5 and 6), as described in Materials and Methods section. (C) Luciferase assay showed that the transcriptional activity of the 5′ non-coding sequence of the mouse HO-1 gene is stimulated by wt-p53. U-2 OS and MCF-7, two cell lines that express wt-p53, were transfected with either pluc.min (control) or pGL3-HO1 (HO-1 sequence) and cotransfected with (+DD) or without (−DD) the plasmid pDDm-TO that encodes a dominant negative mutant of p53. Luciferase activities of cells transfected with pGL3-HO1 were expressed relative to the luciferase activity of cells transfected with pluc.min. The averaged results are of three independent experiments performed in duplicate. Standard deviations are indicated. (D) mHO-1 p53RE confers the ability to be transactivated by wt-p53. U-2OS were transfected with either pGL3-HO1 (wt p53RE) or the plasmid pGL3-HO1mut that is mutated within the p53RE as indicated above (mutant p53RE). Luciferase activities of cells transfected with these two plasmids were expressed relative to the luciferase activity of cells transfected with pluc.min (control). The averaged results are of four independent experiments performed in duplicate.
Mentions: The human HO-1 gene is directly transactivated by p53. (A) Alignment of the 5′ non-coding sequence of mouse, rat and human HO-1 gene. In open boxes, the two adjacent decamers that respond to the definition of El Deiry et al. (41) are shown. The nucleotide variants as compared to the consensus sequence (see legend of Figure 2) were indicated by lower case letters and nucleotide variations in rat and human p53 decamers as compared to mouse are underlined. (B) Stimulation of HO-1 gene expression in irradiated human transformed cells expressing wt-p53 (MCF-7 and SKNSH Neo) but not in cells expressing mutant-p53 (MCF-7/R-A1 and SKNSH DD). Cells were non-irradiated (NI) or irradiated (IR) at a dose of 10 Gy. Total RNA was extracted 3 h post-irradiation. Expression of HO-1 was analysed by real-time quantitative RT-PCR, normalized relative to GAPDH mRNA levels and expressed taking as 1 the HO-1 mRNA level of the non-irradiated cells. (C) ChIP assay of HO-1 and Waf1/p21 in MCF-7 cells. Cross-linked p53 protein/DNA complexes were immunoprecipitated with either anti-p53 or anti-mSim3A (negative control) before PCR amplification as described in Materials and Methods section. Input (positive control) indicates a portion of the sonicated chromatin that has been amplified after immunoprecipitation.

Bottom Line: Results presented in this article illustrate an additional degree of complexity.Moreover, induction of HO-1 occurs later than that of Waf1/p21.Finally, the higher stimulation of HO-1 is reached when Waf1/p21 stimulation starts to decrease.

View Article: PubMed Central - PubMed

Affiliation: Commissariat à l'Energie Atomique (CEA), Centre National de la Recherche Scientifique (CNRS), UMR217, route du Panorama BP6, 92265 Fontenay-aux-Roses Cedex and CNRS FRE2937, Institut André Lwoff, 7, rue Guy Moquet, BP8, 94801 Villejuif Cedex, France.

ABSTRACT
Recent data showed that p53 stimulates the expression of genes encoding not only pro- but also antioxidant enzymes. It was suggested that antioxidant genes could be induced under physiologic levels of stress while the prooxidant ones respond to higher level of stress. Results presented in this article illustrate an additional degree of complexity. We show that the expression of Haeme-oxygenase 1 (HO-1), a stress-inducible gene that codes for an enzyme having antioxidant properties, is stimulated in a p53-dependent manner in the thymus and spleen of irradiated mice. We prove that HO-1 is a direct p53 target gene by showing that the p53RE identified within human and mouse genes is specifically bound by p53. The threshold of irradiation dose required to induce a significant response of HO-1 in the lymphoid organs of the irradiated mice is higher than that for Waf1/p21 that encodes an universal inhibitor of cell cycle. Moreover, induction of HO-1 occurs later than that of Waf1/p21. Finally, the higher stimulation of HO-1 is reached when Waf1/p21 stimulation starts to decrease.

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