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Serum withdrawal up-regulates human SIRT1 gene expression in a p53-dependent manner.

Shang L, Zhou H, Xia Y, Wang H, Gao G, Chen B, Liu Q, Shao C, Gong Y - J. Cell. Mol. Med. (2009)

Bottom Line: In this paper, we characterized a putative p53-binding element in the human SIRT1 promoter that might be required for the up-regulation of SIRT1 in response to nutritional stress.There is a C to A change at the second half site in human SIRT1, thus disrupting the core-binding element CWWG in the canonical RRRCWWGYYY.We found that serum withdrawal also up-regulates human SIRT1 gene expression in a p53-dependent manner and that the p53-binding element in SIRT1 is required for the up-regulation.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for Experimental Teratology of the Ministry of Education and Institute of Medical Genetics, Shandong University School of Medicine, Jinan, Shandong, China.

ABSTRACT
SIRT1, a nicotinamide adenine dinucleotide (NAD(+))-dependent histone/protein deacetylase, has been extensively studied recently for its critical role in the regulation of physiology, calorie restriction and aging. Studies on laboratory mice showed that expression of SIRT1 can be induced by starvation in a p53-dependent manner and requires the p53-binding sites present in the Sirt1 promoter. However, it remains to be determined whether these findings based on rodents apply to human beings. In this paper, we characterized a putative p53-binding element in the human SIRT1 promoter that might be required for the up-regulation of SIRT1 in response to nutritional stress. The p53-binding site in the promoter of human SIRT1 is more deviant from the consensus sequence than the corresponding sequence in the mouse Sirt1. There is a C to A change at the second half site in human SIRT1, thus disrupting the core-binding element CWWG in the canonical RRRCWWGYYY. To test whether such sequence change would affect its binding with p53 and the SIRT1 expression under stress, we studied various human cell lines with different p53 status and cells with ectopic expression of functionally distinct p53. We found that serum withdrawal also up-regulates human SIRT1 gene expression in a p53-dependent manner and that the p53-binding element in SIRT1 is required for the up-regulation. Thus, the mechanism responsible for the regulation of SIRT1 expression by p53 is conserved between mice and human beings.

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A putative p53 response element present in the SIRT1 promoter contributes to the induction of human SIRT1 expression by serum withdrawal. (A) Schematic representation of the putative p53 response elements in the promoter sequence of the SIRT1 gene, where R is purine, Y is pyrimidine, W is A or T, and n is a spacer of 0–13 nt between the two half sites. The deviations from consensus were underlined. Seven and eight deviations exist in mouse and human SIRT1 promoter, respectively. Two point mutations (bold) were introduced to change the core sequence from CGTG to AGAG. (B) Functional comparison of the mouse Sirt1 and human SIRT1 promoters in luciferase reporter assay. Same amount of luciferase constructs containing mouse Sirt1 or human SIRT1 were transfected into HEK293 and U2OS cells and cells were maintained in normal nutrients (C) or under serum starvation (S). Cells were harvested 16 hrs after treatments and luciferase activities were determined. The activity of the construct containing mouse Sirt1 promoter under normal nutrients was arbitrarily set as 100%, and the relative luciferase activity under normal or starved condition was calculated accordingly. Each bar represents the value of mean ± S.E.M. (C) Deletion or mutation of p53 response element in SIRT1 promoter abolishes the expression of luciferase stimulated by serum starvation. Constructs with native (P-158) or mutant p53 response element (P-158mut) or without p53 response element (P-111) were transfected into U2OS and HepG2 cells, and the relative luciferase activity under either normal nutrients (C) or serum-starved condition (S) was determined. The activity of construct P-158 under normal nutrient was arbitrarily set to 100%, and the relative luciferase activity under normal or serum-starved condition was calculated accordingly. Each bar represents the value of mean ± S.E.M. (D) Physical binding of p53 response element with p53. The protein-DNA complexes were resolved by native polyacrylamide gel electrophoresis. The retarded mobility of the oligonucleotide was observed in the presence of nuclear extract, lane 2. The formation of DNA-protein complex was competitively inhibited by 100-fold molar excess of unlabelled (cold) wild-type oligonucleotides, lane 3, but not by the same amount of cold mutant oligonucleotides. The retarded mobility of the probe in the presence of p53 protein was supershifted with p53 antibody, lane 5.
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fig02: A putative p53 response element present in the SIRT1 promoter contributes to the induction of human SIRT1 expression by serum withdrawal. (A) Schematic representation of the putative p53 response elements in the promoter sequence of the SIRT1 gene, where R is purine, Y is pyrimidine, W is A or T, and n is a spacer of 0–13 nt between the two half sites. The deviations from consensus were underlined. Seven and eight deviations exist in mouse and human SIRT1 promoter, respectively. Two point mutations (bold) were introduced to change the core sequence from CGTG to AGAG. (B) Functional comparison of the mouse Sirt1 and human SIRT1 promoters in luciferase reporter assay. Same amount of luciferase constructs containing mouse Sirt1 or human SIRT1 were transfected into HEK293 and U2OS cells and cells were maintained in normal nutrients (C) or under serum starvation (S). Cells were harvested 16 hrs after treatments and luciferase activities were determined. The activity of the construct containing mouse Sirt1 promoter under normal nutrients was arbitrarily set as 100%, and the relative luciferase activity under normal or starved condition was calculated accordingly. Each bar represents the value of mean ± S.E.M. (C) Deletion or mutation of p53 response element in SIRT1 promoter abolishes the expression of luciferase stimulated by serum starvation. Constructs with native (P-158) or mutant p53 response element (P-158mut) or without p53 response element (P-111) were transfected into U2OS and HepG2 cells, and the relative luciferase activity under either normal nutrients (C) or serum-starved condition (S) was determined. The activity of construct P-158 under normal nutrient was arbitrarily set to 100%, and the relative luciferase activity under normal or serum-starved condition was calculated accordingly. Each bar represents the value of mean ± S.E.M. (D) Physical binding of p53 response element with p53. The protein-DNA complexes were resolved by native polyacrylamide gel electrophoresis. The retarded mobility of the oligonucleotide was observed in the presence of nuclear extract, lane 2. The formation of DNA-protein complex was competitively inhibited by 100-fold molar excess of unlabelled (cold) wild-type oligonucleotides, lane 3, but not by the same amount of cold mutant oligonucleotides. The retarded mobility of the probe in the presence of p53 protein was supershifted with p53 antibody, lane 5.

Mentions: The p53 target genes typically contain p53 consensus binding sites in their promoters or other regulatory regions. Mouse Sirt1 gene contains two p53-binding sites in its promoter [19]. However, an analysis using software MatInspector indicated that the p53 response element in human SIRT1 promoter differs from that in the mouse Sirt1 promoter at one position (Fig. 2A). There is a C to A substitution in the second half site. This represents a further deviation from the consensus binding site than the mouse Sirt1 promoter and might impair its binding with p53 and, consequently lead to reduced induction of SIRT1 expression in response to nutritional stress in human cells. To examine this, we first tested if there is a functional difference in the promoters of mouse Sirt1 and human SIRT1. As shown in Fig. 2B, the luciferase reporter containing the human SIRT1 promoter showed about half of the activity of the reporter containing the mouse Sirt1 promoter, under normal growth condition and under nutritional stress. Although human SIRT1 promoter had a lower reporter activity than the mouse counterpart, it was still more than 40 times higher than the promoterless pGL3-basic control. This result indicated that the human SIRT1 promoter may possess a similar function as the one in the mouse, although the p53 responsive element it contains is more deviant from the consensus sequence.


Serum withdrawal up-regulates human SIRT1 gene expression in a p53-dependent manner.

Shang L, Zhou H, Xia Y, Wang H, Gao G, Chen B, Liu Q, Shao C, Gong Y - J. Cell. Mol. Med. (2009)

A putative p53 response element present in the SIRT1 promoter contributes to the induction of human SIRT1 expression by serum withdrawal. (A) Schematic representation of the putative p53 response elements in the promoter sequence of the SIRT1 gene, where R is purine, Y is pyrimidine, W is A or T, and n is a spacer of 0–13 nt between the two half sites. The deviations from consensus were underlined. Seven and eight deviations exist in mouse and human SIRT1 promoter, respectively. Two point mutations (bold) were introduced to change the core sequence from CGTG to AGAG. (B) Functional comparison of the mouse Sirt1 and human SIRT1 promoters in luciferase reporter assay. Same amount of luciferase constructs containing mouse Sirt1 or human SIRT1 were transfected into HEK293 and U2OS cells and cells were maintained in normal nutrients (C) or under serum starvation (S). Cells were harvested 16 hrs after treatments and luciferase activities were determined. The activity of the construct containing mouse Sirt1 promoter under normal nutrients was arbitrarily set as 100%, and the relative luciferase activity under normal or starved condition was calculated accordingly. Each bar represents the value of mean ± S.E.M. (C) Deletion or mutation of p53 response element in SIRT1 promoter abolishes the expression of luciferase stimulated by serum starvation. Constructs with native (P-158) or mutant p53 response element (P-158mut) or without p53 response element (P-111) were transfected into U2OS and HepG2 cells, and the relative luciferase activity under either normal nutrients (C) or serum-starved condition (S) was determined. The activity of construct P-158 under normal nutrient was arbitrarily set to 100%, and the relative luciferase activity under normal or serum-starved condition was calculated accordingly. Each bar represents the value of mean ± S.E.M. (D) Physical binding of p53 response element with p53. The protein-DNA complexes were resolved by native polyacrylamide gel electrophoresis. The retarded mobility of the oligonucleotide was observed in the presence of nuclear extract, lane 2. The formation of DNA-protein complex was competitively inhibited by 100-fold molar excess of unlabelled (cold) wild-type oligonucleotides, lane 3, but not by the same amount of cold mutant oligonucleotides. The retarded mobility of the probe in the presence of p53 protein was supershifted with p53 antibody, lane 5.
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Related In: Results  -  Collection

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fig02: A putative p53 response element present in the SIRT1 promoter contributes to the induction of human SIRT1 expression by serum withdrawal. (A) Schematic representation of the putative p53 response elements in the promoter sequence of the SIRT1 gene, where R is purine, Y is pyrimidine, W is A or T, and n is a spacer of 0–13 nt between the two half sites. The deviations from consensus were underlined. Seven and eight deviations exist in mouse and human SIRT1 promoter, respectively. Two point mutations (bold) were introduced to change the core sequence from CGTG to AGAG. (B) Functional comparison of the mouse Sirt1 and human SIRT1 promoters in luciferase reporter assay. Same amount of luciferase constructs containing mouse Sirt1 or human SIRT1 were transfected into HEK293 and U2OS cells and cells were maintained in normal nutrients (C) or under serum starvation (S). Cells were harvested 16 hrs after treatments and luciferase activities were determined. The activity of the construct containing mouse Sirt1 promoter under normal nutrients was arbitrarily set as 100%, and the relative luciferase activity under normal or starved condition was calculated accordingly. Each bar represents the value of mean ± S.E.M. (C) Deletion or mutation of p53 response element in SIRT1 promoter abolishes the expression of luciferase stimulated by serum starvation. Constructs with native (P-158) or mutant p53 response element (P-158mut) or without p53 response element (P-111) were transfected into U2OS and HepG2 cells, and the relative luciferase activity under either normal nutrients (C) or serum-starved condition (S) was determined. The activity of construct P-158 under normal nutrient was arbitrarily set to 100%, and the relative luciferase activity under normal or serum-starved condition was calculated accordingly. Each bar represents the value of mean ± S.E.M. (D) Physical binding of p53 response element with p53. The protein-DNA complexes were resolved by native polyacrylamide gel electrophoresis. The retarded mobility of the oligonucleotide was observed in the presence of nuclear extract, lane 2. The formation of DNA-protein complex was competitively inhibited by 100-fold molar excess of unlabelled (cold) wild-type oligonucleotides, lane 3, but not by the same amount of cold mutant oligonucleotides. The retarded mobility of the probe in the presence of p53 protein was supershifted with p53 antibody, lane 5.
Mentions: The p53 target genes typically contain p53 consensus binding sites in their promoters or other regulatory regions. Mouse Sirt1 gene contains two p53-binding sites in its promoter [19]. However, an analysis using software MatInspector indicated that the p53 response element in human SIRT1 promoter differs from that in the mouse Sirt1 promoter at one position (Fig. 2A). There is a C to A substitution in the second half site. This represents a further deviation from the consensus binding site than the mouse Sirt1 promoter and might impair its binding with p53 and, consequently lead to reduced induction of SIRT1 expression in response to nutritional stress in human cells. To examine this, we first tested if there is a functional difference in the promoters of mouse Sirt1 and human SIRT1. As shown in Fig. 2B, the luciferase reporter containing the human SIRT1 promoter showed about half of the activity of the reporter containing the mouse Sirt1 promoter, under normal growth condition and under nutritional stress. Although human SIRT1 promoter had a lower reporter activity than the mouse counterpart, it was still more than 40 times higher than the promoterless pGL3-basic control. This result indicated that the human SIRT1 promoter may possess a similar function as the one in the mouse, although the p53 responsive element it contains is more deviant from the consensus sequence.

Bottom Line: In this paper, we characterized a putative p53-binding element in the human SIRT1 promoter that might be required for the up-regulation of SIRT1 in response to nutritional stress.There is a C to A change at the second half site in human SIRT1, thus disrupting the core-binding element CWWG in the canonical RRRCWWGYYY.We found that serum withdrawal also up-regulates human SIRT1 gene expression in a p53-dependent manner and that the p53-binding element in SIRT1 is required for the up-regulation.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for Experimental Teratology of the Ministry of Education and Institute of Medical Genetics, Shandong University School of Medicine, Jinan, Shandong, China.

ABSTRACT
SIRT1, a nicotinamide adenine dinucleotide (NAD(+))-dependent histone/protein deacetylase, has been extensively studied recently for its critical role in the regulation of physiology, calorie restriction and aging. Studies on laboratory mice showed that expression of SIRT1 can be induced by starvation in a p53-dependent manner and requires the p53-binding sites present in the Sirt1 promoter. However, it remains to be determined whether these findings based on rodents apply to human beings. In this paper, we characterized a putative p53-binding element in the human SIRT1 promoter that might be required for the up-regulation of SIRT1 in response to nutritional stress. The p53-binding site in the promoter of human SIRT1 is more deviant from the consensus sequence than the corresponding sequence in the mouse Sirt1. There is a C to A change at the second half site in human SIRT1, thus disrupting the core-binding element CWWG in the canonical RRRCWWGYYY. To test whether such sequence change would affect its binding with p53 and the SIRT1 expression under stress, we studied various human cell lines with different p53 status and cells with ectopic expression of functionally distinct p53. We found that serum withdrawal also up-regulates human SIRT1 gene expression in a p53-dependent manner and that the p53-binding element in SIRT1 is required for the up-regulation. Thus, the mechanism responsible for the regulation of SIRT1 expression by p53 is conserved between mice and human beings.

Show MeSH
Related in: MedlinePlus