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Sp1 is essential for p16 expression in human diploid fibroblasts during senescence.

Wu J, Xue L, Weng M, Sun Y, Zhang Z, Wang W, Tong T - PLoS ONE (2007)

Bottom Line: Both Sp1 RNAi and Mithramycin, a DNA intercalating agent that interferes with Sp1 and Sp3 binding activities, reduced p16(INK4a) gene expression.In addition, the enhanced binding of Sp1 to p16(INK4a) promoter during cellular senescence appeared to be the result of increased Sp1 binding affinity, not an alteration in Sp1 protein level.All these results suggest that GC- II is the key site for Sp1 binding and increase of Sp1 binding activity rather than protein levels contributes to the induction of p16(INK4a) expression during cell aging.

View Article: PubMed Central - PubMed

Affiliation: Peking University Research Center on Aging, Department of Biochemistry and Molecular Biology, Peking University, Health Science Center, Beijing, China.

ABSTRACT

Background: p16(INK4a) tumor suppressor protein has been widely proposed to mediate entrance of the cells into the senescent stage. Promoter of p16(INK4a) gene contains at least five putative GC boxes, named GC-I to V, respectively. Our previous data showed that a potential Sp1 binding site, within the promoter region from -466 to -451, acts as a positive transcription regulatory element. These results led us to examine how Sp1 and/or Sp3 act on these GC boxes during aging in cultured human diploid fibroblasts.

Methodology/principal findings: Mutagenesis studies revealed that GC-I, II and IV, especially GC-II, are essential for p16(INK4a) gene expression in senescent cells. Electrophoretic mobility shift assays (EMSA) and ChIP assays demonstrated that both Sp1 and Sp3 bind to these elements and the binding activity is enhanced in senescent cells. Ectopic overexpression of Sp1, but not Sp3, induced the transcription of p16(INK4a). Both Sp1 RNAi and Mithramycin, a DNA intercalating agent that interferes with Sp1 and Sp3 binding activities, reduced p16(INK4a) gene expression. In addition, the enhanced binding of Sp1 to p16(INK4a) promoter during cellular senescence appeared to be the result of increased Sp1 binding affinity, not an alteration in Sp1 protein level.

Conclusions/significance: All these results suggest that GC- II is the key site for Sp1 binding and increase of Sp1 binding activity rather than protein levels contributes to the induction of p16(INK4a) expression during cell aging.

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Expressions of p16INK4a mRNA and protein are inhibited by MTR treatment.Young (Y) and senescent (S) 2BS cells were either treated with 100 nM MTR (MTR) for 24 hr or left untreated, total RNA and protein were prepared and subjected to analyze the expression of indicated genes by Northern blotting (A) and Western blotting (B), respectively.
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pone-0000164-g006: Expressions of p16INK4a mRNA and protein are inhibited by MTR treatment.Young (Y) and senescent (S) 2BS cells were either treated with 100 nM MTR (MTR) for 24 hr or left untreated, total RNA and protein were prepared and subjected to analyze the expression of indicated genes by Northern blotting (A) and Western blotting (B), respectively.

Mentions: To confirm the effect of Sp1 on p16INK4a promoter activity, mithramycin (MTR), which specifically blocks Sp1 and Sp3 binding to GC boxes, was added to 2BS cells after transfection with pGL3-620. As expected, the p16INK4a promoter activity was inhibited by MTR in a dose-dependent manner in both young (Fig. 5A) and senescent (Fig. 5B) 2BS cells. Further, the p16INK4a expression was also reduced at mRNA (Fig. 6A) and protein (Fig. 6B) levels in 2BS cells, by 66% (mRNA level) and 48% (protein level) respectively, in the senescence group with 24 hours of MTR treatment.


Sp1 is essential for p16 expression in human diploid fibroblasts during senescence.

Wu J, Xue L, Weng M, Sun Y, Zhang Z, Wang W, Tong T - PLoS ONE (2007)

Expressions of p16INK4a mRNA and protein are inhibited by MTR treatment.Young (Y) and senescent (S) 2BS cells were either treated with 100 nM MTR (MTR) for 24 hr or left untreated, total RNA and protein were prepared and subjected to analyze the expression of indicated genes by Northern blotting (A) and Western blotting (B), respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1764714&req=5

pone-0000164-g006: Expressions of p16INK4a mRNA and protein are inhibited by MTR treatment.Young (Y) and senescent (S) 2BS cells were either treated with 100 nM MTR (MTR) for 24 hr or left untreated, total RNA and protein were prepared and subjected to analyze the expression of indicated genes by Northern blotting (A) and Western blotting (B), respectively.
Mentions: To confirm the effect of Sp1 on p16INK4a promoter activity, mithramycin (MTR), which specifically blocks Sp1 and Sp3 binding to GC boxes, was added to 2BS cells after transfection with pGL3-620. As expected, the p16INK4a promoter activity was inhibited by MTR in a dose-dependent manner in both young (Fig. 5A) and senescent (Fig. 5B) 2BS cells. Further, the p16INK4a expression was also reduced at mRNA (Fig. 6A) and protein (Fig. 6B) levels in 2BS cells, by 66% (mRNA level) and 48% (protein level) respectively, in the senescence group with 24 hours of MTR treatment.

Bottom Line: Both Sp1 RNAi and Mithramycin, a DNA intercalating agent that interferes with Sp1 and Sp3 binding activities, reduced p16(INK4a) gene expression.In addition, the enhanced binding of Sp1 to p16(INK4a) promoter during cellular senescence appeared to be the result of increased Sp1 binding affinity, not an alteration in Sp1 protein level.All these results suggest that GC- II is the key site for Sp1 binding and increase of Sp1 binding activity rather than protein levels contributes to the induction of p16(INK4a) expression during cell aging.

View Article: PubMed Central - PubMed

Affiliation: Peking University Research Center on Aging, Department of Biochemistry and Molecular Biology, Peking University, Health Science Center, Beijing, China.

ABSTRACT

Background: p16(INK4a) tumor suppressor protein has been widely proposed to mediate entrance of the cells into the senescent stage. Promoter of p16(INK4a) gene contains at least five putative GC boxes, named GC-I to V, respectively. Our previous data showed that a potential Sp1 binding site, within the promoter region from -466 to -451, acts as a positive transcription regulatory element. These results led us to examine how Sp1 and/or Sp3 act on these GC boxes during aging in cultured human diploid fibroblasts.

Methodology/principal findings: Mutagenesis studies revealed that GC-I, II and IV, especially GC-II, are essential for p16(INK4a) gene expression in senescent cells. Electrophoretic mobility shift assays (EMSA) and ChIP assays demonstrated that both Sp1 and Sp3 bind to these elements and the binding activity is enhanced in senescent cells. Ectopic overexpression of Sp1, but not Sp3, induced the transcription of p16(INK4a). Both Sp1 RNAi and Mithramycin, a DNA intercalating agent that interferes with Sp1 and Sp3 binding activities, reduced p16(INK4a) gene expression. In addition, the enhanced binding of Sp1 to p16(INK4a) promoter during cellular senescence appeared to be the result of increased Sp1 binding affinity, not an alteration in Sp1 protein level.

Conclusions/significance: All these results suggest that GC- II is the key site for Sp1 binding and increase of Sp1 binding activity rather than protein levels contributes to the induction of p16(INK4a) expression during cell aging.

Show MeSH
Related in: MedlinePlus