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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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Related in: MedlinePlus

Knock-down of Sp1 reduces expression of endogenous p16INK4a.Following transfection of 2BS cells with si-Sp1 or a control plasmid, RT-PCR (A) and Western blotting (B) were carried out to analyze the expression of the genes indicated.
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pone-0000164-g007: Knock-down of Sp1 reduces expression of endogenous p16INK4a.Following transfection of 2BS cells with si-Sp1 or a control plasmid, RT-PCR (A) and Western blotting (B) were carried out to analyze the expression of the genes indicated.

Mentions: As the mechanism of MTR treatment is blocking the transcription factors such as Sp family binding to GC-box, in this way, MTR could also affect the binding of other transcriptional factors to p16INK4a promoter. To investigate the role of Sp1 specifically, Sp1 was knocked-down by RNAi to further confirm Sp1 binding to GC boxes is essential for the transcription of p16INK4a (Fig. 7A). Western Blot showed that si-Sp1 remarkably reduced the expression of Sp1, which in turn lead to a reduction of p16INK4a expression (Fig. 7B).


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)

Knock-down of Sp1 reduces expression of endogenous p16INK4a.Following transfection of 2BS cells with si-Sp1 or a control plasmid, RT-PCR (A) and Western blotting (B) were carried out to analyze the expression of the genes indicated.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0000164-g007: Knock-down of Sp1 reduces expression of endogenous p16INK4a.Following transfection of 2BS cells with si-Sp1 or a control plasmid, RT-PCR (A) and Western blotting (B) were carried out to analyze the expression of the genes indicated.
Mentions: As the mechanism of MTR treatment is blocking the transcription factors such as Sp family binding to GC-box, in this way, MTR could also affect the binding of other transcriptional factors to p16INK4a promoter. To investigate the role of Sp1 specifically, Sp1 was knocked-down by RNAi to further confirm Sp1 binding to GC boxes is essential for the transcription of p16INK4a (Fig. 7A). Western Blot showed that si-Sp1 remarkably reduced the expression of Sp1, which in turn lead to a reduction of p16INK4a expression (Fig. 7B).

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