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Tumor suppressor RASSF1A promoter: p53 binding and methylation.

Tian Y, Hou Y, Zhou X, Cheng H, Zhou R - PLoS ONE (2011)

Bottom Line: Further chromatin immunoprecipitation confirmed p53 binding to the RASSF1A.Moreover, p53 binding to the promoter down-regulated RASSF1A expression.These results suggest that p53 protein specifically binds to the RASSF1A promoter and inhibits its expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Center for Developmental Biology, College of Life Science, Wuhan University, Wuhan, China.

ABSTRACT
Oncogenes and tumor suppressors work in concert to regulate cell growth or death, which is a pair of antagonist factors for regulation of tumorigenesis. Here we show promoter characteristic of tumor suppressor RASSF1A, which revealed a p53 binding site in the distal and a GC-rich region in the proximal promoter region of RASSF1A, in despite of TATA box-less. The GC-rich region, which is ∼300 bp upstream from the RASSF1A ATG, showed the strongest promoter activity in an assay of RASSF1A-driving GFP expression. Methylation analysis of the CpG island showed that 78.57% of the GC sties were methylated in testis tumor samples compared with methylation-less in normal testis. Hypermethylation of the GC-rich region is associated with RASSF1A silencing in human testis tumors. In addition, electrophoretic mobility shift assay indicated that p53 protein bound to the RASSF1A promoter. Further chromatin immunoprecipitation confirmed p53 binding to the RASSF1A. Moreover, p53 binding to the promoter down-regulated RASSF1A expression. These results suggest that p53 protein specifically binds to the RASSF1A promoter and inhibits its expression. Our results provide new insight into the mechanism of action of tumor suppressors and may be a starting point for development of new approaches to cancer treatment.

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Down-regulation of RASSF1A by p53.A, RASSF1A promoter activity was inhibited by p53 protein with a dose dependent manner. pRASSF1A-GFP (construction c in Fig. 1) was co-transfected with different concentration of CMV-p53 into COS-7 cells, and transfected cells were analyzed by the flow cytometry. B, pRASSF1A-GFP was transfected into COS-7 cells (a) or co-transfected with p53 (b), and images were taken under fluorescent microscopy. p53 remarkably decreased expression of RASSF1A-GFP. GFP was mainly expressed in the cytoplasm. Nuclei were stained with Hoechst. C, Overexpression of p53 in Siha cells decreased RASSF1A protein levels, revealed by Western blot analysis of RASSF1A and p53 expression using anti-RASSF1A or p53 antibody after transfection of the p53 expression plasmid into Siha cells. Lysates of non-transfected cells and cells transfected with the pcDNA3 vector were used as controls. β-actin was used as an internal control. Molecular weights of the proteins are shown on the right.
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pone-0017017-g004: Down-regulation of RASSF1A by p53.A, RASSF1A promoter activity was inhibited by p53 protein with a dose dependent manner. pRASSF1A-GFP (construction c in Fig. 1) was co-transfected with different concentration of CMV-p53 into COS-7 cells, and transfected cells were analyzed by the flow cytometry. B, pRASSF1A-GFP was transfected into COS-7 cells (a) or co-transfected with p53 (b), and images were taken under fluorescent microscopy. p53 remarkably decreased expression of RASSF1A-GFP. GFP was mainly expressed in the cytoplasm. Nuclei were stained with Hoechst. C, Overexpression of p53 in Siha cells decreased RASSF1A protein levels, revealed by Western blot analysis of RASSF1A and p53 expression using anti-RASSF1A or p53 antibody after transfection of the p53 expression plasmid into Siha cells. Lysates of non-transfected cells and cells transfected with the pcDNA3 vector were used as controls. β-actin was used as an internal control. Molecular weights of the proteins are shown on the right.

Mentions: We then examined the effect of p53 binding on RASSF1A expression using a RASSF1A promoter-EGFP expression reporter construct (pRASSF1A-EGFP). When p53 and pRASSF1A-EGFP were co-transfected into COS-7 cells, expression of the reporter EGFP was significantly inhibited in a dose-dependent manner, reaching saturation when 40 ng of the p53 vector was transfected (Fig. 4A). Fluorescent microscopy analysis showed directly that p53 remarkably decreased expression of RASSF1A-GFP (Fig. 4B). Further, when p53 was overexpressed in endogenous RASSF1A-expressing Siha cells, RASSF1A protein expression was markedly decreased (Fig. 4C). These results indicated that p53 protein not only specifically binds the RASSF1A promoter but also efficiently downregulates RASSF1A expression.


Tumor suppressor RASSF1A promoter: p53 binding and methylation.

Tian Y, Hou Y, Zhou X, Cheng H, Zhou R - PLoS ONE (2011)

Down-regulation of RASSF1A by p53.A, RASSF1A promoter activity was inhibited by p53 protein with a dose dependent manner. pRASSF1A-GFP (construction c in Fig. 1) was co-transfected with different concentration of CMV-p53 into COS-7 cells, and transfected cells were analyzed by the flow cytometry. B, pRASSF1A-GFP was transfected into COS-7 cells (a) or co-transfected with p53 (b), and images were taken under fluorescent microscopy. p53 remarkably decreased expression of RASSF1A-GFP. GFP was mainly expressed in the cytoplasm. Nuclei were stained with Hoechst. C, Overexpression of p53 in Siha cells decreased RASSF1A protein levels, revealed by Western blot analysis of RASSF1A and p53 expression using anti-RASSF1A or p53 antibody after transfection of the p53 expression plasmid into Siha cells. Lysates of non-transfected cells and cells transfected with the pcDNA3 vector were used as controls. β-actin was used as an internal control. Molecular weights of the proteins are shown on the right.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045384&req=5

pone-0017017-g004: Down-regulation of RASSF1A by p53.A, RASSF1A promoter activity was inhibited by p53 protein with a dose dependent manner. pRASSF1A-GFP (construction c in Fig. 1) was co-transfected with different concentration of CMV-p53 into COS-7 cells, and transfected cells were analyzed by the flow cytometry. B, pRASSF1A-GFP was transfected into COS-7 cells (a) or co-transfected with p53 (b), and images were taken under fluorescent microscopy. p53 remarkably decreased expression of RASSF1A-GFP. GFP was mainly expressed in the cytoplasm. Nuclei were stained with Hoechst. C, Overexpression of p53 in Siha cells decreased RASSF1A protein levels, revealed by Western blot analysis of RASSF1A and p53 expression using anti-RASSF1A or p53 antibody after transfection of the p53 expression plasmid into Siha cells. Lysates of non-transfected cells and cells transfected with the pcDNA3 vector were used as controls. β-actin was used as an internal control. Molecular weights of the proteins are shown on the right.
Mentions: We then examined the effect of p53 binding on RASSF1A expression using a RASSF1A promoter-EGFP expression reporter construct (pRASSF1A-EGFP). When p53 and pRASSF1A-EGFP were co-transfected into COS-7 cells, expression of the reporter EGFP was significantly inhibited in a dose-dependent manner, reaching saturation when 40 ng of the p53 vector was transfected (Fig. 4A). Fluorescent microscopy analysis showed directly that p53 remarkably decreased expression of RASSF1A-GFP (Fig. 4B). Further, when p53 was overexpressed in endogenous RASSF1A-expressing Siha cells, RASSF1A protein expression was markedly decreased (Fig. 4C). These results indicated that p53 protein not only specifically binds the RASSF1A promoter but also efficiently downregulates RASSF1A expression.

Bottom Line: Further chromatin immunoprecipitation confirmed p53 binding to the RASSF1A.Moreover, p53 binding to the promoter down-regulated RASSF1A expression.These results suggest that p53 protein specifically binds to the RASSF1A promoter and inhibits its expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Center for Developmental Biology, College of Life Science, Wuhan University, Wuhan, China.

ABSTRACT
Oncogenes and tumor suppressors work in concert to regulate cell growth or death, which is a pair of antagonist factors for regulation of tumorigenesis. Here we show promoter characteristic of tumor suppressor RASSF1A, which revealed a p53 binding site in the distal and a GC-rich region in the proximal promoter region of RASSF1A, in despite of TATA box-less. The GC-rich region, which is ∼300 bp upstream from the RASSF1A ATG, showed the strongest promoter activity in an assay of RASSF1A-driving GFP expression. Methylation analysis of the CpG island showed that 78.57% of the GC sties were methylated in testis tumor samples compared with methylation-less in normal testis. Hypermethylation of the GC-rich region is associated with RASSF1A silencing in human testis tumors. In addition, electrophoretic mobility shift assay indicated that p53 protein bound to the RASSF1A promoter. Further chromatin immunoprecipitation confirmed p53 binding to the RASSF1A. Moreover, p53 binding to the promoter down-regulated RASSF1A expression. These results suggest that p53 protein specifically binds to the RASSF1A promoter and inhibits its expression. Our results provide new insight into the mechanism of action of tumor suppressors and may be a starting point for development of new approaches to cancer treatment.

Show MeSH
Related in: MedlinePlus