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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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p53 protein binds to the RASSF1A promoter.A, EMSA analysis of p53 binding to the RASSF1A promoter. WT, the wild type p53 binding site probe from human RASSF1A promoter; Mut, mutant p53 binding site probe; ds, two complementary oligonucleotide probes; ss, single stranded probe. The supershift band in lane 2 contains the anti-p53/wt DNA/p53 protein complex. Complexes were separated by native gel electrophoresis. Mutant sequence was showed in lower panel. B, ChIP assay of p53 binding to the RASSF1A promoter in Siha cells. Samples of sonicated chromatin from Siha cells cross-linked in 1% formaldehyde were immunoprecipitated with anti-p53 antibody, preimmuno IgG (IgG) and no antibody (beads only). DNA isolated from immunoprecipitated material was amplified by PCR with primers to amplify the 147 bp RASSF1A promoter sequences flanking the p53 binding site (−2799 bp to −2653 bp). A region of 192 bp flanking intron 1 and exon 2 of the RASSF1A was amplified as a control. Input lanes represent sonicated chromatin samples as positive control. The amplified PCR fragments were analysed on 2% agarose gel.
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pone-0017017-g003: p53 protein binds to the RASSF1A promoter.A, EMSA analysis of p53 binding to the RASSF1A promoter. WT, the wild type p53 binding site probe from human RASSF1A promoter; Mut, mutant p53 binding site probe; ds, two complementary oligonucleotide probes; ss, single stranded probe. The supershift band in lane 2 contains the anti-p53/wt DNA/p53 protein complex. Complexes were separated by native gel electrophoresis. Mutant sequence was showed in lower panel. B, ChIP assay of p53 binding to the RASSF1A promoter in Siha cells. Samples of sonicated chromatin from Siha cells cross-linked in 1% formaldehyde were immunoprecipitated with anti-p53 antibody, preimmuno IgG (IgG) and no antibody (beads only). DNA isolated from immunoprecipitated material was amplified by PCR with primers to amplify the 147 bp RASSF1A promoter sequences flanking the p53 binding site (−2799 bp to −2653 bp). A region of 192 bp flanking intron 1 and exon 2 of the RASSF1A was amplified as a control. Input lanes represent sonicated chromatin samples as positive control. The amplified PCR fragments were analysed on 2% agarose gel.

Mentions: As a predicted p53-binding site exists in the RASSF1A promoter (at −2718 bp), although lack of methylation CpG site in the binding site, further functional test for the binding site using gel-shift assay showed that His–p53 specifically and efficiently bound to RASSF1A (Fig. 3A). The specific interaction was confirmed by adding the p53 antibody into the binding reaction, resulting in a supershift band (antibody/p53/RASSF1A) (Fig. 3A, lane 2). Furthermore, a mutant RASSF1A probe decreased the formation of the p53/RASSF1A complex. Further test in vivo of p53 binding to the RASSF1A promoter using Chromatin Immunoprecipitation (ChIP) assay confirmed p53 binding to the RASSF1A (Fig. 3B). These results indicated that the p53 protein specifically binds to the RASSF1A promoter.


Tumor suppressor RASSF1A promoter: p53 binding and methylation.

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

p53 protein binds to the RASSF1A promoter.A, EMSA analysis of p53 binding to the RASSF1A promoter. WT, the wild type p53 binding site probe from human RASSF1A promoter; Mut, mutant p53 binding site probe; ds, two complementary oligonucleotide probes; ss, single stranded probe. The supershift band in lane 2 contains the anti-p53/wt DNA/p53 protein complex. Complexes were separated by native gel electrophoresis. Mutant sequence was showed in lower panel. B, ChIP assay of p53 binding to the RASSF1A promoter in Siha cells. Samples of sonicated chromatin from Siha cells cross-linked in 1% formaldehyde were immunoprecipitated with anti-p53 antibody, preimmuno IgG (IgG) and no antibody (beads only). DNA isolated from immunoprecipitated material was amplified by PCR with primers to amplify the 147 bp RASSF1A promoter sequences flanking the p53 binding site (−2799 bp to −2653 bp). A region of 192 bp flanking intron 1 and exon 2 of the RASSF1A was amplified as a control. Input lanes represent sonicated chromatin samples as positive control. The amplified PCR fragments were analysed on 2% agarose gel.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017017-g003: p53 protein binds to the RASSF1A promoter.A, EMSA analysis of p53 binding to the RASSF1A promoter. WT, the wild type p53 binding site probe from human RASSF1A promoter; Mut, mutant p53 binding site probe; ds, two complementary oligonucleotide probes; ss, single stranded probe. The supershift band in lane 2 contains the anti-p53/wt DNA/p53 protein complex. Complexes were separated by native gel electrophoresis. Mutant sequence was showed in lower panel. B, ChIP assay of p53 binding to the RASSF1A promoter in Siha cells. Samples of sonicated chromatin from Siha cells cross-linked in 1% formaldehyde were immunoprecipitated with anti-p53 antibody, preimmuno IgG (IgG) and no antibody (beads only). DNA isolated from immunoprecipitated material was amplified by PCR with primers to amplify the 147 bp RASSF1A promoter sequences flanking the p53 binding site (−2799 bp to −2653 bp). A region of 192 bp flanking intron 1 and exon 2 of the RASSF1A was amplified as a control. Input lanes represent sonicated chromatin samples as positive control. The amplified PCR fragments were analysed on 2% agarose gel.
Mentions: As a predicted p53-binding site exists in the RASSF1A promoter (at −2718 bp), although lack of methylation CpG site in the binding site, further functional test for the binding site using gel-shift assay showed that His–p53 specifically and efficiently bound to RASSF1A (Fig. 3A). The specific interaction was confirmed by adding the p53 antibody into the binding reaction, resulting in a supershift band (antibody/p53/RASSF1A) (Fig. 3A, lane 2). Furthermore, a mutant RASSF1A probe decreased the formation of the p53/RASSF1A complex. Further test in vivo of p53 binding to the RASSF1A promoter using Chromatin Immunoprecipitation (ChIP) assay confirmed p53 binding to the RASSF1A (Fig. 3B). These results indicated that the p53 protein specifically binds to the RASSF1A promoter.

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