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Probing the functional impact of sequence variation on p53-DNA interactions using a novel microsphere assay for protein-DNA binding with human cell extracts.

Noureddine MA, Menendez D, Campbell MR, Bandele OJ, Horvath MM, Wang X, Pittman GS, Chorley BN, Resnick MA, Bell DA - PLoS Genet. (2009)

Bottom Line: Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs.A group of eight single nucleotide polymorphisms (SNPs) was examined and binding profiles closely matched transactivation capability tested in luciferase constructs.Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s.

View Article: PubMed Central - PubMed

Affiliation: Environmental Genomics Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, USA.

ABSTRACT
The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs). Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation -- including polymorphisms -- and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD) for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt) variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs) was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks.

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Evaluation of p53 binding to response element SNP alleles.Binding for predicted strong (Dark bars) and weak (open bars) alleles of the polymorphic sequence is shown relative to p21 RE binding (100%). Each bar represents the mean of 3 experiments carried out on different days and performed in triplicate (error bars represent SD). Positive and negative control oligonucleotides (left side) were included in each experiment and each sample was tested with a negative control extract (data not shown). This experiment was replicated using p53 extracts generated from a second tissue type (U2OS cells, Figure S8). These results are in close agreement with evaluation of these SNP alleles when cloned into a luciferase construct and transfected into SaOS2 cells expressing p53 (Tomso et al. [5]).
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pgen-1000462-g005: Evaluation of p53 binding to response element SNP alleles.Binding for predicted strong (Dark bars) and weak (open bars) alleles of the polymorphic sequence is shown relative to p21 RE binding (100%). Each bar represents the mean of 3 experiments carried out on different days and performed in triplicate (error bars represent SD). Positive and negative control oligonucleotides (left side) were included in each experiment and each sample was tested with a negative control extract (data not shown). This experiment was replicated using p53 extracts generated from a second tissue type (U2OS cells, Figure S8). These results are in close agreement with evaluation of these SNP alleles when cloned into a luciferase construct and transfected into SaOS2 cells expressing p53 (Tomso et al. [5]).

Mentions: SNPs in 8 putative p53 REs were identified using the bioinformatics approach described in Tomso et al [5]. Sequences are shown in Table S1. Oligonucleotides containing the predicted weak and strong alleles for these p53 REs were placed on microspheres and evaluated for binding to activated p53 in nuclear extracts (Figure 5). Negative and positive control oligonucleotides were hybridized in parallel in the multiplexed reaction and nuclear extract from untreated cells was also tested as a negative control (mean binding to p21 probe for untreated cell extracts was 1.6%+/−0.19%, n = 6 of the Doxo treated value); binding to p21 was consistent with p53 content assessed by western blot and TransAm ELISA. Among these putative p53 REs, the predicted strong alleles for REs in ADARB1, ARHGEF7, and EOMES displayed binding strength that was ∼30% of p21 binding, a level that is similar to PUMA and GADD45 (Figures 2 and 6). With the exception of REs upstream of DCC and SEI1, we observed that the predicted weak alleles showed significantly reduced binding. SNPs that altered the conserved core sequence (ADARB1, EOMES, RRM1 and TLR8) showed dramatic reductions in binding. This approach to functional evaluation of TF binding site SNPs would be useful for rapid screening of candidate SNPs in TF binding sites identified through bioinformatics or whole genome association studies.


Probing the functional impact of sequence variation on p53-DNA interactions using a novel microsphere assay for protein-DNA binding with human cell extracts.

Noureddine MA, Menendez D, Campbell MR, Bandele OJ, Horvath MM, Wang X, Pittman GS, Chorley BN, Resnick MA, Bell DA - PLoS Genet. (2009)

Evaluation of p53 binding to response element SNP alleles.Binding for predicted strong (Dark bars) and weak (open bars) alleles of the polymorphic sequence is shown relative to p21 RE binding (100%). Each bar represents the mean of 3 experiments carried out on different days and performed in triplicate (error bars represent SD). Positive and negative control oligonucleotides (left side) were included in each experiment and each sample was tested with a negative control extract (data not shown). This experiment was replicated using p53 extracts generated from a second tissue type (U2OS cells, Figure S8). These results are in close agreement with evaluation of these SNP alleles when cloned into a luciferase construct and transfected into SaOS2 cells expressing p53 (Tomso et al. [5]).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2667269&req=5

pgen-1000462-g005: Evaluation of p53 binding to response element SNP alleles.Binding for predicted strong (Dark bars) and weak (open bars) alleles of the polymorphic sequence is shown relative to p21 RE binding (100%). Each bar represents the mean of 3 experiments carried out on different days and performed in triplicate (error bars represent SD). Positive and negative control oligonucleotides (left side) were included in each experiment and each sample was tested with a negative control extract (data not shown). This experiment was replicated using p53 extracts generated from a second tissue type (U2OS cells, Figure S8). These results are in close agreement with evaluation of these SNP alleles when cloned into a luciferase construct and transfected into SaOS2 cells expressing p53 (Tomso et al. [5]).
Mentions: SNPs in 8 putative p53 REs were identified using the bioinformatics approach described in Tomso et al [5]. Sequences are shown in Table S1. Oligonucleotides containing the predicted weak and strong alleles for these p53 REs were placed on microspheres and evaluated for binding to activated p53 in nuclear extracts (Figure 5). Negative and positive control oligonucleotides were hybridized in parallel in the multiplexed reaction and nuclear extract from untreated cells was also tested as a negative control (mean binding to p21 probe for untreated cell extracts was 1.6%+/−0.19%, n = 6 of the Doxo treated value); binding to p21 was consistent with p53 content assessed by western blot and TransAm ELISA. Among these putative p53 REs, the predicted strong alleles for REs in ADARB1, ARHGEF7, and EOMES displayed binding strength that was ∼30% of p21 binding, a level that is similar to PUMA and GADD45 (Figures 2 and 6). With the exception of REs upstream of DCC and SEI1, we observed that the predicted weak alleles showed significantly reduced binding. SNPs that altered the conserved core sequence (ADARB1, EOMES, RRM1 and TLR8) showed dramatic reductions in binding. This approach to functional evaluation of TF binding site SNPs would be useful for rapid screening of candidate SNPs in TF binding sites identified through bioinformatics or whole genome association studies.

Bottom Line: Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs.A group of eight single nucleotide polymorphisms (SNPs) was examined and binding profiles closely matched transactivation capability tested in luciferase constructs.Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s.

View Article: PubMed Central - PubMed

Affiliation: Environmental Genomics Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, USA.

ABSTRACT
The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs). Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation -- including polymorphisms -- and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD) for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt) variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs) was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks.

Show MeSH
Related in: MedlinePlus