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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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Related in: MedlinePlus

Microsphere assay for protein-DNA (MAPD) binding.Double-stranded oligonucleotides containing p53 binding sequence of interest and single-stranded tag overhang were hybridized to FlexMap beads bearing complementary anti-tag sequence. Multiplex groups of beads were prepared and incubated with nuclear extracts containing transcription factor. Primary and secondary antibodies were added, beads were sorted and fluorescence signal was quantified for each bead type and normalized by biotin/streptavidin/phycoerythrin signal to adjust for bead-specific signal effects.
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pgen-1000462-g001: Microsphere assay for protein-DNA (MAPD) binding.Double-stranded oligonucleotides containing p53 binding sequence of interest and single-stranded tag overhang were hybridized to FlexMap beads bearing complementary anti-tag sequence. Multiplex groups of beads were prepared and incubated with nuclear extracts containing transcription factor. Primary and secondary antibodies were added, beads were sorted and fluorescence signal was quantified for each bead type and normalized by biotin/streptavidin/phycoerythrin signal to adjust for bead-specific signal effects.

Mentions: The effects of sequence variation among binding sites for a given TF have been difficult to quantify in protein-DNA binding experiments. We developed a fluorescent microsphere-based approach to interrogate the interaction between p53 and its target REs (Figure 1 and S1). Double-stranded DNA fragments bearing p53 RE sequences (ConA, ConC, p21, PUMA, GADD45) and a negative control (WRNC) were uniquely linked to different sets of fluorescent microsphere beads (see Materials and Methods and Supporting Information for oligonucleotide design). Equal quantities of the 6 bead types were combined. The p21 RE, a strong binding sequence, was used as a positive control. ConA and ConC are both perfect matches with the p53 consensus RE and have been demonstrated to function as strong binding REs in a p53 model system [23] and human cells (Menendez, Jordan and Resnick, unpublished) with ConA being more responsive to p53. p53 binding to the strong RE from p21 and to the moderate-to-weak REs from PUMA and GADD45, respectively, are well established [24],[25].


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)

Microsphere assay for protein-DNA (MAPD) binding.Double-stranded oligonucleotides containing p53 binding sequence of interest and single-stranded tag overhang were hybridized to FlexMap beads bearing complementary anti-tag sequence. Multiplex groups of beads were prepared and incubated with nuclear extracts containing transcription factor. Primary and secondary antibodies were added, beads were sorted and fluorescence signal was quantified for each bead type and normalized by biotin/streptavidin/phycoerythrin signal to adjust for bead-specific signal effects.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000462-g001: Microsphere assay for protein-DNA (MAPD) binding.Double-stranded oligonucleotides containing p53 binding sequence of interest and single-stranded tag overhang were hybridized to FlexMap beads bearing complementary anti-tag sequence. Multiplex groups of beads were prepared and incubated with nuclear extracts containing transcription factor. Primary and secondary antibodies were added, beads were sorted and fluorescence signal was quantified for each bead type and normalized by biotin/streptavidin/phycoerythrin signal to adjust for bead-specific signal effects.
Mentions: The effects of sequence variation among binding sites for a given TF have been difficult to quantify in protein-DNA binding experiments. We developed a fluorescent microsphere-based approach to interrogate the interaction between p53 and its target REs (Figure 1 and S1). Double-stranded DNA fragments bearing p53 RE sequences (ConA, ConC, p21, PUMA, GADD45) and a negative control (WRNC) were uniquely linked to different sets of fluorescent microsphere beads (see Materials and Methods and Supporting Information for oligonucleotide design). Equal quantities of the 6 bead types were combined. The p21 RE, a strong binding sequence, was used as a positive control. ConA and ConC are both perfect matches with the p53 consensus RE and have been demonstrated to function as strong binding REs in a p53 model system [23] and human cells (Menendez, Jordan and Resnick, unpublished) with ConA being more responsive to p53. p53 binding to the strong RE from p21 and to the moderate-to-weak REs from PUMA and GADD45, respectively, are well established [24],[25].

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