Author Summary Characterizing the functional roles of gene regulatory sequences and the impact of genetic polymorphism on protein-DNA binding are rapidly growing areas of geno- mics. The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements, and we have developed a multiplex format microsphere assay (MAPD) to probe how sequence variation alters p53 binding. Testing pools of multiplexed oligonucleotides that contain numerous p53 binding targets or different sequence variants, we demonstrate that subtle changes in target sequence can impact p53 binding, and these effects are not captured by commonly used computational binding models. A group of single nucleotide polymorphisms was examined and binding profiles closely matched gene expression measured in a parallel system. We also observed distinct binding patterns that were characteristic of transiently expressed wild type and mutant p53s, suggesting that this method may be useful for probing the impact of protein structural variation on DNA binding. MAPD utilizes biologically meaningful cell extracts in a multiplexed, quantitative format that provides a powerful experimental tool for elucidating the functional impact of sequence polymor- phism and protein variation on protein/DNA binding in transcriptional networks. immunoprecipitation (ChIP) but each of these approaches has experimental limitations. We sought to test p53 binding to a large number of defined sequences, including single nucleotide variants, with cell extracts generated from different experimental conditions using an approach that would be amenable to high sample throughput and replication. Microsphere-based flow cytometric assays utilize fluorescent-dyed beads (,6 micrometer micro- spheres) coated with molecules such as nucleic acids or peptides [14]. Beads are placed in an assay medium containing biological targets (e.g., cell extracts containing p53 or ERa) where interactions can take place. Each bead type has a distinct spectral signature and up to 100 bead types per reaction can be used. A dual laser-equipped flow cytometer detects both the bead identity and a fluorescent reporter dye attached to the target of interest that is captured on the bead. The fluorescence signal intensity reflects relative quantification of target/bead interaction. This technology has been adapted to many assays [15], including RNA expression analysis [16], serum cytokine analysis [17,18] and high- throughput genotyping [18,19,20,21]. Based on this platform, we developed a highly reproducible general approach for performing large-scale quantitative studies of protein-DNA binding using p53 or other transcription factors, such as ERa Importantly, this microsphere assay for protein-DNA binding (MAPD) generates biologically-meaningful binding data using nuclear extracts from human cells, permitting flexible experimental designs. We show that the MAPD approach provides high resolution and utility in the functional evaluation of p53 binding to RE variants, including single nucleotide changes in REs, making it an effective tool in modern genomics research. Comparing experimental and computed binding values based on a position weight matrix (PWM) model, commonly used for evaluating TF binding, we demonstrate that subtle changes in the binding sequence have functional consequences that are not captured by PWM models. Furthermore, using a panel of p53 REs and p53 RE SNPs, we observed patterns of binding that are highly correlated with luciferase-based transactivation measurements in mammalian cells. The p53 mutants, S121F and R175H, displayed Sequence Variation and p53 DNA Binding a change in binding specificity and a loss of binding respectively. The binding properties of these mutants are consistent with their previously described effects on gene expression [22]. While in vitro binding measurements are not a surrogate for ChIP or gene expression studies, this method allows quantification of a necessary step in transcription. This work demonstrates that MAPD can not only be used to evaluate RE sequence variation and SNPs, but may also be useful for probing functional changes in the protein structure of DNA binding proteins. Results Multiplex Binding Measurements of Activated p53 with Known REs 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 quan- tities 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]. Figure 2 displays p53 binding after incubating the 6-plex beads with nuclear extracts from human lymphoblastoid cells that were either not treated (NT), containing small amounts of activated p53, or treated with doxorubicin (DOXO), inducing high levels of activated p53 [26]. MAPD could detect p53 binding in both. The Figure 1. 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. doi:10.1371/journal.pgen.1000462.g001 PLoS Genetics | www.plosgenetics.org 2 May 2009 | Volume 5 | Issue 5 | e1000462