Sequence Variation and p53 DNA Binding Figure 6. Functional interrogation of bona fide p53 REs. A multiplex oligonucleotide panel of previously validated p53 REs [23] was assembled (each oligonucleotide type on a unique microsphere bead) and tested for p53 binding. A) RE panel was treated with nuclear extracts from DOXO- treated lymphoblastoid cells or SaOS-2 cells (p53-null) that were transiently transfected with plasmid pC53-SN3 (coding for human wt p53 cDNA under control of the CMV promoter; see Materials and Methods). Equivalent quantities of p53 were used in each assay (as measured by Western (shown in insert panel 6B) and ELISA). Following normalization to p21LWT value (p21 = 1), patterns of binding are similar for DOXO activated p53 and ectopically expressed wt p53 (see also Figure S7A). The insert in Figure 6A displays the correlation between binding and luciferase expression for several elements. Figure S7 displays the relationship between DOXO activated p53 binding and p53 ChIP. The panel included ConA and WRNC as positive and negative controls, respectively. B) RE panel of beads was treated with nuclear extracts from either SaOS-2 cells (p53-null) that have been transiently transfected with empty plasmid (p53 null, not shown), plasmid expressing p53 wt (p53 wt), plasmids expressing p53 mutant S121F or R175H. Equivalent quantities of p53 were used in each assay (as measured by Western (shown in insert panel 6B); no binding was observed for p532/ 2 SaOS-2 cells. Symbol (#) indicates RE retained binding in the S121F mutant. Bar values are mean6SEM (n = 3). Note both the numerical difference and the breaks in the scale of horizontal axes. doi:10.1371/journal.pgen.1000462.g006 binding could provide insight into tumor biology, carcinogenesis and treatment response [35,36]. TF binding to REs is a necessary step in regulating transcriptional networks [8]. EMSA and chromatin immunopre- cipitation (ChIP) have been essential in advancing the study of gene regulation. However, both approaches are low in sample throughput, labor–intensive, difficult to quantify and have a limited dynamic range. More recently, ChIP combined with microarrays [34] or sequencing, and protein binding arrays [47,48] have allowed parallel analyses of TF binding to thousands of sequences, and these techniques are highly effective for qualitative discovery of binding sites. However, currently these approaches have experimental constraints, including relatively low sample throughput, uncharacterized variability, and are expensive. Surface plasmon resonance and fluorescence anisotropy are useful for determining detailed biochemical parameters, but like protein binding arrays are currently restricted to purified proteins. MAPD complements existing approaches for the functional examination of candidate binding sequences because this system permits relative quantification and allows multiple endpoints (up to 100 DNA REs per experiment including internal reference controls). In addition, the use of cell extracts in a rapid, low-cost 96-well format enables complex experimental designs with large numbers of samples and replication. An automated format is possible and screening for inhibitors of binding or compounds that selectively restore binding could be accomplished. A strength of the MAPD approach over other binding methods is the inclusion of an internal reference positive control (for example, p21, a strong binding RE) and a negative control (WRNC, a nonbinding sequence) within the multiplexed assay which provide for quality control and normalization across experiments. MAPD is a general methodology as demonstrated by detection of not only p53 binding, but also the nuclear hormone receptor ERa binding its RE (Figure S5). MAPD could be adapted to detect the impact of multiple proteins in a transcription complex or to evaluate multiple binding sites within native or artificially PLoS Genetics | www.plosgenetics.org 8 May 2009 | Volume 5 | Issue 5 | e1000462