phycoerythrin-conjugated secondary antibodies (1:75 dilution, R- phycoerythrin-conjugated goat anti-mouse (p53) or 1:75 dilution, R-phycoerythrin-conjugated goat anti-rabbit (both purchased from Invitrogen) for 30 min and washed 3 times with 50 mL Wash Buffer 2. Dilutions were prepared using a buffer made up of PBS (5 mM Phosphate, 1.35 mM KCl, 68 mM NaCl) supple- mented with 0.5% Tween 20 and 1% BSA. For signal normalization, DNA-conjugated beads were treated separately with phycoerythrin-conjugated streptavidin, 16 streptavidin-PE diluted in Wash Buffer 1 for 20 min in the absence of any nuclear extracts, then washed 3 times with 50 mL Wash Buffer 2. For signal detection, beads were then re-suspended in 150 mL Wash Buffer 1 and subjected to flow analysis using a Bio-Plex 200 (Bio- Rad Laboratories) by following the manufacturer’s recommenda- tion for instrument settings and calibration. All binding reactions were conducted in triplicate. Measurement of p53 Binding to SNP Alleles The effect of SNPs on p53 binding to putative REs was examined using the microsphere assay for protein-DNA binding as described above. Each bead type was conjugated to double- stranded oligonucleotides that contained either the predicted strong or weak allele in separate annealing reactions (i.e., ADARB1 Strong and ADARB1 Weak were conjugated to bead 12). Multiplexed allele sets were tested in parallel binding reactions. To assess p53 binding, beads were incubated with nuclear extracts derived from human either human lymphoblastoid (Figure 5) or U2OS cells (Figure S8) that were treated with doxorubicin or untreated (not shown). Negative and positive control oligonucle- otide-bead targets were examined in each parallel reaction, and untreated nuclear extracts were used as an additional negative control. Bead-Specific Corrections Oligonucleotide density on beads varies among bead types[14,52]. Therefore, bead-specific contribution to overall signal must be taken into account. To account for bead-specific effects, we used a 59 biotin moiety on the reverse oligonucleotide bound to a phycoerythrin-conjugated streptavidin to measure the maximal signal from each bead type used. For each bead type, the protein-binding signal was normalized by the bead-specific signal, independent of nuclear extracts and reported as relative binding. In addition, FlexMap bead tag/anti-tag oligonucleotide sequences should be evaluated for their similarity to target sequences and binding to protein of interest. Bioinformatics We implemented a position weight matrix (PWM) for quantitatively estimating DNA-protein binding and for predicting novel REs [40]. In a standard PWM model, entries are the probability of observing nucleotides at each position for the set of sequences examined. The p53 RE PWM was computed from 59 published p53 REs (118 halfsites) (Table S2) [43]. The PWM score for a p53 RE was calculated by scoring each half-site independently, then summing the two. The ERa PWM was based on 28 published ERa REs (Table S1). The binding PWM and sequence logo was calculated by modifying the approach of Veprintsev and Fersht [30]. Briefly, a binding difference matrix was generated by subtracting the binding value for each of 60 ConA variants from the ConA binding value. Equations 2, A.1, A.2 from the Appendix in Veprintsev and Fersht [30] were used to generate a binding-based information content (IC) position weight matrix. This IC PWM was used to create the sequence logo shown in Figure 4B using the enoLOGOS software (http://biodev.hgen. Sequence Variation and p53 DNA Binding pitt.edu/cgi-bin/enologos/enologos.cgi), and was used to calculate the predicted binding scores for the RE sequences plotted in Figures 4D. These REs plotted in Figure 4D are the same as those in Figure 6 and the sequences are listed in Table S1. Supporting Information Figure S1 Microsphere assay procedure. A) Double stranded oligonucleotides containing sequences of interest are hybridized to FlexMap beads and multiplex groups of beads are prepared. B) Beads are incubated with nuclear extracts containing transcription factor. C) Primary and secondary antibodies are added. D) Beads are sorted and fluorescence signal is quantified for each bead type. A critical technical feature of the assay is the use of FlexMAP oligonucleotide-coated microspheres which allow for the attach- ment of desired sequences to the bead. However, because the density of oligonucleotide tags present on the surface of the beads varies between bead types and bead batches, a bead type-specific signal correction must be made in each experiment [14,52]. Each double-stranded oligonucleotide tested had a biotin incorporated at the free 59end (not shown in all drawings) and following the hybridization, was conjugated with streptavidin-phycoerythrin and read on the Bioplex 200 instrument. These biotin-streptavidin- phycoerythrin values provide a readout for how many double- stranded target oligonucleotides are present on the surface of each bead type, and were used to normalize bead signals in each experiment. Found at: doi:10.1371/journal.pgen.1000462.s001 (0.07 MB PDF) Figure S2 Tests of system components. A multiplex set of six oligonucleotide-conjugated beads, each carrying a p53 binding site (ConA, ConC, p21, PUMA, GADD45) and a negative control (WRNC), was incubated under various conditions as shown and analyzed for p53 binding as described in Supplemental Materials and Methods. Lanes 1–5,7 and 8 show background and non- specific interaction. A modest signal is detected following incubation for 60 minutes with nuclear extracts from untreated lymphoblastoid cells in the presence of primary and secondary antibodies (lane 6). This signal was slightly enhanced in the presence of free noncompeting oligo (lane 10) and attenuated in the presence of a competing oligo (lane 11). A strong signal was detected (lane 9) following incubation for 60 minutes with nuclear extracts from lymphoblastoid cells treated with Doxo (30 mg/ml for 18 hrs). In addition the signal was strongly enhanced in the presence of free NC oligo (lane 12) for all p53 REs present in the 6-plex. The increased signal in the presences of noncompeting oligonucleotide is likely due to blocking of nonspecific DNA- binding proteins in the extract thereby enhancing specific p53 binding to target oligonucleotides. The signal was greatly attenuated in the presence of 150 pmol competing oligonucleotide (lane 13). The minimal signal for lane 13 indicates there is little if any nonspecific binding of p53 to beads or antitag sequences, even in the absence of noncompeting oligonucleotides. Note the two scales of vertical axis. Values shown are mean for each bead type+/2SD (n = 3). Found at: doi:10.1371/journal.pgen.1000462.s002 (0.77 MB PDF) Figure S3 p53 binding in the presence of non-competing and competing oligonucleotides. Specificity was ascertained by varying amounts of competing oligonucleotide which was observed to reduce specific binding in a concentration-dependent manner. Similarly, the use of a noncompeting (or nonspecific) oligonucle- otide, which blocks binding of nonspecific DNA binding proteins to the target sequences, enhances the sensitivity as well as detection limit and dynamic range of the assay. A multiplex set of 6 PLoS Genetics | www.plosgenetics.org 10 May 2009 | Volume 5 | Issue 5 | e1000462