constructed promoters. Ongoing studies are examining the functional evolution of p53 response elements across mammalian species [43,49], co-evolution of the p53 protein, as well as rules for p53 transactivation [50]. Because of MAPD’s sensitivity and dynamic range, the impact of SNPs or mutations on binding can be assessed providing a valuable tool in genomics research. We found that subtle changes in binding sequence can have effects that are not captured with existing computational models. Using nuclear extracts from human cells expressing p53 mutants, R175H and S121F, we observe a loss of binding and a change of binding specificity, respectively, consistent with their effect on gene expression. The correlation between binding in this nuclear extract-based in vitro system and transactivation in mammalian cells suggests the important role that sequence-specific p53 interactions play in transcriptional activation. MAPD will facilitate studies of how variation in target binding sites affects binding and fine-tuning the regulation of transcriptional networks as well as the impact of polymorphic variation on master regulatory networks. Materials and Methods Cell Culture and Treatments Human lymphoblastoid cells were grown in RPMI 1640 media supplemented with 15% heat-inactivated fetal bovine serum (Invitrogen Corp., Carlsbad, CA) and incubated at 5% CO2 at 37uC with 1% penicillin-streptomycin antibiotics (Invitrogen). The cell lines GM12824 and GM12825 used for exposure were purchased from Coriell Cell Repositories (Camden, NJ). Cells were grown to ,8.56105 cells/mL before being exposed to 0.6 mg/mL (1 mM) doxorubicin (Sigma, St. Louis, MO) for 18 hours at 37uC. Human osteosarcoma cell lines SaOS-2 (HTB- 85, ATCC, Manassas, VA) and U2OS (HTB-96, ATCC) were grown in McCoy’s A5 medium supplemented with 10% FBS and 100 mg/ml of penicillin and streptomycin. Cells were incubated at 37uC with 5% CO2. Transient Transfection For transient transfections, 16106 SaOS2 cells were seeded in a 100 mm plate. After 24 hours cells were transfected with 2 mg of purified endotoxin-free p53 expression vectors in the presence of non-liposomal transfection reagent, FuGENE 6 (Roche Diagnos- tics, Indianapolis, IN) according to manufacturer’s protocol. U2OS cells were transfected with 2 mg pSG5-ERa expression vector, provided by Dr. Gilbert Schonfelder, 24 hours prior to preparation of extracts. Plasmids pC53-SN3[51] coding for human p53 cDNA under the control of CMV promoter and pCMV-Neo-Bam were provided by Dr. Bert Vogelstein. p53 mutants were made by site-directed mutagenesis according to the manufacturer’s protocol (QuickChange Site-Directed Mutagenesis kit, Stratagene, La Jolla, CA). p53 constructs were confirmed by sequencing. Nuclear Protein Extraction and Quantification The presence of p53 in extracts was assessed by western blot and TransAM p53 Transcription Factor assay (Active Motif). Nuclear protein was extracted from non-treated and doxorubicin- treated cells according to manufacturer’s protocol (Active Motif, Carlsbad, CA) for ,8.86106 cells with the following modification. Cells were transferred to 15 mL centrifuge tubes, spun at 4uC (2006g, 5 min) and washed in 16 PBS/phosphatase. Protein concentration was measured in triplicate using the BCA Protein Assay kit (Pierce Biochemical, Rockford, IL), followed by a fluorescence plate read using a HTS 7000 Bio Assay Reader (Perkin Elmer, Waltham, MA). Sequence Variation and p53 DNA Binding Western Blot Analysis Equal amounts of whole cell or nuclear extracts were separated on NuPAGE BisTris gels and transferred to polyvinylidene difluoride membranes (Invitrogen). The blots were probed with primary antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for p53 (PAb1801 and DO-1) and actin (C-11). For ERa detection we used rabbit polyclonal anti-ERa (H-184, Santa Cruz). Bands were detected using horseradish peroxide-conjugated secondary antibodies (Santa Cruz) and the ECL Western Blotting System (Amersham Biosciences, Piscataway, NJ). General Oligonucleotide Design The double-stranded DNA fragments incorporated a forward oligonucleotide with a 24-nt ‘‘tag’’ at the 59 end linked to a 50-nt fragment containing a sequence of interest. The forward oligonucleotide was hybridized to a unique 24-nt ‘‘anti-tag’’ present on each FlexMAP bead type (Luminex, Austin, TX). The 50-nt modular fragment typically contained a 20-nt RE in the middle of a 30-nt common backbone sequence (sequences are described in Supporting Information). For p53 REs with a 1-nt spacer (MDM2RE2, IGFBP3 and BAXA), the backbone length was decreased to 29-nt. For ERa REs (17-nt in length), the backbone sequence was increased to 33-nt. Reverse oligonucleo- tides complementary to the 50-nt modular fragment were synthesized with a 59 biotin moiety. All oligonucleotides were synthesized by Invitrogen and are listed in the Supporting tables. Preparation of Oligonucleotide-Bead Targets We used 15 mL each bead type (approximately 2,500 beads) per reaction. Beads were vortexed for 30 seconds and added to double-stranded DNA containing the corresponding tag sequence in an annealing buffer containing 5 mM Tris (pH 7.5), 10 mM NaCl, and 0.5 mM EDTA. The final primer concentration was 1.5 mM. Annealing was performed in a PCR cycler (95uC for 5 min, followed by an incremental 5uC decrease, holding for 1 minute at each temperature, until reaching 45uC). Beads were then placed in a filter plate (MultiScreenHTS-BV filter plate, Millipore Corp., Bedford, MA) and washed several times with Wash Buffer 1 (Assay Buffer A, Cytokine Reagent Kit, Bio-Rad Laboratories, Hercules, CA). Microsphere Protein-DNA Binding Washed beads with conjugated DNA were then resuspended in the appropriate volume of protein binding buffer (0.05 M KCl, 0.1 mM EDTA, 5 mM DDT, 0.05% Triton X-100, 10 mM MgCl2, 1.0 mg/mL BSA, 0.5 mM ATP and 25 mM HEPES, pH 7.6) supplemented with 150 pmoles non-competing double- stranded oligo, WRNC. The noncompeting oligo from the TransAm p53 kit (Active Motif, Carlbad, CA) was also effective. In a typical binding assay (detection of p53 and ERa), beads were resuspended in 50 mL protein binding buffer and supplemented with 1.75 mg nuclear extracts from either treated or non-treated cells, followed by thorough mixing for 15 seconds. Beads were incubated with nuclear extracts for 1 hour at room temperature in the dark, transferred onto a filter plate, washed 3 times with 50 mL Wash Buffer 2 and incubated for 30 min with primary antibody. For p53 detection, we used mouse monoclonal anti-p53 primary antibody (1:500 dilution, clone DO-7, BD Biosciences, San Jose, CA). For ERa detection, we used rabbit polyclonal anti-ERa (1:500 H-184, Santa Cruz). Beads were washed 3 times with 50 mL Wash Buffer 2 (50 mM KCl, 0.1 mM EDTA, 5 mM DTT, 0.5 mg/mL BSA, 0.05% Triton X-100, 10 mM MgCl2 and 25 mM HEPES, pH 7.6). The beads were then incubated with PLoS Genetics | www.plosgenetics.org 9 May 2009 | Volume 5 | Issue 5 | e1000462