Probing the Functional Impact of Sequence Variation on p53-DNA Interactions Using a Novel Microsphere Assay for Protein-DNA Binding with Human Cell Extracts Maher A. Noureddine1., Daniel Menendez2., Michelle R. Campbell1., Omari J. Bandele1, Monica M. Horvath1, Xuting Wang 1 , Gary S. Pittman1, Brian N. Chorley1, Michael A. Resnick2, Douglas A. Bell1* 1 Environmental Genomics Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States of America, 2 Chromosome Stability Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States of America 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 polymor- phisms—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. Citation: Noureddine MA, Menendez D, Campbell MR, Bandele OJ, Horvath MM, et al. (2009) Probing the Functional Impact of Sequence Variation on p53-DNA Interactions Using a Novel Microsphere Assay for Protein-DNA Binding with Human Cell Extracts. PLoS Genet 5(5): e1000462. doi:10.1371/journal.pgen.1000462 Editor: Gregory S. Barsh, Stanford University School of Medicine, United States of America Received September 4, 2008; Accepted March 30, 2009; Published May 8, 2009 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This research was funded entirely by the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health (projects: Z01-ES-100475-M-0001 and Z01 ES065079-15). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: BELL1@niehs.nih.gov . These authors contributed equally to this work. Introduction The prominent tumor suppressor p53 is a transcription factor (TF) that regulates a vast network of genes. Functional evaluation of binding to its target DNA response elements (REs) is a key step in understanding the many events that can lead to tumorigenesis. Following activation, p53 localizes to the nucleus, forms a tetramer, binds to REs and transactivates or suppresses target genes[1,2]. The well-established p53 RE consensus sequence consists of two 10-nt half-sites (RRRCWWGYYY-N*- RRRCWWGYYY, R = purine, Y = pyrimidine, W = A/T and N* = 0 to 13-nt spacer) [3] and p53 binding to these DNA elements is a necessary event for p53-regulated gene expression. The relationship between p53 activation, binding and in vivo gene transactivation remains an important area of investigation [4]. Experimental studies have identified numerous bona fide REs necessary for p53-regulated transactivation, and thousands of potential sites have been implicated by bioinformatics [5,6]. The presence of single nucleotide polymorphisms (SNPs) in REs may impact transcription factor binding and result in altered gene expression. Genetic variation in TF binding is a significant evolutionary mechanism in mammals [7] and directs species- specific gene expression [8]. In human populations, response element variation is known to alter cellular processes including diabetes [9], development [10], angiogenesis [11] and susceptibil- ity to many other diseases including cancer [12]. With the accelerating identification of noncoding, disease- and expression- associated SNPs through genome-wide association studies [13], evaluating the functional and mechanistic consequences of regulatory SNPs is a general problem in human genetics. We have identified several hundred SNPs in the human genome that reside within putative p53 REs [5]. Assessing the impact of these RE SNPs is of direct relevance to p53 function in the cell cycle, DNA repair, apoptosis and subsequent tumorigenesis. Binding of p53 or its mutants to REs has been assessed by in vitro techniques such as electrophoretic mobility shift (EMSA), surface plasmon resonance, fluorescent anisotropy and chromatin PLoS Genetics | www.plosgenetics.org 1 May 2009 | Volume 5 | Issue 5 | e1000462