Probing the Functional Impact of Sequence Variation on p53-DNA Interactions Using a Novel Microsphere Assay for Protein-DNA Binding with Human Cell Extracts

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 polymorphisms—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.

[1]  J. Moss,et al.  The C/A(-18) polymorphism in the surfactant protein B gene influences transcription and protein levels of surfactant protein B. , 2007, American journal of physiology. Lung cellular and molecular physiology.

[2]  I. Simon,et al.  Chromatin immunoprecipitation-on-chip reveals stress-dependent p53 occupancy in primary normal cells but not in established cell lines. , 2008, Cancer research.

[3]  A. Børresen-Dale,et al.  TP53 mutations in human cancers: functional selection and impact on cancer prognosis and outcomes , 2007, Oncogene.

[4]  A. Fersht,et al.  Comparative binding of p53 to its promoter and DNA recognition elements. , 2005, Journal of molecular biology.

[5]  C. Prives,et al.  p53 and p73 display common and distinct requirements for sequence specific binding to DNA , 2006, Nucleic acids research.

[6]  M. Olivier,et al.  Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database , 2007, Human mutation.

[7]  D. Bell,et al.  Correction: Noncanonical DNA Motifs as Transactivation Targets by Wild Type and Mutant p53 , 2008, PLoS Genetics.

[8]  Marie A. Iannone,et al.  Multiplexed Microsphere‐Based Flow Cytometric Assays , 2003 .

[9]  K. Kinzler,et al.  Oncogenic forms of p53 inhibit p53-regulated gene expression. , 1992, Science.

[10]  D. Bell,et al.  Divergent Evolution of Human p53 Binding Sites: Cell Cycle Versus Apoptosis , 2007, PLoS genetics.

[11]  Francesca Storici,et al.  Differential Transactivation by the p53 Transcription Factor Is Highly Dependent on p53 Level and Promoter Target Sequence , 2002, Molecular and Cellular Biology.

[12]  A. Fersht,et al.  Algorithm for prediction of tumour suppressor p53 affinity for binding sites in DNA , 2008, Nucleic acids research.

[13]  T. Omi,et al.  Identification of a regulatory SNP in the retinol binding protein 4 gene associated with type 2 diabetes in Mongolia , 2007, Human Genetics.

[14]  E. Appella,et al.  Phosphorylation Site Interdependence of Human p53 Post-translational Modifications in Response to Stress* , 2003, Journal of Biological Chemistry.

[15]  P. Bryant,et al.  A multiplex microsphere bead assay for comparative RNA expression analysis using flow cytometry. , 2004, Journal of biotechnology.

[16]  Alberto Inga,et al.  The Biological Impact of the Human Master Regulator p53 Can Be Altered by Mutations That Change the Spectrum and Expression of Its Target Genes , 2006, Molecular and Cellular Biology.

[17]  W. Wasserman,et al.  Identification of functional SNPs in the 5-prime flanking sequences of human genes , 2005, BMC Genomics.

[18]  N. Perkins,et al.  Regulation of p53 tumour suppressor target gene expression by the p52 NF‐κB subunit , 2006, The EMBO journal.

[19]  J. Ott,et al.  The p53MH algorithm and its application in detecting p53-responsive genes , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[20]  M. Berger,et al.  Protein binding microarrays (PBMs) for rapid, high-throughput characterization of the sequence specificities of DNA binding proteins. , 2006, Methods in molecular biology.

[21]  Yan Song,et al.  Nuclear extracellular signal-regulated kinase 2 phosphorylates p53 at Thr55 in response to doxorubicin. , 2001, Biochemical and biophysical research communications.

[22]  A. Oliphant,et al.  BeadArray technology: enabling an accurate, cost-effective approach to high-throughput genotyping. , 2002, BioTechniques.

[23]  D. Bell,et al.  Single nucleotide polymorphism in transcriptional regulatory regions and expression of environmentally responsive genes. , 2005, Toxicology and applied pharmacology.

[24]  K. Pearce,et al.  Multiplexed molecular interactions of nuclear receptors using fluorescent microspheres. , 2001, Cytometry.

[25]  B. Aronow,et al.  Functional evolution of the p53 regulatory network through its target response elements , 2008, Proceedings of the National Academy of Sciences.

[26]  T. Consler,et al.  Effect of microsphere binding site density on the apparent affinity of an interaction partner , 2006, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[27]  J. Jordan,et al.  Changing the p53 master regulatory network: ELEMENTary, my dear Mr Watson , 2007, Oncogene.

[28]  A. Levine,et al.  MDM2 SNP309 accelerates tumor formation in a gender-specific and hormone-dependent manner. , 2006, Cancer research.

[29]  A. Philippakis,et al.  Compact, universal DNA microarrays to comprehensively determine transcription-factor binding site specificities , 2006, Nature Biotechnology.

[30]  Peter J. F. Snijders,et al.  Bead-Based Multiplex Genotyping of Human Papillomaviruses , 2006, Journal of Clinical Microbiology.

[31]  A. Inga,et al.  Functional mutants of the sequence-specific transcription factor p53 and implications for master genes of diversity , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[32]  J. Espinosa.  Mechanisms of regulatory diversity within the p53 transcriptional network , 2008, Oncogene.

[33]  E. Appella,et al.  Phosphorylation of murine p53 at ser-18 regulates the p53 responses to DNA damage. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[34]  H. Hill,et al.  Development of a multiplexed fluorescent immunoassay for the quantitation of antibody responses to group A streptococci. , 2006, Journal of immunological methods.

[35]  K. Kinzler,et al.  Definition of a consensus binding site for p53 , 1992, Nature Genetics.

[36]  R. Verdun,et al.  p53 functions through stress- and promoter-specific recruitment of transcription initiation components before and after DNA damage. , 2003, Molecular cell.

[37]  Brian N Chorley,et al.  Identification of polymorphic antioxidant response elements in the human genome. , 2007, Human molecular genetics.

[38]  Z. Weng,et al.  A Global Map of p53 Transcription-Factor Binding Sites in the Human Genome , 2006, Cell.

[39]  M. Passos-Bueno,et al.  A functional SNP in the promoter region of TCOF1 is associated with reduced gene expression and YY1 DNA-protein interaction. , 2005, Gene.

[40]  E. Appella,et al.  Acetylation of Mouse p53 at Lysine 317 Negatively Regulates p53 Apoptotic Activities after DNA Damage , 2006, Molecular and Cellular Biology.

[41]  M. Weiner,et al.  Microsphere-based single nucleotide polymorphism genotyping. , 2003, Methods in molecular biology.

[42]  P. Tegtmeyer,et al.  Interaction of p53 with its consensus DNA-binding site , 1995, Molecular and cellular biology.

[43]  Eduardo Sontag,et al.  Transcriptional control of human p53-regulated genes , 2008, Nature Reviews Molecular Cell Biology.

[44]  R. Iggo,et al.  Chromatin immunoprecipitation analysis fails to support the latency model for regulation of p53 DNA binding activity in vivo , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[45]  Michael D. Wilson,et al.  Species-Specific Transcription in Mice Carrying Human Chromosome 21 , 2008, Science.

[46]  Michelle R. Campbell,et al.  Functionally distinct polymorphic sequences in the human genome that are targets for p53 transactivation. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[47]  E. Nieschlag,et al.  Single-nucleotide polymorphisms in the promoter region influence the expression of the human follicle-stimulating hormone receptor. , 2005, Fertility and sterility.

[48]  D. Bell,et al.  Noncanonical DNA Motifs as Transactivation Targets by Wild Type and Mutant p53 , 2008, PLoS genetics.

[49]  Gerhard Walzl,et al.  An Evaluation of Commercial Fluorescent Bead-Based Luminex Cytokine Assays , 2008, PloS one.

[50]  D. Menendez,et al.  A SNP in the flt-1 promoter integrates the VEGF system into the p53 transcriptional network , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[51]  J. Espinosa,et al.  Transcriptional regulation by p53 through intrinsic DNA/chromatin binding and site-directed cofactor recruitment. , 2001, Molecular cell.

[52]  A. Clark,et al.  Evolution of transcription factor binding sites in Mammalian gene regulatory regions: conservation and turnover. , 2002, Molecular biology and evolution.