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author:("Wang, luting")
1.  Interactions of Chromatin Context, Binding Site Sequence Content, and Sequence Evolution in Stress-Induced p53 Occupancy and Transactivation 
PLoS Genetics  2015;11(1):e1004885.
Cellular stresses activate the tumor suppressor p53 protein leading to selective binding to DNA response elements (REs) and gene transactivation from a large pool of potential p53 REs (p53REs). To elucidate how p53RE sequences and local chromatin context interact to affect p53 binding and gene transactivation, we mapped genome-wide binding localizations of p53 and H3K4me3 in untreated and doxorubicin (DXR)-treated human lymphoblastoid cells. We examined the relationships among p53 occupancy, gene expression, H3K4me3, chromatin accessibility (DNase 1 hypersensitivity, DHS), ENCODE chromatin states, p53RE sequence, and evolutionary conservation. We observed that the inducible expression of p53-regulated genes was associated with the steady-state chromatin status of the cell. Most highly inducible p53-regulated genes were suppressed at baseline and marked by repressive histone modifications or displayed CTCF binding. Comparison of p53RE sequences residing in different chromatin contexts demonstrated that weaker p53REs resided in open promoters, while stronger p53REs were located within enhancers and repressed chromatin. p53 occupancy was strongly correlated with similarity of the target DNA sequences to the p53RE consensus, but surprisingly, inversely correlated with pre-existing nucleosome accessibility (DHS) and evolutionary conservation at the p53RE. Occupancy by p53 of REs that overlapped transposable element (TE) repeats was significantly higher (p<10−7) and correlated with stronger p53RE sequences (p<10−110) relative to nonTE-associated p53REs, particularly for MLT1H, LTR10B, and Mer61 TEs. However, binding at these elements was generally not associated with transactivation of adjacent genes. Occupied p53REs located in L2-like TEs were unique in displaying highly negative PhyloP scores (predicted fast-evolving) and being associated with altered H3K4me3 and DHS levels. These results underscore the systematic interaction between chromatin status and p53RE context in the induced transactivation response. This p53 regulated response appears to have been tuned via evolutionary processes that may have led to repression and/or utilization of p53REs originating from primate-specific transposon elements.
Author Summary
It is well established that p53 binds DNA elements near p53 target genes to regulate the response to cellular stress. To assess factors influencing binding to response elements and subsequent gene expression, we have analyzed 2932 p53-occupied response elements (p53REs) in the context of genome-wide chromatin state, DNA accessibility and dynamics, and considered roles for binding-sequence specificity and evolutionary conservation. While p53 occupancy level shows little apparent direct relationship to gene expression change, after grouping expressed genes by their chromatin status at baseline, a relationship between occupancy of p53REs and gene expression change emerged. Analysis of p53RE sequences demonstrated that p53 occupancy was strongly correlated with sequence similarity to p53RE consensus, but surprisingly, was inversely correlated with nucleosome accessibility (DHS) and evolutionary conservation. These data revealed a systematic interaction between p53RE content and chromatin context that affects both quantitative p53 occupancy and the induced transactivation response to exposure. Moreover, this interaction appears to have been tuned via evolutionary events involving transposable elements, which strongly bind p53, but in only a few instances affect gene expression levels. Models of p53-regulated gene expression response that consider both chromatin state and sequence context may prove useful in guiding strategies for cancer prevention or therapy.
doi:10.1371/journal.pgen.1004885
PMCID: PMC4287438  PMID: 25569532
2.  A Polymorphic p53 Response Element in KIT Ligand Influences Cancer Risk and Has Undergone Natural Selection 
Cell  2013;155(2):410-422.
SUMMARY
The ability of p53 to regulate transcription is crucial for tumor suppression and implies that inherited polymorphisms in functional p53-binding sites could influence cancer. Here, we identify a polymorphic p53 responsive element and demonstrate its influence on cancer risk using genome-wide data sets of cancer susceptibility loci, genetic variation, p53 occupancy, and p53-binding sites. We uncover a single-nucleotide polymorphism (SNP) in a functional p53-binding site and establish its influence on the ability of p53 to bind to and regulate transcription of the KITLG gene. The SNP resides in KITLG and associates with one of the largest risks identified among cancer genome-wide association studies. We establish that the SNP has undergone positive selection throughout evolution, signifying a selective benefit, but go on to show that similar SNPs are rare in the genome due to negative selection, indicating that polymorphisms in p53-binding sites are primarily detrimental to humans.
doi:10.1016/j.cell.2013.09.017
PMCID: PMC4171736  PMID: 24120139
3.  Targeted Deletion of Nrf2 Impairs Lung Development and Oxidant Injury in Neonatal Mice 
Antioxidants & Redox Signaling  2012;17(8):1066-1082.
Abstract
Aims: Nrf2 is an essential transcription factor for protection against oxidant disorders. However, its role in organ development and neonatal disease has received little attention. Therapeutically administered oxygen has been considered to contribute to bronchopulmonary dysplasia (BPD) in prematurity. The current study was performed to determine Nrf2-mediated molecular events during saccular-to-alveolar lung maturation, and the role of Nrf2 in the pathogenesis of hyperoxic lung injury using newborn Nrf2-deficient (Nrf2−/−) and wild-type (Nrf2+/+) mice. Results: Pulmonary basal expression of cell cycle, redox balance, and lipid/carbohydrate metabolism genes was lower while lymphocyte immunity genes were more highly expressed in Nrf2−/− neonates than in Nrf2+/+ neonates. Hyperoxia-induced phenotypes, including mortality, arrest of saccular-to-alveolar transition, and lung edema, and inflammation accompanying DNA damage and tissue oxidation were significantly more severe in Nrf2−/− neonates than in Nrf2+/+ neonates. During lung injury pathogenesis, Nrf2 orchestrated expression of lung genes involved in organ injury and morphology, cellular growth/proliferation, vasculature development, immune response, and cell–cell interaction. Bioinformatic identification of Nrf2 binding motifs and augmented hyperoxia-induced inflammation in genetically deficient neonates supported Gpx2 and Marco as Nrf2 effectors. Innovation: This investigation used lung transcriptomics and gene targeted mice to identify novel molecular events during saccular-to-alveolar stage transition and to elucidate Nrf2 downstream mechanisms in protection from hyperoxia-induced injury in neonate mouse lungs. Conclusion: Nrf2 deficiency augmented lung injury and arrest of alveolarization caused by hyperoxia during the newborn period. Results suggest a therapeutic potential of specific Nrf2 activators for oxidative stress-associated neonatal disorders including BPD. Antioxid. Redox Signal. 00, 000–000.
doi:10.1089/ars.2011.4288
PMCID: PMC3423869  PMID: 22400915
4.  Correction: CSF1 Is a Novel p53 Target Gene Whose Protein Product Functions in a Feed-Forward Manner to Suppress Apoptosis and Enhance p53-Mediated Growth Arrest 
PLoS ONE  2013;8(9):10.1371/annotation/8deafa94-628f-4b03-9dbc-2ec78f5bf737.
doi:10.1371/annotation/8deafa94-628f-4b03-9dbc-2ec78f5bf737
PMCID: PMC3779272
5.  CSF1 Is a Novel p53 Target Gene Whose Protein Product Functions in a Feed-Forward Manner to Suppress Apoptosis and Enhance p53-Mediated Growth Arrest 
PLoS ONE  2013;8(9):e74297.
The p53 tumor suppressor gene has a common polymorphism at codon 72 that alters its function. We previously reported that the proline 72 polymorphic variant of p53 (P72) demonstrates increased ability to transactivate a subset of genes, relative to arginine 72 (R72); one of these genes is macrophage colony stimulating factor (CSF1). At present, the mechanism(s) underlying the increased transcriptional activity of P72 toward genes like CSF1 have not been completely elucidated. Additionally, the consequences of increased transcription of genes like CSF1 by the P72 variant to the downstream p53 pathway are unknown. In this report, we address these issues. We show that the CSF1 gene contains a conserved binding site for p53, and interestingly that the P72 variant shows increased ability to bind to this site. Moreover, we show that increased CSF1/CSF1R signaling in P72 cells feeds back on the p53 pathway to enhance p53 phosphorylation, levels, and transactivation of target genes, particularly the cyclin-dependent kinase inhibitor p21 (CDKN1A). This leads to an increase in p53-mediated growth arrest, along with a concomitant decrease in apoptosis. Notably, the CSF1/CSF1R signaling axis is overexpressed in several epithelial cancers, and there is clinical evidence that this pathway plays a role in radio-resistance of some cancers. We show that cells expressing CSF1 and CSF1R are indeed radio-resistant, and further, that this effect requires p53. These combined data are the first to implicate the CSF1/CSF1R pathway in the decision between p53-mediated growth arrest and apoptosis. They are also the first to highlight a cytokine as influential in cell fate determined by p53 in epithelial cells. Finally, these data may explain the association of the P72 variant and the CSF1/CSF1R pathway with increased senescence and radio-resistance in some epithelial tumor types.
doi:10.1371/journal.pone.0074297
PMCID: PMC3760869  PMID: 24019961
6.  Novel Hematopoietic Target Genes in the NRF2-Mediated Transcriptional Pathway 
Nuclear factor- (erythroid-derived 2) like 2 (NFE2L2, NRF2) is a key transcriptional activator of the antioxidant response pathway and is closely related to erythroid transcription factor NFE2. Under oxidative stress, NRF2 heterodimerizes with small Maf proteins and binds cis-acting enhancer sequences found near oxidative stress response genes. Using the dietary isothiocyanate sulforaphane (SFN) to activate NRF2, chromatin immunoprecipitation sequencing (ChIP-seq) identified several hundred novel NRF2-mediated targets beyond its role in oxidative stress. Activated NRF2 bound the antioxidant response element (ARE) in promoters of several known and novel target genes involved in iron homeostasis and heme metabolism, including known targets FTL and FTH1, as well as novel binding in the globin locus control region. Five novel NRF2 target genes were chosen for followup: AMBP, ABCB6, FECH, HRG-1 (SLC48A1), and TBXAS1. SFN-induced gene expression in erythroid K562 and lymphoid cells were compared for each target gene. NRF2 silencing showed reduced expression in lymphoid, lung, and hepatic cells. Furthermore, stable knockdown of NRF2 negative regulator KEAP1 in K562 cells resulted in increased NQO1, AMBP, and TBXAS1 expression. NFE2 binding sites in K562 cells revealed similar binding profiles as lymphoid NRF2 sites in all potential NRF2 candidates supporting a role for NRF2 in heme metabolism and erythropoiesis.
doi:10.1155/2013/120305
PMCID: PMC3677633  PMID: 23766848
7.  450K Epigenome-Wide Scan Identifies Differential DNA Methylation in Newborns Related to Maternal Smoking during Pregnancy 
Environmental Health Perspectives  2012;120(10):1425-1431.
Background: Epigenetic modifications, such as DNA methylation, due to in utero exposures may play a critical role in early programming for childhood and adult illness. Maternal smoking is a major risk factor for multiple adverse health outcomes in children, but the underlying mechanisms are unclear.
Objective: We investigated epigenome-wide methylation in cord blood of newborns in relation to maternal smoking during pregnancy.
Methods: We examined maternal plasma cotinine (an objective biomarker of smoking) measured during pregnancy in relation to DNA methylation at 473,844 CpG sites (CpGs) in 1,062 newborn cord blood samples from the Norwegian Mother and Child Cohort Study (MoBa) using the Infinium HumanMethylation450 BeadChip (450K).
Results: We found differential DNA methylation at epigenome-wide statistical significance (p-value < 1.06 × 10–7) for 26 CpGs mapped to 10 genes. We replicated findings for CpGs in AHRR, CYP1A1, and GFI1 at strict Bonferroni-corrected statistical significance in a U.S. birth cohort. AHRR and CYP1A1 play a key role in the aryl hydrocarbon receptor signaling pathway, which mediates the detoxification of the components of tobacco smoke. GFI1 is involved in diverse developmental processes but has not previously been implicated in responses to tobacco smoke.
Conclusions: We identified a set of genes with methylation changes present at birth in children whose mothers smoked during pregnancy. This is the first study of differential methylation across the genome in relation to maternal smoking during pregnancy using the 450K platform. Our findings implicate epigenetic mechanisms in the pathogenesis of the adverse health outcomes associated with this important in utero exposure.
doi:10.1289/ehp.1205412
PMCID: PMC3491949  PMID: 22851337
epigenetics; epigenome-wide; in utero; maternal smoking; methylation
8.  Identification of novel NRF2-regulated genes by ChIP-Seq: influence on retinoid X receptor alpha 
Nucleic Acids Research  2012;40(15):7416-7429.
Cellular oxidative and electrophilic stress triggers a protective response in mammals regulated by NRF2 (nuclear factor (erythroid-derived) 2-like; NFE2L2) binding to deoxyribonucleic acid-regulatory sequences near stress-responsive genes. Studies using Nrf2-deficient mice suggest that hundreds of genes may be regulated by NRF2. To identify human NRF2-regulated genes, we conducted chromatin immunoprecipitation (ChIP)-sequencing experiments in lymphoid cells treated with the dietary isothiocyanate, sulforaphane (SFN) and carried out follow-up biological experiments on candidates. We found 242 high confidence, NRF2-bound genomic regions and 96% of these regions contained NRF2-regulatory sequence motifs. The majority of binding sites were near potential novel members of the NRF2 pathway. Validation of selected candidate genes using parallel ChIP techniques and in NRF2-silenced cell lines indicated that the expression of about two-thirds of the candidates are likely to be directly NRF2-dependent including retinoid X receptor alpha (RXRA). NRF2 regulation of RXRA has implications for response to retinoid treatments and adipogenesis. In mouse, 3T3-L1 cells’ SFN treatment affected Rxra expression early in adipogenesis, and knockdown of Nrf2-delayed Rxra expression, both leading to impaired adipogenesis.
doi:10.1093/nar/gks409
PMCID: PMC3424561  PMID: 22581777
9.  Nrf2-regulated PPARγ Expression Is Critical to Protection against Acute Lung Injury in Mice 
Rationale: The NF-E2 related factor 2 (Nrf2)–antioxidant response element (ARE) pathway is essential for protection against oxidative injury and inflammation including hyperoxia-induced acute lung injury. Microarray expression profiling revealed that lung peroxisome proliferator activated receptor γ (PPARγ) induction is suppressed in hyperoxia-susceptible Nrf2-deficient (Nrf2−/−) mice compared with wild-type (Nrf2+/+) mice. PPARγ has pleiotropic beneficial effects including antiinflammation in multiple tissues.
Objectives: We tested the hypothesis that PPARγ is an important determinant of pulmonary responsivity to hyperoxia regulated by Nrf2.
Methods: A computational bioinformatic method was applied to screen potential AREs in the Pparg promoter for Nrf2 binding. The functional role of a potential ARE was investigated by in vitro promoter analysis. A role for PPARγ in hyperoxia-induced acute lung injury was determined by temporal silencing of PPARγ via intranasal delivery of PPARγ-specific interference RNA and by administration of a PPARγ ligand 15-deoxy-Δ12,14-prostaglandin J2 in mice.
Measurements and Main Results: Deletion or site-directed mutagenesis of a potential ARE spanning -784/-764 sequence significantly attenuated hyperoxia-increased Pparg promoter activity in airway epithelial cells overexpressing Nrf2, indicating that the -784/-764 ARE is critical for Nrf2-regulated PPARγ expression. Mice with decreased lung PPARγ by specific interference RNA treatment had significantly augmented hyperoxia-induced pulmonary inflammation and injury. 15 Deoxy-Δ12,14-prostaglandin J2 administration significantly reduced hyperoxia-induced lung inflammation and edema in Nrf2+/+, but not in Nrf2−/− mice.
Conclusions: Results indicate for the first time that Nrf2-driven PPARγ induction has an essential protective role in pulmonary oxidant injury. Our observations provide new insights into the therapeutic potential of PPARγ in airway oxidative inflammatory disorders.
doi:10.1164/rccm.200907-1047OC
PMCID: PMC2913232  PMID: 20224069
antioxidant response element; hyperoxia; inflammation; siRNA; 15d-PGJ2
10.  Human single-nucleotide polymorphisms alter p53 sequence-specific binding at gene regulatory elements 
Nucleic Acids Research  2010;39(1):178-189.
p53 coordinates the expression of an intricate network of genes in response to stress signals. Sequence-specific DNA binding is essential for p53-mediated tumor suppression. We evaluated the impact of single-nucleotide polymorphisms (SNPs) in p53 response elements (p53RE) on DNA binding and gene expression in response to DNA damage. Using a bioinformatics approach based on incorporating p53 binding strength into a position weight matrix, we selected 32 SNPs in putative and validated p53REs. The microsphere assay for protein–DNA binding (MAPD) and allele-specific expression analysis was employed to assess the impact of SNPs on p53-DNA binding and gene expression, respectively. Comparing activated p53 binding in nuclear extracts from doxorubicin- or ionizing radiation (IR)-treated human cells, we observed little difference in binding profiles. Significant p53 binding was observed for most polymorphic REs and several displayed binding comparable to the p21 RE. SNP alleles predicted to lower p53 binding indeed reduced binding in 25 of the 32 sequences. Chromatin immunoprecipitation-sequencing in lymphoblastoid cells confirmed p53 binding to seven polymorphic p53 REs in response to doxorubicin. In addition, five polymorphisms were associated with altered gene expression following doxorubicin treatment. Our findings demonstrate an effective strategy to identify and evaluate SNPs that may alter p53-mediated stress responses.
doi:10.1093/nar/gkq764
PMCID: PMC3017610  PMID: 20817676
11.  Genetic Variation and Antioxidant Response Gene Expression in the Bronchial Airway Epithelium of Smokers at Risk for Lung Cancer 
PLoS ONE  2010;5(8):e11934.
Prior microarray studies of smokers at high risk for lung cancer have demonstrated that heterogeneity in bronchial airway epithelial cell gene expression response to smoking can serve as an early diagnostic biomarker for lung cancer. As a first step in applying functional genomic analysis to population studies, we have examined the relationship between gene expression variation and genetic variation in a central molecular pathway (NRF2-mediated antioxidant response) associated with smoking exposure and lung cancer. We assessed global gene expression in histologically normal airway epithelial cells obtained at bronchoscopy from smokers who developed lung cancer (SC, n = 20), smokers without lung cancer (SNC, n = 24), and never smokers (NS, n = 8). Functional enrichment analysis showed that the NRF2-mediated, antioxidant response element (ARE)-regulated genes, were significantly lower in SC, when compared with expression levels in SNC. Importantly, we found that the expression of MAFG (a binding partner of NRF2) was correlated with the expression of ARE genes, suggesting MAFG levels may limit target gene induction. Bioinformatically we identified single nucleotide polymorphisms (SNPs) in putative ARE genes and to test the impact of genetic variation, we genotyped these putative regulatory SNPs and other tag SNPs in selected NRF2 pathway genes. Sequencing MAFG locus, we identified 30 novel SNPs and two were associated with either gene expression or lung cancer status among smokers. This work demonstrates an analysis approach that integrates bioinformatics pathway and transcription factor binding site analysis with genotype, gene expression and disease status to identify SNPs that may be associated with individual differences in gene expression and/or cancer status in smokers. These polymorphisms might ultimately contribute to lung cancer risk via their effect on the airway gene expression response to tobacco-smoke exposure.
doi:10.1371/journal.pone.0011934
PMCID: PMC2914741  PMID: 20689807
12.  Identification of polymorphic antioxidant response elements (AREs) in the human genome 
Human molecular genetics  2007;16(10):1188-1200.
Single nucleotide polymorphisms (SNPs) in transcription factor binding sites (TFBSs) may affect the binding of transcription factors, lead to differences in gene expression and phenotypes, and therefore affect susceptibility to environmental exposure. We developed an integrated computational system for discovering functional SNPs in TFBSs in the human genome and predicting their impact on the expression of target genes. In this system we: (1) construct a position weight matrix (PWM) from a collection of experimentally discovered TFBSs; (2) predict TFBSs in SNP sequences using the PWM and map SNPs to the upstream regions of genes; (3) examine the evolutionary conservation of putative TFBSs by phylogenetic footprinting; (4) prioritize candidate SNPs based on microarray expression profiles from tissues in which the transcription factor of interest is either deleted or over-expressed; and (5) finally, analyze association of SNP genotypes with gene expression phenotypes. The application of our system has been tested to identify functional polymorphisms in the antioxidant response element (ARE), a cis-acting enhancer sequence found in the promoter region of many genes that encode antioxidant and Phase II detoxification enzymes/proteins. In response to oxidative stress, the transcription factor NRF2 (nuclear factor erythroid-derived 2-like 2) binds to AREs, mediating transcriptional activation of its responsive genes and modulating in vivo defense mechanisms against oxidative damage. Using our novel computational tools, we have identified a set of polymorphic AREs with functional evidence, showing the utility of our system to direct further experimental validation of genomic sequence variations that could be useful for identifying high-risk individuals.
doi:10.1093/hmg/ddm066
PMCID: PMC2805149  PMID: 17409198
13.  Discovery and verification of functional single nucleotide polymorphisms in regulatory genomic regions: Current and developing technologies 
Mutation research  2008;659(1-2):147-157.
The most common form of genetic variation, single nucleotide polymorphisms or SNPs, can affect the way an individual responds to the environment and modify disease risk. Although most of the millions of SNPs have little or no effect on gene regulation and protein activity, there are many circumstances where base changes can have deleterious effects. Non-synonymous SNPs that result in amino acid changes in proteins have been studied because of their obvious impact on protein activity. It is well known that SNPs within regulatory regions of the genome can result in disregulation of gene transcription. However, the impact of SNPs located in putative regulatory regions, or rSNPs, is harder to predict for two primary reasons. First, the mechanistic roles of non-coding genomic sequence remain poorly defined. Second, experimental validation of the functional consequences of rSNPs is often slow and laborious. In this review, we summarize traditional and novel methodologies for candidate rSNPs selection, in particular in silico techniques that aid in candidate rSNP selection. Additionally we will discuss molecular biological techniques that assess the impact of rSNPs on binding of regulatory machinery, as well as functional consequences on transcription. Standard techniques such as EMSA and luciferase reporter constructs are still widely used to assess effects of rSNPs on binding and gene transcription; however, these protocols are often bottlenecks in the discovery process. Therefore, we highlight novel and developing high-throughput protocols that promise to aid in shortening the process of rSNP validation. Given the large amount of genomic information generated from a multitude of re-sequencing and genome-wide SNP array efforts, future focus should be to develop validation techniques that will allow greater understanding of the impact these polymorphisms have on human health and disease.
doi:10.1016/j.mrrev.2008.05.001
PMCID: PMC2676583  PMID: 18565787
polymorphism; SNPs; gene regulation; functional genomics; microsphere assay
14.  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 Genetics  2009;5(5):e1000462.
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.
Author Summary
Characterizing the functional roles of gene regulatory sequences and the impact of genetic polymorphism on protein-DNA binding are rapidly growing areas of genomics. The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements, and we have developed a multiplex format microsphere assay (MAPD) to probe how sequence variation alters p53 binding. Testing pools of multiplexed oligonucleotides that contain numerous p53 binding targets or different sequence variants, we demonstrate that subtle changes in target sequence can impact p53 binding, and these effects are not captured by commonly used computational binding models. A group of single nucleotide polymorphisms was examined and binding profiles closely matched gene expression measured in a parallel system. We also observed distinct binding patterns that were characteristic of transiently expressed wild type and mutant p53s, suggesting that this method may be useful for probing the impact of protein structural variation on DNA binding. MAPD utilizes biologically meaningful cell extracts in a multiplexed, quantitative 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.
doi:10.1371/journal.pgen.1000462
PMCID: PMC2667269  PMID: 19424414
15.  Divergent Evolution of Human p53 Binding Sites: Cell Cycle Versus Apoptosis  
PLoS Genetics  2007;3(7):e127.
The p53 tumor suppressor is a sequence-specific pleiotropic transcription factor that coordinates cellular responses to DNA damage and stress, initiating cell-cycle arrest or triggering apoptosis. Although the human p53 binding site sequence (or response element [RE]) is well characterized, some genes have consensus-poor REs that are nevertheless both necessary and sufficient for transactivation by p53. Identification of new functional gene regulatory elements under these conditions is problematic, and evolutionary conservation is often employed. We evaluated the comparative genomics approach for assessing evolutionary conservation of putative binding sites by examining conservation of 83 experimentally validated human p53 REs against mouse, rat, rabbit, and dog genomes and detected pronounced conservation differences among p53 REs and p53-regulated pathways. Bona fide NRF2 (nuclear factor [erythroid-derived 2]-like 2 nuclear factor) and NFκB (nuclear factor of kappa light chain gene enhancer in B cells) binding sites, which direct oxidative stress and innate immunity responses, were used as controls, and both exhibited high interspecific conservation. Surprisingly, the average p53 RE was not significantly more conserved than background genomic sequence, and p53 REs in apoptosis genes as a group showed very little conservation. The common bioinformatics practice of filtering RE predictions by 80% rodent sequence identity would not only give a false positive rate of ∼19%, but miss up to 57% of true p53 REs. Examination of interspecific DNA base substitutions as a function of position in the p53 consensus sequence reveals an unexpected excess of diversity in apoptosis-regulating REs versus cell-cycle controlling REs (rodent comparisons: p < 1.0 e−12). While some p53 REs show relatively high levels of conservation, REs in many genes such as BAX, FAS, PCNA, CASP6, SIVA1, and P53AIP1 show little if any homology to rodent sequences. This difference suggests that among mammalian species, evolutionary conservation differs among p53 REs, with some having ancient ancestry and others of more recent origin. Overall our results reveal divergent evolutionary pressure among the binding targets of p53 and emphasize that comparative genomics methods must be used judiciously and tailored to the evolutionary history of the targeted functional regulatory regions.
Author Summary
The p53 tumor suppressor is a transcription factor that coordinates cellular responses to DNA damage and stress, initiating cell-cycle arrest or triggering apoptosis. Evolutionary conservation is often employed to separate the functional “wheat” from the nonfunctional “chaff” when identifying binding sites of transcription factors like p53. We evaluated evolutionary conservation of 83 experimentally validated human p53 binding sites against mouse, rat, rabbit, and dog genomes, and similarly examined binding sites for two other transcription factors as controls, NRF2 (nuclear factor [erythroid-derived 2]-like 2 nuclear factor) and NFκB (nuclear factor of kappa light chain gene enhancer in B cells), which direct oxidative stress and innate immunity responses, respectively. NRF2 and NFκB binding sites both exhibited high interspecific conservation, indicative of purifying natural selection, but surprisingly, human p53 response elements on average displayed a lack of conservation. Thus conservation is not useful in the prediction of p53 binding sites. After grouping p53 REs by gene ontology, we observed that binding sites in cell-cycle genes like CDKN1A displayed high conservation, while p53 binding sites in apoptosis and DNA repair genes showed an unexpected excess of diversity and very little homology with rodent sequences. Overall these results reveal divergent evolutionary pressure among the binding targets of p53 and suggest caution in generalizing about the similarity of regulation of the p53 pathway between humans and rodents.
doi:10.1371/journal.pgen.0030127
PMCID: PMC1934401  PMID: 17677004
16.  Surface Immobilization Antigen of the Parasitic Ciliate Ichthyophthirius multifiliis Elicits Protective Immunity in Channel Catfish (Ictalurus punctatus) 
Channel catfish (Ictalurus punctatus) that survive infection with the parasitic ciliate Ichthyophthirius multifiliis acquire immunity to subsequent challenge and produce specific antibodies in serum that immobilize the parasite in vitro. Cellular surface protein antigens targeted by these antibodies are referred to as immobilization antigens (i-antigens). By using an immobilizing mouse monoclonal antibody as a ligand, the i-antigen of I. multifiliis isolate G5 was purified to homogeneity by immunoaffinity chromatography, and its immunogenicity was confirmed by inoculating rabbit and channel catfish to produce immobilizing antisera. To test the purified i-antigen as a subunit vaccine, channel catfish fingerlings were injected intraperitoneally (i.p.) with purified i-antigen at a dose of 10 μg/fish in complete Freund’s adjuvant on day 1, followed by a second i.p. injection of the same amount of i-antigen in incomplete Freund’s adjuvant on day 15. Negative control fish were immunized similarly with either bovine serum albumin (BSA) or an immobilization-irrelevant I. multifiliis protein. On day 84, the fish were challenged with live I. multifiliis G5 theronts at a dose of 15,000 cells per fish. Seventy-two percent of the fish immunized with i-antigen survived the challenge. All negative control fish died within 16 days of exposure. There was a significant difference in the median days to death between the negative control fish injected with BSA and the fish that died following vaccination with i-antigen. Fish injected with i-antigen developed high immobilizing antibody titers in serum. This is the first demonstration of a direct role for i-antigens in the elicitation of protective immunity, suggesting that these proteins by themselves serve as effective subunit vaccines against I. multifiliis.
doi:10.1128/CDLI.9.1.176-181.2002
PMCID: PMC119907  PMID: 11777850

Results 1-16 (16)