A polymorphic p53 response element in KIT ligand influences cancer risk and has undergone natural selection.

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.

Epigenome-wide association study of lung cancer among never smokers in two prospective cohorts in Shanghai, China.

BACKGROUND: The aetiology of lung cancer among individuals who never smoked remains elusive, despite 15% of lung cancer cases in men and 53% in women worldwide being unrelated to smoking. Epigenetic alterations, particularly DNA methylation (DNAm) changes, have emerged as potential drivers. Yet, few prospective epigenome-wide association studies (EWAS), primarily focusing on peripheral blood DNAm with limited representation of never smokers, have been conducted. METHODS: We conducted a nested case-control study of 80 never-smoking incident lung cancer cases and 83 never-smoking controls within the Shanghai Women's Health Study and Shanghai Men's Health Study. DNAm was measured in prediagnostic oral rinse samples using Illumina MethylationEPIC array. Initially, we conducted an EWAS to identify differentially methylated positions (DMPs) associated with lung cancer in the discovery sample of 101 subjects. The top 50 DMPs were further evaluated in a replication sample of 62 subjects, and results were pooled using fixed-effect meta-analysis. RESULTS: Our study identified three DMPs significantly associated with lung cancer at the epigenome-wide significance level of p<8.22×10-8. These DMPs were identified as cg09198866 (MYH9; TXN2), cg01411366 (SLC9A10) and cg12787323. Furthermore, examination of the top 1000 DMPs indicated significant enrichment in epithelial regulatory regions and their involvement in small GTPase-mediated signal transduction pathways. Additionally, GrimAge acceleration was identified as a risk factor for lung cancer (OR=1.19 per year; 95% CI 1.06 to 1.34). CONCLUSIONS: While replication in a larger sample size is necessary, our findings suggest that DNAm patterns in prediagnostic oral rinse samples could provide novel insights into the underlying mechanisms of lung cancer in never smokers.

An African-specific polymorphism in the TP53 gene impairs p53 tumor suppressor function in a mouse model.

A nonsynonymous single-nucleotide polymorphism at codon 47 in TP53 exists in African-descent populations (P47S, rs1800371; referred to here as S47). Here we report that, in human cell lines and a mouse model, the S47 variant exhibits a modest decrease in apoptosis in response to most genotoxic stresses compared with wild-type p53 but exhibits a significant defect in cell death induced by cisplatin. We show that, compared with wild-type p53, S47 has nearly indistinguishable transcriptional function but shows impaired ability to transactivate a subset of p53 target genes, including two involved in metabolism:Gls2(glutaminase 2) and Sco2 We also show that human and mouse cells expressing the S47 variant are markedly resistant to cell death by agents that induce ferroptosis (iron-mediated nonapoptotic cell death). We show that mice expressing S47 in homozygous or heterozygous form are susceptible to spontaneous cancers of diverse histological types. Our data suggest that the S47 variant may contribute to increased cancer risk in individuals of African descent, and our findings highlight the need to assess the contribution of this variant to cancer risk in these populations. These data also confirm the potential relevance of metabolism and ferroptosis to tumor suppression by p53.

Age-specific survival rates, causes of death, and allowable take of golden eagles in the western United States.

In the United States, the Bald and Golden Eagle Protection Act prohibits take of golden eagles (Aquila chrysaetos) unless authorized by permit, and stipulates that all permitted take must be sustainable. Golden eagles are unintentionally killed in conjunction with many lawful activities (e.g., electrocution on power poles, collision with wind turbines). Managers who issue permits for incidental take of golden eagles must determine allowable take levels and manage permitted take accordingly. To aid managers in making these decisions in the western United States, we used an integrated population model to obtain estimates of golden eagle vital rates and population size, and then used those estimates in a prescribed take level (PTL) model to estimate the allowable take level. Estimated mean annual survival rates for golden eagles ranged from 0.70 (95% credible interval = 0.66-0.74) for first-year birds to 0.90 (0.88-0.91) for adults. Models suggested a high proportion of adult female golden eagles attempted to breed and breeding pairs fledged a mean of 0.53 (0.39-0.72) young annually. Population size in the coterminous western United States has averaged ~31,800 individuals for several decades, with λ = 1.0 (0.96-1.05). The PTL model estimated a median allowable take limit of ~2227 (708-4182) individuals annually given a management objective of maintaining a stable population. We estimate that take averaged 2572 out of 4373 (59%) deaths annually, based on a representative sample of transmitter-tagged golden eagles. For the subset of golden eagles that were recovered and a cause of death determined, anthropogenic mortality accounted for an average of 74% of deaths after their first year; leading forms of take over all age classes were shooting (~670 per year), collisions (~611), electrocutions (~506), and poisoning (~427). Although observed take overlapped the credible interval of our allowable take estimate and the population overall has been stable, our findings indicate that additional take, unless mitigated for, may not be sustainable. Our analysis demonstrates the utility of the joint application of integrated population and prescribed take level models to management of incidental take of a protected species.

Does taxonomic and numerical resolution affect the assessment of invertebrate community structure in New World freshwater wetlands?

The efficiency of biodiversity assessments and biomonitoring studies is commonly challenged by limitations in taxonomic identification and quantification approaches. In this study, we assessed the effects of different taxonomic and numerical resolutions on a range of community structure metrics in invertebrate compositional data sets from six regions distributed across North and South America. We specifically assessed the degree of similarity in the metrics (richness, equitability, beta diversity, heterogeneity in community composition and congruence) for data sets identified to a coarse resolution (usually family level) and the finest taxonomic resolution practical (usually genus level, sometimes species or morphospecies) and by presence-absence and relative abundance numerical resolutions. Spearman correlations showed highly significant and positive associations between univariate metrics (richness and equitability) calculated for coarse- and finest-resolution datasets. Procrustes analysis detected significant congruence between composition datasets. Higher correlation coefficients were found for datasets with the same numerical resolutions regardless of the taxonomic level (about 90%), while the correlations for comparisons across numerical resolutions were consistently lower. Our findings indicate that family-level resolution can be used as a surrogate of finer taxonomic resolutions to calculate a range of biodiversity metrics commonly used to describe invertebrate community structure patterns in New World freshwater wetlands without significant loss of information. However, conclusions on biodiversity patterns derived from datasets with different numerical resolutions should be critically considered in studies on wetland invertebrates.

Epigenome-wide meta-analysis of DNA methylation and childhood asthma.

BACKGROUND: Epigenetic mechanisms, including methylation, can contribute to childhood asthma. Identifying DNA methylation profiles in asthmatic patients can inform disease pathogenesis. OBJECTIVE: We sought to identify differential DNA methylation in newborns and children related to childhood asthma. METHODS: Within the Pregnancy And Childhood Epigenetics consortium, we performed epigenome-wide meta-analyses of school-age asthma in relation to CpG methylation (Illumina450K) in blood measured either in newborns, in prospective analyses, or cross-sectionally in school-aged children. We also identified differentially methylated regions. RESULTS: In newborns (8 cohorts, 668 cases), 9 CpGs (and 35 regions) were differentially methylated (epigenome-wide significance, false discovery rate < 0.05) in relation to asthma development. In a cross-sectional meta-analysis of asthma and methylation in children (9 cohorts, 631 cases), we identified 179 CpGs (false discovery rate < 0.05) and 36 differentially methylated regions. In replication studies of methylation in other tissues, most of the 179 CpGs discovered in blood replicated, despite smaller sample sizes, in studies of nasal respiratory epithelium or eosinophils. Pathway analyses highlighted enrichment for asthma-relevant immune processes and overlap in pathways enriched both in newborns and children. Gene expression correlated with methylation at most loci. Functional annotation supports a regulatory effect on gene expression at many asthma-associated CpGs. Several implicated genes are targets for approved or experimental drugs, including IL5RA and KCNH2. CONCLUSION: Novel loci differentially methylated in newborns represent potential biomarkers of risk of asthma by school age. Cross-sectional associations in children can reflect both risk for and effects of disease. Asthma-related differential methylation in blood in children was substantially replicated in eosinophils and respiratory epithelium.

Crohn's disease IRGM risk alleles are associated with altered gene expression in human tissues.

Crohn's disease (CD) is a chronic inflammatory gastrointestinal disorder. Genetic association studies have implicated dysregulated autophagy in CD. Among risk loci identified are a promoter single nucleotide polymorphism (SNP)( rs13361189 ) and two intragenic SNPs ( rs9637876 , rs10065172 ) in immunity-related GTPase family M ( IRGM) a gene that encodes a protein of the autophagy initiation complex. All three SNPs have been proposed to modify IRGM expression, but reports have been divergent and largely derived from cell lines. Here, analyzing RNA-Sequencing data of human tissues from the Genotype-Tissue Expression Project, we found that rs13361189 minor allele carriers had reduced IRGM expression in whole blood and terminal ileum, and upregulation in ileum of ZNF300P1, a locus adjacent to IRGM on chromosome 5q33.1 that encodes a long noncoding RNA. Whole blood and ileum from minor allele carriers had altered expression of multiple additional genes that have previously been linked to colitis and/or autophagy. Notable among these was an increase in ileum of LTF (lactoferrin), an established fecal inflammatory biomarker of CD, and in whole blood of TNF, a key cytokine in CD pathogenesis. Last, we confirmed that risk alleles at all three loci associated with increased risk for CD but not ulcerative colitis in a case-control study. Taken together, our findings suggest that genetically encoded IRGM deficiency may predispose to CD through dysregulation of inflammatory gene networks. Gene expression profiling of disease target tissues in genetically susceptible populations is a promising strategy for revealing new leads for the study of molecular pathogenesis and, potentially, for precision medicine. NEW & NOTEWORTHY Single nucleotide polymorphisms in immunity-related GTPase family M ( IRGM), a gene that encodes an autophagy initiation protein, have been linked epidemiologically to increased risk for Crohn's disease (CD). Here, we show for the first time that subjects with risk alleles at two such loci, rs13361189 and rs10065172 , have reduced IRGM expression in whole blood and terminal ileum, as well as dysregulated expression of a wide array of additional genes that regulate inflammation and autophagy.

Sulforaphane enriched transcriptome of lung mitochondrial energy metabolism and provided pulmonary injury protection via Nrf2 in mice.

Nrf2 is essential to antioxidant response element (ARE)-mediated host defense. Sulforaphane (SFN) is a phytochemical antioxidant known to affect multiple cellular targets including Nrf2-ARE pathway in chemoprevention. However, the role of SFN in non-malignant airway disorders remain unclear. To test if pre-activation of Nrf2-ARE signaling protects lungs from oxidant-induced acute injury, wild-type (Nrf2+/+) and Nrf2-deficient (Nrf2-/-) mice were given SFN orally or as standardized broccoli sprout extract diet (SBE) before hyperoxia or air exposure. Hyperoxia-induced pulmonary injury and oxidation indices were significantly reduced by SFN or SBE in Nrf2+/+ mice but not in Nrf2-/- mice. SFN upregulated a large cluster of basal lung genes that are involved in mitochondrial oxidative phosphorylation, energy metabolism, and cardiovascular protection only in Nrf2+/+ mice. Bioinformatic analysis elucidated ARE-like motifs on these genes. Transcript abundance of the mitochondrial machinery genes remained significantly higher after hyperoxia exposure in SFN-treated Nrf2+/+ mice than in SFN-treated Nrf2-/- mice. Nuclear factor-κB was suggested to be a central molecule in transcriptome networks affected by SFN. Minor improvement of hyperoxia-caused lung histopathology and neutrophilia by SFN in Nrf2-/- mice implies Nrf2-independent or alternate effector mechanisms. In conclusion, SFN is suggested to be as a preventive intervention in a preclinical model of acute lung injury by linking mitochondria and Nrf2. Administration of SFN alleviated acute lung injury-like pathogenesis in a Nrf2-dependent manner. Potential AREs in the SFN-inducible transcriptome for mitochondria bioenergetics provided a new insight into the downstream mechanisms of Nrf2-mediated pulmonary protection.

Elevated Heterozygosity in Adults Relative to Juveniles Provides Evidence of Viability Selection on Eagles and Falcons.

Viability selection yields adult populations that are more genetically variable than those of juveniles, producing a positive correlation between heterozygosity and survival. Viability selection could be the result of decreased heterozygosity across many loci in inbred individuals and a subsequent decrease in survivorship resulting from the expression of the deleterious alleles. Alternatively, locus-specific differences in genetic variability between adults and juveniles may be driven by forms of balancing selection, including heterozygote advantage, frequency-dependent selection, or selection across temporal and spatial scales. We use a pooled-sequencing approach to compare genome-wide and locus-specific genetic variability between 74 golden eagle (Aquila chrysaetos), 62 imperial eagle (Aquila heliaca), and 69 prairie falcon (Falco mexicanus) juveniles and adults. Although genome-wide genetic variability is comparable between juvenile and adult golden eagles and prairie falcons, imperial eagle adults are significantly more heterozygous than juveniles. This evidence of viability selection may stem from a relatively smaller imperial eagle effective population size and potentially greater genetic load. We additionally identify ~2000 single-nucleotide polymorphisms across the 3 species with extreme differences in heterozygosity between juveniles and adults. Many of these markers are associated with genes implicated in immune function or olfaction. These loci represent potential targets for studies of how heterozygote advantage, frequency-dependent selection, and selection over spatial and temporal scales influence survivorship in avian species. Overall, our genome-wide data extend previous studies that used allozyme or microsatellite markers and indicate that viability selection may be a more common evolutionary phenomenon than often appreciated.

New insights into the phylogenetics and population structure of the prairie falcon (Falco mexicanus).

BACKGROUND: Management requires a robust understanding of between- and within-species genetic variability, however such data are still lacking in many species. For example, although multiple population genetics studies of the peregrine falcon (Falco peregrinus) have been conducted, no similar studies have been done of the closely-related prairie falcon (F. mexicanus) and it is unclear how much genetic variation and population structure exists across the species' range. Furthermore, the phylogenetic relationship of F. mexicanus relative to other falcon species is contested. We utilized a genomics approach (i.e., genome sequencing and assembly followed by single nucleotide polymorphism genotyping) to rapidly address these gaps in knowledge. RESULTS: We sequenced the genome of a single female prairie falcon and generated a 1.17 Gb (gigabases) draft genome assembly. We generated maximum likelihood phylogenetic trees using complete mitochondrial genomes as well as nuclear protein-coding genes. This process provided evidence that F. mexicanus is an outgroup to the clade that includes the peregrine falcon and members of the subgenus Hierofalco. We annotated > 16,000 genes and almost 600,000 high-quality single nucleotide polymorphisms (SNPs) in the nuclear genome, providing the raw material for a SNP assay design featuring > 140 gene-associated markers and a molecular-sexing marker. We subsequently genotyped ~ 100 individuals from California (including the San Francisco East Bay Area, Pinnacles National Park and the Mojave Desert) and Idaho (Snake River Birds of Prey National Conservation Area). We tested for population structure and found evidence that individuals sampled in California and Idaho represent a single panmictic population. CONCLUSIONS: Our study illustrates how genomic resources can rapidly shed light on genetic variability in understudied species and resolve phylogenetic relationships. Furthermore, we found evidence of a single, randomly mating population of prairie falcons across our sampling locations. Prairie falcons are highly mobile and relatively rare long-distance dispersal events may promote gene flow throughout the range. As such, California's prairie falcons might be managed as a single population, indicating that management actions undertaken to benefit the species at the local level have the potential to influence the species as a whole.

Interactions of chromatin context, binding site sequence content, and sequence evolution in stress-induced p53 occupancy and transactivation.

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.

Global analysis of methylation profiles from high resolution CpG data.

New high throughput technologies are now enabling simultaneous epigenetic profiling of DNA methylation at hundreds of thousands of CpGs across the genome. A problem of considerable practical interest is identification of large scale, global changes in methylation that are associated with environmental variables, clinical outcomes, or other experimental conditions. However, there has been little statistical research on methods for global methylation analysis using technologies with individual CpG resolution. To address this critical gap in the literature, we develop a new strategy for global analysis of methylation profiles using a functional regression approach wherein we approximate either the density or the cumulative distribution function (CDF) of the methylation values for each individual using B-spline basis functions. The spline coefficients for each individual are allowed to summarize the individual's overall methylation profile. We then test for association between the overall distribution and a continuous or dichotomous outcome variable using a variance component score test that naturally accommodates the correlation between spline coefficients. Simulations indicate that our proposed approach has desirable power while protecting type I error. The method was applied to detect methylation differences, both genome wide and at LINE1 elements, between the blood samples from rheumatoid arthritis patients and healthy controls and to detect the epigenetic changes of human hepatocarcinogenesis in the context of alcohol abuse and hepatitis C virus infection. A free implementation of our methods in the R language is available in the Global Analysis of Methylation Profiles (GAMP) package at http://research.fhcrc.org/wu/en.html.

A genetic model of differential susceptibility to human respiratory syncytial virus (RSV) infection.

Respiratory syncytial virus (RSV) is the primary cause of lower respiratory tract infection during childhood and causes severe symptoms in some patients, which may cause hospitalization and death. Mechanisms for differential responses to RSV are unknown. Our objective was to develop an in vitro model of RSV infection to evaluate interindividual variation in response to RSV and identify susceptibility genes. Populations of human-derived HapMap lymphoblastoid cell lines (LCLs) were infected with RSV. Compared with controls, RSV-G mRNA expression varied from ~1- to 400-fold between LCLs. Basal expression of a number of gene transcripts, including myxovirus (influenza virus) resistance 1 (MX1), significantly correlated with RSV-G expression in HapMap LCLs. Individuals in a case-control population of RSV-infected children who were homozygous (n=94) or heterozygous (n=172) for the predicted deleterious A allele in a missense G/A SNP in MX1 had significantly greater risk for developing severe RSV disease relative to those with the major allele (n=108) (χ(2)=5.305, P=0.021; OR: 1.750, 95% CI: 1.110, 2.758, P=0.021). We conclude that genetically diverse human LCLs enable identification of susceptibility genes (e.g., MX1) for RSV disease severity in children, providing insight for disease risk.

Mange Caused by a Novel Micnemidocoptes Mite in a Golden Eagle ( Aquila chrysaetos ).

A second-year, female golden eagle ( Aquila chrysaetos ) was live trapped in northern California because of severe feather loss and crusting of the skin on the head and legs. On physical examination, the bird was lethargic, dehydrated, and thin, with severe feather loss and diffuse hyperemia and crusting on the head, ventral wings, ventrum, dorsum, and pelvic limbs. Mites morphologically similar to Micnemidocoptes derooi were identified with scanning electron microscopy. The eagle was treated with ivermectin (0.4 mg/kg) once weekly for 7 weeks, as well as pyrethrin, meloxicam, ceftiofur crystalline free acid, and voriconazole. Although the eagle's condition improved, and live mites or eggs were not evident on skin scrapings at the time of completion of ivermectin treatment, evidence of dead mites and mite feces were present after the last dose of ivermectin. Two additional doses of ivermectin and 2 doses of topical selamectin (23 mg/kg) were administered 2 and 4 weeks apart, respectively. No mite eggs, feces, or adults were evident after treatment was completed. A second golden eagle found in the same region was also affected with this mite but died soon after presentation. This is the first report, to our knowledge, of successful treatment, as well as treatment with selamectin, of mites consistent with Micnemidocoptes species in any raptorial species.

Knemidocoptic mange in wild golden eagles, California, USA.

During 2012-2013 in California, USA, 3 wild golden eagles were found with severe skin disease; 2 died. The cause was a rare mite, most closely related to Knemidocoptes derooi mites. Cautionary monitoring of eagle populations, habitats, and diseases is warranted.

Linking polymorphic p53 response elements with gene expression in airway epithelial cells of smokers and cancer risk.

Chronic cigarette smoking exposes airway epithelial cells to thousands of carcinogens, oxidants and DNA-damaging agents, creating a field of molecular injury in the airway and altering gene expression. Studies of cytologically normal bronchial epithelial cells from smokers have identified transcription-based biomarkers that may prove useful in early diagnosis of lung cancer, including a number of p53-regulated genes. The ability of p53 to regulate transcription is critical for tumor suppression, and this suggests that single-nucleotide polymorphisms (SNPs) in functional p53 binding sites (p53 response elements, or p53REs) that affect gene expression could influence susceptibility to cancer. To connect p53RE SNP genotype with gene expression and cancer risk, we identified a set of 204 SNPs in putative p53REs, and performed cis expression quantitative trait loci (eQTL) analysis, assessing associations between SNP genotypes and mRNA levels of adjacent genes in bronchial epithelial cells obtained from 44 cigarette smokers. To further test and validate these genotype-expression associations, we searched published eQTL studies from independent populations and determined that 53% (39/74) of the bronchial epithelial eQTLs were observed in at least one of other studies. SNPs in p53REs were also evaluated for effects on p53-DNA binding using a quantitative in vitro protein-DNA binding assay. Last, based on linkage disequilibrium, we found 6 p53RE SNPs associated with gene expression were identified as cancer risk SNPs by either genome-wide association studies or candidate gene studies. We provide an approach for identifying and evaluating potentially functional SNPs that may modulate the airway gene expression response to smoking and may influence susceptibility to cancers.

Assessing predictions of population viability analysis: Peregrine Falcon populations in California.

Population viability analysis (PVA) has been an important tool for evaluating species extinction risk and alternative management strategies, but there is little information on how well PVA predicts population trajectories following changes in management actions. We tested previously published predictions from a stage-structured PVA of Peregrine Falcons (Falco peregrinus) in California, USA (Wootton and Bell 1992), against population trajectories following the 1992 termination of statewide, active management (population supplementation of captive-reared young). In the absence of extensive post-management monitoring, we developed surrogate estimates of breeding population size by calibrating several citizen science data sets (Christmas Bird Count, CBC; and North American Breeding Bird Survey, BBS) to intensive population surveys taken primarily during the active management period. CBC abundance data standardized by observer effort exhibited a strong relationship to intensive survey data (r2 = 0.971), indicated significantly reduced annual population growth rates after management was terminated (λ = 0.023 ± 0.013 SE) than when supplementation occurred (λ = 0.089 ± 0.023 SE), and demonstrated an increasing population as predicted by the PVA. The population trajectory fell within the 95% CI of stochastic simulations of the model either with or without density dependence and assuming either measurement error or process error, but models with process error were most strongly supported by the data. These results indicate that PVA can quantitatively anticipate population trajectories following changes in management, highlight the importance of post-management monitoring of species of concern, and illustrate the benefits of using management changes as large-scale experiments to more rigorously test PVA.

Identification of novel NRF2-regulated genes by ChIP-Seq: influence on retinoid X receptor alpha.

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.

Human single-nucleotide polymorphisms alter p53 sequence-specific binding at gene regulatory elements.

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.

Formation of stress-specific p53 binding patterns is influenced by chromatin but not by modulation of p53 binding affinity to response elements.

The p53 protein is crucial for adapting programs of gene expression in response to stress. Recently, we revealed that this occurs partly through the formation of stress-specific p53 binding patterns. However, the mechanisms that generate these binding patterns remain largely unknown. It is not established whether the selective binding of p53 is achieved through modulation of its binding affinity to certain response elements (REs) or via a chromatin-dependent mechanism. To shed light on this issue, we used a microsphere assay for protein-DNA binding to measure p53 binding patterns on naked DNA. In parallel, we measured p53 binding patterns within chromatin using chromatin immunoprecipitation and DNase I coupled to ligation-mediated polymerase chain reaction footprinting. Through this experimental approach, we revealed that UVB and Nutlin-3 doses, which lead to different cellular outcomes, induce similar p53 binding patterns on naked DNA. Conversely, the same treatments lead to stress-specific p53 binding patterns on chromatin. We show further that altering chromatin remodeling using an histone acetyltransferase inhibitor reduces p53 binding to REs. Altogether, our results reveal that the formation of p53 binding patterns is not due to the modulation of sequence-specific p53 binding affinity. Rather, we propose that chromatin and chromatin remodeling are required in this process.