Petr Ponomarenko

An experimental verification of the predicted effects of promoter TATA-box polymorphisms associated with human diseases on interactions between the TATA boxes and TATA-binding protein.

Human genome sequencing has resulted in a great body of data, including a stunningly large number of single nucleotide polymorphisms (SNPs) with unknown phenotypic manifestations. Identification and comprehensive analysis of regulatory SNPs in human gene promoters will help quantify the effects of these SNPs on human health. Based on our experimental and computer-aided study of SNPs in TATA boxes and the use of literature data, we have derived an equation for TBP/TATA equilibrium binding in three successive steps: TATA-binding protein (TBP) sliding along DNA due to their nonspecific affinity for each other ↔ recognition of the TATA box ↔ stabilization of the TBP/TATA complex. Using this equation, we have analyzed TATA boxes containing SNPs associated with human diseases and made in silico predictions of changes in TBP/TATA affinity. An electrophoretic mobility shift assay (EMSA)-based experimental study performed under the most standardized conditions demonstrates that the experimentally measured values are highly correlated with the predicted values: the coefficient of linear correlation, r, was 0.822 at a significance level of α<10−7 for equilibrium K D values, (-ln K D), and 0.785 at a significance level of α<10−3 for changes in equilibrium K D (δ) due to SNPs in the TATA boxes (). It has been demonstrated that the SNPs associated with increased risk of human diseases such as α-, β- and δ-thalassemia, myocardial infarction and thrombophlebitis, changes in immune response, amyotrophic lateral sclerosis, lung cancer and hemophilia B Leyden cause 2–4-fold changes in TBP/TATA affinity in most cases. The results obtained strongly suggest that the TBP/TATA equilibrium binding equation derived can be used for analysis of TATA-box sequences and identification of SNPs with a potential of being functionally important.

Obesity-related known and candidate SNP markers can significantly change affinity of TATA-binding protein for human gene promoters

BackgroundObesity affects quality of life and life expectancy and is associated with cardiovascular disorders, cancer, diabetes, reproductive disorders in women, prostate diseases in men, and congenital anomalies in children. The use of single nucleotide polymorphism (SNP) markers of diseases and drug responses (i.e., significant differences of personal genomes of patients from the reference human genome) can help physicians to improve treatment. Clinical research can validate SNP markers via genotyping of patients and demonstration that SNP alleles are significantly more frequent in patients than in healthy people. The search for biomedical SNP markers of interest can be accelerated by computer-based analysis of hundreds of millions of SNPs in the 1000 Genomes project because of selection of the most meaningful candidate SNP markers and elimination of neutral SNPs.ResultsWe cross-validated the output of two computer-based methods: DNA sequence analysis using Web service SNP_TATA_Comparator and keyword search for articles on comorbidities of obesity. Near the sites binding to TATA-binding protein (TBP) in human gene promoters, we found 22 obesity-related candidate SNP markers, including rs10895068 (male breast cancer in obesity); rs35036378 (reduced risk of obesity after ovariectomy); rs201739205 (reduced risk of obesity-related cancers due to weight loss by diet/exercise in obese postmenopausal women); rs183433761 (obesity resistance during a high-fat diet); rs367732974 and rs549591993 (both: cardiovascular complications in obese patients with type 2 diabetes mellitus); rs200487063 and rs34104384 (both: obesity-caused hypertension); rs35518301, rs72661131, and rs562962093 (all: obesity); and rs397509430, rs33980857, rs34598529, rs33931746, rs33981098, rs34500389, rs63750953, rs281864525, rs35518301, and rs34166473 (all: chronic inflammation in comorbidities of obesity). Using an electrophoretic mobility shift assay under nonequilibrium conditions, we empirically validated the statistical significance (α < 0.00025) of the differences in TBP affinity values between the minor and ancestral alleles of 4 out of the 22 SNPs: rs200487063, rs201381696, rs34104384, and rs183433761. We also measured half-life (t1/2), Gibbs free energy change (ΔG), and the association and dissociation rate constants, ka and kd, of the TBP-DNA complex for these SNPs.ConclusionsValidation of the 22 candidate SNP markers by proper clinical protocols appears to have a strong rationale and may advance postgenomic predictive preventive personalized medicine.

How to Use SNP_TATA_Comparator to Find a Significant Change in Gene Expression Caused by the Regulatory SNP of This Gene's Promoter via a Change in Affinity of the TATA-Binding Protein for This Promoter

The use of biomedical SNP markers of diseases can improve effectiveness of treatment. Genotyping of patients with subsequent searching for SNPs more frequent than in norm is the only commonly accepted method for identification of SNP markers within the framework of translational research. The bioinformatics applications aimed at millions of unannotated SNPs of the “1000 Genomes” can make this search for SNP markers more focused and less expensive. We used our Web service involving Fishers Z-score for candidate SNP markers to find a significant change in a genes expression. Here we analyzed the change caused by SNPs in the genes promoter via a change in affinity of the TATA-binding protein for this promoter. We provide examples and discuss how to use this bioinformatics application in the course of practical analysis of unannotated SNPs from the “1000 Genomes” project. Using known biomedical SNP markers, we identified 17 novel candidate SNP markers nearby: rs549858786 (rheumatoid arthritis); rs72661131 (cardiovascular events in rheumatoid arthritis); rs562962093 (stroke); rs563558831 (cyclophosphamide bioactivation); rs55878706 (malaria resistance, leukopenia), rs572527200 (asthma, systemic sclerosis, and psoriasis), rs371045754 (hemophilia B), rs587745372 (cardiovascular events); rs372329931, rs200209906, rs367732974, and rs549591993 (all four: cancer); rs17231520 and rs569033466 (both: atherosclerosis); rs63750953, rs281864525, and rs34166473 (all three: malaria resistance, thalassemia).

HOW MULTIPLE AUXIN RESPONSIVE ELEMENTS MAY INTERACT IN PLANT PROMOTERS: A REVERSE PROBLEM SOLUTION

Plant hormone auxin is a key regulator of growth and development. Auxin affects gene expression through ARF transcription factors, which bind specifically auxin responsive elements (AuxREs). Auxin responsive genes usually have more than one AuxRE, for example, a widely used auxin sensor DR5 contains seven AuxREs. Auxin responsive regions of several plant genes have been studied using sets of transgenic constructions in which the activity of one or several AuxREs were abolished. Here we present the method for analysis of the datasets on promoter activity assays having promoter sequences, namely, number and sequences of AuxREs, altogether with their measured auxin induction level. The method for a reverse problem solution considers two extreme models of AuxRE cooperation. Additive model describes auxin induction level of a gene as a sum of the individual AuxREs impacts. Multiplicative model considers pure cooperation between the AuxREs, where the combined effect is the multiplication of the individual AuxRE impacts. The reverse problem solution allows estimating the impact of an individual AuxRE into the induction level and the model for their cooperation. For promoters of three genes belonging to different plant species we showed that the multiplicative model fits better than additive. The reverse problem solution also suggests repressive state of auxin responsive promoters before auxin induction. The developed method provides possibility to investigate AuxRE structure-activity relationship and may be used as the basis for a novel approach for AuxRE recognition.

Toward high-resolution population genomics using archaeological samples

The term ‘ancient DNA’ (aDNA) is coming of age, with over 1,200 hits in the PubMed database, beginning in the early 1980s with the studies of ‘molecular paleontology’. Rooted in cloning and limited sequencing of DNA from ancient remains during the pre-PCR era, the field has made incredible progress since the introduction of PCR and next-generation sequencing. Over the last decade, aDNA analysis ushered in a new era in genomics and became the method of choice for reconstructing the history of organisms, their biogeography, and migration routes, with applications in evolutionary biology, population genetics, archaeogenetics, paleo-epidemiology, and many other areas. This change was brought by development of new strategies for coping with the challenges in studying aDNA due to damage and fragmentation, scarce samples, significant historical gaps, and limited applicability of population genetics methods. In this review, we describe the state-of-the-art achievements in aDNA studies, with particular focus on human evolution and demographic history. We present the current experimental and theoretical procedures for handling and analysing highly degraded aDNA. We also review the challenges in the rapidly growing field of ancient epigenomics. Advancement of aDNA tools and methods signifies a new era in population genetics and evolutionary medicine research.

Candidate SNP markers of aggressiveness-related complications and comorbidities of genetic diseases are predicted by a significant change in the affinity of TATA-binding protein for human gene promoters

BackgroundAggressiveness in humans is a hereditary behavioral trait that mobilizes all systems of the body—first of all, the nervous and endocrine systems, and then the respiratory, vascular, muscular, and others—e.g., for the defense of oneself, children, family, shelter, territory, and other possessions as well as personal interests. The level of aggressiveness of a person determines many other characteristics of quality of life and lifespan, acting as a stress factor. Aggressive behavior depends on many parameters such as age, gender, diseases and treatment, diet, and environmental conditions. Among them, genetic factors are believed to be the main parameters that are well-studied at the factual level, but in actuality, genome-wide studies of aggressive behavior appeared relatively recently. One of the biggest projects of the modern science—1000 Genomes—involves identification of single nucleotide polymorphisms (SNPs), i.e., differences of individual genomes from the reference genome. SNPs can be associated with hereditary diseases, their complications, comorbidities, and responses to stress or a drug. Clinical comparisons between cohorts of patients and healthy volunteers (as a control) allow for identifying SNPs whose allele frequencies significantly separate them from one another as markers of the above conditions. Computer-based preliminary analysis of millions of SNPs detected by the 1000 Genomes project can accelerate clinical search for SNP markers due to preliminary whole-genome search for the most meaningful candidate SNP markers and discarding of neutral and poorly substantiated SNPs.ResultsHere, we combine two computer-based search methods for SNPs (that alter gene expression) {i} Web service SNP_TATA_Comparator (DNA sequence analysis) and {ii} PubMed-based manual search for articles on aggressiveness using heuristic keywords. Near the known binding sites for TATA-binding protein (TBP) in human gene promoters, we found aggressiveness-related candidate SNP markers, including rs1143627 (associated with higher aggressiveness in patients undergoing cytokine immunotherapy), rs544850971 (higher aggressiveness in old women taking lipid-lowering medication), and rs10895068 (childhood aggressiveness-related obesity in adolescence with cardiovascular complications in adulthood).ConclusionsAfter validation of these candidate markers by clinical protocols, these SNPs may become useful for physicians (may help to improve treatment of patients) and for the general population (a lifestyle choice preventing aggressiveness-related complications).

Hypothetical SNP markers that significantly affect the affinity of the TATA-binding protein to VEGFA, ERBB2, IGF1R, FLT1, KDR, and MET oncogene promoters as chemotherapy targets

The following hypothesis has been proposed: IF an SNP can significantly increase the expression of an oncogene by increasing the affinity of the TATA-binding protein (TBP) to its promoter, THEN this SNP can also reduce the apparent bioactivity of inhibitors of this oncogene during antitumor chemotherapy and vice versa. In the context of this hypothesis, the previously proposed method (http://beehive.bionet.nsc. ru/cgi-bin/mgs/tatascan/start.pl) was applied to analyze all SNPs found within the [-70; -20] regions (which harbor all proven TBP-binding sites) of the promoters of VEGFA, EGFR, ERBB2, IGF1R, FLT1, KDR, and MET oncogenes according to the human reference genome, hg19. For 83% of these SNPs, their effect on TBP affinity to the oncogene promoters required for assembly of preinitiation complexes was not significant. rs36208385, rs36208384, rs370995111, rs372731987, rs111811434, rs369547510, rs76407893, rs369728300, and rs72001900 can potentially serve as SNP markers to reduce the apparent bioactivity of oncogene inhibitors, while rs141092704, rs184083669, rs145139616, rs200697953, rs187746433, rs199730913, rs377370642, rs114484350, rs374921120, rs146790957, rs376727645, and rs72001900 can be the markers for enhancing this activity.

Candidate SNP Markers of Familial and Sporadic Alzheimer's Diseases Are Predicted by a Significant Change in the Affinity of TATA-Binding Protein for Human Gene Promoters

While year after year, conditions, quality, and duration of human lives have been improving due to the progress in science, technology, education, and medicine, only eight diseases have been increasing in prevalence and shortening human lives because of premature deaths according to the retrospective official review on the state of US health, 1990-2010. These diseases are kidney cancer, chronic kidney diseases, liver cancer, diabetes, drug addiction, poisoning cases, consequences of falls, and Alzheimers disease (AD) as one of the leading pathologies. There are familial AD of hereditary nature (~4% of cases) and sporadic AD of unclear etiology (remaining ~96% of cases; i.e., non-familial AD). Therefore, sporadic AD is no longer a purely medical problem, but rather a social challenge when someone asks oneself: “What can I do in my own adulthood to reduce the risk of sporadic AD at my old age to save the years of my lifespan from the destruction caused by it?” Here, we combine two computational approaches for regulatory SNPs: Web service SNP_TATA_Comparator for sequence analysis and a PubMed-based keyword search for articles on the biochemical markers of diseases. Our purpose was to try to find answers to the question: “What can be done in adulthood to reduce the risk of sporadic AD in old age to prevent the lifespan reduction caused by it?” As a result, we found 89 candidate SNP markers of familial and sporadic AD (e.g., rs562962093 is associated with sporadic AD in the elderly as a complication of stroke in adulthood, where natural marine diets can reduce risks of both diseases in case of the minor allele of this SNP). In addition, rs768454929, and rs761695685 correlate with sporadic AD as a comorbidity of short stature, where maximizing stature in childhood and adolescence as an integral indicator of health can minimize (or even eliminate) the risk of sporadic AD in the elderly. After validation by clinical protocols, these candidate SNP markers may become interesting to the general population [may help to choose a lifestyle (in childhood, adolescence, and adulthood) that can reduce the risks of sporadic AD, its comorbidities, and complications in the elderly].

Candidate SNP markers of social dominance, which may affect the affinity of the TATA-binding protein for human gene promoters

The following heuristic hypothesis has been proposed: if an excess of a protein in several animal organs was experimentally identified as a physiological marker of increased aggressiveness, and if a single nucleotide polymorphism (SNP) can cause the overexpression of a human gene homologous to the animal gene encoding this protein, then this polymorphism can be a candidate SNP marker of social dominance. In turn, a deficient expression would correspond to subordinate behavior. Within this hypothesis, we analyzed 21 human genes, ADORA2A, BDNF, CC2D1A, CC2D1B, ESR2, FEV, FOS, GH1, GLTSCR2, GRIN1, HTR1B, HTR1A, HTR2A, HTR2C, LGI4, LEP, MAOA, SLC17A7, SLC6A3, SNCA, and TH, which determine the functions of the proteins known as the physiological markers of aggressive behavior in animals. These genes encode for hormones and their receptors, biosynthetic enzymes, receptors of neurotransmitters, and transcription and neurotrophic factors. These proteins have been postulated to play important roles in determining the hierarchical relationships in social animals. Using our previously developed Web-service SNP_TATA_Comparator (http://beehive.bionet.nsc.ru/cgi-bin/mgs/tatascan/start.pl), we analyzed 381 SNPs within the [–70;–20] region preceding the start of the protein-coding transcripts, obtained from the database dbSNP, v.147. This is the region for all the known TATA-binding protein (TBP) binding sites. As a result, we found 45 and 47 candidate SNP markers of dominance and subordination, respectively (e.g., rs373600960 and rs747572588). Within the proposed heuristic hypotheses and dbSNP database v.147, we found statistically significant (α < 10–5) evidence of the effects of natural selection against the deficient expression of genes, which can affect the predisposition to dominate. We also obtained evidence favoring the hypothesis that both subordinate and dominant behavior can be the norm of reaction of aggressiveness (difference not significant: α > 0.35). The proposed hypothesis, the candidate SNP markers obtained on its basis, and the observed regularities of the effects of their natural selection on the human genome are discussed in comparison with the published data with respect to whether these markers can have an effect on the expression of social dominance in human society. We conclude that our candidate SNPs, identified with a computational model, require further experimental verification.

Prediction and verification of the influence of the rs367781716 SNP on the interaction of the ТАТА-binding protein with the promoter of the human АВСА9 gene

The high-throughput sequencing project 1000 Genomes made it possible to catalog and utilize genetic loci and single nucleotide polymorphisms (SNPs) in medicine. The analysis of SNP markers allows physicians to optimize treatment. However, tens of millions of unannotated SNPs correspond to a gigantic number of false positive (false negative) candidate SNP markers that are selected by computer methods for comparing their frequency in patients with that in healthy people. This approach contributes to the undervaluation of clinically relevant SNPs and to unnecessary computational expenses for the verification of neutral SNPs. The preclinical empirical verification of possible candidate SNP markers may eliminate neutral SNPs from the dataset. In the present study, using the SNP_TATA_Comparator web service, we found the unannotated SNP rs367781716: the substitution of ancestral T (health) with a minor C at position–37 before the transcription initiation site of the ABCA9 gene. This SNP significantly reduces the affinity of TATA-binding protein (TBP) for this gene’s promoter and corresponds to the deficiency (low protein level) of the ABCA9 gene product (the transporter ATP-binding cassette A9) in patients with the–37C allele. For preclinical empirical verification of rs367781716, we used an electrophoretic mobility shift assay (EMSA) to measure the rates of association (ka) and dissociation (kd) of the complexes of TBP with an oligonucleotide matching either allele–37C or–37T of the ABCA9 gene. We found that the rate of association (ka) of the TBP/TATA complex for the minor allele is lower by a factor of 2.4 than that for the ancestral allele. We calculated the empirical value of the change in the equilibrium constant of dissociation (KD = kd/ka), which characterizes the binding affinity of TBP for a promoter containing the TATA box. This empirical value matched the value predicted by the SNP_TATA_Comparator within the allowable margin of error of the measurements and calculations. We also determined the half-life and Gibbs free energy of the complex of TBP with the ABCA9 promoter. Possible phenotypic manifestations of the candidate SNP marker rs367781716 are discussed.