L. K. Savinkova

TATA box polymorphisms in human gene promoters and associated hereditary pathologies

TATA-binding protein (TBP) is the first basal factor that recognizes and binds a TATA box on TATA-containing gene promoters transcribed by RNA polymerase II. Data available in the literature are indicative of admissible variability of the TATA box. The TATA box flanking sequences can influence TBP affinity as well as the level of basal and activated tran-scription. The possibility of mediated involvement in in vivo gene expression regulation of the TBP interactions with variant TATA boxes is supported by data on TATA box polymorphisms and associated human hereditary pathologies. A table containing data on TATA element polymorphisms in human gene promoters (about 40 mutations have been described), associated with particular pathologies, their short functional characteristics, and manifestation mechanisms of TATA-box SNPs is presented. Four classes of polymorphisms are considered: TATA box polymorphisms that weaken and enhance promoter, polymorphisms causing TATA box emergence and disappearance, and human virus TATA box polymorphisms. The described examples are indicative of the polymorphism-associated severe pathologies like thalassemia, the increased risk of hepatocellular carcinoma, sensitivity to H. pylori infection, oral cavity and lung cancers, arterial hypertension, etc.

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.

A Step-by-step Model of TBP/TATA Box Binding Allows Predicting Human Hereditary Diseases by Single Nucleotide Polymorphism

The eukaryotic transcription on TATA-containing promoters usually is enucleated by binding of the TATA-binding protein (TBP) to the TATA box, a signal in DNA located at a distance of 30 bp from the transcription start site. So far, about 15000 papers describe TATA boxes in over 2500 genes as well as over 40 single nucleotide polymorphisms (SNPs, mutations) within and in the vicinity of TATA boxes that are associated with sclerosis, leukemia, cancer, thalassemia, thrombosis, hypertension, cataract, anemia, and other hereditary diseases. In this work, we have compared our measurements of the TBP affinity for the TATA box [1] with the data obtained by PWM [2], which is a generally accepted standard for recognition of the TATA box, and proposed on this basis a model of step-by-step TBP/TATA binding. Using this model, we for the first time succeeded in predicting the hereditary diseases caused by the TATA box damage or emergence as well as by change in the TATA box influence on transcription based on the mutations within this box or around it. Table 1 lists ten double-stranded DNAs (dsDNA) 26 bp long that are synthetic copies of natural TATA boxes and their affinities for TBP, ranging from 18.02 to 21.54 natural logarithm units (ln units), − Ln( K D , TATA ) , which we measured earlier [1] and studied here. First and foremost, we have compared our measurements [1] with the data obtained by PWM [2], a generally accepted standard for TATA box recognition:

The mechanism by which TATA-box polymorphisms associated with human hereditary diseases influence interactions with the TATA-binding protein.

SNPs in ТАТА boxes are the cause of monogenic diseases, contribute to a large number of complex diseases, and have implications for human sensitivity to external and internal environmental signals. The aim of this work was to explore the kinetic characteristics of the formation of human ТВР complexes with ТАТА boxes, in which the SNPs are associated with β‑thalassemias of diverse severity, immunosuppression, neurological disorders, and so on. It has for the first time been demonstrated, using an electrophoretic mobility shift assay, that TBP interacts with SNP‐containing ТАТА boxes with a significant (8–36‐fold) decrease in TBP/ТАТА association rate constant (ka) as compared with that in healthy people, a smaller decrease in dissociation rate constant (kd) and changes in the half‐lives of TBP/ТАТА complexes. Carriers of the −24G allele (rs 1800202T>G) in the TATA box of the triosephosphate isomerase gene promoter, associated with neurological and muscular disorders, were observed to have a 36‐fold decrease in TBP/TATA association rate constant that are consistent with TPI deficiency shown for patients who carry this defective allele. The kinetic characteristics of TBP/ТАТА complexes obtained suggest that, at a molecular level, hereditary diseases are largely caused by changes in TBP/ТАТА association rates and these changes have a bearing on disease severity.

Prediction of the affinity of the TATA-binding protein to TATA boxes with single nucleotide polymorphisms

The TATA-binding protein (TBP) is the subunit of basal transcription factor TFIID that recognizes the TATA boxes of TATA-containing promoters in class II genes, binds to them, and starts the assemblage of the RNA polymerase II basal transcription complex. The sequence of the TATA box with its flanking regions affects the levels of basal and activated transcription. The association of polymorphic TATA boxes with human hereditary diseases supports the hypothesis that TBP-TATA interaction modulates gene expression in vivo. The objective of this work is to determine changes in the TBP/TATA affinity related to polymorphisms in TATA boxes of human promoters. Changes in TBP/TATA affinities were assessed in silico by using our equation for equilibrium TBP/TATA binding in four consecutive steps: nonspecific binding ↔ sliding ↔ braking (stopping) ↔ stabilization. Our predictions agree with known examples of TATA-box polymorphisms and human hereditary diseases associated with them.

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).

TATA box polymorphisms in genes of commercial and laboratory animals and plants associated with selectively valuable traits

Most of more than 11 million experimentally established polymorphisms, accumulated in dbSNP, were identified in the intergenic spacers or coding DNA regions. This fact enables interpretation of the former polymorphisms as neutral, while the latter makes clear the biological sense of the associated mutant phenotypes, “the defect of certain proteins”. The association of polymorphisms in regulatory DNA regions with mutant phenotypes is poorly studied. Specifically, the defects in certain DNA/protein binding sites were identified in less than 500 cases. In TATA-containing genes of eukaryotes the TATA box, the TBP (TATA-binding protein) binding site, is located about 30 bp upstream from the transcription start site. Interaction between DNA and TBP triggers assemblage of the preinitiation complex. For 38 TATA box polymorphisms in the genes of commercial and laboratory animals and plants, the effect on TBP-binding activity was evaluated using the equilibrium equation for the four subsequent steps of TBP/TATA box binding (nonspecific binding ↔ sliding ↔ recognition ↔ stabilization). According to the GenBank data, these 38 polymorphisms were associated with the change in a number of selectively valuable traits. Statistically significant congruence of in silico analysis performed with mutant phenotypes (α < 0.05, binomial law) provides suggestion of the mechanism of phenotypic manifestation of these polymorphisms (changing of the TBP-binding activity), as well a validates the possibility of developing the universal test system for experimental-computer prediction of the effects of TATA box mutations in specified genes on selectively valuable traits of the species, varieties, and breeds.

A precise equation of equilibrium of four steps of TBP binding with the TATA box for prognosis of phenotypic manifestation of mutations

Among the central events of transcription initiation of TATA-containing genes in eukaryotes are the recognition and binding of the TATA box by the TATA-binding protein (TBP) to start the preinitiation complex formation on nucleosomal DNA. Using the equation of equilibrium for step-by-step TBP/TATA binding, we have analyzed 69 experimental datasets for the characteristics of biologically important features altered by TATA-box mutations. Among these features, the TBP/TATA-complex parameters, the transcription level, the activity of gene products, yeast colony growth at a dose of growth inhibitor (phenotype), and the heterogeneity of the response of a population to unspecific environmental stress have been described. Significant correlations were found between in silico prediction for TBP/TATA affinity and experimental data for in vivo and in vitro test systems based on 15 cell types of 19 species, RNA polymerases II and III, and natural, recombinant or mutant TBP. Such an invariant impact of the step-by-step TBP/TATA binding on the biological activity of complex systems, from a molecule to a population, might be due to the fact that TBP/TATA-complex formation precedes specific steps of transcription machinery assembly, which provide the multivariant jigsaw puzzle according to the expression pattern of each eukaryotic gene.

Candidate SNP Markers of Gender-Biased Autoimmune Complications of Monogenic Diseases Are Predicted by a Significant Change in the Affinity of TATA-Binding Protein for Human Gene Promoters

Some variations of human genome [for example, single nucleotide polymorphisms (SNPs)] are markers of hereditary diseases and drug responses. Analysis of them can help to improve treatment. Computer-based analysis of millions of SNPs in the 1000 Genomes project makes a search for SNP markers more targeted. Here, we combined two computer-based approaches: DNA sequence analysis and keyword search in databases. In the binding sites for TATA-binding protein (TBP) in human gene promoters, we found candidate SNP markers of gender-biased autoimmune diseases, including rs1143627 [cachexia in rheumatoid arthritis (double prevalence among women)]; rs11557611 [demyelinating diseases (thrice more prevalent among young white women than among non-white individuals)]; rs17231520 and rs569033466 [both: atherosclerosis comorbid with related diseases (double prevalence among women)]; rs563763767 [Hughes syndrome-related thrombosis (lethal during pregnancy)]; rs2814778 [autoimmune diseases (excluding multiple sclerosis and rheumatoid arthritis) underlying hypergammaglobulinemia in women]; rs72661131 and rs562962093 (both: preterm delivery in pregnant diabetic women); and rs35518301, rs34166473, rs34500389, rs33981098, rs33980857, rs397509430, rs34598529, rs33931746, rs281864525, and rs63750953 (all: autoimmune diseases underlying hypergammaglobulinemia in women). Validation of these predicted candidate SNP markers using the clinical standards may advance personalized medicine.