T. V. Arshinova

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.

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.

Effect of TATA Box polymorphisms in human β-globin gene promoter associated with β-thalassemia on interaction with TATA-binding protein

Single nucleotide polymorphisms (SNPs) are the most common type of genetic variability in humans. Hereditary disorders in the hemoglobin synthesis caused by SNPs in the TATA boxes of the human β-globin gene promoter are associated with β-thalassemia of varying severity. The TATA-binding protein (TBP) is the first basal transcription factor that recognizes and binds TATA boxes in TATA-box-containing promoters and initiates the assembly of RNA polymerase II transcription complexes. This report is the first to present quantitative parameters (Kd) of human TBP interaction with SNP-containing TATA boxes of the β-globin gene promoter. These data are in agreement with literature data on the decrease in normal β-globin RNA synthesis in patients with β-thalassemia.

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.

Change of TATA-binding protein affinity to oligonucleotides corresponding to TATA boxes in human gene promoters bearing polymorphisms associated with hereditary diseases

Single-nucleotide polymorphisms (SNPs) in the regulatory regions of human genes and, in particular, TATA boxes can alter the sensitivity to bacterial and viral infections and to drugs used for their treatment. These SNPs are associated with the risk of widespread multifactorial human diseases, such as arthritis, hypertension, cancer, Alzheimer’s disease, and others. Interaction of the TATA-binding protein (TBP) with TATA boxes on TATA-containing promoters nucleates the preinitiation complex assemblage, which is the initial stage of transcription regulation. This study considers the change of TBP affinity to the TATA boxes of human MBL, CYP 2A6, SOD1, and TPI genes bearing SNPs associated with autoimmune diseases, cardiovascular diseases, amyotrophic lateral sclerosis, neurodegenerative disorders, hypersensitivity to bacterial infections, etc. In all cases a decrease in the affinity of TBP to TATA-containing oligonucleotides was shown.