An experimental study of the effects of SNPs in the TATA boxes of the <i>GRIN1, ASCL3</i> and <i>NOS1</i> genes on interactions with the TATA-binding protein

The GRIN1, ASCL3, and NOS1 genes are associated with various phenotypes of neuropsychiatric disorders. For instance, these genes contribute to the development of schizophrenia, Alzheimer’s and Parkinson’s diseases, and epilepsy. These genes are also associated with various cancers. For example, ASCL3 is overexpressed in breast cancer, and NOS1, in ovarian cancer cell lines. Based on our findings and literature data, we had previously obtained results suggesting that the single-nucleotide polymorphisms (SNPs) that disrupt erythropoiesis are highly likely to be associated with cognitive and neuropsychiatric disorders in humans. In the present work, using SNP_TATA_Z-tester, we investigated the influence of unannotated SNPs in the TATA boxes of the promoters of the GRIN1, ASCL3, and NOS1 genes (which are involved in neuropsychiatric disorders and cancers) on the interaction of the TATA boxes with the TATA-binding protein (TBP). Double-stranded oligodeoxyribonucleotides identical to the TATA-containing promoter regions of the GRIN1, ASCL3, and NOS1 genes (reference and minor alleles) and recombinant human TBP were employed to study in vitro (by an electrophoretic mobility shift assay) kinetic characteristics of the formation of TBP–TATA complexes and their affinity. It was found, for example, that allele A of rs1402667001 in the GRIN1 promoter increases TBP–TATA affinity 1.4-fold, whereas allele C in the TATA box of the ASCL3 promoter decreases the affinity 1.4-fold. The lifetime of the complexes in both cases decreased by ~20 % due to changes in the rates of association and dissociation of the complexes (ka and kd, respectively). Our experimental results are consistent with the literature showing GRIN1 underexpression in schizophrenic disorders as well as an increased risk of cervical, bladder, and kidney cancers and lymphoma during ASCL3 underexpression. The effect of allele A of the –27G&gt;A SNP (rs1195040887) in the NOS1 promoter is suggestive of an increased risk of ischemic damage to the brain in carriers. A comparison of experimental TBP–TATA affinity values (KD) of wild-type and minor alleles with predicted ones showed that the data correlate well (linear correlation coefficient r = 0.94, p &lt; 0.01)

Candidate SNP-markers altering TBP binding affinity for promoters of the Y-linked genes CDY2A, SHOX, and ZFY are lowering many indexes of reproductive potential in men

Reproductive potential is the most important conditional indicator reflecting the ability of individuals in a population to reproduce, survive and develop under optimal environmental conditions. As for humans, the concept of reproductive potential can include the level of the individual’s mental and physical state, which allows them to reproduce healthy offspring when they reach social and physical maturity. Female reproductive potential has been investigated in great detail, whereas the male reproductive potential (MRP) has not received the equal amount of attention as yet. Therefore, here we focused on the human Y chromosome and found candidate single-nucleotide polymorphism (SNP) markers of MRP. With our development named Web-service SNP_TATA_Z-tester, we examined in silico all 35 unannotated SNPs within 70-bp proximal promoters of the three Y-linked genes, CDY2A, SHOX and ZFY, which represent all types of human Y-chromosome genes, namely: unique, pseudo-autosomal, and human X-chromosome gene paralogs, respectively. As a result, we found 11 candidate SNP markers for MRP, which can significantly alter the TATA-binding protein (TBP) binding affinity for promoters of these genes. First of all, we selectively verified in vitro the values of the TBP-promoter affinity under this study, Pearson’s linear correlation between predicted and measured values of which were r = 0.94 (significance p &lt; 0.005). Next, as a discussion, using keyword search tools of the PubMed database, we found clinically proven physiological markers of human pathologies, which correspond to a change in the expression of the genes carrying the candidate SNP markers predicted here. These were markers for spermatogenesis disorders (ZFY: rs1388535808 and rs996955491), for male maturation arrest (CDY2A: rs200670724) as well as for disproportionate short stature at Madelung deformity (e. g., SHOX: rs1452787381) and even for embryogenesis disorders (e. g., SHOX: rs28378830). This indicates a wide range of MRI indicators, alterations in which should be expected in the case of SNPs in the promoters of the human Y-chromosome genes and which can go far beyond changes in male fertility

AN EXPERIMENTAL STUDY OF THE EFFECT OF RARE POLYMORPHISMS OF HUMAN HBB, HBD AND F9 PROMOTER TATA BOXES ON THE KINETICS OF INTERACTION WITH THE TATA-BINDING PROTEIN

Human genes HBB, HBD and F9 belong to the hematopoiesis system. The deficiency or excess of these genes’ products is the cause of hereditary thalassemias of various severity and haemophilia B Leyden. Previously, it was shown that a number of annotated single-nucleotide polymorphisms of TATA boxes of these genes associated with the occurrence of ß- and δ-thalassemia affect the interaction with the TATAbinding protein, the interaction changing proportionally with the change in the number of gene products. In the present work, we investigate the effect of rare not annotated single-nucleotide polymorphisms (SNPs) of TATA boxes of these genes with an unknown manifestation on the TATA-binding protein interaction. To study the kinetic characteristics of TBP/TATA complex formation in vitro, doublestranded oligodeoxynucleotides identical to the TATA-containing portions of the promoters of the HBB, HBD and F9 genes (“normal” and minor alleles) and recombinant human TBP were used. It was shown that the TATA-box SNP of –25A &gt; C (rs281864525) and the deletion of the –25AA (rs63750953) TATA-box of the β-globin gene have the same effect on the TBP/TATA affinity, which decreases 3-folds in both cases. However, the effect of these substitutions on the rate of the TBP/TATA complex formation is significantly different: SNP –25A &gt; C decreases the rate 5-fold, and the deletion decreases the rate more than 7-fold. The influence of substitutions on the strength of the TBP/TATA complexes has a different effect. If in the case of SNP –25A &gt; C the strength of the complexes increases 1.8-fold, then in the case of the –25AA deletion, the strength of the complexes increases 2.4-fold, even though the affinity of the TATAbinding protein to the TATA box decreases. A comparison of experimental values of affinity (KD) of the TBP/T complexes of “normal” and minor alleles with the predicted has shown that data correlate well with each other. The coefficient of linear correlation r = 0.94 (α &lt; 0.0001). A comprehensive approach to the study of rare polymorphisms may lead to the identification of the most sensitive markers of orphan diseases, which will contribute to the development of reliable and rapid methods for their diagnosis and treatment

Сandidate SNP-markers of rheumatoid arthritis that can significantly alter the affinity of the TATA-binding protein for human gene promoters

Rheumatoid polyarthritis (RA) is an autoimmune disease with autoantibodies, including antibodies to citrullant antigens and proinflammatory cytokines, such as TNF-α and IL-6, which are involved in the induction of chronic synovitis, bone erosion, followed by deformity. Immunopathogenesis is based on the mechanisms of the breakdown of immune tolerance to its own antigens, which is characterized by an increase in the activity of T-effector cells, causing RA symptomatology. At the same time, against the background of such increased activity of effector lymphocytes, a decrease in the activity of a number of regulatory cells, including regulatory T-cells (Treg) and myeloid suppressor cells, is recorded. There is reason to say that it is the change in the activity of suppressor cells that is the leading element in RA pathogenesis. That is why only periods of weakening (remission) of RA are spoken of. According to the more powerful female immune system compared to the male one, the risk of developing RA in women is thrice as high, this risk decreases during breastfeeding and grows during pregnancy as well as after menopause in proportion to the level of sex hormones. It is believed that 50 % of the risk of developing RA depends on the conditions and lifestyle, while the remaining 50 % is dependent on genetic predisposition. That is why, RA fits the main idea of postgenomic predictive-preventive personalized medicine that is to give a chance to those who would like to reduce his/her risk of diseases by bringing his/her conditions and lifestyle in line with the data on his/her genome sequenced. This is very important, since doctors consider RA as one of the most frequent causes of disability. Using the Web service SNP_TATA_Z-tester (http://beehive.bionet.nsc.ru/cgi-bin/mgs/tatascan_fox/start.pl), 227 variants of single nucleotide polymorphism (SNP) of the human gene promoters were studied. As a result, 43 candidate SNP markers for RA that can alter the affinity of the TATA-binding protein (TBP) for the promoters of these genes were predicted

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

The high-throughput sequencing project “1 000 Genomes” made it possible to catalog and utilize genetic loci and single nucleotide polymorphisms (SNPs) in medicine. Analysis of SNP markers (significantly frequent differences of individual genomes of patients from the reference human genome) allows physicians to optimize treatment. On the other hand, 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 comparison of their frequency in patients with that in healthy people. This approach contributes to undervaluation of clinically relevant SNPs and to unnecessary computational expenses (on verification of neutral SNPs). Preclinical empirical verification of possible candidate SNP markers may eliminate neutral SNPs from the dataset. In the present study, we found, using the SNP_TATA_Comparator web service, the unannotated SNP rs367781716: the substitution of ancestral T (health) with minor C at position –37 before the transcription initiation site of the АВСА9 gene. This SNP significantly reduces affinity of TATAbinding protein (TBP) for this gene’s promoter and corresponds to a deficiency (low protein level) of the АВСА9 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 formation (ka) and decay (kd) of the complexes of TBP with an oligonucleotide matching either allele –37C or –37T of the АВСА9 gene. We found that the rate of formation (ka) of the TBP/TATA complex for the minor allele is 2.4-fold lower 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 binding affinity of TBP for a promoter containing the ТАТА box. This empirical value matched the value predicted by SNP_ТАТА _Comparator within the 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 АВСА9 promoter. Possible phenotypic manifestations of the candidate SNP marker rs367781716 are discussed

SURFACE PLASMON RESONANCE STUDY OF THE INTERACTION BETWEEN THE HUMAN TATA-BOX BINDING PROTEIN AND THE TATA ELEMENT OF THE NOS2A GENE PROMOTER

Interaction ofthe TATA-binding protein (ТВР) with the TATA box and formation ofthe TBP/TATA complex is the first step in the transcription complex assembly and the central event in transcription regulation. It is known that some single nucleotide polymorphisms (SNPs) in the human TATA box and its flanks are associated with elevated risk ofvarious disorders (Savinkova etal., 2009). Little is known about changes in the TBP/TATA interaction leading to the elevated risk ofany disease. Thus, we have studied the influence ofSNP (–21 t &gt; c), adjoining to the TATA box ofthe NOS2A promoter, on the interaction by surface plasmon resonance (SPR) on biosensor ProteOn XPR36 (BioRad). The sensograms obtained in the experiment indicate that the constant for the interaction with TATA bearing SNP –21 t &gt; c differ little from the constant with the wild-type box and the dissociation constants differ approximately twofold. Experiments utilizing the SPR method and classical EMSA have shown that ТВР immobilization on the microchip dextran surface does not alter its affinity to TATA-containing oligonucleotides

Apurinic/apyrimidinic (AP) site recognition by the 5′-dRP/AP lyase in poly(ADP-ribose) polymerase-1 (PARP-1)

The capacity of human poly(ADP-ribose) polymerase-1 (PARP-1) to interact with intact apurinic/apyrimidinic (AP) sites in DNA has been demonstrated. In cell extracts, sodium borohydride reduction of the PARP-1/AP site DNA complex resulted in covalent cross-linking of PARP-1 to DNA; the identity of cross-linked PARP-1 was confirmed by mass spectrometry. Using purified human PARP-1, the specificity of PARP-1 binding to AP site-containing DNA was confirmed in competition binding experiments. PARP-1 was only weakly activated to conduct poly(ADP-ribose) synthesis upon binding to AP site-containing DNA, but was strongly activated for poly(ADP-ribose) synthesis upon strand incision by AP endonuclease 1 (APE1). By virtue of its binding to AP sites, PARP-1 could be poised for its role in base excision repair, pending DNA strand incision by APE1 or the 5′-dRP/AP lyase activity in PARP-1