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 of social dominance, which may affect the affinity of the TATAbinding 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 physiological marker of increased aggressiveness and if a polymorphism (SNP) can cause superexpression of the human gene homologous of the animal gene encoding this protein, then this polymorphism can be a candidate SNP marker of social dominance, whereas a deficient expression corresponds to subordinate and vice versa. 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, TH – which represent the functions of proteins known as physiological markers of aggressive behavior in animals: hormones and their receptors, biosynthetic enzymes and receptors of neurotransmitters, transcription and neurotrophic factors. These proteins may play an important role in determining 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 region of [–70; –20] relative to the start protein-coding transcripts, which is the region of the all known TATA-binding protein (TBP) binding sites. We took them from the database dbSNP, v.147 As a result, we found 45 and 47 candidate SNP markers of dominance and submission, respectively (e. g., rs373600960 and rs747572588). Within the framework of the proposed heuristic hypotheses and database dbSNP v.147, we found statistically significant (α &lt; 10-5) evidence of the effects of natural selection against the deficient expression of genes, which can affect the predisposition to dominate, as well as in favor of both subordination and domination behavior as a norm of reaction of aggressiveness (difference not significant: α &gt; 0.35). The proposed hypothesis, the candidate SNP markers predicted and the observed regularities of effects of natural selection for the human genome are discussed in comparison with published data: whether they can have any relation to social dominance in human. It was concluded that these results require experimental verification

The effects of SN Ps in the regions of positioning RNA polymerase II on the TBP/promoter affinity in the genes of human circadian clock

Genetic variability in the genes of circadian clock is manifested as the phenotypic variability of physiological functions and behavior as well as disorders of the function of not only the clock but also other systems, leading to the development of a pathologies. We analyzed the influence of SNPs localized in the [–70, –20] region from the transcription start site of the gene on TBP / promoter affinity in two groups of genes that are components of the system of human circadian clock. The first group comprises the genes of the circadian oscillator core (11 genes); the second, the genes of the nearest regulatory environment of the circadian oscillator (21 genes). A group for comparison included genes with another function (31 genes). The SNP_TATA_Comparator web service was used for prediction of the effect of SNPs in the regions of positioning of RNA polymerase II on the dissociation constant for TBP / promoter. It was shown that the number of SNP markers reducing the TBP / promoter affinity in the first group of genes significantly lower than the number of SNP markers increasing affinity (α &lt; 10–3). The reverse was true of the comparison group: SNP markers reduced TBP / promoter affinity to a significantly greater extent than the SNP marker increased affinity (α &lt; 10–6). This property may be a characteristic feature of genes  of the circadian oscillator. These predictions are important for identification of candidate SNP markers of various pathologies associated with the dysfunction of circadian clock genes for further testing them in experimental and clinical studies, as well as for verification of mathematical models of the circadian oscillator

Affinity Labeling of RNA Polymerase II in the Transcriptionally Active Complex by a Phosphorylating Analog of the Initiation Substrate

RNA polymerase II is composed of 12 subunits Substrate derivatives with various chemical groups were used to study the active site of RNA polymerase II. Reagents with arylazide groups were activated by irradi ation at the appropriate wavelength. An additional reagent was required for detection of the products of pro tein interaction with other reagents. For example, when carbonyl containing reagents were used for irreversible attachment of an affinity reagent to the protein, the gen erated Schiff bases were reduced with NaBH 4 . These studies were performed with highly purified enzyme preparations , poly[d(A T)] ACCELERATED PUBLICATION 0006 2979/00/6510 1129$25.00 ©2000 MAIK &quot;Nauka / Interperiodica&quot; * To whom correspondence should be addressed. Vol. 65, No. 10, 2000, pp. 1129 1134. Translated from Biokhimiya, Vol. 65, No. 10, 2000, pp. 1334 1340. Original Russian Text Copyright © 2000 Abstract-Affinity modification of RNA polymerase II by a phosphorylating analog of the initiation substrate carrying a zwitterionic 5′ terminal phosphate group with a 4 N,N dimethylaminopyridine residue (DMAP pA) was studied during specific transcription initiation controlled by the late adenoviral promotor. Super selective affinity labeling and standard conditions of affinity modification resulted in labeling a polypeptide with molecular weight corresponding to that of the third subunit of the enzyme, RPB3 (45 kD). The initiation substrate (ATP) protects RNA polymerase II from modification. The third subunit may be involved in the formation of the substrate binding site of the enzyme

Biomedical and candidate SN P markers of chronopathologies can significantly change affinity of ТАТА -binding protein for human gene promoters

Computational analysis of millions of unannotated SNPs from the 1000 Genomes Project may speed up the search for biomedical SNP markers. We combined the analysis of SNPs in the binding sites of ТАТА - binding protein (ТВР) using a previously described W eb service (http://beehive.bionet.nsc.ru/cgi-bin/mgs/ tatascan/start.pl) with a keyword search for biochemicalmarkers of chronopathologies, which correspond to clinical manifestations of these SNPs. In the [–70; –20] region of promoters of 14 human genes (location of proven binding sites of ТВР), we found 32 known and candidate SNP markers of circadian- rhythm disturbances, including rs17231520 and rs569033466 (both: risk of chronopathologies in liver); rs35036378 (behavioral chronoaberrations); rs549858786 (rheumatoid arthritis with a chronoaberration of IL1B expression); rs563207167, rs11557611, and rs5505 (all three: chronopathologies of the tumor – host balance, blood pressure, and the reproductive system); rs1143627 (bipolar disorder with circadian dependence of diagnosis and treatment); rs16887226 and rs544850971 (both: lowered resistance to endotoxins because of the imbalance between the circadian and immune systems); rs367732974 and rs549591993 (both: circadian dependence of heart attacks); rs563763767 (circadian dependence of myocardial infarction); rs2276109 and rs572527200 (both: circadian dependence of asthma attacks); rs34223104, rs563558831, and rs10168 (circadian optima of treatment with methotrexate and cyclophosphamide); and rs397509430, rs33980857,rs34598529, rs33931746, rs33981098, rs34500389, rs63750953, rs281864525, rs35518301, and rs34166473 (all: neurosensory hearing loss and restless legs syndrome). For these SNPs, we evaluated α (significance) of changes in the affinity of ТВР for promoters, where increased affinity corresponds to overexpression of the genes, and decreased affinity to deficient expression (Z-test). Verification of these 32 SNP markers according to clinical standards and protocols may advance the field of predictive preventive personalized medicine

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

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

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

Влияние герминальных мутаций в гене CHEK2 на выживаемость до биохимического рецидива и безметастатическую выживаемость после радикального лечения у больных раком предстательной железы

Objective: to evaluate the prognostic value of pathogenic germline BRCA1, BRCA2 and CHEK2 mutations on biochemical relapse-free survival (BRFS) and metastasis-free survival (MFS) following radical treatment in patients with localized and locally advanced prostate cancer (PCa).Materials and methods. Tumor features and outcomes of 102 patients with PCa were analyzed. In all patients nadir prostate-specific antigen (PSA) have been achieved: radical prostatectomy was undergone by 85 patients; 17 patients received radical radiotherapy. Exclusion criteria were postoperative nadir PSA &gt;0.2 ng/mL, adjuvant hormone therapy. During follow-up a total of 65 (63.7 %) patients developed biochemical relapse (BCR), and 39 (38.2 %) patients developed metastatic progression of PCa. All patients were genotyped for clinically significant pathogenic germline mutations 1100delC, I157Tand IVS2+1G&gt;A in the CHEK2gene, 185delAG, 4153delA, 5382insC, 3875del4, 3819del5, C61G, 2080delA in the BRCA1 gene, 6174delT in the BRCA2 gene by polymerase chain reaction real-time using a set “OncoGenetics” (LLC “Research and Production Company DNA-Technology”, Russia, registration certificate № 2010/08415). The second step was the determination of the coding part of the BRCA1 and BRCA2 genes by the Sanger sequencing using a set “Beckman Coulter enomeLab GeXP”.Results. Pathogenic germline mutations in the CHEK2 gene were identified in 16 (15.7 %) patients: heterozygous missense mutation I157T (c.470T&gt;C, rs17879961) was identified in 15 (14.7 %) patients, heterozygous mutation IVS2+1G&gt;A (c.319+1G&gt;A, rs765080766) was identified in 1 (0.9 %) patient. No cases of the 1100delC mutation in the CHEK2 gene and clinically significant mutations in the BRCA1 and BRCA2 genes were detected. Germline mutations I157TandIVS2+1G&gt;A in the CHEK2gene are statistically significant independent unfavorable prognostic factor for BRFS (hazard ratio (HR) 3.272; 95 % confidence interval (CI) 1.688—6.341, p &lt;0.001) and marginally significant independent unfavorable prognostic factor for MFS (HR 2.186; 95 % CI 0.932—5.126, p = 0.072). Subgroup analysis confirm independent prognostic value of germline CHEK2 mutations in patients with localized PCa (for BRFS HR 3.048; 95 % CI 1.024—9.078; p = 0.045; for MFS HR 5.168; 95 % CI 1.231—21.699; p = 0,025), and its marginally significant prognostic value in patient with locally advanced PCa T3-T4N0M0 (for BRFS HR 3.099; 95 % CI 0.991-9.689; р = 0.052) and TanyN1M0 stage (for MFS HR 5.089; 95 % CI 0.724-35.755; p = 0.102). Germline mutations I157T and IVS2+1G&gt;A in the CHEK2 gene are associated with increased risk of early BCR during 12 months (HR 3.795; 95 % CI 2.06-6.98; p &lt;0.001) and early metastatic progression during 24 months (HR 6.72; 95 % CI 2.02-22.34; p = 0.004) following radical treatment. This study has certain limitations due to its retrospective recruitment and a small sample of patients. Conclusions. Our results confirm that germline CHEK2 mutations I157T and IVS2+1G&gt;A are an unfavorable prognostic factor for patients with PCa, associated with increased risk of early biochemical relapse and metastatic progression, worse BRFS and MFS.Цель исследования — оценить прогностическое влияние герминальных патогенных мутаций в генах BRCA1, BRCA2 и CHEK2 на выживаемость до биохимического рецидива и безметастатическую выживаемость у больных локализованным и местно-распространенным раком предстательной железы, получивших радикальное лечение.Материалы и методы. В ретроспективный анализ включены данные 102 больных раком предстательной железы, у которых был достигнут надир простатического специфического антигена после радикальной простатэктомии (n = 85) или лучевой терапии (n = 17). Критериями исключения были надир послеоперационного простатического специфического антигена &gt;0,2 нг/мл, наличие адъювантной гормональной терапии. При наблюдении биохимический рецидив выявлен у 65 (63,7 %) больных, метастатическое прогрессирование заболевания — у 39 (38,2 %). Всем пациентам выполнена ДНК-диагностика герминальных клинически значимых патогенных мутаций 1100delC, I157T и IVS2+1G&gt;A в гене CHEK2, мутаций 185delAG, 4153delA, 5382insC, 3875del4, 3819del5, C61G, 2080delA в гене BRCA1, 6174delT в гене BRCA2 с использованием метода полимеразной цепной реакции в режиме реального времени (панель «Онкогенетика», регистрационное удостоверение № ФСР 2010/08415), вторым этапом проведено определение кодирующей части генов BRCA1 и BRCA2 с использованием метода секвенирования по Сэнгеру на платформе Beckman Coulter enomeLab GeXP.Результаты. Патогенные герминальные мутации в гене CHEK2 выявлены в 16 (15,7 %) из 102 случаев: миссенс-мутация I157T (c. 470T&gt;C, rs17879961) в гетерозиготном состоянии у 15 (14,7 %) пациентов, мутация IVS2+1G&gt;A (c. 319+1G&gt;A, rs765080 766) в гетерозиготном состоянии у 1 (0,9 %).Случаев наследования мутации 1100delC в гене CHEK2 и клинически значимых мутаций в генах BRCA1 и BRCA2 не обнаружено. Герминальные мутации I157T и IVS2+1G&gt;A в гене CHEK2являются достоверным независимым маркером неблагоприятного прогноза выживаемости без биохимического рецидива (отношение рисков (ОР) 3,272; 95 % доверительный интервал (ДИ) 1,688—6,341; p &lt;0,001) и имеют тенденцию значимости фактора неблагоприятного прогноза безметастатической выживаемости (ОР 2,186; 95 % ДИ 0,932—5,126; p = 0,072). Данные подгруппового анализа подтверждают независимую прогностическую значимость патогенных герминальных мутаций в гене CHEK2 при локализованной стадии рака предстательной железы (выживаемость без биохимического рецидива: ОР 3,048; 95 % ДИ 1,024—9,078; p = 0,045; безметастатическая выживаемость: ОР 5,168; 95 % ДИ 1,231—21,699;p = 0,025), а также тенденцию значимости для больных с местно-распространенными стадиями T3—T4N0M0 (выживаемость без биохимического рецидива ОР 3,099; 95 % ДИ 0,991—9,689; р = 0,052) и ТлюбаяN1M0 (безметастатическая выживаемость: ОР 5,089; 95 % ДИ 0,724—35,755; p = 0,102). Герминальные мутации I157T и IVS2+1G&gt;A в гене CHEK2 ассоциированы с повышенным риском раннего биохимического рецидива в течение 12 мес (ОР 3,795; 95 % ДИ2,06—6,98; p &lt;0,001) и раннего метастатического прогрессирования в течение 24 мес (ОР 6,72; 95 % ДИ2,02—22,34; p = 0,004) после радикального лечения. Настоящее исследование имеет определенные ограничения, связанные с ретроспективным набором и малой выборкой пациентов. Заключение. Герминальные мутации I157T и IVS2+1G&gt;A в гене CHEK2являются фактором неблагоприятного прогноза для больных раком предстательной железы, ассоциированным с повышенным риском раннего биохимического рецидива и метастатического прогрессирования заболевания после радикального лечения, снижением выживаемости без биохимического рецидива и безметастатической выживаемости