Gennady V. Vasiliev

Point mutations within 663-666 bp of intron 6 of the human TDO2 gene, associated with a number of psychiatric disorders, damage the YY-1 transcription factor binding site.

Single base mutations G→A at position 663 and G→T at position 666 of intron 6 of the human tryptophan oxygenase gene (TDO2) are associated with a variety of psychiatric disorders [Comings, D.E. et al. (1996) Pharmacogenetics 6, 307–318]. Binding of rat liver nuclear extract proteins to synthetic double‐strand oligonucleotides corresponding to three allelic states of the region between 651 bp and 680 bp of human TDO2 intron 6 has been studied by gel shift assay. It has been demonstrated that to each allelic state of the region there corresponds a specific set of proteins that interacts with it. With the aid of computer analysis and using specific anti‐YY‐1 antibodies it has been shown that both mutations damage the YY‐1 transcription factor binding site.

rSNP_Guide, a database system for analysis of transcription factor binding to target sequences: application to SNPs and site-directed mutations

rSNP_Guide is a novel curated database system for analysis of transcription factor (TF) binding to target sequences in regulatory gene regions altered by mutations. It accumulates experimental data on naturally occurring site variants in regulatory gene regions and site-directed mutations. This database system also contains the web tools for SNP analysis, i.e., active applet applying weight matrices to predict the regulatory site candidates altered by a mutation. The current version of the rSNP_Guide is supplemented by six sub-databases: (i) rSNP_DB, on DNA-protein interaction caused by mutation; (ii) SYSTEM, on experimental systems; (iii) rSNP_BIB, on citations to original publications; (iv) SAMPLES, on experimentally identified sequences of known regulatory sites; (v) MATRIX, on weight matrices of known TF sites; (vi) rSNP_Report, on characteristic examples of successful rSNP_Tools implementation. These databases are useful for the analysis of natural SNPs and site-directed mutations. The databases are available through the Web, http://wwwmgs.bionet.nsc.ru/mgs/systems/rsnp/.

Species‐specific effects of the hepatocarcinogens 3′‐methyl‐4‐dimethyl‐aminoazobenzene and ortho‐aminoazotoluene in mouse and rat liver

The effects of rat‐specific hepatocarcinogen 3′‐methyl‐4‐dimethylaminoazobenzene (3′‐MeDAB), mouse‐specific hepatocarcinogen ortho‐aminoazotoluene (OAT), non‐species‐specific hepatocarcinogen diethylnitrosamine (DENA), and non‐carcinogenic 4′‐methyl‐4‐dimethylaminoazobenzene (4′‐MeDAB) on glucocorticoid induction of tyrosine aminotransferase (TAT) and DNA‐binding activity of hepatocyte nuclear factor 3 (HNF3) family of transcription factors were investigated with carcinogen‐susceptible and ‐resistant animals. Species‐specific hepatocarcinogens 3′‐MeDAB and OAT strongly inhibited glucocorticoid induction of TAT in the liver of susceptible but not resistant animals. DENA, which is highly carcinogenic for the liver of both rats and mice inhibited glucocorticoid induction of TAT in both species, while non‐carcinogenic 4′‐MeDAB was absolutely ineffective both in rats and mice. The inhibition of TAT activity by the carcinogens was due to reduced levels of TAT mRNA, which is most likely to be a result of the reduced rate of transcription initiation of the TAT gene. In all cases, the TAT inhibition was accompanied by significant reduction of DNA‐binding activity of the HNF3 transcription factor, which is known to be critical to glucocorticoid regulation of TAT gene. We also demonstrated that the described species‐specific effects of OAT and of 3′‐MeDAB on HNF3 DNA‐binding activity may be initiated not only by administration in vivo, but also by their direct administration to homogenate, intact nuclei or nuclear lysate, but not to nuclear extract fraction, obtained by precipitation with 0.32 g/mL of ammonium sulfate (Fraction I). We showed, that a factor responsible for this effect might be precipitated in 0.32–0.47g/mL interval of ammonium sulfate concentration. In contrast, non‐specific hepatocarcinogen DENA was effective upon being added directly to Fraction I, implying a different mechanism of its action.

Identification of microRNA Genes in Three Opisthorchiids

Background Opisthorchis felineus, O. viverrini, and Clonorchis sinensis (family Opisthorchiidae) are parasitic flatworms that pose a serious threat to humans in some countries and cause opisthorchiasis/clonorchiasis. Chronic disease may lead to a risk of carcinogenesis in the biliary ducts. MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression at post-transcriptional level and are implicated in the regulation of various cellular processes during the parasite- host interplay. However, to date, the miRNAs of opisthorchiid flukes, in particular those essential for maintaining their complex biology and parasitic mode of existence, have not been satisfactorily described. Methodology/Principal Findings Using a SOLiD deep sequencing-bioinformatic approach, we identified 43 novel and 18 conserved miRNAs for O. felineus (miracidia, metacercariae and adult worms), 20 novel and 16 conserved miRNAs for O. viverrini (adult worms), and 33 novel and 18 conserved miRNAs for C. sinensis (adult worms). The analysis of the data revealed differences in the expression level of conserved miRNAs among the three species and among three the developmental stages of O. felineus. Analysis of miRNA genes revealed two gene clusters, one cluster-like region and one intronic miRNA in the genome. The presence and structure of the two gene clusters were validated using a PCR-based approach in the three flukes. Conclusions This study represents a comprehensive description of miRNAs in three members of the family Opistorchiidae, significantly expands our knowledge of miRNAs in multicellular parasites and provides a basis for understanding the structural and functional evolution of miRNAs in these metazoan parasites. Results of this study also provides novel resources for deeper understanding the complex parasite biology, for further research on the pathogenesis and molecular events of disease induced by the liver flukes. The present data may also facilitate the development of novel approaches for the prevention and treatment of opisthorchiasis/clonorchiasis.

Bioinformatical and experimental approaches to investigation of transcription factor binding sites in vertebrate genes

The development of computer-assisted methods for transcription factor binding sites (TFBS) recognition is necessary for study the DNA regulatory transcription code. There are a great number of experimental methods that enable TFBS identification in genome sequences. The experimental data can be used to elaborate multiple computer approaches to recognition of TFBS, each of which has its own advantages and limitations. A short review of the characteristics of computer methods of TFBS prediction based on various principles is presented. Methods used for experimental monitoring of predicted sites are analyzed. Data concerning DNA regulatory potential and its realization at the chromatin level, obtained using these methods, are discussed along with approaches to recognition of target genes of certain transcription factors in the genome sequences.

Adult Opisthorchis felineus major protein fractions deduced from transcripts: comparison with liver flukes Opisthorchis viverrini and Clonorchis sinensis.

The epidemiologically important liver flukes Opisthorchis felineus, Opisthorchis viverrini, and Clonorchis sinensis are of interest to health professionals, epidemiologists, pharmacologists, and molecular biologists. Recently the transcriptomes of the latter two species were intensively investigated. However our knowledge on molecular biology of O. felineus is scarce. We report the first results of the O. felineus transcriptome analysis. We isolated and annotated a total of 2560 expressed sequence tag (EST) sequences from adult O. felineus (deposited within the database of expressed sequence tags (dbEST), under accession numbers GenBank: JK624271-JK626790, JK006511-JK006547, JK649790-JK649792). Clustering and analysis resulted in the detection of 267 contigs. Of the protein sequences deduced from these, 82% had homologs in the NCBI (nr) protein database and 63% contained conserved domains, allowing the functions to be interpreted using the Gene Ontology terms. Comprehensive analysis of Opisthorchiidae- and Trematoda-specific substitutions within amino acid sequences deduced for the proteins myoglobin, vitelline precursor protein, cathepsin F, and 28kDa glutathione transferase was carried out. The gene set of the 32 ribosomal proteins for the three Opisthorchiidae species with the addition of available Schistosoma and Fasciola orthologs was created and is provided in the supplementary. The orthologous gene set created was used for inferring phylogeny within the Trematoda with special attention to interrelations within the Opisthorchiidae. The phylogenetic analysis revealed a closer relationship between C. sinensis and O. viverrini and some divergence of O. felineus from either O. viverrini or C. sinensis.

Potential binding sites for SF-1: Recognition by the SiteGA method, experimental verification, and search for new target genes

The transcription factor SF-1 (steroidogenic factor 1) regulates the expression of the steroidogenesis genes, coordinates the development and function of the hypothalamic-pituitary-gonadal and adrenal systems, and plays an important role in the development and function of the reproductive system. SF-1 belongs to the superfamily of nuclear receptors and activates gene expression via binding as a monomer to DNA. The SiteGA method was developed for recognizing the binding sites for SF-1. The method utilizes a genetic algorithm and discriminant analysis to identify the context features of extended (93-bp) regions harboring the SF-1 sites in a training sample. Recognition of the SF-1 sites showed that the SiteGA method allows more reliable predictions as compared to the common weight matrix method. Experimental verification of 18 putative SF-1 sites predicted for the regulatory regions of the steroidogenesis genes showed that 15 (83%) of them did indeed interact with SF-1. The density of putative SF-1 sites was analyzed in the regulatory regions of genes from various functional groups, and new target genes of SF-1 were sought in the human genome. The potential targets of SF-1 include the genes coding for cytokine receptors, growth factor receptors, and proteins involved in the corresponding signal transduction pathways, as well as genes expressed in the epididymis. Expression of SF-1 in the epididymis was predicted and verified experimentally.

Binding Sites for Transcription Factor SF-1 in Promoter Regions of Genes Encoding Mouse Steroidogenesis Enzymes 3βHSDI and P450c17

Using gel retardation of DNA samples and specific antibodies, binding sites for the transcription factor SF-1 were found in positions −53/−44-and −285/−270 in the promoter region of the mouse Cyp17 gene and in position −117/−108 of the promoter region of the mouse 3βHSDI gene.

Hidden heterogeneity of transcription factor binding sites

Steroidogenic factor 1 (SF-1) belongs to a small group of the transcription factors that bind DNA only as a monomer. Three different approaches-Sitecon, SiteGA, and oPWM-constructed using the same training sample of experimentally confirmed SF-1 binding sites have been used to recognize these sites. The appropriate prediction thresholds for recognition models have been selected. Namely, the thresholds concordant by false positive or negative rates for various methods were used to optimize the discrimination of steroidogenic gene promoters from the datasets of non-specific promoters. After experimental verification, the models were used to analyze the ChIP-seq data for SF-1. It has been shown that the sets of sites recognized by different models overlap only partially and that an integration of these models allows for identification of SF-1 sites in up to 80% of the ChIP-seq loci. The structures of the sites detected using the three recognition models in the ChIP-seq peaks falling within the [-5000, +5000] region relative to the transcription start sites (TSS) extracted from the FANTOM5 project have been analyzed. The MATLIGN classified the frequency matrices for the sites predicted by oPWM, Sitecon, and SiteGA into two groups. The first group is described by oPWM/Sitecon and the second, by SiteGA. Gene ontology (GO) analysis has been used to clarify the differences between the sets of genes carrying different variants of SF-1 binding sites. Although this analysis in general revealed a considerable overlap in GO terms for the genes carrying the binding sites predicted by oPWM, Sitecon, or SiteGA, only the last method elicited notable trend to terms related to negative regulation and apoptosis. The results suggest that the SF-1 binding sites are different in both their structure and the functional annotation of the set of target genes correspond to the predictions by oPWM+Sitecon and SiteGA. Further application of Homer software for de novo identification of enriched motifs in ChIP-Seq data for SF-1ChIP-seq dataset gave the data similar to oPWM+Sitecon.

The Sitega Tool for Recognition and Context Analysis of Transcription Factor Binding Sites: Significant Dinucleotide Features Besides the Canonical Consensus Exemplified By SF-1 Binding Site

Development of computational methods to search for transcription factor binding sites (TFBSs) is important in investigation of regulatory regions of eukaryotic genes and in genome annotation. We propose a SiteGA method for recognition of TFBSs, providing the search of SF-1 binding sites as an example. The SiteGA method was implemented using a genetic algorithm (GA) involving an iterative discriminant analysis of local dinucleotide context characteristics. These characteristics were compiled not only over the core binding site (BS) region, but over its flanks as well. The major advancement of this approach is an improvement in accuracy by a large window capturing the meaningful context features besides the canonical consensus. The experimental verification confirmed the majority of predicted sites. The program SiteGA is available at http://wwwmgs2.bionet.nsc.ru/mgs/programs/sitega/.