T. I. Merkulova

Transcription Regulatory Regions Database (TRRD): its status in 2002

Transcription Regulatory Regions Database (TRRD) is an informational resource containing an integrated description of the gene transcription regulation. An entry of the database corresponds to a gene and contains the data on localization and functions of the transcription regulatory regions as well as gene expression patterns. TRRD contains only experimental data that are inputted into the database through annotating scientific publication. TRRD release 6.0 comprises the information on 1167 genes, 5537 transcription factor binding sites, 1714 regulatory regions, 14 locus control regions and 5335 expression patterns obtained through annotating 3898 scientific papers. This information is arranged in seven databases: TRRDGENES (general gene description), TRRDLCR (locus control regions); TRRDUNITS (regulatory regions: promoters, enhancers, silencers, etc.), TRRDSITES (transcription factor binding sites), TRRDFACTORS (transcription factors), TRRDEXP (expression patterns) and TRRDBIB (experimental publications). Sequence Retrieval System (SRS) is used as a basic tool for navigating and searching TRRD and integrating it with external informational and software resources. The visualization tool, TRRD Viewer, provides the information representation in a form of maps of gene regulatory regions. The option allowing nucleotide sequences to be searched for according to their homology using BLAST is also included. TRRD is available at http://www.bionet.nsc.ru/trrd/.

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/.

Application of experimentally verified transcription factor binding sites models for computational analysis of ChIP-Seq data.

BackgroundChIP-Seq is widely used to detect genomic segments bound by transcription factors (TF), either directly at DNA binding sites (BSs) or indirectly via other proteins. Currently, there are many software tools implementing different approaches to identify TFBSs within ChIP-Seq peaks. However, their use for the interpretation of ChIP-Seq data is usually complicated by the absence of direct experimental verification, making it difficult both to set a threshold to avoid recognition of too many false-positive BSs, and to compare the actual performance of different models.ResultsUsing ChIP-Seq data for FoxA2 binding loci in mouse adult liver and human HepG2 cells we compared FoxA binding-site predictions for four computational models of two fundamental classes: pattern matching based on existing training set of experimentally confirmed TFBSs (oPWM and SiteGA) and de novo motif discovery (ChIPMunk and diChIPMunk). To properly select prediction thresholds for the models, we experimentally evaluated affinity of 64 predicted FoxA BSs using EMSA that allows safely distinguishing sequences able to bind TF. As a result we identified thousands of reliable FoxA BSs within ChIP-Seq loci from mouse liver and human HepG2 cells. It was found that the performance of conventional position weight matrix (PWM) models was inferior with the highest false positive rate. On the contrary, the best recognition efficiency was achieved by the combination of SiteGA & diChIPMunk/ChIPMunk models, properly identifying FoxA BSs in up to 90% of loci for both mouse and human ChIP-Seq datasets.ConclusionsThe experimental study of TF binding to oligonucleotides corresponding to predicted sites increases the reliability of computational methods for TFBS-recognition in ChIP-Seq data analysis. Regarding ChIP-Seq data interpretation, basic PWMs have inferior TFBS recognition quality compared to the more sophisticated SiteGA and de novo motif discovery methods. A combination of models from different principles allowed identification of proper TFBSs.

Involvement of Transcription Factor HNF3γ in the Effect of o-Aminoazotoluene on Glucocorticoid Induction of Tyrosine Aminotransferase in Mice Sensitive to its Hepatocarcinogenic Action

In the rodent liver, hepatocarcinogens inhibit the glucocorticoid induction of several liver‐specific genes, including tyrosine aminotransferase (TAT). A distinct positive correlation exists in mice between the extent of inhibition of TAT induction after acute administration of o‐aminoazotoluene (OAT) and the frequency of liver tumors after chronic exposure to the carcinogen. To elucidate the mechanism of the carcinogenic action, the effects of OAT on the DNA‐binding activity of several transcription factors participating in the glucocorticoid regulation of TAT gene expression were studied. The experimental inbred male mice were sensitive (A/He and SWR/J, tumor induction frequency of 75–100%, TAT induction inhibition of 35–50%) and resistant (CC57BR/Mv and AKR/J, 0–6% and 10–15%, respectively) to OAT. Gel retardation experiments showed that hepatocyte nuclear factor 3 (HNF3)γ DNA‐binding activity was strongly reduced in nuclear extracts from the livers of OAT‐treated A/He and SWR/J mice but only slightly reduced in CC57Br/Mv and AKR/J mice. The DNA‐binding activities of Ets, AP1 family members, and GME binding proteins were unaffected. HNF3γ DNA‐binding activity was reduced by 1 h after OAT administration and remained low for 1 mo, as did inhibition of TAT induction in the liver. These results suggested that the inhibitory effect of OAT on the glucocorticoid induction of TAT is mediated by reduced HNF3γ DNA‐binding activity.

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.

Mining DNA sequences to predict sites which mutations cause genetic diseases

Currently single nucleotide polymorphism (SNP) analysis becomes the crossroad of bioinformatics and medicine. We have developed a data mining system, http://wwwmgs.bionet.nsc.ru/mgs/systems/rsnp/, called rSNP_Guide, to discover regulatory sites in DNA sequences, which mutations could be the cause of genetic diseases. During the first step, we estimate the abilities of the proteins considered to bind to genomic DNA, which alterations by mutations are associated with a genetic disease under study. During the second step, we formalize the disease-associated experimental data on the SNP-referred alterations in DNA binding to unknown protein. During the third step, we cluster fuzzily all known proteins examined so that to determine one of them, which specific site is altered by mutations in consistence with that of the unknown protein experimentally associated with genetic disease. During the fourth step, we predict the known protein, which binding site is (i) resent on DNA and (ii) altered by mutations associated with genetic disease. Finally, during the last step, we estimate the robustness of this prediction. The rSNP_Guide has been tested on the SNPs with the known relationships between regulatory site alterations and genetic disease penetration. Besides, the novel SNPs-referred regulatory sites associated with the genetic disease penetrations were discovered and, then, successfully confirmed experimentally.

Correlation between hepatocarcinogenic effect of estragole and its influence on glucocorticoid induction of liver-specific enzymes and activities of FOXA and HNF4 transcription factors in mouse and rat liver

It is known that the carcinogenic effect of estragole, a component of essential oils of many spicy plants, is characterized by species, tissue, and sex specificity. It causes mainly liver tumors in female mice but is not carcinogenic for male mice and for rats. In this work, the estragole hepatocarcinogenicity was shown for female mice of previously not studied ICR line. The strict correlation between estragole hepatocarcinogenicity and its ability to decrease the level of glucocorticoid induction of liver-specific enzymes tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO) was found. Inhibition of TAT and TO inducibility by estragole takes place only in female mice but not in male mice and in rats. Studying the estragole effect on DNA-binding activity of transcription factors, present mainly in liver and regulating expression of genes encoding liver-specific proteins, has shown that estragole decreases FOXA and HNF4 activities but not activities of C/EBP and HNF1, and this happens only in female mice, for which this substance is hepatocarcinogen, but not in male mice and in rats. Pentachlorophenol, preventing hepatocarcinogenic effect of estragole, abolishes inhibitory influence of the latter on the TAT and TO glucocorticoid induction and restores DNA-binding activity of FOXA and HNF4. Thus, a correlation was revealed between the estragole hepatocarcinogenic effect and decrease in DNA-binding activity of transcription factors FOXA and HNF4, which might be indicative of the role of these factors in tumor suppression mechanisms in liver.

The immediate vicinity of mouse metallothionein-I gene contains two sites conferring glucocorticoid inducibility to the heterologous promoter

Glucocorticoid responsive elements (GREs) located −252 to −209 bp upstream and +1011 to +1054 bp downstream of the transcription initiation site of the mouse metallotionein‐I (mMT‐I) gene were identified in transient transfection eperiments. However, the promoter region of the mMT‐I gene (−330 to +70 bp) was found to provide low, if any, glucocorticoid induction of the linked CAT gene, while showing strong cadmium regulation, comparable with the in vivo level.

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.