D. Yu. Oshchepkov

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.

Sitecon—A Tool for Analysis of DNA Physicochemical and Conformational Properties: E2F/DP Transcription Factor Binding Site Analysis and Recognition

Research into molecular mechanisms underlying DNA-protein interactions using statistical analysis of nucleotide sequences of binding sites is most important for understanding the principles of gene expression regulation. The local conformation of transcription factor binding sites determined by their context is a factor responsible for specificity of the DNA-protein interactions. Analysis of the local conformations of a set of functional DNA sequences allows the conservative context-dependent conformational and physicochemical properties (CDCPP) reflecting molecular mechanisms of interactions to be determined. A set of conservative CDCPP specific of sites for binding a particular transcriptional factor can be effectively used for their recognition. The methods for determining the conservative CDCPP in short regions of aligned functional DNA sequences and recognizing potential transcription factor binding sites basing on a set of these conservative CDCPP determined are proposed in this work. To demonstrate implementation of the method, the binding sites of the heterodimeric complex E2F/DP were analyzed as an example The discovered specific CDCPP for a set of these binding sites reflect the molecular mechanism of their interaction.

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.

Detection of target genes of FOXA transcription factors involved in proliferation control

To reveal the mechanism of tumor-suppressing activity of FOXA proteins in liver, a search for potential target genes of these transcription factors involved in proliferation control was carried out. In the first step, we have used data from the literature concerning gene expression in mouse liver (high content of FOXA proteins) and kidney (FOXA expression is absent) obtained by hybridization on microchips. A search for FOXA binding sites in regulatory regions of forty differentially expressing genes involved in proliferation control was carried out using the computer method SITECON. Eleven genes containing clusters of potential FOXA sites incorporating 3–6-fold repeats of TTTG were revealed. The FOXA-specific interaction with such microsatellite sites was confirmed by gel-retardation technique using the GST-fused protein containing the DNA-binding domain of FOXA2. Six genes containing clusters of confirmed binding sites—Cul2, Cdc73, Ptk, Pdcd, Creb, and Ppp2r5d—were selected. The effect of hepatocarcinogen orthoaminoazotoluene (OAT), which lowers the FOXA activity, on expression of these genes was studied by the real-time PCR. OAT was shown to increase sharply the level of mRNA of the Cul2 and Cdc73 genes.

Search for new binding sites for the transcriptional factor SF-1 by the SITECON method: Experimental verification and analysis of regulatory regions of orthologous genes

165 Recognition of transcriptional factor binding sites in genomic sequences is a promising approach to deciphering the regulatory code of DNA. Today, the methods of recognition of transcriptional factor binding sites based of various principles (consensus, weight matrices, etc.) are known and widely used [1]. However, in most cases, the recognition accuracy of transcriptional factor binding sites is insufficient for analysis of extended genomic sequences [2, 3], which makes it necessary to use additional criteria that allow the percent of false predictions to be decreased [3–5]. In addition, in different situations, the existing methods of recognition of transcriptional factor binding sites may differ significantly with respect to recognition quality even within the same approach (e.g., weight matrixes) [6]. For this reason, development of new approaches to recognizing transcriptional factor binding sites remains a topical problem. Another key problem is determination whether the transcriptional factor binding sites predicted by computer methods are functional in the regulatory regions of different genes in vivo (i.e., in chromosomes in living cells).

Extension of Cell Cycle Gene Network Description Based on Prediction of Potential Binding Sites for E2F Transcription Factor

Annotation of scientific papers using the GeneNet technology allowed for reconstruction of the gene network regulating the cell cycle (the G1/S transition). To extend the network, we searched for potential sites for E2F transcription factor binding in promoter regions of genes whose expression depends on the cell cycle phase, however, any experimental evidence for the presence of E2F binding sites is lacking. New potential target genes for E2F factor were found. Products of these genes are involved in DNA replication and cell cycle regulation as well as in chromatin compacting, repair, and apoptosis. Combination of GeneNet-compiled data with the results of prediction of new potential E2F sites in regulatory regions enriched the reconstructed cell cycle gene network with new regulatory linkages.

Effect of flanking sequences on the accuracy of the recognition of transcription factor binding sites

The development of in vitro technologies has produced new experimental information on protein binding onto DNA, which is accumulated in databases and used in studies of mechanisms regulating gene expression and in the development of computer-assisted methods of binding site recognition in pro- and eukaryotic genomes. However, it is still questionable to what extent in vitro selected sequences reflect the actual structures of the real transcription factor (TF) binding sites. The Kullback–Leibler divergence has been applied to the comparison of frequency matrices of TF binding sites constructed on sets of artificially selected sequences and real sites. The similarity of core sequences of real and artificial sites has been observed for 80% of all TFs studied. For 20% of TFs, in vitro selected binding site sequences have a broader range of permissible significant nucleotides not found in real sites. The optimal lengths of DNA sequences containing real binding sites, at which the sites are recognized most accurately, are estimated by the weight matrix method. For approximately 80% of the TFs studied, the optimal binding site length notably exceeds the lengths of the core sequences, as well as the lengths of in vitro selected sites. The detected features of in vitro selected TF binding sites impose constraints on their use in the development of computer-assisted methods of the recognition of candidate sites in genomic sequences.

Reconstruction of the mechanisms that regulate the expression of the Escherihia coli yfiA gene under stress conditions

The regulatory region of the Escherichia coli yfiA gene was reconstructed by using the SITECON web resource and mathematical modeling; the complexity of its expression under oxidative stress was assessed. Simulation of the response of E. coli cells transformed with the pYfi-gfp plasmid to oxidative stress indicated that the maximum agreement with experimental data was achieved in a model implying complex action, apparently mediated by several transcription factors (TFs). The regulatory region of the yfiA gene was inspected for potential TF binding sites, and highly reliable predictions were made for TFs MarA, IscR, MetJ, PurR, and SoxS, which directly or indirectly participate in the response of the gene to oxidative stress, and for CRP, a global regulator of carbohydrate catabolism. The presence of binding sites for CRP, MarA, and SoxS in the E. coli yfiA promoter was confirmed by a electrophoretic mobility-shift assay with purified recombinant TFs. This fact explains the sensitivity of yfiA to mitomycin and radical-forming agents.

Analysis of data on large-scale chromatin immunoprecipitation by recognition of transcription factor binding sites

Chromatin immunoprecipitation followed by mass parallel sequencing of the precipitated fragments (ChIP-Seq) is broadly used for detailed investigation of the distribution of various transcription factor binding sites over the whole genome. The ChIP-Seq profiles obtained by immunoprecipitation of mouse liver chromatin with antibodies against the FoxA2 transcription factor [Wederell et al., 2008] were analyzed by our methods of recognition of FoxA binding sites. The following classification of locus profiles was proposed on the basis of analysis. (1)Unimodal loci (possessing a single peak) with length below 600 bp. These loci are likely to be formed by a single FoxA binding site. (2) Multimodal loci (possessing two or more distinct peaks) of lengths over 600 bp and all longer loci. Each locus of this group is likely to be composed of numerous single sites.

Computer Analysis of Conformational and Physicochemical Properties of Nucleotide Sequences Cleavable by DNA Topoisomerase I

DNA binding with enzymes is followed by specific adaptation of the DNA structure, including partial or almost complete melting, structural changes in the sugar-phosphate backbone, stretching, compressing, bending or kinking, base flipping, etc. The set of conformational changes is individual for each enzyme and is aimed at efficiently adjusting the orbitals of the reacting groups of the enzyme and the specific DNA site to 10°–15°. The efficiency of nucleotide sequence adaptation determined by the enzyme depends on several structural characteristics. Optimal adjustment is achieved only in the case of specific DNA sequences; as a result, the reaction rate is four to eight orders of magnitude higher with specific than with nonspecific sequences. DNA topoisomerase I (Topo) is a sequence-dependent enzyme. Although less efficiently, Topo cleaves sequences which differ considerably from the optimal sequence. A method based on the analysis of conformational and physicochemical properties of the DNA helix was used to examine many nucleotide sequences cleavable by Topo. The method yields detailed information on similarity or difference of DNA structural units. The cleavable sequences proved to be similar in roll, slide, twist, and rise. In addition, all sequences had sterically disadvantageous contacts between N3 and NH2 of guanines and N3 of adenines in the minor groove, which corresponded to the presence of dinucleotides Py-Pu in the cleavage site. DNA bending towards the major groove is easier in the case of the optimal sequence. This method is promising for analyzing the efficiency of nucleic acid cleavage by various DNA- and RNA-dependent enzymes.