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Most of the position weight matrix (PWM) based bioinformatics methods developed to predict transcription factor binding sites (TFBS) assume each nucleotide in the sequence motif contributes independently to the interaction between protein and DNA sequence, usually producing high false positive predictions. The increasing availability of TF enrichment profiles from recent ChIP-Seq methodology facilitates the investigation of dependent structure and accurate prediction of TFBSs. We develop a novel Tree-based PWM (TPWM) approach to accurately model the interaction between TF and its binding site. The whole tree-structured PWM could be considered as a mixture of different conditional-PWMs. We propose a discriminative approach, called TPD (TPWM based Discriminative Approach), to construct the TPWM from the ChIP-Seq data with a pre-existing PWM. To achieve the maximum discriminative power between the positive and negative datasets, the cutoff value is determined based on the Matthew Correlation Coefficient (MCC). The resulting TPWMs are evaluated with respect to accuracy on extensive synthetic datasets. We then apply our TPWM discriminative approach on several real ChIP-Seq datasets to refine the current TFBS models stored in the TRANSFAC database. Experiments on both the simulated and real ChIP-Seq data show that the proposed method starting from existing PWM has consistently better performance than existing tools in detecting the TFBSs. The improved accuracy is the result of modelling the complete dependent structure of the motifs and better prediction of true positive rate. The findings could lead to better understanding of the mechanisms of TF-DNA interactions.

Correct interactions between transcription factors (TFs) and their binding sites (TFBSs) are of central importance to gene regulation. Recently developed chromatin-immunoprecipitation DNA chip (ChIP-chip) techniques and the phylogenetic footprinting method provide ways to identify TFBSs with high precision. In this study, we constructed a user-friendly interactive platform for dynamic binding site mapping using ChIP-chip data and phylogenetic footprinting as two filters. MYBS (Mining Yeast Binding Sites) is a comprehensive web server that integrates an array of both experimentally verified and predicted position weight matrixes (PWMs) from eleven databases, including 481 binding motif consensus sequences and 71 PWMs that correspond to 183 TFs. MYBS users can search within this platform for motif occurrences (possible binding sites) in the promoters of genes of interest via simple motif or gene queries in conjunction with the above two filters. In addition, MYBS enables users to visualize in parallel the potential regulators for a given set of genes, a feature useful for finding potential regulatory associations between TFs. MYBS also allows users to identify target gene sets of each TF pair, which could be used as a starting point for further explorations of TF combinatorial regulation. MYBS is available at http://cg1.iis.sinica.edu.tw/~mybs/.

By binding to short and highly conserved DNA sequences in genomes, DNA-binding proteins initiate, enhance or repress biological processes. Accurately identifying such binding sites, often represented by position weight matrices (PWMs), is an important step in understanding the control mechanisms of cells. When given coordinates of a DNA-binding domain (DBD) bound with DNA, a potential function can be used to estimate the change of binding affinity after base substitutions, where the changes can be summarized as a PWM. This technique provides an effective alternative when the chromatin immunoprecipitation data are unavailable for PWM inference. To facilitate the procedure of predicting PWMs based on protein–DNA complexes or even structures of the unbound state, the web server, DBD2BS, is presented in this study. The DBD2BS uses an atom-level knowledge-based potential function to predict PWMs characterizing the sequences to which the query DBD structure can bind. For unbound queries, a list of 1066 DBD–DNA complexes (including 1813 protein chains) is compiled for use as templates for synthesizing bound structures. The DBD2BS provides users with an easy-to-use interface for visualizing the PWMs predicted based on different templates and the spatial relationships of the query protein, the DBDs and the DNAs. The DBD2BS is the first attempt to predict PWMs of DBDs from unbound structures rather than from bound ones. This approach increases the number of existing protein structures that can be exploited when analyzing protein–DNA interactions. In a recent study, the authors showed that the kernel adopted by the DBD2BS can generate PWMs consistent with those obtained from the experimental data. The use of DBD2BS to predict PWMs can be incorporated with sequence-based methods to discover binding sites in genome-wide studies.

Available at: http://dbd2bs.csie.ntu.edu.tw/, http://dbd2bs.csbb.ntu.edu.tw/, and http://dbd2bs.ee.ncku.edu.tw.

Background

Development of high-throughput methods for measuring DNA interactions of transcription factors together with computational advances in short motif inference algorithms is expanding our understanding of transcription factor binding site motifs. The consequential growth of sequence motif data sets makes it important to systematically group and categorise regulatory motifs. It has been shown that there are familial tendencies in DNA sequence motifs that are predictive of the family of factors that binds them. Further development of methods that detect and describe familial motif trends has the potential to help in measuring the similarity of novel computational motif predictions to previously known data and sensitively detecting regulatory motifs similar to previously known ones from novel sequence.

Results

We propose a probabilistic model for position weight matrix (PWM) sequence motif families. The model, which we call the 'metamotif' describes recurring familial patterns in a set of motifs. The metamotif framework models variation within a family of sequence motifs. It allows for simultaneous estimation of a series of independent metamotifs from input position weight matrix (PWM) motif data and does not assume that all input motif columns contribute to a familial pattern. We describe an algorithm for inferring metamotifs from weight matrix data. We then demonstrate the use of the model in two practical tasks: in the Bayesian NestedMICA model inference algorithm as a PWM prior to enhance motif inference sensitivity, and in a motif classification task where motifs are labelled according to their interacting DNA binding domain.

Conclusions

We show that metamotifs can be used as PWM priors in the NestedMICA motif inference algorithm to dramatically increase the sensitivity to infer motifs. Metamotifs were also successfully applied to a motif classification problem where sequence motif features were used to predict the family of protein DNA binding domains that would interact with it. The metamotif based classifier is shown to compare favourably to previous related methods. The metamotif has great potential for further use in machine learning tasks related to especially de novo computational sequence motif inference. The metamotif methods presented have been incorporated into the NestedMICA suite.

Accurate prediction of transcription factor binding sites (TFBSs) is a prerequisite for identifying cis-regulatory modules that underlie transcriptional regulatory circuits encoded in the genome. Here, we present a computational framework for detecting TFBSs, when multiple position weight matrices (PWMs) for a transcription factor are available. Grouping multiple PWMs of a transcription factor (TF) based on their sequence similarity improves the specificity of TFBS prediction, which was evaluated using multiple genome-wide ChIP-Seq data sets from 26 TFs. The Z-scores of the area under a receiver operating characteristic curve (AUC) values of 368 TFs were calculated and used to statistically identify co-occurring regulatory motifs in the TF bound ChIP loci. Motifs that are co-occurring along with the empirical bindings of E2F, JUN or MYC have been evaluated, in the basal or stimulated condition. Results prove our method can be useful to systematically identify the co-occurring motifs of the TF for the given conditions.

Scanning through genomes for potential transcription factor binding sites (TFBSs) is becoming increasingly important in this post-genomic era. The position weight matrix (PWM) is the standard representation of TFBSs utilized when scanning through sequences for potential binding sites. However, many transcription factor (TF) motifs are short and highly degenerate, and methods utilizing PWMs to scan for sites are plagued by false positives. Furthermore, many important TFs do not have well-characterized PWMs, making identification of potential binding sites even more difficult. One approach to the identification of sites for these TFs has been to use the 3D structure of the TF to predict the DNA structure around the TF and then to generate a PWM from the predicted 3D complex structure. However, this approach is dependent on the similarity of the predicted structure to the native structure. We introduce here a novel approach to identify TFBSs utilizing structure information that can be applied to TFs without characterized PWMs, as long as a 3D complex structure (TF/DNA) exists. This approach utilizes an energy function that is uniquely trained on each structure. Our approach leads to increased prediction accuracy and robustness compared with those using a more general energy function. The software is freely available upon request.

Direct binding by a transcription factor (TF) to the proximal promoter of a gene is a strong evidence that the TF regulates the gene. Assaying the genome-wide binding of every TF in every cell type and condition is currently impractical. Histone modifications correlate with tissue/cell/condition-specific (‘tissue specific’) TF binding, so histone ChIP-seq data can be combined with traditional position weight matrix (PWM) methods to make tissue-specific predictions of TF–promoter interactions.

Results: We use supervised learning to train a naïve Bayes predictor of TF–promoter binding. The predictor's features are the histone modification levels and a PWM-based score for the promoter. Training and testing uses sets of promoters labeled using TF ChIP-seq data, and we use cross-validation on 23 such datasets to measure the accuracy. A PWM+histone naïve Bayes predictor using a single histone modification (H3K4me3) is substantially more accurate than a PWM score or a conservation-based score (phylogenetic motif model). The naïve Bayes predictor is more accurate (on average) at all sensitivity levels, and makes only half as many false positive predictions at sensitivity levels from 10% to 80%. On average, it correctly predicts 80% of bound promoters at a false positive rate of 20%. Accuracy does not diminish when we test the predictor in a different cell type (and species) from training. Accuracy is barely diminished even when we train the predictor without using TF ChIP-seq data.

Availability: Our tissue-specific predictor of promoters bound by a TF is called Dr Gene and is available at http://bioinformatics.org.au/drgene.

Contact: t.bailey@imb.uq.edu.au

Supplementary information: Supplementary data are available at Bioinformatics online.

Networks of regulatory relations between transcription factors (TF) and their target genes (TG)- implemented through TF binding sites (TFBS)- are key features of biology. An idealized approach to solving such networks consists of starting from a consensus TFBS or a position weight matrix (PWM) to generate a high accuracy list of candidate TGs for biological validation. Developing and evaluating such approaches remains a formidable challenge in regulatory bioinformatics. We perform a benchmark study on 34 Drosophila TFs to assess existing TFBS and cis-regulatory module (CRM) detection methods, with a strong focus on the use of multiple genomes. Particularly, for CRM-modelling we investigate the addition of orthologous sites to a known PWM to construct phyloPWMs and we assess the added value of phylogenentic footprinting to predict contextual motifs around known TFBSs. For CRM-prediction, we compare motif conservation with network-level conservation approaches across multiple genomes. Choosing the optimal training and scoring strategies strongly enhances the performance of TG prediction for more than half of the tested TFs. Finally, we analyse a 35th TF, namely Eyeless, and find a significant overlap between predicted TGs and candidate TGs identified by microarray expression studies. In summary we identify several ways to optimize TF-specific TG predictions, some of which can be applied to all TFs, and others that can be applied only to particular TFs. The ability to model known TF-TG relations, together with the use of multiple genomes, results in a significant step forward in solving the architecture of gene regulatory networks.

Single nucleotide polymorphisms (SNPs) in transcription factor binding sites (TFBSs) may affect the binding of transcription factors, lead to differences in gene expression and phenotypes, and therefore affect susceptibility to environmental exposure. We developed an integrated computational system for discovering functional SNPs in TFBSs in the human genome and predicting their impact on the expression of target genes. In this system we: (1) construct a position weight matrix (PWM) from a collection of experimentally discovered TFBSs; (2) predict TFBSs in SNP sequences using the PWM and map SNPs to the upstream regions of genes; (3) examine the evolutionary conservation of putative TFBSs by phylogenetic footprinting; (4) prioritize candidate SNPs based on microarray expression profiles from tissues in which the transcription factor of interest is either deleted or over-expressed; and (5) finally, analyze association of SNP genotypes with gene expression phenotypes. The application of our system has been tested to identify functional polymorphisms in the antioxidant response element (ARE), a cis-acting enhancer sequence found in the promoter region of many genes that encode antioxidant and Phase II detoxification enzymes/proteins. In response to oxidative stress, the transcription factor NRF2 (nuclear factor erythroid-derived 2-like 2) binds to AREs, mediating transcriptional activation of its responsive genes and modulating in vivo defense mechanisms against oxidative damage. Using our novel computational tools, we have identified a set of polymorphic AREs with functional evidence, showing the utility of our system to direct further experimental validation of genomic sequence variations that could be useful for identifying high-risk individuals.

Computational identification of transcription factor binding sites is an important research area of computational biology. Positional weight matrix (PWM) is a model to describe the sequence pattern of binding sites. Usually, transcription factor binding sites prediction methods based on PWMs require user-defined thresholds. The arbitrary threshold and also the relatively low specificity of the algorithm prevent the result of such an analysis from being properly interpreted. In this study, a method was developed to identify over-represented cis-elements with PWM-based similarity scores. Three sets of closely related promoters were analyzed, and only over- represented motifs with high PWM similarity scores were reported. The thresholds to evaluate the similarity scores to the PWMs of putative transcription factors binding sites can also be automatically determined during the analysis, which can also be used in further research with the same PWMs. The online program is available on the website: http://www.bioinfo.tsinghua.edu.cn/∼zhengjsh/OTFBS/.

DNA-binding proteins such as transcription factors use DNA-binding domains (DBDs) to bind to specific sequences in the genome to initiate many important biological functions. Accurate prediction of such target sequences, often represented by position weight matrices (PWMs), is an important step to understand many biological processes. Recent studies have shown that knowledge-based potential functions can be applied on protein-DNA co-crystallized structures to generate PWMs that are considerably consistent with experimental data. However, this success has not been extended to DNA-binding proteins lacking co-crystallized structures. This study aims at investigating the possibility of predicting the DNA sequences bound by DNA-binding proteins from the proteins' unbound structures (structures of the unbound state). Given an unbound query protein and a template complex, the proposed method first employs structure alignment to generate synthetic protein-DNA complexes for the query protein. Once a complex is available, an atomic-level knowledge-based potential function is employed to predict PWMs characterizing the sequences to which the query protein can bind. The evaluation of the proposed method is based on seven DNA-binding proteins, which have structures of both DNA-bound and unbound forms for prediction as well as annotated PWMs for validation. Since this work is the first attempt to predict target sequences of DNA-binding proteins from their unbound structures, three types of structural variations that presumably influence the prediction accuracy were examined and discussed. Based on the analyses conducted in this study, the conformational change of proteins upon binding DNA was shown to be the key factor. This study sheds light on the challenge of predicting the target DNA sequences of a protein lacking co-crystallized structures, which encourages more efforts on the structure alignment-based approaches in addition to docking- and homology modeling-based approaches for generating synthetic complexes.

Transcription factor-DNA interactions, central to cellular regulation and control, are commonly described by position weight matrices (PWMs). These matrices are frequently used to predict transcription factor binding sites in regulatory regions of DNA to complement and guide further experimental investigation. The DNA sequence preferences of transcription factors, encoded in PWMs, are dictated primarily by select residues within the DNA binding domain(s) that interact directly with DNA. Therefore, the DNA binding properties of homologous transcription factors with identical DNA binding domains may be characterized by PWMs derived from different species. Accordingly, we have implemented a fully automated domain-level homology searching method for identical DNA binding sequences.

By applying the domain-level homology search to transcription factors with existing PWMs in the JASPAR and TRANSFAC databases, we were able to significantly increase coverage in terms of the total number of PWMs associated with a given species, assign PWMs to transcription factors that did not previously have any associations, and increase the number of represented species with PWMs over an order of magnitude. Additionally, using protein binding microarray (PBM) data, we have validated the domain-level method by demonstrating that transcription factor pairs with matching DNA binding domains exhibit comparable DNA binding specificity predictions to transcription factor pairs with completely identical sequences.

The increased coverage achieved herein demonstrates the potential for more thorough species-associated investigation of protein-DNA interactions using existing resources. The PWM scanning results highlight the challenging nature of transcription factors that contain multiple DNA binding domains, as well as the impact of motif discovery on the ability to predict DNA binding properties. The method is additionally suitable for identifying domain-level homology mappings to enable utilization of additional information sources in the study of transcription factors. The domain-level homology search method, resulting PWM mappings, web-based user interface, and web API are publicly available at http://dodoma.systemsbiology.netdodoma.systemsbiology.net.

Predicting how and where proteins, especially transcription factors (TFs), interact with DNA is an important problem in biology. We present here a systematic study of predictive modeling approaches to the TF–DNA binding problem, which have been frequently shown to be more efficient than those methods only based on position-specific weight matrices (PWMs). In these approaches, a statistical relationship between genomic sequences and gene expression or ChIP-binding intensities is inferred through a regression framework; and influential sequence features are identified by variable selection. We examine a few state-of-the-art learning methods including stepwise linear regression, multivariate adaptive regression splines, neural networks, support vector machines, boosting and Bayesian additive regression trees (BART). These methods are applied to both simulated datasets and two whole-genome ChIP-chip datasets on the TFs Oct4 and Sox2, respectively, in human embryonic stem cells. We find that, with proper learning methods, predictive modeling approaches can significantly improve the predictive power and identify more biologically interesting features, such as TF–TF interactions, than the PWM approach. In particular, BART and boosting show the best and the most robust overall performance among all the methods.

Background

The identifying of binding sites for transcription factors is a key component of gene regulatory network analysis. This is often done using position-weight matrices (PWMs). Because of the importance of in silico mapping of tentative binding sites, we previously developed an approach for PWM optimization that substantially improves the accuracy of such mapping.

Results

The present work implements the optimization algorithm applied to the existing PWM for GATA-3 transcription factor and builds a new di-nucleotide PWM. The existing available PWM is based on experimental data adopted from Jaspar. The optimized PWM substantially improves the sensitivity and specificity of the TF mapping compared to the conventional applications. The refined PWM also facilitates in silico identification of novel binding sites that are supported by experimental data. We also describe uncommon positioning of binding motifs for several T-cell lineage specific factors in human promoters.

Conclusion

Our proposed di-nucleotide PWM approach outperforms the conventional mono-nucleotide PWM approach with respect to GATA-3. Therefore our new di-nucleotide PWM provides new insight into plausible transcriptional regulatory interactions in human promoters.

Typical approaches for predicting transcription factor binding sites (TFBSs) involve use of a position-specific weight matrix (PWM) to statistically characterize the sequences of the known sites. Recently, an alternative physicochemical approach, called SiteSleuth, was proposed. In this approach, a linear support vector machine (SVM) classifier is trained to distinguish TFBSs from background sequences based on local chemical and structural features of DNA. SiteSleuth appears to generally perform better than PWM-based methods. Here, we improve the SiteSleuth approach by considering both new physicochemical features and algorithmic modifications. New features are derived from Gibbs energies of amino acid–DNA interactions and hydroxyl radical cleavage profiles of DNA. Algorithmic modifications consist of inclusion of a feature selection step, use of a nonlinear kernel in the SVM classifier, and use of a consensus-based post-processing step for predictions. We also considered SVM classification based on letter features alone to distinguish performance gains from use of SVM-based models versus use of physicochemical features. The accuracy of each of the variant methods considered was assessed by cross validation using data available in the RegulonDB database for 54 Escherichia coli TFs, as well as by experimental validation using published ChIP-chip data available for Fis and Lrp.

Background

An important step in annotation of sequenced genomes is the identification of transcription factor binding sites. More than a hundred different computational methods have been proposed, and it is difficult to make an informed choice. Therefore, robust assessment of motif discovery methods becomes important, both for validation of existing tools and for identification of promising directions for future research.

Results

We use a machine learning perspective to analyze collections of transcription factors with known binding sites. Algorithms are presented for finding position weight matrices (PWMs), IUPAC-type motifs and mismatch motifs with optimal discrimination of binding sites from remaining sequence. We show that for many data sets in a recently proposed benchmark suite for motif discovery, none of the common motif models can accurately discriminate the binding sites from remaining sequence. This may obscure the distinction between the potential performance of the motif discovery tool itself versus the intrinsic complexity of the problem we are trying to solve. Synthetic data sets may avoid this problem, but we show on some previously proposed benchmarks that there may be a strong bias towards a presupposed motif model. We also propose a new approach to benchmark data set construction. This approach is based on collections of binding site fragments that are ranked according to the optimal level of discrimination achieved with our algorithms. This allows us to select subsets with specific properties. We present one benchmark suite with data sets that allow good discrimination between positive and negative instances with the common motif models. These data sets are suitable for evaluating algorithms for motif discovery that rely on these models. We present another benchmark suite where PWM, IUPAC and mismatch motif models are not able to discriminate reliably between positive and negative instances. This suite could be used for evaluating more powerful motif models.

Conclusion

Our improved benchmark suites have been designed to differentiate between the performance of motif discovery algorithms and the power of motif models. We provide a web server where users can download our benchmark suites, submit predictions and visualize scores on the benchmarks.

Identification of DNA motifs from ChIP-seq/ChIP-chip [chromatin immunoprecipitation (ChIP)] data is a powerful method for understanding the transcriptional regulatory network. However, most established methods are designed for small sample sizes and are inefficient for ChIP data. Here we propose a new k-mer occurrence model to reflect the fact that functional DNA k-mers often cluster around ChIP peak summits. With this model, we introduced a new measure to discover functional k-mers. Using simulation, we demonstrated that our method is more robust against noises in ChIP data than available methods. A novel word clustering method is also implemented to group similar k-mers into position weight matrices (PWMs). Our method was applied to a diverse set of ChIP experiments to demonstrate its high sensitivity and specificity. Importantly, our method is much faster than several other methods for large sample sizes. Thus, we have developed an efficient and effective motif discovery method for ChIP experiments.

Genome-wide analyses of protein binding sites generate large amounts of data; a ChIP dataset might contain 10,000 sites. Unbiased motif discovery in such datasets is not generally feasible using current methods that employ probabilistic models. We propose an efficient method, GADEM, which combines spaced dyads and an expectation-maximization (EM) algorithm. Candidate words (four to six nucleotides) for constructing spaced dyads are prioritized by their degree of overrepresentation in the input sequence data. Spaced dyads are converted into starting position weight matrices (PWMs). GADEM then employs a genetic algorithm (GA), with an embedded EM algorithm to improve starting PWMs, to guide the evolution of a population of spaced dyads toward one whose entropy scores are more statistically significant. Spaced dyads whose entropy scores reach a pre-specified significance threshold are declared motifs. GADEM performed comparably with MEME on 500 sets of simulated “ChIP” sequences with embedded known P53 binding sites. The major advantage of GADEM is its computational efficiency on large ChIP datasets compared to competitors. We applied GADEM to six genome-wide ChIP datasets. Approximately, 15 to 30 motifs of various lengths were identified in each dataset. Remarkably, without any prior motif information, the expected known motif (e.g., P53 in P53 data) was identified every time. GADEM discovered motifs of various lengths (6–40 bp) and characteristics in these datasets containing from 0.5 to >13 million nucleotides with run times of 5 to 96 h. GADEM can be viewed as an extension of the well-known MEME algorithm and is an efficient tool for de novo motif discovery in large-scale genome-wide data. The GADEM software is available at http://www.niehs.nih.gov/research/resources/software/GADEM/.

Abstract

Genome-wide analyses of protein binding sites generate large amounts of data; a ChIP dataset might contain 10,000 sites. Unbiased motif discovery in such datasets is not generally feasible using current methods that employ probabilistic models. We propose an efficient method, gadem, which combines spaced dyads and an expectation-maximization (EM) algorithm. Candidate words (four to six nucleotides) for constructing spaced dyads are prioritized by their degree of overrepresentation in the input sequence data. Spaced dyads are converted into starting position weight matrices (PWMs). gadem then employs a genetic algorithm (GA), with an embedded EM algorithm to improve starting PWMs, to guide the evolution of a population of spaced dyads toward one whose entropy scores are more statistically significant. Spaced dyads whose entropy scores reach a pre-specified significance threshold are declared motifs. gadem performed comparably with meme on 500 sets of simulated “ChIP” sequences with embedded known P53 binding sites. The major advantage of gadem is its computational efficiency on large ChIP datasets compared to competitors. We applied gadem to six genome-wide ChIP datasets. Approximately, 15 to 30 motifs of various lengths were identified in each dataset. Remarkably, without any prior motif information, the expected known motif (e.g., P53 in P53 data) was identified every time. gadem discovered motifs of various lengths (6–40 bp) and characteristics in these datasets containing from 0.5 to >13 million nucleotides with run times of 5 to 96 h. gadem can be viewed as an extension of the well-known meme algorithm and is an efficient tool for de novo motif discovery in large-scale genome-wide data. The gadem software is available at www.niehs.nih.gov/research/resources/software/GADEM/.

We identified binding sites for Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) in the human genome using chromatin immunoprecipitation and microarrays. The sequences for these newly identified sites were used to generate a position-weighted matrix (PWM) for EBNA1's DNA-binding sites. This PWM helped identify additional DNA-binding sites for EBNA1 in the genomes of EBV, Kaposi's sarcoma-associated herpesvirus, and cercopithecine herpesvirus 15 (CeHV-15) (also called herpesvirus papio 15). In particular, a homologue of the Rep* locus in EBV was predicted in the genome of CeHV-15, which is notable because Rep* of EBV was not predicted by the previously developed consensus sequence for EBNA1's binding DNA. The Rep* of CeHV-15 functions as an origin of DNA synthesis in the EBV-positive cell line Raji; this finding thus builds on a set of DNA-binding sites for EBNA1 predicted in silico.

Background

Identifying transcription factor binding sites (TFBS) in silico is key in understanding gene regulation. TFBS are string patterns that exhibit some variability, commonly modelled as “position weight matrices” (PWMs). Though convenient, the PWM has significant limitations, in particular the assumed independence of positions within the binding motif; and predictions based on PWMs are usually not very specific to known functional sites. Analysis here on binding sites in yeast suggests that correlation of dinucleotides is not limited to near-neighbours, but can extend over considerable gaps.

Methodology/Principal Findings

I describe a straightforward generalization of the PWM model, that considers frequencies of dinucleotides instead of individual nucleotides. Unlike previous efforts, this method considers all dinucleotides within an extended binding region, and does not make an attempt to determine a priori the significance of particular dinucleotide correlations. I describe how to use a “dinucleotide weight matrix” (DWM) to predict binding sites, dealing in particular with the complication that its entries are not independent probabilities. Benchmarks show, for many factors, a dramatic improvement over PWMs in precision of predicting known targets. In most cases, significant further improvement arises by extending the commonly defined “core motifs” by about 10bp on either side. Though this flanking sequence shows no strong motif at the nucleotide level, the predictive power of the dinucleotide model suggests that the “signature” in DNA sequence of protein-binding affinity extends beyond the core protein-DNA contact region.

Conclusion/Significance

While computationally more demanding and slower than PWM-based approaches, this dinucleotide method is straightforward, both conceptually and in implementation, and can serve as a basis for future improvements.

Motivation: Genome-wide mRNA profiling provides a snapshot of the global state of cells under different conditions. However, mRNA levels do not provide direct understanding of upstream regulatory mechanisms. Here, we present a new approach called Expression2Kinases (X2K) to identify upstream regulators likely responsible for observed patterns in genome-wide gene expression. By integrating chromatin immuno-precipitation (ChIP)-seq/chip and position weight matrices (PWMs) data, protein–protein interactions and kinase–substrate phosphorylation reactions, we can better identify regulatory mechanisms upstream of genome-wide differences in gene expression. We validated X2K by applying it to recover drug targets of food and drug administration (FDA)-approved drugs from drug perturbations followed by mRNA expression profiling; to map the regulatory landscape of 44 stem cells and their differentiating progeny; to profile upstream regulatory mechanisms of 327 breast cancer tumors; and to detect pathways from profiled hepatic stellate cells and hippocampal neurons. The X2K approach can advance our understanding of cell signaling and unravel drugs mechanisms of action.

Availability: The software and source code are freely available at: http://www.maayanlab.net/X2K.

Contact: avi.maayan@mssm.edu

Supplementary information: Supplementary data are available at Bioinformatics online.

Motivation: Transcription factors (TFs) are crucial during the lifetime of the cell. Their functional roles are defined by the genes they regulate. Uncovering these roles not only sheds light on the TF at hand but puts it into the context of the complete regulatory network.

Results: Here, we present an alignment- and threshold-free comparative genomics approach for assigning functional roles to DNA regulatory motifs. We incorporate our approach into the Gomo algorithm, a computational tool for detecting associations between a user-specified DNA regulatory motif [expressed as a position weight matrix (PWM)] and Gene Ontology (GO) terms. Incorporating multiple species into the analysis significantly improves Gomo's ability to identify GO terms associated with the regulatory targets of TFs. Including three comparative species in the process of predicting TF roles in Saccharomyces cerevisiae and Homo sapiens increases the number of significant predictions by 75 and 200%, respectively. The predicted GO terms are also more specific, yielding deeper biological insight into the role of the TF. Adjusting motif (binding) affinity scores for individual sequence composition proves to be essential for avoiding false positive associations. We describe a novel DNA sequence-scoring algorithm that compensates a thermodynamic measure of DNA-binding affinity for individual sequence base composition. Gomo's prediction accuracy proves to be relatively insensitive to how promoters are defined. Because Gomo uses a threshold-free form of gene set analysis, there are no free parameters to tune. Biologists can investigate the potential roles of DNA regulatory motifs of interest using Gomo via the web (http://meme.nbcr.net).

Contact: t.bailey@uq.edu.au

Supplementary information: Supplementary data are available at Bioinformatics online.

We present the webserver 3D transcription factor (3DTF) to compute position-specific weight matrices (PWMs) of transcription factors using a knowledge-based statistical potential derived from crystallographic data on protein–DNA complexes. Analysis of available structures that can be used to construct PWMs shows that there are hundreds of 3D structures from which PWMs could be derived, as well as thousands of proteins homologous to these. Therefore, we created 3DTF, which delivers binding matrices given the experimental or modeled protein–DNA complex. The webserver can be used by biologists to derive novel PWMs for transcription factors lacking known binding sites and is freely accessible at http://www.gene-regulation.com/pub/programs/3dtf/.

Background

Knowledge of transcription factor-DNA binding patterns is crucial for understanding gene transcription. Numerous DNA-binding proteins are annotated as transcription factors in the literature, however, for many of them the corresponding DNA-binding motifs remain uncharacterized.

Results

The position weight matrices (PWMs) of transcription factors from different structural classes have been determined using a knowledge-based statistical potential. The scoring function calibrated against crystallographic data on protein-DNA contacts recovered PWMs of various members of widely studied transcription factor families such as p53 and NF-κB. Where it was possible, extensive comparison to experimental binding affinity data and other physical models was made. Although the p50p50, p50RelB, and p50p65 dimers belong to the same family, particular differences in their PWMs were detected, thereby suggesting possibly different in vivo binding modes. The PWMs of p63 and p73 were computed on the basis of homology modeling and their performance was studied using upstream sequences of 85 p53/p73-regulated human genes. Interestingly, about half of the p63 and p73 hits reported by the Match algorithm in the altogether 126 promoters lay more than 2 kb upstream of the corresponding transcription start sites, which deviates from the common assumption that most regulatory sites are located more proximal to the TSS. The fact that in most of the cases the binding sites of p63 and p73 did not overlap with the p53 sites suggests that p63 and p73 could influence the p53 transcriptional activity cooperatively. The newly computed p50p50 PWM recovered 5 more experimental binding sites than the corresponding TRANSFAC matrix, while both PWMs showed comparable receiver operator characteristics.

Conclusions

A novel algorithm was developed to calculate position weight matrices from protein-DNA complex structures. The proposed algorithm was extensively validated against experimental data. The method was further combined with Homology Modeling to obtain PWMs of factors for which crystallographic complexes with DNA are not yet available. The performance of PWMs obtained in this work in comparison to traditionally constructed matrices demonstrates that the structure-based approach presents a promising alternative to experimental determination of transcription factor binding properties.