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1.  Optimizing the GATA-3 position weight matrix to improve the identification of novel binding sites 
BMC Genomics  2012;13:416.
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.
doi:10.1186/1471-2164-13-416
PMCID: PMC3481455  PMID: 22913572
Transcription factor; Binding sites; GATA-3; Human promoter; Position weight matrix; Optimization
2.  Optimized Position Weight Matrices in Prediction of Novel Putative Binding Sites for Transcription Factors in the Drosophila melanogaster Genome 
PLoS ONE  2013;8(8):e68712.
Position weight matrices (PWMs) have become a tool of choice for the identification of transcription factor binding sites in DNA sequences. DNA-binding proteins often show degeneracy in their binding requirement and thus the overall binding specificity of many proteins is unknown and remains an active area of research. Although existing PWMs are more reliable predictors than consensus string matching, they generally result in a high number of false positive hits. Our previous study introduced a promising approach to PWM refinement in which known motifs are used to computationally mine putative binding sites directly from aligned promoter regions using composition of similar sites. In the present study, we extended this technique originally tested on single examples of transcription factors (TFs) and showed its capability to optimize PWM performance to predict new binding sites in the fruit fly genome. We propose refined PWMs in mono- and dinucleotide versions similarly computed for a large variety of transcription factors of Drosophila melanogaster. Along with the addition of many auxiliary sites the optimization includes variation of the PWM motif length, the binding sites location on the promoters and the PWM score threshold. To assess the predictive performance of the refined PWMs we compared them to conventional TRANSFAC and JASPAR sources. The results have been verified using performed tests and literature review. Overall, the refined PWMs containing putative sites derived from real promoter content processed using optimized parameters had better general accuracy than conventional PWMs.
doi:10.1371/journal.pone.0068712
PMCID: PMC3735551  PMID: 23936309
3.  DBD2BS: connecting a DNA-binding protein with its binding sites 
Nucleic Acids Research  2012;40(Web Server issue):W173-W179.
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.
doi:10.1093/nar/gks564
PMCID: PMC3394304  PMID: 22693214
4.  3DTF: a web server for predicting transcription factor PWMs using 3D structure-based energy calculations 
Nucleic Acids Research  2012;40(Web Server issue):W180-W185.
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/.
doi:10.1093/nar/gks551
PMCID: PMC3394331  PMID: 22693215
5.  Dinucleotide Weight Matrices for Predicting Transcription Factor Binding Sites: Generalizing the Position Weight Matrix 
PLoS ONE  2010;5(3):e9722.
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.
doi:10.1371/journal.pone.0009722
PMCID: PMC2842295  PMID: 20339533
6.  The XXmotif web server for eXhaustive, weight matriX-based motif discovery in nucleotide sequences 
Nucleic Acids Research  2012;40(Web Server issue):W104-W109.
The discovery of regulatory motifs enriched in sets of DNA or RNA sequences is fundamental to the analysis of a great variety of functional genomics experiments. These motifs usually represent binding sites of proteins or non-coding RNAs, which are best described by position weight matrices (PWMs). We have recently developed XXmotif, a de novo motif discovery method that is able to directly optimize the statistical significance of PWMs. XXmotif can also score conservation and positional clustering of motifs. The XXmotif server provides (i) a list of significantly overrepresented motif PWMs with web logos and E-values; (ii) a graph with color-coded boxes indicating the positions of selected motifs in the input sequences; (iii) a histogram of the overall positional distribution for selected motifs and (iv) a page for each motif with all significant motif occurrences, their P-values for enrichment, conservation and localization, their sequence contexts and coordinates. Free access: http://xxmotif.genzentrum.lmu.de.
doi:10.1093/nar/gks602
PMCID: PMC3394272  PMID: 22693218
7.  Position Weight Matrix, Gibbs Sampler, and the Associated Significance Tests in Motif Characterization and Prediction 
Scientifica  2012;2012:917540.
Position weight matrix (PWM) is not only one of the most widely used bioinformatic methods, but also a key component in more advanced computational algorithms (e.g., Gibbs sampler) for characterizing and discovering motifs in nucleotide or amino acid sequences. However, few generally applicable statistical tests are available for evaluating the significance of site patterns, PWM, and PWM scores (PWMS) of putative motifs. Statistical significance tests of the PWM output, that is, site-specific frequencies, PWM itself, and PWMS, are in disparate sources and have never been collected in a single paper, with the consequence that many implementations of PWM do not include any significance test. Here I review PWM-based methods used in motif characterization and prediction (including a detailed illustration of the Gibbs sampler for de novo motif discovery), present statistical and probabilistic rationales behind statistical significance tests relevant to PWM, and illustrate their application with real data. The multiple comparison problem associated with the test of site-specific frequencies is best handled by false discovery rate methods. The test of PWM, due to the use of pseudocounts, is best done by resampling methods. The test of individual PWMS for each sequence segment should be based on the extreme value distribution.
doi:10.6064/2012/917540
PMCID: PMC3820676  PMID: 24278755
8.  Increasing Coverage of Transcription Factor Position Weight Matrices through Domain-level Homology 
PLoS ONE  2012;7(8):e42779.
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.
doi:10.1371/journal.pone.0042779
PMCID: PMC3428306  PMID: 22952610
9.  An approach to identify over-represented cis-elements in related sequences 
Nucleic Acids Research  2003;31(7):1995-2005.
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/.
PMCID: PMC152803  PMID: 12655017
10.  A Bayesian Search for Transcriptional Motifs 
PLoS ONE  2010;5(11):e13897.
Identifying transcription factor (TF) binding sites (TFBSs) is an important step towards understanding transcriptional regulation. A common approach is to use gaplessly aligned, experimentally supported TFBSs for a particular TF, and algorithmically search for more occurrences of the same TFBSs. The largest publicly available databases of TF binding specificities contain models which are represented as position weight matrices (PWM). There are other methods using more sophisticated representations, but these have more limited databases, or aren't publicly available. Therefore, this paper focuses on methods that search using one PWM per TF. An algorithm, MATCHTM, for identifying TFBSs corresponding to a particular PWM is available, but is not based on a rigorous statistical model of TF binding, making it difficult to interpret or adjust the parameters and output of the algorithm. Furthermore, there is no public description of the algorithm sufficient to exactly reproduce it. Another algorithm, MAST, computes a p-value for the presence of a TFBS using true probabilities of finding each base at each offset from that position. We developed a statistical model, BaSeTraM, for the binding of TFs to TFBSs, taking into account random variation in the base present at each position within a TFBS. Treating the counts in the matrices and the sequences of sites as random variables, we combine this TFBS composition model with a background model to obtain a Bayesian classifier. We implemented our classifier in a package (SBaSeTraM). We tested SBaSeTraM against a MATCHTM implementation by searching all probes used in an experimental Saccharomyces cerevisiae TF binding dataset, and comparing our predictions to the data. We found no statistically significant differences in sensitivity between the algorithms (at fixed selectivity), indicating that SBaSeTraM's performance is at least comparable to the leading currently available algorithm. Our software is freely available at: http://wiki.github.com/A1kmm/sbasetram/building-the-tools.
doi:10.1371/journal.pone.0013897
PMCID: PMC2987817  PMID: 21124986
11.  Jaccard index based similarity measure to compare transcription factor binding site models 
Background
Positional weight matrix (PWM) remains the most popular for quantification of transcription factor (TF) binding. PWM supplied with a score threshold defines a set of putative transcription factor binding sites (TFBS), thus providing a TFBS model.
TF binding DNA fragments obtained by different experimental methods usually give similar but not identical PWMs. This is also common for different TFs from the same structural family. Thus it is often necessary to measure the similarity between PWMs. The popular tools compare PWMs directly using matrix elements. Yet, for log-odds PWMs, negative elements do not contribute to the scores of highly scoring TFBS and thus may be different without affecting the sets of the best recognized binding sites. Moreover, the two TFBS sets recognized by a given pair of PWMs can be more or less different depending on the score thresholds.
Results
We propose a practical approach for comparing two TFBS models, each consisting of a PWM and the respective scoring threshold. The proposed measure is a variant of the Jaccard index between two TFBS sets. The measure defines a metric space for TFBS models of all finite lengths. The algorithm can compare TFBS models constructed using substantially different approaches, like PWMs with raw positional counts and log-odds. We present the efficient software implementation: MACRO-APE (MAtrix CompaRisOn by Approximate P-value Estimation).
Conclusions
MACRO-APE can be effectively used to compute the Jaccard index based similarity for two TFBS models. A two-pass scanning algorithm is presented to scan a given collection of PWMs for PWMs similar to a given query.
Availability and implementation
MACRO-APE is implemented in ruby 1.9; software including source code and a manual is freely available at http://autosome.ru/macroape/ and in supplementary materials.
doi:10.1186/1748-7188-8-23
PMCID: PMC3851813  PMID: 24074225
Transcription factor binding site; TFBS; Transcription factor binding site model; Binding motif; Jaccard similarity; Position weight matrix; PWM; P-value; Position specific frequency matrix; PSFM; Macroape
12.  Measuring similarities between transcription factor binding sites 
BMC Bioinformatics  2005;6:237.
Background
Collections of transcription factor binding profiles (Transfac, Jaspar) are essential to identify regulatory elements in DNA sequences. Subsets of highly similar profiles complicate large scale analysis of transcription factor binding sites.
Results
We propose to identify and group similar profiles using two independent similarity measures: χ2 distances between position frequency matrices (PFMs) and correlation coefficients between position weight matrices (PWMs) scores.
Conclusion
We show that these measures complement each other and allow to associate Jaspar and Transfac matrices. Clusters of highly similar matrices are identified and can be used to optimise the search for regulatory elements. Moreover, the application of the measures is illustrated by assigning E-box matrices of a SELEX experiment and of experimentally characterised binding sites of circadian clock genes to the Myc-Max cluster.
doi:10.1186/1471-2105-6-237
PMCID: PMC1261160  PMID: 16191190
13.  Identification of co-occurring transcription factor binding sites from DNA sequence using clustered position weight matrices 
Nucleic Acids Research  2011;40(5):e38.
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.
doi:10.1093/nar/gkr1252
PMCID: PMC3300004  PMID: 22187154
14.  Transcription Factor Binding Sites Prediction Based on Modified Nucleosomes 
PLoS ONE  2014;9(2):e89226.
In computational methods, position weight matrices (PWMs) are commonly applied for transcription factor binding site (TFBS) prediction. Although these matrices are more accurate than simple consensus sequences to predict actual binding sites, they usually produce a large number of false positive (FP) predictions and so are impoverished sources of information. Several studies have employed additional sources of information such as sequence conservation or the vicinity to transcription start sites to distinguish true binding regions from random ones. Recently, the spatial distribution of modified nucleosomes has been shown to be associated with different promoter architectures. These aligned patterns can facilitate DNA accessibility for transcription factors. We hypothesize that using data from these aligned and periodic patterns can improve the performance of binding region prediction. In this study, we propose two effective features, “modified nucleosomes neighboring” and “modified nucleosomes occupancy”, to decrease FP in binding site discovery. Based on these features, we designed a logistic regression classifier which estimates the probability of a region as a TFBS. Our model learned each feature based on Sp1 binding sites on Chromosome 1 and was tested on the other chromosomes in human CD4+T cells. In this work, we investigated 21 histone modifications and found that only 8 out of 21 marks are strongly correlated with transcription factor binding regions. To prove that these features are not specific to Sp1, we combined the logistic regression classifier with the PWM, and created a new model to search TFBSs on the genome. We tested the model using transcription factors MAZ, PU.1 and ELF1 and compared the results to those using only the PWM. The results show that our model can predict Transcription factor binding regions more successfully. The relative simplicity of the model and capability of integrating other features make it a superior method for TFBS prediction.
doi:10.1371/journal.pone.0089226
PMCID: PMC3931712  PMID: 24586611
15.  Improved predictions of transcription factor binding sites using physicochemical features of DNA 
Nucleic Acids Research  2012;40(22):e175.
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.
doi:10.1093/nar/gks771
PMCID: PMC3526315  PMID: 22923524
16.  GADEM: A Genetic Algorithm Guided Formation of Spaced Dyads Coupled with an EM Algorithm for Motif Discovery 
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/.
doi:10.1089/cmb.2008.16TT
PMCID: PMC2756050  PMID: 19193149
ChIP; de novo motif discovery; expectation-maximization; genetic algorithm; k-mer; spaced dyad
17.  gadem: A Genetic Algorithm Guided Formation of Spaced Dyads Coupled with an EM Algorithm for Motif Discovery 
Journal of Computational Biology  2009;16(2):317-329.
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/.
doi:10.1089/cmb.2008.16TT
PMCID: PMC2756050  PMID: 19193149
ChIP; de novo motif discovery; expectation-maximization; genetic algorithm; k-mer; spaced dyad
18.  A General Pairwise Interaction Model Provides an Accurate Description of In Vivo Transcription Factor Binding Sites 
PLoS ONE  2014;9(6):e99015.
The identification of transcription factor binding sites (TFBSs) on genomic DNA is of crucial importance for understanding and predicting regulatory elements in gene networks. TFBS motifs are commonly described by Position Weight Matrices (PWMs), in which each DNA base pair contributes independently to the transcription factor (TF) binding. However, this description ignores correlations between nucleotides at different positions, and is generally inaccurate: analysing fly and mouse in vivo ChIPseq data, we show that in most cases the PWM model fails to reproduce the observed statistics of TFBSs. To overcome this issue, we introduce the pairwise interaction model (PIM), a generalization of the PWM model. The model is based on the principle of maximum entropy and explicitly describes pairwise correlations between nucleotides at different positions, while being otherwise as unconstrained as possible. It is mathematically equivalent to considering a TF-DNA binding energy that depends additively on each nucleotide identity at all positions in the TFBS, like the PWM model, but also additively on pairs of nucleotides. We find that the PIM significantly improves over the PWM model, and even provides an optimal description of TFBS statistics within statistical noise. The PIM generalizes previous approaches to interdependent positions: it accounts for co-variation of two or more base pairs, and predicts secondary motifs, while outperforming multiple-motif models consisting of mixtures of PWMs. We analyse the structure of pairwise interactions between nucleotides, and find that they are sparse and dominantly located between consecutive base pairs in the flanking region of TFBS. Nonetheless, interactions between pairs of non-consecutive nucleotides are found to play a significant role in the obtained accurate description of TFBS statistics. The PIM is computationally tractable, and provides a general framework that should be useful for describing and predicting TFBSs beyond PWMs.
doi:10.1371/journal.pone.0099015
PMCID: PMC4057186  PMID: 24926895
19.  PiDNA: predicting protein–DNA interactions with structural models 
Nucleic Acids Research  2013;41(Web Server issue):W523-W530.
Predicting binding sites of a transcription factor in the genome is an important, but challenging, issue in studying gene regulation. In the past decade, a large number of protein–DNA co-crystallized structures available in the Protein Data Bank have facilitated the understanding of interacting mechanisms between transcription factors and their binding sites. Recent studies have shown that both physics-based and knowledge-based potential functions can be applied to protein–DNA complex structures to deliver position weight matrices (PWMs) that are consistent with the experimental data. To further use the available structural models, the proposed Web server, PiDNA, aims at first constructing reliable PWMs by applying an atomic-level knowledge-based scoring function on numerous in silico mutated complex structures, and then using the PWM constructed by the structure models with small energy changes to predict the interaction between proteins and DNA sequences. With PiDNA, the users can easily predict the relative preference of all the DNA sequences with limited mutations from the native sequence co-crystallized in the model in a single run. More predictions on sequences with unlimited mutations can be realized by additional requests or file uploading. Three types of information can be downloaded after prediction: (i) the ranked list of mutated sequences, (ii) the PWM constructed by the favourable mutated structures, and (iii) any mutated protein–DNA complex structure models specified by the user. This study first shows that the constructed PWMs are similar to the annotated PWMs collected from databases or literature. Second, the prediction accuracy of PiDNA in detecting relatively high-specificity sites is evaluated by comparing the ranked lists against in vitro experiments from protein-binding microarrays. Finally, PiDNA is shown to be able to select the experimentally validated binding sites from 10 000 random sites with high accuracy. With PiDNA, the users can design biological experiments based on the predicted sequence specificity and/or request mutated structure models for further protein design. As well, it is expected that PiDNA can be incorporated with chromatin immunoprecipitation data to refine large-scale inference of in vivo protein–DNA interactions. PiDNA is available at: http://dna.bime.ntu.edu.tw/pidna.
doi:10.1093/nar/gkt388
PMCID: PMC3692134  PMID: 23703214
20.  Assessment of clusters of transcription factor binding sites in relationship to human promoter, CpG islands and gene expression 
BMC Genomics  2004;5:16.
Background
Gene expression is regulated mainly by transcription factors (TFs) that interact with regulatory cis-elements on DNA sequences. To identify functional regulatory elements, computer searching can predict TF binding sites (TFBS) using position weight matrices (PWMs) that represent positional base frequencies of collected experimentally determined TFBS. A disadvantage of this approach is the large output of results for genomic DNA. One strategy to identify genuine TFBS is to utilize local concentrations of predicted TFBS. It is unclear whether there is a general tendency for TFBS to cluster at promoter regions, although this is the case for certain TFBS. Also unclear is the identification of TFs that have TFBS concentrated in promoters and to what level this occurs. This study hopes to answer some of these questions.
Results
We developed the cluster score measure to evaluate the correlation between predicted TFBS clusters and promoter sequences for each PWM. Non-promoter sequences were used as a control. Using the cluster score, we identified a PWM group called PWM-PCP, in which TFBS clusters positively correlate with promoters, and another PWM group called PWM-NCP, in which TFBS clusters negatively correlate with promoters. The PWM-PCP group comprises 47% of the 199 vertebrate PWMs, while the PWM-NCP group occupied 11 percent. After reducing the effect of CpG islands (CGI) against the clusters using partial correlation coefficients among three properties (promoter, CGI and predicted TFBS cluster), we identified two PWM groups including those strongly correlated with CGI and those not correlated with CGI.
Conclusion
Not all PWMs predict TFBS correlated with human promoter sequences. Two main PWM groups were identified: (1) those that show TFBS clustered in promoters associated with CGI, and (2) those that show TFBS clustered in promoters independent of CGI. Assessment of PWM matches will allow more positive interpretation of TFBS in regulatory regions.
doi:10.1186/1471-2164-5-16
PMCID: PMC375527  PMID: 15053842
promoter; tissue-specific gene expression; position weight matrix; regulatory motif
21.  Structure-based prediction of C2H2 zinc-finger binding specificity: sensitivity to docking geometry 
Nucleic Acids Research  2007;35(4):1085-1097.
Predicting the binding specificity of transcription factors is a critical step in the characterization and computational identification and of cis-regulatory elements in genomic sequences. Here we use protein–DNA structures to predict binding specificity and consider the possibility of predicting position weight matrices (PWM) for an entire protein family based on the structures of just a few family members. A particular focus is the sensitivity of prediction accuracy to the docking geometry of the structure used. We investigate this issue with the goal of determining how similar two docking geometries must be for binding specificity predictions to be accurate. Docking similarity is quantified using our recently described interface alignment score (IAS). Using a molecular-mechanics force field, we predict high-affinity nucleotide sequences that bind to the second zinc-finger (ZF) domain from the Zif268 protein, using different C2H2 ZF domains as structural templates. We identify a strong relationship between IAS values and prediction accuracy, and define a range of IAS values for which accurate structure-based predictions of binding specificity is to be expected. The implication of our results for large-scale, structure-based prediction of PWMs is discussed.
doi:10.1093/nar/gkl1155
PMCID: PMC1851644  PMID: 17264128
22.  ScerTF: a comprehensive database of benchmarked position weight matrices for Saccharomyces species 
Nucleic Acids Research  2011;40(D1):D162-D168.
Saccharomyces cerevisiae is a primary model for studies of transcriptional control, and the specificities of most yeast transcription factors (TFs) have been determined by multiple methods. However, it is unclear which position weight matrices (PWMs) are most useful; for the roughly 200 TFs in yeast, there are over 1200 PWMs in the literature. To address this issue, we created ScerTF, a comprehensive database of 1226 motifs from 11 different sources. We identified a single matrix for each TF that best predicts in vivo data by benchmarking matrices against chromatin immunoprecipitation and TF deletion experiments. We also used in vivo data to optimize thresholds for identifying regulatory sites with each matrix. To correct for biases from different methods, we developed a strategy to combine matrices. These aligned matrices outperform the best available matrix for several TFs. We used the matrices to predict co-occurring regulatory elements in the genome and identified many known TF combinations. In addition, we predict new combinations and provide evidence of combinatorial regulation from gene expression data. The database is available through a web interface at http://ural.wustl.edu/ScerTF. The site allows users to search the database with a regulatory site or matrix to identify the TFs most likely to bind the input sequence.
doi:10.1093/nar/gkr1180
PMCID: PMC3245033  PMID: 22140105
23.  AthaMap-assisted transcription factor target gene identification in Arabidopsis thaliana 
The AthaMap database generates a map of potential transcription factor binding sites (TFBS) and small RNA target sites in the Arabidopsis thaliana genome. The database contains sites for 115 different transcription factors (TFs). TFBS were identified with positional weight matrices (PWMs) or with single binding sites. With the new web tool ‘Gene Identification’, it is possible to identify potential target genes for selected TFs. For these analyses, the user can define a region of interest of up to 6000 bp in all annotated genes. For TFBS determined with PWMs, the search can be restricted to high-quality TFBS. The results are displayed in tables that identify the gene, position of the TFBS and, if applicable, individual score of the TFBS. In addition, data files can be downloaded that harbour positional information of TFBS of all TFs in a region between −2000 and +2000 bp relative to the transcription or translation start site. Also, data content of AthaMap was increased and the database was updated to the TAIR8 genome release.
Database URL: http://www.athamap.de/gene_ident.php
doi:10.1093/database/baq034
PMCID: PMC3011983  PMID: 21177332
24.  Predicting Target DNA Sequences of DNA-Binding Proteins Based on Unbound Structures 
PLoS ONE  2012;7(2):e30446.
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.
doi:10.1371/journal.pone.0030446
PMCID: PMC3270014  PMID: 22312425
25.  Tree-Based Position Weight Matrix Approach to Model Transcription Factor Binding Site Profiles 
PLoS ONE  2011;6(9):e24210.
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.
doi:10.1371/journal.pone.0024210
PMCID: PMC3166302  PMID: 21912677

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