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1.  AthaMap web tools for the analysis and identification of co-regulated genes 
Nucleic Acids Research  2006;35(Database issue):D857-D862.
The AthaMap database generates a map of cis-regulatory elements for the whole Arabidopsis thaliana genome. This database has been extended by new tools to identify common cis-regulatory elements in specific regions of user-provided gene sets. A resulting table displays all cis-regulatory elements annotated in AthaMap including positional information relative to the respective gene. Further tables show overviews with the number of individual transcription factor binding sites (TFBS) present and TFBS common to the whole set of genes. Over represented cis-elements are easily identified. These features were used to detect specific enrichment of drought-responsive elements in cold-induced genes. For identification of co-regulated genes, the output table of the colocalization function was extended to show the closest genes and their relative distances to the colocalizing TFBS. Gene sets determined by this function can be used for a co-regulation analysis in microarray gene expression databases such as Genevestigator or PathoPlant. Additional improvements of AthaMap include display of the gene structure in the sequence window and a significant data increase. AthaMap is freely available at .
doi:10.1093/nar/gkl1006
PMCID: PMC1761422  PMID: 17148485
2.  AthaMap, integrating transcriptional and post-transcriptional data 
Nucleic Acids Research  2008;37(Database issue):D983-D986.
The AthaMap database generates a map of predicted transcription factor binding sites (TFBS) for the whole Arabidopsis thaliana genome. AthaMap has now been extended to include data on post-transcriptional regulation. A total of 403 173 genomic positions of small RNAs have been mapped in the A. thaliana genome. These identify 5772 putative post-transcriptionally regulated target genes. AthaMap tools have been modified to improve the identification of common TFBS in co-regulated genes by subtracting post-transcriptionally regulated genes from such analyses. Furthermore, AthaMap was updated to the TAIR7 genome annotation, a graphic display of gene analysis results was implemented, and the TFBS data content was increased. AthaMap is freely available at http://www.athamap.de/.
doi:10.1093/nar/gkn709
PMCID: PMC2686474  PMID: 18842622
3.  ‘MicroRNA Targets’, a new AthaMap web-tool for genome-wide identification of miRNA targets in Arabidopsis thaliana 
BioData Mining  2012;5:7.
Background
The AthaMap database generates a genome-wide map for putative transcription factor binding sites for A. thaliana. When analyzing transcriptional regulation using AthaMap it may be important to learn which genes are also post-transcriptionally regulated by inhibitory RNAs. Therefore, a unified database for transcriptional and post-transcriptional regulation will be highly useful for the analysis of gene expression regulation.
Methods
To identify putative microRNA target sites in the genome of A. thaliana, processed mature miRNAs from 243 annotated miRNA genes were used for screening with the psRNATarget web server. Positional information, target genes and the psRNATarget score for each target site were annotated to the AthaMap database. Furthermore, putative target sites for small RNAs from seven small RNA transcriptome datasets were used to determine small RNA target sites within the A. thaliana genome.
Results
Putative 41,965 genome wide miRNA target sites and 10,442 miRNA target genes were identified in the A. thaliana genome. Taken together with genes targeted by small RNAs from small RNA transcriptome datasets, a total of 16,600 A. thaliana genes are putatively regulated by inhibitory RNAs. A novel web-tool, ‘MicroRNA Targets’, was integrated into AthaMap which permits the identification of genes predicted to be regulated by selected miRNAs. The predicted target genes are displayed with positional information and the psRNATarget score of the target site. Furthermore, putative target sites of small RNAs from selected tissue datasets can be identified with the new ‘Small RNA Targets’ web-tool.
Conclusions
The integration of predicted miRNA and small RNA target sites with transcription factor binding sites will be useful for AthaMap-assisted gene expression analysis. URL: http://www.athamap.de/
doi:10.1186/1756-0381-5-7
PMCID: PMC3410767  PMID: 22800758
Arabidopsis thaliana; AthaMap; MicroRNAs; Small RNAs; Post-transcriptional regulation
4.  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
5.  AthaMap: an online resource for in silico transcription factor binding sites in the Arabidopsis thaliana genome 
Nucleic Acids Research  2004;32(Database issue):D368-D372.
Gene expression is controlled mainly by the binding of transcription factors to regulatory sequences. To generate a genomic map for regulatory sequences, the Arabidopsis thaliana genome was screened for putative transcription factor binding sites. Using publicly available data from the TRANSFAC database and from publications, alignment matrices for 23 transcription factors of 13 different factor families were used with the pattern search program Patser to determine the genomic positions of more than 2.4 × 106 putative binding sites. Due to the dense clustering of genes and the observation that regulatory sequences are not restricted to upstream regions, the prediction of binding sites was performed for the whole genome. The genomic positions and the underlying data were imported into the newly developed AthaMap database. This data can be accessed by positional information or the Arabidopsis Genome Initiative identification number. Putative binding sites are displayed in the defined region. Data on the matrices used and on the thresholds applied in these screens are given in the database. Considering the high density of sites it will be a valuable resource for generating models on gene expression regulation. The data are available at http://www.athamap.de.
doi:10.1093/nar/gkh017
PMCID: PMC308752  PMID: 14681436
6.  PathoPlant®: a platform for microarray expression data to analyze co-regulated genes involved in plant defense responses 
Nucleic Acids Research  2006;35(Database issue):D841-D845.
Plants react to pathogen attack by expressing specific proteins directed toward the infecting pathogens. This involves the transcriptional activation of specific gene sets. PathoPlant®, a database on plant–pathogen interactions and signal transduction reactions, has now been complemented by microarray gene expression data from Arabidopsis thaliana subjected to pathogen infection and elicitor treatment. New web tools enable identification of plant genes regulated by specific stimuli. Sets of genes co-regulated by multiple stimuli can be displayed as well. A user-friendly web interface was created for the submission of gene sets to be analyzed. This results in a table, listing the stimuli that act either inducing or repressing on the respective genes. The search can be restricted to certain induction factors to identify, e.g. strongly up- or down-regulated genes. Up to three stimuli can be combined with the option of induction factor restriction to determine similarly regulated genes. To identify common cis-regulatory elements in co-regulated genes, a resulting gene list can directly be exported to the AthaMap database for analysis. PathoPlant is freely accessible at .
doi:10.1093/nar/gkl835
PMCID: PMC1669748  PMID: 17099232
7.  Internet Resources for Gene Expression Analysis in Arabidopsis thaliana 
Current Genomics  2008;9(6):375-380.
The number of online databases and web-tools for gene expression analysis in Arabidopsis thaliana has increased tremendously during the last years. These resources permit the database-assisted identification of putative cis-regulatory DNA sequences, their binding proteins, and the determination of common cis-regulatory motifs in coregulated genes. DNA binding proteins may be predicted by the type of cis-regulatory motif. Further questions of combinatorial control based on the interaction of DNA binding proteins and the colocalization of cis-regulatory motifs can be addressed. The database-assisted spatial and temporal expression analysis of DNA binding proteins and their target genes may help to further refine experimental approaches. Signal transduction pathways upstream of regulated genes are not yet fully accessible in databases mainly because they need to be manually annotated. This review focuses on the use of the AthaMap and PathoPlant® databases for gene expression regulation analysis and discusses similar and complementary online databases and web-tools. Online databases are helpful for the development of working hypothesis and for designing subsequent experiments.
doi:10.2174/138920208785699535
PMCID: PMC2691667  PMID: 19506727
Bioinformatics; databases; gene expression; plants; transcription; web-server.
8.  TTS Mapping: integrative WEB tool for analysis of triplex formation target DNA Sequences, G-quadruplets and non-protein coding regulatory DNA elements in the human genome 
BMC Genomics  2009;10(Suppl 3):S9.
Background
DNA triplexes can naturally occur, co-localize and interact with many other regulatory DNA elements (e.g. G-quadruplex (G4) DNA motifs), specific DNA-binding proteins (e.g. transcription factors (TFs)), and micro-RNA (miRNA) precursors. Specific genome localizations of triplex target DNA sites (TTSs) may cause abnormalities in a double-helix DNA structure and can be directly involved in some human diseases. However, genome localization of specific TTSs, their interconnection with regulatory DNA elements and physiological roles in a cell are poor defined. Therefore, it is important to identify comprehensive and reliable catalogue of specific potential TTSs (pTTSs) and their co-localization patterns with other regulatory DNA elements in the human genome.
Results
"TTS mapping" database is a web-based search engine developed here, which is aimed to find and annotate pTTSs within a region of interest of the human genome. The engine provides descriptive statistics of pTTSs in a given region and its sequence context. Different annotation tracks of TTS-overlapping gene region(s), G4 motifs, CpG Island, miRNA precursors, miRNA targets, transcription factor binding sites (TFBSs), Single Nucleotide Polymorphisms (SNPs), small nucleolar RNAs (snoRNA), and repeat elements are also mapped based onto a sequence location provided by UCSC genome browser, G4 database http://www.quadruplex.org and several other datasets. The results pages provide links to UCSC genome browser annotation tracks and relative DBs. BLASTN program was included to check the uniqueness of a given pTTS in the human genome. Recombination- and mutation-prone genes (e.g. EVI-1, MYC) were found to be significantly enriched by TTSs and multiple co-occurring with our regulatory DNA elements. TTS mapping reveals that a high-complementary and evolutionarily conserved polypurine and polypyrimidine DNA sequence pair linked by a non-conserved short DNA sequence can form miR-483 transcribed from intron 2 of IGF2 gene and bound double-strand nucleic acid TTSs forming natural triplex structures.
Conclusion
TTS mapping provides comprehensive visual and analytical tools to help users to find pTTSs, G-quadruplets and other regulatory DNA elements in various genome regions. TTS Mapping not only provides sequence visualization and statistical information, but also integrates knowledge about co-localization TTS with various DNA elements and facilitates that data analysis. In particular, TTS Mapping reveals complex structural-functional regulatory module of gene IGF2 including TF MZF1 binding site and ncRNA precursor mir-483 formed by the high-complementary and evolutionarily conserved polypurine- and polypyrimidine-rich DNA pair. Such ncRNAs capable of forming helical triplex structures with a polypurine strand of a nucleic acid duplexes (DNA or RNA) via Hoogsteen or reverse Hoogsteen hydrogen bonds. Our web tool could be used to discover biologically meaningful genome modules and to optimize experimental design of anti-gene treatment.
doi:10.1186/1471-2164-10-S3-S9
PMCID: PMC2788396  PMID: 19958507
9.  PlantPAN: Plant promoter analysis navigator, for identifying combinatorial cis-regulatory elements with distance constraint in plant gene groups 
BMC Genomics  2008;9:561.
Background
The elucidation of transcriptional regulation in plant genes is important area of research for plant scientists, following the mapping of various plant genomes, such as A. thaliana, O. sativa and Z. mays. A variety of bioinformatic servers or databases of plant promoters have been established, although most have been focused only on annotating transcription factor binding sites in a single gene and have neglected some important regulatory elements (tandem repeats and CpG/CpNpG islands) in promoter regions. Additionally, the combinatorial interaction of transcription factors (TFs) is important in regulating the gene group that is associated with the same expression pattern. Therefore, a tool for detecting the co-regulation of transcription factors in a group of gene promoters is required.
Results
This study develops a database-assisted system, PlantPAN (Plant Promoter Analysis Navigator), for recognizing combinatorial cis-regulatory elements with a distance constraint in sets of plant genes. The system collects the plant transcription factor binding profiles from PLACE, TRANSFAC (public release 7.0), AGRIS, and JASPER databases and allows users to input a group of gene IDs or promoter sequences, enabling the co-occurrence of combinatorial transcription factor binding sites (TFBSs) within a defined distance (20 bp to 200 bp) to be identified. Furthermore, the new resource enables other regulatory features in a plant promoter, such as CpG/CpNpG islands and tandem repeats, to be displayed. The regulatory elements in the conserved regions of the promoters across homologous genes are detected and presented.
Conclusion
In addition to providing a user-friendly input/output interface, PlantPAN has numerous advantages in the analysis of a plant promoter. Several case studies have established the effectiveness of PlantPAN. This novel analytical resource is now freely available at .
doi:10.1186/1471-2164-9-561
PMCID: PMC2633311  PMID: 19036138
10.  Identifying Functional Transcription Factor Binding Sites in Yeast by Considering Their Positional Preference in the Promoters 
PLoS ONE  2013;8(12):e83791.
Transcription factor binding site (TFBS) identification plays an important role in deciphering gene regulatory codes. With comprehensive knowledge of TFBSs, one can understand molecular mechanisms of gene regulation. In the recent decades, various computational approaches have been proposed to predict TFBSs in the genome. The TFBS dataset of a TF generated by each algorithm is a ranked list of predicted TFBSs of that TF, where top ranked TFBSs are statistically significant ones. However, whether these statistically significant TFBSs are functional (i.e. biologically relevant) is still unknown. Here we develop a post-processor, called the functional propensity calculator (FPC), to assign a functional propensity to each TFBS in the existing computationally predicted TFBS datasets. It is known that functional TFBSs reveal strong positional preference towards the transcriptional start site (TSS). This motivates us to take TFBS position relative to the TSS as the key idea in building our FPC. Based on our calculated functional propensities, the TFBSs of a TF in the original TFBS dataset could be reordered, where top ranked TFBSs are now the ones with high functional propensities. To validate the biological significance of our results, we perform three published statistical tests to assess the enrichment of Gene Ontology (GO) terms, the enrichment of physical protein-protein interactions, and the tendency of being co-expressed. The top ranked TFBSs in our reordered TFBS dataset outperform the top ranked TFBSs in the original TFBS dataset, justifying the effectiveness of our post-processor in extracting functional TFBSs from the original TFBS dataset. More importantly, assigning functional propensities to putative TFBSs enables biologists to easily identify which TFBSs in the promoter of interest are likely to be biologically relevant and are good candidates to do further detailed experimental investigation. The FPC is implemented as a web tool at http://santiago.ee.ncku.edu.tw/FPC/.
doi:10.1371/journal.pone.0083791
PMCID: PMC3873331  PMID: 24386279
11.  AtPAN: an integrated system for reconstructing transcriptional regulatory networks in Arabidopsis thaliana 
BMC Genomics  2012;13:85.
Background
Construction of transcriptional regulatory networks (TRNs) is of priority concern in systems biology. Numerous high-throughput approaches, including microarray and next-generation sequencing, are extensively adopted to examine transcriptional expression patterns on the whole-genome scale; those data are helpful in reconstructing TRNs. Identifying transcription factor binding sites (TFBSs) in a gene promoter is the initial step in elucidating the transcriptional regulation mechanism. Since transcription factors usually co-regulate a common group of genes by forming regulatory modules with similar TFBSs. Therefore, the combinatorial interactions of transcription factors must be modeled to reconstruct the gene regulatory networks.
Description For systems biology applications, this work develops a novel database called Arabidopsis thaliana Promoter Analysis Net (AtPAN), capable of detecting TFBSs and their corresponding transcription factors (TFs) in a promoter or a set of promoters in Arabidopsis. For further analysis, according to the microarray expression data and literature, the co-expressed TFs and their target genes can be retrieved from AtPAN. Additionally, proteins interacting with the co-expressed TFs are also incorporated to reconstruct co-expressed TRNs. Moreover, combinatorial TFs can be detected by the frequency of TFBSs co-occurrence in a group of gene promoters. In addition, TFBSs in the conserved regions between the two input sequences or homologous genes in Arabidopsis and rice are also provided in AtPAN. The output results also suggest conducting wet experiments in the future.
Conclusions
The AtPAN, which has a user-friendly input/output interface and provide graphical view of the TRNs. This novel and creative resource is freely available online at http://AtPAN.itps.ncku.edu.tw/.
doi:10.1186/1471-2164-13-85
PMCID: PMC3314555  PMID: 22397531
12.  rVISTA 2.0: evolutionary analysis of transcription factor binding sites 
Nucleic Acids Research  2004;32(Web Server issue):W217-W221.
Identifying and characterizing the transcription factor binding site (TFBS) patterns of cis-regulatory elements represents a challenge, but holds promise to reveal the regulatory language the genome uses to dictate transcriptional dynamics. Several studies have demonstrated that regulatory modules are under positive selection and, therefore, are often conserved between related species. Using this evolutionary principle, we have created a comparative tool, rVISTA, for analyzing the regulatory potential of noncoding sequences. Our ability to experimentally identify functional noncoding sequences is extremely limited, therefore, rVISTA attempts to fill this great gap in genomic analysis by offering a powerful approach for eliminating TFBSs least likely to be biologically relevant. The rVISTA tool combines TFBS predictions, sequence comparisons and cluster analysis to identify noncoding DNA regions that are evolutionarily conserved and present in a specific configuration within genomic sequences. Here, we present the newly developed version 2.0 of the rVISTA tool, which can process alignments generated by both the zPicture and blastz alignment programs or use pre-computed pairwise alignments of several vertebrate genomes available from the ECR Browser and GALA database. The rVISTA web server is closely interconnected with the TRANSFAC database, allowing users to either search for matrices present in the TRANSFAC library collection or search for user-defined consensus sequences. The rVISTA tool is publicly available at http://rvista.dcode.org/.
doi:10.1093/nar/gkh383
PMCID: PMC441521  PMID: 15215384
13.  AGRIS: Arabidopsis Gene Regulatory Information Server, an information resource of Arabidopsis cis-regulatory elements and transcription factors 
BMC Bioinformatics  2003;4:25.
Background
The gene regulatory information is hardwired in the promoter regions formed by cis-regulatory elements that bind specific transcription factors (TFs). Hence, establishing the architecture of plant promoters is fundamental to understanding gene expression. The determination of the regulatory circuits controlled by each TF and the identification of the cis-regulatory sequences for all genes have been identified as two of the goals of the Multinational Coordinated Arabidopsis thaliana Functional Genomics Project by the Multinational Arabidopsis Steering Committee (June 2002).
Results
AGRIS is an information resource of Arabidopsis promoter sequences, transcription factors and their target genes. AGRIS currently contains two databases, AtTFDB (Arabidopsis thaliana transcription factor database) and AtcisDB (Arabidopsis thaliana cis-regulatory database). AtTFDB contains information on approximately 1,400 transcription factors identified through motif searches and grouped into 34 families. AtTFDB links the sequence of the transcription factors with available mutants and, when known, with the possible genes they may regulate. AtcisDB consists of the 5' regulatory sequences of all 29,388 annotated genes with a description of the corresponding cis-regulatory elements. Users can search the databases for (i) promoter sequences, (ii) a transcription factor, (iii) a direct target genes for a specific transcription factor, or (vi) a regulatory network that consists of transcription factors and their target genes.
Conclusion
AGRIS provides the necessary software tools on Arabidopsis transcription factors and their putative binding sites on all genes to initiate the identification of transcriptional regulatory networks in the model dicotyledoneous plant Arabidopsis thaliana. AGRIS can be accessed from .
doi:10.1186/1471-2105-4-25
PMCID: PMC166152  PMID: 12820902
14.  LASAGNA: A novel algorithm for transcription factor binding site alignment 
BMC Bioinformatics  2013;14:108.
Background
Scientists routinely scan DNA sequences for transcription factor (TF) binding sites (TFBSs). Most of the available tools rely on position-specific scoring matrices (PSSMs) constructed from aligned binding sites. Because of the resolutions of assays used to obtain TFBSs, databases such as TRANSFAC, ORegAnno and PAZAR store unaligned variable-length DNA segments containing binding sites of a TF. These DNA segments need to be aligned to build a PSSM. While the TRANSFAC database provides scoring matrices for TFs, nearly 78% of the TFs in the public release do not have matrices available. As work on TFBS alignment algorithms has been limited, it is highly desirable to have an alignment algorithm tailored to TFBSs.
Results
We designed a novel algorithm named LASAGNA, which is aware of the lengths of input TFBSs and utilizes position dependence. Results on 189 TFs of 5 species in the TRANSFAC database showed that our method significantly outperformed ClustalW2 and MEME. We further compared a PSSM method dependent on LASAGNA to an alignment-free TFBS search method. Results on 89 TFs whose binding sites can be located in genomes showed that our method is significantly more precise at fixed recall rates. Finally, we described LASAGNA-ChIP, a more sophisticated version for ChIP (Chromatin immunoprecipitation) experiments. Under the one-per-sequence model, it showed comparable performance with MEME in discovering motifs in ChIP-seq peak sequences.
Conclusions
We conclude that the LASAGNA algorithm is simple and effective in aligning variable-length binding sites. It has been integrated into a user-friendly webtool for TFBS search and visualization called LASAGNA-Search. The tool currently stores precomputed PSSM models for 189 TFs and 133 TFs built from TFBSs in the TRANSFAC Public database (release 7.0) and the ORegAnno database (08Nov10 dump), respectively. The webtool is available at http://biogrid.engr.uconn.edu/lasagna_search/.
doi:10.1186/1471-2105-14-108
PMCID: PMC3747862  PMID: 23522376
15.  CTF: a CRF-based transcription factor binding sites finding system 
BMC Genomics  2012;13(Suppl 8):S18.
Background
Identifying the location of transcription factor bindings is crucial to understand transcriptional regulation. Currently, Chromatin Immunoprecipitation followed with high-throughput Sequencing (ChIP-seq) is able to locate the transcription factor binding sites (TFBSs) accurately in high throughput and it has become the gold-standard method for TFBS finding experimentally. However, due to its high cost, it is impractical to apply the method in a very large scale. Considering the large number of transcription factors, numerous cell types and various conditions, computational methods are still very valuable to accurate TFBS identification.
Results
In this paper, we proposed a novel integrated TFBS prediction system, CTF, based on Conditional Random Fields (CRFs). Integrating information from different sources, CTF was able to capture patterns of TFBSs contained in different features (sequence, chromatin and etc) and predicted the TFBS locations with a high accuracy. We compared CTF with several existing tools as well as the PWM baseline method on a dataset generated by ChIP-seq experiments (TFBSs of 13 transcription factors in mouse genome). Results showed that CTF performed significantly better than existing methods tested.
Conclusions
CTF is a powerful tool to predict TFBSs by integrating high throughput data and different features. It can be a useful complement to ChIP-seq and other experimental methods for TFBS identification and thus improve our ability to investigate functional elements in post-genomic era.
Availability: CTF is freely available to academic users at: http://cbb.sjtu.edu.cn/~ccwei/pub/software/CTF/CTF.php
doi:10.1186/1471-2164-13-S8-S18
PMCID: PMC3535700  PMID: 23282203
16.  Selective Constraints in Experimentally Defined Primate Regulatory Regions 
PLoS Genetics  2008;4(8):e1000157.
Changes in gene regulation may be important in evolution. However, the evolutionary properties of regulatory mutations are currently poorly understood. This is partly the result of an incomplete annotation of functional regulatory DNA in many species. For example, transcription factor binding sites (TFBSs), a major component of eukaryotic regulatory architecture, are typically short, degenerate, and therefore difficult to differentiate from randomly occurring, nonfunctional sequences. Furthermore, although sites such as TFBSs can be computationally predicted using evolutionary conservation as a criterion, estimates of the true level of selective constraint (defined as the fraction of strongly deleterious mutations occurring at a locus) in regulatory regions will, by definition, be upwardly biased in datasets that are a priori evolutionarily conserved. Here we investigate the fitness effects of regulatory mutations using two complementary datasets of human TFBSs that are likely to be relatively free of ascertainment bias with respect to evolutionary conservation but, importantly, are supported by experimental data. The first is a collection of almost >2,100 human TFBSs drawn from the literature in the TRANSFAC database, and the second is derived from several recent high-throughput chromatin immunoprecipitation coupled with genomic microarray (ChIP-chip) analyses. We also define a set of putative cis-regulatory modules (pCRMs) by spatially clustering multiple TFBSs that regulate the same gene. We find that a relatively high proportion (∼37%) of mutations at TFBSs are strongly deleterious, similar to that at a 2-fold degenerate protein-coding site. However, constraint is significantly reduced in human and chimpanzee pCRMS and ChIP-chip sequences, relative to macaques. We estimate that the fraction of regulatory mutations that have been driven to fixation by positive selection in humans is not significantly different from zero. We also find that the level of selective constraint in our TFBSs, pCRMs, and ChIP-chip sequences is negatively correlated with the expression breadth of the regulated gene, whereas the opposite relationship holds at that gene's nonsynonymous and synonymous sites. Finally, we find that the rate of protein evolution in a transcription factor appears to be positively correlated with the breadth of expression of the gene it regulates. Our study suggests that strongly deleterious regulatory mutations are considerably more likely (1.6-fold) to occur in tissue-specific than in housekeeping genes, implying that there is a fitness cost to increasing “complexity” of gene expression.
Author Summary
Changes in gene expression have been suggested to play a major role in mammalian evolution. In eukaryotes, gene expression is primarily controlled by sites, such as transcription factor binding sites (TFBSs), located in the noncoding region of the genome. The majority of these TFBSs remain unannotated, however, because they are typically short, degenerate, and laborious to identify experimentally. As a result, the effects of mutations in TFBSs on organism fitness remain poorly understood. We collected a dataset of TFBSs derived from the experimental biology literature and recent high-throughput studies to estimate the proportions of new mutations in TFBSs that have strongly deleterious and strongly beneficial effects upon organism fitness. We find that a relatively high proportion of new mutations in TFBSs are strongly deleterious, although it appears that relatively few are adaptive. We also demonstrate that the fraction of strongly deleterious regulatory mutations is correlated with the breadth of expression of the regulated gene. Thus, ubiquitously expressed genes are likely to experience fewer deleterious regulatory mutations than those expressed in a small number of tissues.
doi:10.1371/journal.pgen.1000157
PMCID: PMC2490716  PMID: 18704158
17.  Genomic Reconstruction of the Transcriptional Regulatory Network in Bacillus subtilis 
Journal of Bacteriology  2013;195(11):2463-2473.
The adaptation of microorganisms to their environment is controlled by complex transcriptional regulatory networks (TRNs), which are still only partially understood even for model species. Genome scale annotation of regulatory features of genes and TRN reconstruction are challenging tasks of microbial genomics. We used the knowledge-driven comparative-genomics approach implemented in the RegPredict Web server to infer TRN in the model Gram-positive bacterium Bacillus subtilis and 10 related Bacillales species. For transcription factor (TF) regulons, we combined the available information from the DBTBS database and the literature with bioinformatics tools, allowing inference of TF binding sites (TFBSs), comparative analysis of the genomic context of predicted TFBSs, functional assignment of target genes, and effector prediction. For RNA regulons, we used known RNA regulatory motifs collected in the Rfam database to scan genomes and analyze the genomic context of new RNA sites. The inferred TRN in B. subtilis comprises regulons for 129 TFs and 24 regulatory RNA families. First, we analyzed 66 TF regulons with previously known TFBSs in B. subtilis and projected them to other Bacillales genomes, resulting in refinement of TFBS motifs and identification of novel regulon members. Second, we inferred motifs and described regulons for 28 experimentally studied TFs with previously unknown TFBSs. Third, we discovered novel motifs and reconstructed regulons for 36 previously uncharacterized TFs. The inferred collection of regulons is available in the RegPrecise database (http://regprecise.lbl.gov/) and can be used in genetic experiments, metabolic modeling, and evolutionary analysis.
doi:10.1128/JB.00140-13
PMCID: PMC3676070  PMID: 23504016
18.  SNP@lincTFBS: An Integrated Database of Polymorphisms in Human LincRNA Transcription Factor Binding Sites 
PLoS ONE  2014;9(7):e103851.
Large intergenic non-coding RNAs (lincRNAs) are a new class of functional transcripts, and aberrant expression of lincRNAs was associated with several human diseases. The genetic variants in lincRNA transcription factor binding sites (TFBSs) can change lincRNA expression, thereby affecting the susceptibility to human diseases. To identify and annotate these functional candidates, we have developed a database SNP@lincTFBS, which is devoted to the exploration and annotation of single nucleotide polymorphisms (SNPs) in potential TFBSs of human lincRNAs. We identified 6,665 SNPs in 6,614 conserved TFBSs of 2,423 human lincRNAs. In addition, with ChIPSeq dataset, we identified 139,576 SNPs in 304,517 transcription factor peaks of 4,813 lincRNAs. We also performed comprehensive annotation for these SNPs using 1000 Genomes Project datasets across 11 populations. Moreover, one of the distinctive features of SNP@lincTFBS is the collection of disease-associated SNPs in the lincRNA TFBSs and SNPs in the TFBSs of disease-associated lincRNAs. The web interface enables both flexible data searches and downloads. Quick search can be query of lincRNA name, SNP identifier, or transcription factor name. SNP@lincTFBS provides significant advances in identification of disease-associated lincRNA variants and improved convenience to interpret the discrepant expression of lincRNAs. The SNP@lincTFBS database is available at http://bioinfo.hrbmu.edu.cn/SNP_lincTFBS.
doi:10.1371/journal.pone.0103851
PMCID: PMC4116217  PMID: 25075616
19.  Identification of novel regulatory modules in dicotyledonous plants using expression data and comparative genomics 
Genome Biology  2006;7(11):R103.
A strategy combining classical motif overrepresentation in co-regulated genes with comparative footprinting is applied to identify 80 transcription factor binding sites and 139 regulatory modules in Arabidopsis thaliana.
Background
Transcriptional regulation plays an important role in the control of many biological processes. Transcription factor binding sites (TFBSs) are the functional elements that determine transcriptional activity and are organized into separable cis-regulatory modules, each defining the cooperation of several transcription factors required for a specific spatio-temporal expression pattern. Consequently, the discovery of novel TFBSs in promoter sequences is an important step to improve our understanding of gene regulation.
Results
Here, we applied a detection strategy that combines features of classic motif overrepresentation approaches in co-regulated genes with general comparative footprinting principles for the identification of biologically relevant regulatory elements and modules in Arabidopsis thaliana, a model system for plant biology. In total, we identified 80 TFBSs and 139 regulatory modules, most of which are novel, and primarily consist of two or three regulatory elements that could be linked to different important biological processes, such as protein biosynthesis, cell cycle control, photosynthesis and embryonic development. Moreover, studying the physical properties of some specific regulatory modules revealed that Arabidopsis promoters have a compact nature, with cooperative TFBSs located in close proximity of each other.
Conclusion
These results create a starting point to unravel regulatory networks in plants and to study the regulation of biological processes from a systems biology point of view.
doi:10.1186/gb-2006-7-11-r103
PMCID: PMC1794593  PMID: 17090307
20.  EZ-Retrieve: a web-server for batch retrieval of coordinate-specified human DNA sequences and underscoring putative transcription factor-binding sites 
Nucleic Acids Research  2002;30(21):e121.
The availability of a draft human genome sequence and ability to monitor the transcription of thousands of genes with DNA microarrays has necessitated the need for new computational tools that can analyze cis-regulatory elements controlling genes that display similar expression patterns. We have developed a tool designated EZ-Retrieve that can: (i) retrieve any particular region of human genome sequence from the NCBI database and (ii) analyze retrieved sequences for putative transcription factor-binding sites (TFBSs) as they appear on the TRANSFAC database. The tool is web-based, user-friendly and offers both batch sequence retrieval and batch TFBS prediction. A major application of EZ-Retrieve is the analysis of co-expressed genes that are highlighted as expression clusters in DNA microarray experiments.
PMCID: PMC135846  PMID: 12409480
21.  A Machine Learning Approach for Identifying Novel Cell Type–Specific Transcriptional Regulators of Myogenesis 
PLoS Genetics  2012;8(3):e1002531.
Transcriptional enhancers integrate the contributions of multiple classes of transcription factors (TFs) to orchestrate the myriad spatio-temporal gene expression programs that occur during development. A molecular understanding of enhancers with similar activities requires the identification of both their unique and their shared sequence features. To address this problem, we combined phylogenetic profiling with a DNA–based enhancer sequence classifier that analyzes the TF binding sites (TFBSs) governing the transcription of a co-expressed gene set. We first assembled a small number of enhancers that are active in Drosophila melanogaster muscle founder cells (FCs) and other mesodermal cell types. Using phylogenetic profiling, we increased the number of enhancers by incorporating orthologous but divergent sequences from other Drosophila species. Functional assays revealed that the diverged enhancer orthologs were active in largely similar patterns as their D. melanogaster counterparts, although there was extensive evolutionary shuffling of known TFBSs. We then built and trained a classifier using this enhancer set and identified additional related enhancers based on the presence or absence of known and putative TFBSs. Predicted FC enhancers were over-represented in proximity to known FC genes; and many of the TFBSs learned by the classifier were found to be critical for enhancer activity, including POU homeodomain, Myb, Ets, Forkhead, and T-box motifs. Empirical testing also revealed that the T-box TF encoded by org-1 is a previously uncharacterized regulator of muscle cell identity. Finally, we found extensive diversity in the composition of TFBSs within known FC enhancers, suggesting that motif combinatorics plays an essential role in the cellular specificity exhibited by such enhancers. In summary, machine learning combined with evolutionary sequence analysis is useful for recognizing novel TFBSs and for facilitating the identification of cognate TFs that coordinate cell type–specific developmental gene expression patterns.
Author Summary
The development of multicellular organisms requires the formation of a diversity of cell types. Each cell has a unique genetic program that is orchestrated by regulatory sequences called enhancers, comprising multiple short DNA sequences that bind distinct transcription factors. Understanding developmental regulatory networks requires knowledge of the sequence features of functionally related enhancers. We developed an integrated evolutionary and computational approach for deciphering enhancer regulatory codes and applied this method to discover new components of the transcriptional network controlling muscle development in the fruit fly, Drosophila melanogaster. Our method involves assembling known muscle enhancers, expanding this set with evolutionarily conserved sequences, computationally classifying these enhancers based on their shared sequence features, and scanning the entire Drosophila genome to predict additional related enhancers. Using this approach, we created a map of 5,500 putative muscle enhancers, identified candidate transcription factors to which they bind, observed a strong correlation between mapped enhancers and muscle gene expression, and uncovered extensive heterogeneity among combinations of transcription factor binding sites in validated muscle enhancers, a feature that may contribute to the individual cellular specificities of these regulatory elements. Our strategy can readily be generalized to study transcriptional networks in other organisms and developmental contexts.
doi:10.1371/journal.pgen.1002531
PMCID: PMC3297574  PMID: 22412381
22.  ReAlignerV: Web-based genomic alignment tool with high specificity and robustness estimated by species-specific insertion sequences 
BMC Bioinformatics  2008;9:112.
Background
Detecting conserved noncoding sequences (CNSs) across species highlights the functional elements. Alignment procedures combined with computational prediction of transcription factor binding sites (TFBSs) can narrow down key regulatory elements. Repeat masking processes are often performed before alignment to mask insertion sequences such as transposable elements (TEs). However, recently such TEs have been reported to influence the gene regulatory network evolution. Therefore, an alignment approach that is robust to TE insertions is meaningful for finding novel conserved TFBSs in TEs.
Results
We constructed a web server 'ReAlignerV' for complex alignment of genomic sequences. ReAlignerV returns ladder-like schematic alignments that integrate predicted TFBSs and the location of TEs. It also provides pair-wise alignments in which the predicted TFBS sites and their names are shown alongside each sequence. Furthermore, we evaluated false positive aligned sites by focusing on the species-specific TEs (SSTEs), and found that ReAlignerV has a higher specificity and robustness to insertions for sequences having more than 20% TE content, compared to LAGAN, AVID, MAVID and BLASTZ.
Conclusion
ReAlignerV can be applied successfully to TE-insertion-rich sequences without prior repeat masking, and this increases the chances of finding regulatory sequences hidden in TEs, which are important sources of the regulatory network evolution. ReAlignerV can be accessed through and downloaded from .
doi:10.1186/1471-2105-9-112
PMCID: PMC2267439  PMID: 18294369
23.  Large-scale computational identification of regulatory SNPs with rSNP-MAPPER 
BMC Genomics  2012;13(Suppl 4):S7.
Background
The computational analysis of regulatory SNPs (rSNPs) is an essential step in the elucidation of the structure and function of regulatory networks at the cellular level. In this work we focus in particular on SNPs that potentially affect a Transcription Factor Binding Site (TFBS) to a significant extent, possibly resulting in changes to gene expression patterns or alternative splicing. The application described here is based on the MAPPER platform, a previously developed web-based system for the computational detection of TFBSs in DNA sequences.
Methods
rSNP-MAPPER is a computational tool that analyzes SNPs lying within predicted TFBSs and determines whether the allele substitution results in a significant change in the TFBS predictive score. The application's simple and intuitive interface supports several usage modes. For example, the user may search for potential rSNPs in the promoters of one or more genes, specified as a list of identifiers or chosen among the members of a pathway. Alternatively, the user may specify a set of SNPs to be analyzed by uploading a list of SNP identifiers or providing the coordinates of a genomic region. Finally, the user can provide two alternative sequences (wildtype and mutant), and the system will determine the location of variants to be analyzed by comparing them.
Results
In this paper we outline the architecture of rSNP-MAPPER, describing its intuitive and powerful user interface in detail. We then present several examples of the use of rSNP-MAPPER to reproduce and confirm experimental studies aimed at identifying regulatory SNPs in human genes, that show how rSNP-MAPPER is able to detect and characterize rSNPs with high accuracy. Results are richly annotated and can be displayed online or downloaded in a number of different formats.
Conclusions
rSNP-MAPPER is optimized for large scale work, allowing for the efficient annotation of thousands of SNPs, and is designed to assist in the genome-wide investigation of transcriptional regulatory networks, prioritizing potential rSNPs for subsequent experimental validation. rSNP-MAPPER is freely available at http://genome.ufl.edu/mapper/.
doi:10.1186/1471-2164-13-S4-S7
PMCID: PMC3303742  PMID: 22759655
24.  Phylogenetic simulation of promoter evolution: estimation and modeling of binding site turnover events and assessment of their impact on alignment tools 
Genome Biology  2007;8(10):R225.
Phylogenetic simulation of promoter evolution were used to analyze functional site turnover in regulatory sequences.
Background
The phenomenon of functional site turnover has important implications for the study of regulatory region evolution, such as for promoter sequence alignments and transcription factor binding site (TFBS) identification. At present, it remains difficult to estimate TFBS turnover rates on real genomic sequences, as reliable mappings of functional sites across related species are often not available. As an alternative, we introduce a flexible new simulation system, Phylogenetic Simulation of Promoter Evolution (PSPE), designed to study functional site turnovers in regulatory sequences.
Results
Using PSPE, we study replacement turnover rates of different individual TFBSs and simple modules of two sites under neutral evolutionary functional constraints. We find that TFBS replacement turnover can happen rapidly in promoters, and turnover rates vary significantly among different TFBSs and modules. We assess the influence of different constraints such as insertion/deletion rate and translocation distances. Complementing the simulations, we give simple but effective mathematical models for TFBS turnover rate prediction. As one important application of PSPE, we also present a first systematic evaluation of multiple sequence aligners regarding their capability of detecting TFBSs in promoters with site turnovers.
Conclusion
PSPE allows researchers for the first time to investigate TFBS replacement turnovers in promoters systematically. The assessment of alignment tools points out the limitations of current approaches to identify TFBSs in non-coding sequences, where turnover events of functional sites may happen frequently, and where we are interested in assessing the similarity on the functional level. PSPE is freely available at the authors' website.
doi:10.1186/gb-2007-8-10-r225
PMCID: PMC2246299  PMID: 17956628
25.  CisMols Analyzer: identification of compositionally similar cis-element clusters in ortholog conserved regions of coordinately expressed genes 
Nucleic Acids Research  2005;33(Web Server issue):W408-W411.
Combinatorial interactions of sequence-specific trans-acting factors with localized genomic cis-element clusters are the principal mechanism for regulating tissue-specific and developmental gene expression. With the emergence of expanding numbers of genome-wide expression analyses, the identification of the cis-elements responsible for specific patterns of transcriptional regulation represents a critical area of investigation. Computational methods for the identification of functional cis-regulatory modules are difficult to devise, principally because of the short length and degenerate nature of individual cis-element binding sites and the inherent complexity that is generated by combinatorial interactions within cis-clusters. Filtering candidate cis-element clusters based on phylogenetic conservation is helpful for an individual ortholog gene pair, but combining data from cis-conservation and coordinate expression across multiple genes is a more difficult problem. To approach this, we have extended an ortholog gene-pair database with additional analytical architecture to allow for the analysis and identification of maximal numbers of compositionally similar and phylogenetically conserved cis-regulatory element clusters from a list of user-selected genes. The system has been successfully tested with a series of functionally related and microarray profile-based co-expressed ortholog pairs of promoters and genes using known regulatory regions as training sets and co-expressed genes in the olfactory and immunohematologic systems as test sets. CisMols Analyzer is accessible via a Web interface at .
doi:10.1093/nar/gki486
PMCID: PMC1160246  PMID: 15980500

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