Nucleic Acids Research Advance Access published online on June 21, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm376
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
WebMOTIFS: automated discovery, filtering and scoring of DNA sequence motifs using multiple programs and Bayesian approaches
Katherine A. Romer1,
Guy-Richard Kayombya1 and
Ernest Fraenkel2,3,*
1Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 2MIT Computer Science and Artificial Intelligence Laboratory, 32 Vassar Street, Cambridge, MA 02139, USA and 3Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
*To whom correspondence should be addressed. Tel: 617 258 8702; Fax: 617 395 2652; Email: fraenkel-admin{at}mit.edu
Received January 16, 2007. Revised April 27, 2007. Accepted April 30, 2007.
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ABSTRACT
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WebMOTIFS provides a web interface that facilitates the discovery
and analysis of DNA-sequence motifs. Several studies have shown
that the accuracy of motif discovery can be significantly improved
by using multiple
de novo motif discovery programs and using
randomized control calculations to identify the most significant
motifs or by using Bayesian approaches. WebMOTIFS makes it easy
to apply these strategies. Using a single submission form, users
can run several motif discovery programs and score, cluster
and visualize the results. In addition, the Bayesian motif discovery
program THEME can be used to determine the class of transcription
factors that is most likely to regulate a set of sequences.
Input can be provided as a list of gene or probe identifiers.
Used with the default settings, WebMOTIFS accurately identifies
biologically relevant motifs from diverse data in several species.
WebMOTIFS is freely available at
http://fraenkel.mit.edu/webmotifs.
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INTRODUCTION
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One of the principal challenges in analyzing genomic sequences
is to identify patterns, or ‘motifs,’ that represent
functional elements (
1). An important use of sequence motifs
is to represent sites where transcriptional regulatory proteins
bind and modulate expression of genes. There are many algorithms
for finding such motifs. Given the same input data, these algorithms
often discover different motifs, with no one algorithm consistently
recovering all biologically significant patterns. Several studies
have demonstrated that it is possible to achieve higher accuracy
and sensitivity by combining the results from multiple motif
discovery programs (
2–4). However, this approach requires
considerable computational overhead for managing data in a variety
of formats and for clustering and scoring the large number of
discovered motifs.
WebMOTIFS is a user-friendly web-based program that makes it easy to follow the current ‘best practice’ in motif discovery. A single web-based form facilitates data entry. WebMOTIFS automatically performs motif discovery on these data with several programs. The results from these programs are scored and integrated, and the most significant motifs are provided in an easily interpreted graphic output. WebMOTIFS also offers users the opportunity to analyze their data with THEME (5), a Bayesian motif discovery program that incorporates prior knowledge about the biochemical properties of many DNA-binding domains. The THEME approach is much more powerful than de novo motif discovery programs in mammalian species, and reveal both the motif and the class of DNA-binding protein that regulates a set of sequences.
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PROGRAM DESCRIPTION AND ORGANIZATION
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WebMOTIFS is designed to automate the identification of regulatory
sequence motifs using multiple motif discovery algorithms. Users
may provide gene names (RefSeq or yeast ORF names) or probe
identifiers from one of the several microarray platforms for
Saccharomyces cerevisiae,
Mus musculus and
Homo sapiens. WebMOTIFS
automates all the remaining steps and sends the user an email
with a link to the results, which are kept on our server for
30 days. In addition to graphical output, the data can be downloaded
in text-based formats. An overview of the processing is shown
in
Figure 1.
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Figure 1. Overview of the WebMOTIFS analysis package. The user provides a set of gene or probe identifiers that WebMOTIFS converts to sequences. In the default mode, the sequences are analyzed by four motif discovery programs. The outputs of each program are tested for statistical significance and clustered to reveal a small set of likely motifs. In the advanced mode, the Bayesian motif discovery program THEME is also used to find motifs consistent with particular DNA-binding domain families. THEME includes its own principled scoring algorithms, eliminating the need for post-processing. The motifs discovered using ChIP-chip data for Fkh2 in high-H2O2 conditions are shown. The motifs discovered using the default settings match the known specificity of Fkh2 and of the interacting protein Mcm1. THEME also finds the Fkh2 and Mcm1 motifs, and in both cases correctly identifies the DNA-binding domain family. Note that many motif discovery algorithms are non-deterministic. Therefore, results may very among repeated runs of WebMOTIFS using the same input.
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Run with the default options, WebMOTIFS reports integrated results
from four motif discovery programs: MEME (
6), AlignACE (
7),
MDscan (
8) and Weeder (
9,
10). WebMOTIFS retrieves sequences
corresponding to each input gene or probe name. If a gene name
is provided or the probe is from a microarray designed for transcriptional
profiling, the sequences are chosen based on the corresponding
transcriptional start site. The sequence surrounding a probe
is used for arrays designed for ChIP-chip. These sequences are
automatically passed to the requested motif discovery programs
without masking.
To combine the results from different motif discovery programs, WebMOTIFS objectively evaluates the significance of each motif. It compares the hypergeometric enrichment score for each motif to the distribution of scores for motifs found by the same program in sets of randomly selected promoters (2). Next, since motif discovery programs may discover very similar motifs, the significant motifs are clustered and a single representative motif for each cluster is computed. Currently, WebMOTIFS uses the clustering algorithm reported in Harbison et al. (2), although more sophisticated algorithms have been developed (11).
WebMOTIFS also provides the option of Bayesian motif discovery with THEME. The THEME algorithm searches for motifs consistent with proteins from specified DNA-binding domain families. The significance of each discovered motif is determined using cross-validation. THEME is particularly powerful in revealing motifs in mammalian promoters that are often missed by other methods (5). The user can specify which DNA-binding domains are expected to be involved in the regulation of the input sequences or test all the available DNA-binding domain families.
WebMOTIFS offers a unique combination of features. First, WebMOTIFS is completely web-based, with all jobs running on our server, so it can work on any operating system. Although many motif discovery programs have web interfaces, it is difficult to merge the results of these programs. Other available tools for running multiple motif discovery programs, such as BEST (the Binding-site Estimation Suite of Tools) (12) and TAMO (Tools for Analysis of Motifs, the software package on which WebMOTIFS is based) (13), are downloadable software packages. Second, WebMOTIFS analyzes the results of motif discovery automatically with default values that typically produce useful results. WebMOTIFS has few adjustable parameters, which makes it less flexible than TAMO and BEST, but also makes it easier to use and less vulnerable to user error. Third, WebMOTIFS facilitates both input and output. It automatically extracts sequences corresponding to gene and probe names, clusters the discovered motifs and produces sequence logos representing the results. Clustering and visualizing the results of motif discovery with multiple programs helps make sense of the large number of discovered motifs, providing a quick summary of the results.
Example
We evaluated WebMOTIFS by analyzing previously reported genome-wide chromatin-immunoprecipitation experiments in S. cerevisiae (2), taking the list of bound genes for each transcription factor in each condition as input to WebMOTIFS. We compared the results from WebMOTIFS with the motifs previously reported by MacIsaac et al. (14). Run with the default settings (without Bayesian motif discovery), WebMOTIFS discovers the correct motif in 51 out of the 64 transcription factors. These results are particularly striking, because, in contrast to MacIsaac et al. (14), the programs currently incorporated in WebMOTIFS do not take advantage of information from evolutionary conservation.
The significance filtering and clustering steps provided by WebMOTIFS reveal the most statistically significant motifs, which are frequently also the most biologically relevant. For example, applying WebMOTIFS to the genes bound by the transcription factor Fkh2 in high-H2O2 conditions produced 163 motifs. Significance filtering eliminated most of these results, and clustering grouped the remaining 32 motifs into two clusters. The highest-ranked cluster is a good match to the known specificity of Fkh2, and the second cluster is a good match to the known specificity of Mcm1. The Mcm1 motif is the most significant motif identified by THEME, which correctly attributes it to the SRF-TF family (15). The next most significant motif matches the known specificity and the DNA-binding domain family of Fkh2 (Figure 1). Fkh2 and Mcm1 have previously been reported to bind cooperatively to a number of promoters in S. cerevisiae (16,17).
We also tested WebMOTIFS on chromatin-immunoprecipitation data from mouse and human (5), taking up to 200 bound probes for each transcription factor as input. In general, motif discovery is more difficult on human and mouse data than on yeast sequences (4). Nevertheless, WebMOTIFS is often able to identify the correct motif as significant when run with the default settings. For instance, applying WebMOTIFS to binding data for Hnf4
in human hepatocytes produced 640 motifs. After significance filtering and clustering, the only remaining motif matches the known specificity of this protein, as reported in TRANSFAC (18) (Figure 2). Running WebMOTIFS with the THEME option also reveals the Hnf4 motif, which is correctly attributed to the nuclear hormone receptor family (19).
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CONCLUSION
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WebMOTIFS is an easy-to-use motif discovery tool that provides
an interface to integrated motif discovery and automatically
does clustering, scoring, and visualization of the results.
WebMOTIFS automatically selects significant motifs, taking the
best results from different motif discovery programs and combining
them in an intuitive output format. It also offers the opportunity
to incorporate prior knowledge of transcription factor structure
using Bayesian motif discovery. Run with the default settings,
it provides accurate results on a variety of data.
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ACKNOWLEDGEMENTS
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We thank Kenzie MacIsaac and other members of the Fraenkel laboratory
for helpful discussions. We are grateful to the authors of AlignACE,
MDscan, MEME and Weeder for permission to include their programs.
Supported by funding from the Whitaker Foundation, the John
S. Reed (1961) Fund and the MIT Undergraduate Research Opportunities
Program. Funding to pay the Open Access publication charges
for this article was provided by the Whitaker Foundation.
Conflict of interest statement. None declared.
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