 Research
 Open Access
 Published:
Extracting transcription factor binding sites from unaligned gene sequences with statistical models
BMC Bioinformatics volume 9, Article number: S7 (2008)
Abstract
Background
Transcription factor binding sites (TFBSs) are crucial in the regulation of gene transcription. Recently, chromatin immunoprecipitation followed by cDNA microarray hybridization (ChIPchip array) has been used to identify potential regulatory sequences, but the procedure can only map the probable proteinDNA interaction loci within 1–2 kb resolution. To find out the exact binding motifs, it is necessary to build a computational method to examine the ChIPchip array binding sequences and search for possible motifs representing the transcription factor binding sites.
Results
We developed a program to find out accurate motif sites from a set of unaligned DNA sequences in the yeast genome. Compared with MDscan, the prediction results suggest that, overall, our algorithm outperforms MDscan since the predicted motifs are more consistent with previously known specificities reported in the literature and have better prediction ranks. Our program also outperforms the constraintless Cosmo program, especially in the elimination of false positives.
Conclusion
In this study, an improved sampling algorithm is proposed to incorporate the binomial probability model to build significant initial candidate motif sets. By investigating the statistical dependence between base positions in TFBSs, the method of dependency graphs and their expanded Bayesian networks is combined. The results show that our program satisfactorily extract transcription factor binding sites from unaligned gene sequences.
Background
Understanding transcription is central to understanding genetic regulatory mechanisms. The transcription of a gene is generally dependent on the presence of specific signals located at upstream regions of the corepromoter. These specific signals derive from their use as binding sites by transcription factors (TFs), and are therefore termed transcription factor binding sites (TFBSs). Recently, chromatin immunoprecipitation followed by cDNA microarray hybridization (ChIPchip array) has been used to identify potential regulatory sequences, but the procedure can only map the probable proteinDNA interaction loci within 1–2 kilobases resolution [1]. To find out the exact binding motifs, it is necessary to build a computational method to examine the ChIPchip array binding sequences and search for possible motifs representing the TFBSs (motif discovery).
There are many computational TFBS motif finding tools available [2–4]. The traditional approach for finding TFBSs is to collect and align a set of promoter sequences of coregulated genes from either the literature or systematic experiments. Numerous computational tools, such as CONSENSUS [5], EM [6], MEME [7] and the Gibbs sampler [8], have utilized the approach to identify short DNA sequence motifs which are statistically overrepresented in the promoter sequences.
Other than the alignmentbased motif finding algorithms in above, many approaches have tried to extend to the use of evolutionary conservation information such as phylogenetic footprinting or the detection of combinations of binding sites (termed as cisregulatory modules; CRMs) [2, 3]. Phylogenetic footprinting methods [9–11] is an approach that seeks to identify conserved regulatory elements by comparing genomic sequences between related species. However, due to the statistical nature of the approach, e.g., a small amount of closely related species, not all transcription binding sites can be found by using phylogenetic footprinting. Hence, some algorithms have emerged to combine the alignmentbased motif prediction with phylogenetic footprinting such as PhyloGibbs [12] and MY sampler [13]. On the other hand, by the detection of CRMs due to the cooperative interactions between TFs, algorithms like those in [14–16] can produce predictions of substantially better specificity than those of isolated sites.
Recently, more effective motif finders, e.g., MDscan [1], ANNSpec [17], DMOTIFS [18], DME [19] and Cosmo [20], have taken the advantage of a background set, serving as a negative control. The goal of these discriminant motif finders is to search only for motifs that are most discriminating, that is, only those enriched in the foreground set relative to the background set [2]. Although these motif finders have improved the performance of TFBS prediction, it is still a trouble to have a satisfactory solution. How to find out accurate binding motifs may require much attention in the computational biology community. In this study, an improved sampling algorithm is proposed to incorporate the binomial probability model to build significant initial motif sets. By investigating the statistical dependence between base positions in TFBSs, it appears feasible to use statistical models to formulate the structural dependence of a motif in the identification of TFBSs. In light of this observation, the method of dependency graphs and their expanded Bayesian networks [21] is combined and prediction results show that our algorithm is able to find out motifs more consistent with previously known evidence.
Methods
Let TF be one of the transcription factors to be investigated. The binding dataset of the transcription factor TF, denoted as B_{ TF }, consists of the sequences with low binding pvalue (< 0.001) to the TF in the ChIPchip array data [22]. A sliding window of size w is used to extract segments of length w when sliding through each of the sequences in B_{ TF }.
Let S_{ TF }be the collection of all extracted segments from B_{ TF }, M the number of sequences in the binding dataset B_{ TF }, L_{ i }the length of the i th sequence in the binding dataset B_{ TF }, and T_{ TF }the total number of segments in S_{ TF }. Then
To discover the binding motifs of the transcription factor TF, a number of initial candidate motif sets for TF is subsequently built from the collection S_{ TF }of extracted segments. Note that the contents of segments, called patterns, in S_{ TF }may not be distinct.
Most of early motif finding algorithms, such as Gibbs sampler [8] and MEME [23], have a weakness, where initial candidate motif sets are built by randomly extracting segments from sequences in the binding dataset B_{ TF }(i.e. randomly selecting segments from the set S_{ TF }). To improve the deficiency, the binomial probability distribution model is firstly utilized in the establishment of a number of initial candidate motif sets in our algorithm.
Then in the process of iterative sampling in our algorithm to expand and/or trim each of the initial candidate motif sets, the method of dependency graphs and their expanded Bayesian networks [21] is used to develop a statistical model for the background motif set identified as the union S = ∪_{ TF }S_{ TF }of segments extracted from all transcription factor binding datasets.
The basic procedure to find the binding motifs of the transcription factor TF is as follows:

1.
Build N initial candidate motif sets.

(a)
Take N distinct patterns from the set S_{ TF }with the most highest significance scores as the candidates by the binomial distribution model (see the Binomial probability distribution model subsection).

(b)
Then for each of the N significant binding site candidates for the transcription factor TF, in view of evolution, collect all segments in S_{ TF }whose patterns have no more than d Hamming distance matching to the candidate pattern to form an initial candidate motif set.
At this stage, N initial candidate motif sets for the transcription factor TF are built.

2.
Iteratively sample through the binding dateset B_{ TF }to expand and/or trim each of the N initial candidate motif sets so that their approximate maximum a posteriori (AMAP) scores [1, 24] can keep increasing until the N candidate motif sets are invariant in K consecutive iterations (see the Iterative sampling subsection).

(a)
In the calculation of AMAP scores in this stage, the background model for the background motif set S = ∪_{ TF }S_{ TF }is established under the method of dependency graphs and their expanded Bayesian networks (see the Method of dependency graphs and their expanded Bayesian networks subsection).

3.
Refine each of the N candidate motif sets by reexamining all the segments already included in the motif set. A segment is removed from the motif set if doing so increases the AMAP score.
A simple flowchart for our algorithm is shown in Figure 1. The following subsections will expatiate on each stage of our algorithm. As an illustration of the dynamics of the PWM and the rank of different candidate motif sets at different stages of our algorithm, a summary of the prediction process for the motif of the transcription factor CBF1 is given in Figure 2.
Initial motif sets building
Our method begins by enumerating those patterns in S_{ TF }that appear most often in the binding dataset B_{ TF }than in others. What we want to do first is to calculate the appearance probability of a pattern in S_{ TF }, which is the probability that the pattern appears no less than n times in the binding dataset B_{ TF }. If a pattern b appears more often than other patterns in S_{ TF }and its occurrence probability in a generic intergenic region is comparatively low, the calculated significance score of b would be relatively high. We will take patterns with the most highest significance scores as the candidates to build a number of initial candidate motif sets.
Binomial probability distribution model
The probability to observe exactly j occurrences of pattern b in the collection S_{ TF }of segments extracted from the binding dataset B_{ TF }is estimated by the binomial distribution
where occ(b) is the occurrence times of pattern b in S_{ TF }and f (b) is the probability that pattern b occurs in the intergenic region and is estimated as the relative frequency of pattern b in the union S = ∪_{ TF }S_{ TF }of segments extracted from all transcription factor binding datasets. The probability to observe n or more occurrences of the pattern b in S_{ TF }is
We define the significance score sig_{ TF }(b) of a pattern b to TF as
sig_{ TF }(b) = log_{10}(P_{ TF }(occ(b) ≥ n)).
The less probable pattern b in S appears more than n times in S_{ TF }, the more probable will it be a binding site candidate for the transcription factor TF. We will take N distinct patterns with the most highest significance scores as the candidates.
For each of the N significant binding site candidates for the transcription factor TF, in view of evolution, collect all segments in S_{ TF }whose patterns have no more than d Hamming distance matching to the candidate pattern to form an initial candidate motif set. Thus N initial candidate motif sets for the transcription factor TF are built at the end of this stage. As an example, the PWM and the rank of five initial candidate motif sets for the motif prediction of the transcription factor CBF1 are shown in Figure 2.
Iterative sampling
In this stage, a sampling method is used to expand and/or trim each of the N initial candidate motif sets M_{1}, M_{2},.., M_{ N }. For our purpose, a false motif set M_{N+1}is created by randomly selecting e_{0} (e_{0} is equal to the maximum size of the N initial candidate motif sets) segments from the collection S_{ TF }such that M_{ i }∩ M_{N+1}= ∅, for all i = 1, 2,..., N. In addition, let the collection S = ∪_{ TF }S_{ TF }of segments extracted from all transcription factor binding datasets represent the intergenic background and here be denoted as M_{ BG }.
Approximate maximum a posteriori (AMAP) measure
The score $ama{p}_{{M}_{i}}$ of the approximate maximum a posteriori (AMAP) measure of the candidate motif set M_{ i }is defined as [1, 24]
where p_{s, j}is the frequency of nucleotide j at base position s in the candidate motif set M_{ i }(which can be retrieved from the position specific scoring matrix (PSSM) of M_{ i }), n_{ i }is the number of segments in M_{ i }, and P(mM_{ BG }) is the probability of the pattern of segment m in the motif set M_{ i }under an expanded Bayesian network (EBN) model [21] developed from the background motif set M_{ BG }(EBN model will be discussed shortly).
The first part of the AMAP score is a negative entropy, which is higher if there are more similar patterns in the candidate motif set M_{ i }. A motif set M_{ i }with all identical patterns has the maximum negative entropy 0, whereas equal nucleotide frequencies at every position in the PSSM of M_{ i }has the minimum negative entropy. And a segment m in the candidate motif set M_{ i }which has a pattern much different from the background motif model built from M_{ BG }would have lower appearance probability P (mM_{ BG }) and hence increases the score $ama{p}_{{M}_{i}}$ of the AMAP measure of M_{ i }.
Sampling strategy
In each iteration, there are two steps for the sampler, the Sstep and the Mstep.
In the Sstep, the sampler samples a site by randomly selecting a sequence from B_{ TF }and then randomly picking up a site in the selected sequence to extract a segment m_{ s }of length w. For 1 ≤ i ≤ N, if the sampled segment m_{ s }appears in M_{ i }, segment m_{ s }will be removed from M_{ i }if the AMAP score $ama{p}_{{M}_{i}}$ of the candidate motif set M_{ i }increases after its removal; otherwise, segment m_{ s }will be kept in M_{ i }. Note that the PSSM of the motif model M_{ i }should be retrained if the sampled segment m_{ s }is removed from M_{ i }.
Which one of the N + 1 motif sets would be the best motif set for the sampled segment m_{ s }will depend on the appendant score $ap{p}_{{M}_{i}}$ that the segment m_{ s }is derived from M_{ i }[24, 25]
where n_{ i }is the size of current motif set M_{ i }, P(n_{ i }) equals $\frac{{n}_{i}}{{T}_{TF}}$, P(m_{ s }M_{ i }) and P(m_{ s }M_{ BG }) are the probabilities of the content of the sampled segment m_{ s }under the PSSM model developed from the current motif set M_{ i }and under an EBN model developed from the background M_{ BG }, respectively. The sampled segment m_{ s }will be considered to append into the motif set M_{ i }with the highest appendant score $ap{p}_{{M}_{i}}$. If $ap{p}_{{M}_{N+1}}$ is the highest score, then the sampled segment m_{ s }is appended into the false motif set M_{N+1}unless m_{ s }is already there and the current iteration stops here. If for some i, 1 ≤ i ≤ N, $ap{p}_{{M}_{i}}$ is the highest score, the sampled segment m_{ s }will be further checked in the Mstep to see if we really want to append m_{ s }into M_{ i }unless we have processed m_{ s }for M_{ i }at the beginning of this Sstep as in above and the current iteration stops here.
In the Mstep, the sampler has to decide whether the newly sampled segment m_{ s }should be appended into the candidate motif set M_{ i }or not. The AMAP measure again will be used to evaluate our decision. The sampled segment m_{ s }is appended into the candidate motif set M_{ i }if and only if the score $ama{p}_{{M}_{i}}$ of the motif model M_{ i }is increased once the sampled segment m_{ s }is appended to M_{ i }. Note that the PSSM of the motif model M_{ i }should be retrained after the sampled segment b_{ s }is appended to M_{ i }. Now the Mstep is done and the current iteration stops here.
The sampler will iteratively sample through the binding dataset B_{ TF }to expand and/or trim the N candidate motif sets M_{1}, M_{2},.., M_{ N }so that their AMAP scores $ama{p}_{{M}_{i}}$ will keep increasing. The N candidate motif sets will tend to be invariant after a (larger) number of iterations. The stopping criterion of the sampling process is that all the N candidate motif sets are invariant in K consecutive iterations. The parameter K is usually set to be 1% of the size of S_{ TF }.
Alternative sampling strategy
There is an alternative sampling strategy as follows.
In the Sstep, the new sampler also randomly samples a site from a sequence in B_{ TF }to extract a segment m_{ s }of length w. For 1 ≤ i ≤ N, if the pattern of the sampled segment m_{ s }appears in M_{ i }, all the segments in M_{ i }whose pattern is the same as that of m_{ s }will be removed if the AMAP score $ama{p}_{{M}_{i}}$ of the motif set M_{ i }increases after their removal. Otherwise, these segments will be kept in M_{ i }.
Also in the Sstep, if $ap{p}_{{M}_{N+1}}$ is the highest among all $ap{p}_{{M}_{i}}$, 1 ≤ i ≤ N + 1, then all segments in the set S_{ TF }having the same pattern as that of the sampled segment m_{ s }will be appended into the false motif set M_{N+1}unless these segments are already there and the current iteration stops here. If $ap{p}_{{M}_{i}}$ is the highest for some i, 1 ≤ i ≤ N, the sampled segment m_{ s }will be further checked in the Mstep to see if we really want to append those segments in the set S_{ TF }having the same pattern as that of the sampled segment m_{ s }into M_{ i }unless we have already processed those segments for M_{ i }at the beginning of this Sstep as in above and the current iteration stops here.
In the Mstep, all the segments in the set S_{ TF }having the same pattern as that of the sampled segment m_{ s }are decided to append to the candidate motif set M_{ i }if and only if the AMAP score $ama{p}_{{M}_{i}}$ of M_{ i }increases after these segaments are appended into M_{ i }.
Method of dependency graphs and their expanded Bayesian networks
Considering the binding mechanism of transcription factors to specific DNA sites (motifs), there must be distinctive features for the specific motif regions from other intergenic regions which represent the background DNA sequence. Hence, it is conceivable that we can use a statistical model to capture the feature of a specific DAN site (motif) or a generic DNA intergenic region (background). Since the size of a candidate motif set M_{ i }is often small, a PSSM model is commonly used for M_{ i }instead of any other more sophisticated statistical model. However, the size of the background motif set M_{ BG }is usually large enough to be equipped with a more sophisticated one.
As reported in [21], a dependency graph model is used to fully capture the intrinsic interdependency between base positions in a motif or region. The establishment of dependency between two positions is based on a χ^{2}test from known sample data. An edge is established between two nodes (a node represents a base position) in the graph if the two corresponding base positions of the motif or region are dependent. After all dependent edges have being established completely, a dependency graph for the motif or region is constructed. An example of a dependency graph with 7 nodes is shown in Figure 3.
As reported in [21], although the dependency graph can fully capture the intrinsic interdependency between base positions in a motif or region, it is difficult, if not impossible, to perform statistical inference based on the dependency graph. To resolve the dilemma, the dependency graph is expanded to form a Bayesian network (which is a directed acyclic graph that facilitates statistical reasoning) by allowing a base position in the dependency graph to appear more than once in the Bayesian network as nominally distinct nodes. Figure 4 shows an example of an expanded Bayesian network of the dependency graph in Figure 3. For the detailed procedure of constructing an expanded Bayesian network (EBN) from a dependency graph, please see [21]. In this paper, we use EBNs to model the background motif set M_{ BG }.
Continued with the same example of the motif prediction of the transcription factor CBF1, the PWM and the rank of the five candidate motif sets at the end of the iterative sampling stage are also shown in Figure 2, together with the final results at the end of the refinement stage.
Results and discussion
Data
In order to search for the transcription factor binding sites that regulate gene expressions, we collected binding promotor sequences from the cDNA microarray hybridization (ChIpchip array) of yeast genome [22]. Each of the binding sequences may contain some unknown motifs that are implanted at unknown positions. These data represent the binding affinity of a target transcription factor to the promoter region of a gene in vivo. The experiment protocol assigns a binding pvalue to each binding promoter sequence of the corresponding transcription factor. A sequence with binding pvalue less than 0.001 is considered to be bound by the corresponding transcription factor. The threshold of 0.001 is set up to reduce the false positive identification in yeast genomewide screening.
We collected the ChIpchip array sequence data from the "Motif discovery results – Discovered motifs, version 24" at [26]. For a transcription factor TF to be investigated, we collected all sequences with binding pvalue less than the threshold 0.001 to TF into the binding dataset B_{ TF }. There are 65 binding datasets B_{ TF }being able to be collected from Harbison's website.
Accuracy measurement and comparison
To evaluate the performance of our program, we collected known specificities from many famous websites, such as YPD, SCPD, Transfac and from the literature with experimental evidence [27] to compare with the discovered specificities predicted by our program.
Among the 65 binding datasets B_{ TF }collected from Harbison's website, we chose 36 transcription factor binding datasets which have known specificities with experimental evidence to evaluate the performance of our program. The results of our program for the 36 transcription factor binding datasets are listed in Figure 5. It is deserved to be mentioned that the specificity reported for transcription factor PHO2 in Harbrison et al's website is "GTGCGsyGCG", while the predicted result of our program is "ATTATC". In this case, the newly found motif by our program is more consistent with the results reported by Barbaric et al [28] that PHO2 binds to an ATrich region than the specificity reported in Harbrison et al's website.
In this study, we compared our program with two online programs, MDscan [29] and Cosmo [30]. MDscan is a famous program that can be used to examine the ChIParray selected sequences and search for DNA sequence motifs representing the proteinDNA interaction sites. It takes the advantage of combining two widely adopted motif search strategies, word enumeration and positionspecific weight matrix updating, and incorporates the ChIP enrichment information to accelerate the search and enhance its success rate. The comparison of MDscan with our program is shown in Table 1. Also reported in Table 1 is the performance of our algorithm when the the PSSM model [8] instead of the EBN model [21] is used to model the background motif set M_{ BG }in the calculation of the AMAP scores of the N candidate motif sets and the appendant scores of the N + 1 motif sets. In Table 1, for each transcription factor, the number in each 'Rank' column indicates the rank of the predicted motif which is most consistent with the known evidence from the top ten predicted candidate motifs.
As shown in Table 1, our approach with EBN background model outperforms the other two methods. Our approach with EBN background model gives 30 out of the 36 most predicted motifs for the corresponding 36 transcription factors with the 1st rank, while MDscan and our approach with PSSM background model give only 20 out of 36 and 15 out of 36 most predicted motifs with the 1st rank, respectively. Moreover, MDscan fails in discovering a motif for three transcription factor binding datasets, while our approach in this study is still able to predict a motif consistent with the known evidence.
Cosmo (constrained search for motifs) is a general purpose algorithm for conserved motif detection that allows the search to be supervised by specifying a set of constraints that the PWM of the unknown motif must satisfy. Such constraints may be formulated derived from prior biological knowledge about the structure of the transcription factor, such as the length of the motif intervals. Although Cosmo is based on the same twocomponent multinomial mixture model used in MEME, it employs the likelihood principle instead of the Evalue criterion in MEME. In addition, three model types (OOPS, ZOOPS, or TCM) can dataadaptively be selected in Cosmo to achieve better performance. Since there is no prior knowledge used in our program, we compared it to the constraintless version of the Cosmo program. On the other hand, since the Cosmo program reports only one motif PWM for a dataset, instead of a list of ranked candidate motif PWMs as in MDscan, we adopted only the rank 1 results of our program in this comparison. To evaluate the performance of both programs, we used the statistics proposed by Tompa et al. [4]. For a (computational) tool at the site level, the performance statistics on a dataset are defined as follows:
where TP is the number of known sites overlapped by predicted sites, FN is the number of known sites not overlapped by predicted sites, and FP is the number of predicted sites not overlapped by known sites. To summarize the performance of a given tool over a collection $\mathcal{C}$ of datasets, we compute the "combined" statistics as though C were one large dataset by adding TP, FP and FN respectively over the datasets in $\mathcal{C}$. Then the combined statistics of our program are Sn = 0.6698, PPV = 0.8206, and ASP = 0.7452, while those of Cosmo are Sn = 0.6573, PPV = 0.5134 and ASP = 0.5854. For the detailed Cosmo prediction results and the comparison of the two programs, please see Figure S1 (see Additional file 1). The comparison shows that our program can offer better performance than Cosmo, especially in the elimination of false positives.
The parameters used in our program include the sliding window size w used to extract segments from binding datasets B_{ TF }to form S_{ TF }, the Hamming distance d used to collect segments from S_{ TF }to establish initial candidate motif sets, the number of most dependent edges used to form a dependency graph for the background motif model and the number of parents used in the construction of an expanded Bayesian network from the dependency graph [21]. The parameters used in our program to give the best predicted motifs for each of the 36 transcription factors are listed in Table S1 (see Additional file 2). Comparing the performance of the two sampling strategies discussed in the Method section, as shown in Figure S2 (see Additional file 3), we found that the alternative sampler is faster and has almost identical best predicted motifs with those by the primary sampler, except that transcription factors GCN4, HAP4 and PHO4 have the best predicted motifs one nucleotide position shift from those by the primary sampler. In addition, the alternative sampler is slightly better than the primary sampler in the sense that the best predicted motif for the transcription factor DIG1 promotes its rank from the 2nd place by the primary sampler to the 1st place by the alternative sampler.
Conclusion
In this study, we employed the binomial probability model to establish a number of initial candidate motif sets, and used the method of dependence graphs and their expanded Bayesian networks to model the background motif set as a control to predict TFBSs (motifs) from a set of unaligned DNA sequences. The prediction results suggest that, overall, our algorithm outperforms MDscan since the predicted motifs are more consistent with previously known specificities reported in the literature and have better prediction ranks. And when compared with the constraintless Cosmo program, our algorithm has a slightly higher combined sensitivity Sn, a much higher positive predictive value PPV and a higher average site performance ASP. However, the performance of our algorithm is not much better if the length of possible binding sites are too long (more than 12 bps). Further research is needed to discover long motifs.
Furthermore, variable spacing within binding sites is legitimate for some transcription factors while this study focuses on ungapped motif discovery. Programs such as BIPAD [31] and spaced dyad [32] have investigated into such a bipartitie sequence element discovery problem. Therefore another direction for our future research is to investigate into gapped motifs.
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Acknowledgements
We thank YiSian Liao for helping the execution of our prediction program. We also thank the reviewers for their valuable suggestions which improve the presentation of this paper. This work was supported by the National Science Council, Taiwan, under Contracts NSC 933112B007004 and NSC953114P002005Y.
This article has been published as part of BMC Bioinformatics Volume 9 Supplement 12, 2008: Asia Pacific Bioinformatics Network (APBioNet) Seventh International Conference on Bioinformatics (InCoB2008). The full contents of the supplement are available online at http://www.biomedcentral.com/14712105/9?issue=S12.
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The authors declare that they have no competing interests.
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CL and WY developed and implemented the method. All authors participated in discussions and writing of the paper.
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Lu, CC., Yuan, WH. & Chen, TM. Extracting transcription factor binding sites from unaligned gene sequences with statistical models. BMC Bioinformatics 9, S7 (2008). https://doi.org/10.1186/147121059S12S7
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Keywords
 Transcription Factor Binding Site
 Position Specific Score Matrix
 Phylogenetic Footprinting
 Candidate Motif
 Motif Finding Algorithm