## 5 Discovery of functional relevant TFBSs
### Phylogenetic footprinting  
With the advent of novel high-throughput technologies, a large number of genomic sequences of different species have been sequenced, catalogued and annotated, making it possible to explore the conserved information among orthologous genes in an effort to enhance TFBS prediction. The basic underlying assumption of comparative genomics, or phylogenetic footprinting, is that functional regions evolve under constraints and thus at a lower rate than non-functional regions. Therefore, it is hypothesized that well conserved regions in a set of orthologous sequences survived due to their special functional implications, making them become promising candidates for functional _cis_-regulatory elements [75]. Preliminary evidence seems to support the hypothesis that conservation does imply so kind of, yet to be determined, significance. For instance, [PERSON] et al. [76] sequenced six _Saccharomyces_ species and verified that many TFBSs are conserved across species and also located in conserved blocks although the blocks are often times much longer than the binding sites. Similarly, [PERSON] et al. [77] demonstrated that regions with high-scoring PWM matches that are conserved across human-mouse-rat genomic alignment provided a 44-fold increase in the specificity of the predictions compared to those that are not conserved. Therefore, utilizing the information from orthologous genes across multiple species is becoming a useful paradigm in predicting putatively functional binding sites as well as reducing the false positive matches in motif discovering methods. Now given a gene of interest, one begins constructing a global [78, 79] or local multiple sequence alignment [80-82] of orthologous promoter sequences and then identifies conserved regions which are considered as regulatory regions of the gene. However, all these tools assume that all nucleotides are alike or use a well-established substitution matrix to penalize the insertion, deletion, or substitution in the alignment, and thus they may not align properly non-coding DNA sequences of orthologous genes[82]. Another critical limitation is that for closely related species, the alignment is obvious but impossible to distinguish functional elements from the surrounding non-functional regions like the case of four _Saccharomyces_ species from the sensu stricto group, two species from the sensu lato group and one petite-negative species for example [83]. On the other hand, when the species are too far apart evolutionarily as the case of \(rbcS\) gene in 10 plants shown span \(\sim\) 760 million years of evolution, only 3 conserved sites that are known regulatory elements each of 9 bp long are present in around 500 bp in the 5' upstream regions [84]. Therefore, given that _cis_-regulatory elements are short, degenerate fragments, and present on such a large number of non-functional, diverged regions, regular sequence alignment methods are usually failed to detect properly these short conserved regions [85]. To overcome the problems associated with alignment, motif discovery algorithms e.g. Consensus, Gibbs sampling, MEME have been utilized along with the set of orthologous promoter sequences as the input data, [83, 86]. As a result, it is more likely to have a functional binding site as well as reducing false positive matches if some TF profile is located on a conserved region in the set of promoter sequences; especially, if the region is conserved among sequences from distantly related species. As such the match would be subject to selection pressure and more likely to be functional. A number of available tools such as PhyloCon [87] and CONREAL [88], explored those ideas. However, in the aforementioned approaches phylogenetic relationships of the given sequences are not explored. Therefore, results are still highly biased to favor relationships between sequences located on closely related species [85].