## 5 Discovery of functional relevant TFBSs
### Context-specific search  
While it is recognized that not all binding sites found on a promoter will be functional elements, it is also recognized that functional sites are not activated simultaneously or independently of condition, or environment, since the cooperation of TFs is highly dependent on context [102-106]. Human RANTES/CCL5, a member of the CC- or \(\beta\)-subfamily chemotactic cytokines for instance, appears to have six functionally characterized short regulatory elements on its promoter that mediate its transcription initiation. However, not all six elements are activated simultaneously in any specific tissue in five cell types analyzed and the elements are also highly selective under different stimulating signals regulating gene expression [107]. Consequently, a critical question is to establish a relationship between binding sites and the context in which these sites become functional. The term 'context' here is used in a way that implicitly refers to a set of potentially co-regulated genes e.g. genes that appear either to exhibit correlation in their expression patterns or to be involved in similar functions in a specific condition and/or tissue [103, 108, 109]. Two elements become critical in this direction: (i) knowledge of the set of potentially co-regulated genes, and (ii) the context-specific nature of functionality. We must, however, realize that TFs in higher organisms are more likely to cooperate with nearby bound factors in a combinatorial manner to regulate the transcription process rather than function isolation. As an example, gene _even skipped_ (_eve_) is known to be regulated by at least five TFs (Twi, Tin, dTcf, Mad, and Pnt) binding to a 312 bp MHE enhancer located \(\sim\)6 kbp downstream of the _eve_ coding region. The corresponding binding sites of these TFs form a _cis_-regulatory modules that has been shown to occur significantly less than randomly expected and also more likely responsible for the regulation of other genes if present on their promoters. Therefore, the field has been shifting from the single motif detection to the discovery of composite motifs i.e. _cis_-regulatory modules. From a computational point of view, the concept of _cis_-regulatory modules also helps to reduce the number of false positive matches and make the search strategy more efficient. A _cis_-regulatory module is in general defined as a _cis_-regulatory element which is the smallest functional unit to have a biological role in transcriptional regulation [53]. It consists of a set of individual binding sites of TFs on the proximal promoter region of a gene. A module is mainly characterized by two factors: composition and structural constraints. Composition is a set of non-overlapping binding sites, whereas structural constraints are the strand orientation to which the corresponding TF binds, the order and the distance between binding sites [110]. A variety of tools have been developed to search for modules in a set of promoter sequences without taking into account the structural constraints (also called composite motifs) e.g. Cluster-Buster [111], CisModule [112], MSCAN [113], CisPlusFinder [114], ModuleMiner [115], DiRE [116]. Alternatively, when _cis_-regulatory modules are associated with their structural constraints (now so-called TF-modules), it refers to methods that detect TFBSs using known TF profiles (FrameWorker [110], CMA [117]). The main idea in this direction is to use prior knowledge to identify the set of potentially co-regulated genes and then search the corresponding promoter set for common and/or significant _cis_-regulatory modules (**Figure 3**). Earlier studies assumed that a cluster of coexpressed genes could be under the same regulatory mechanism, e.g. co-regulation [118, 119] or co-function [120]. However, more recent evidence suggests that co-expression alone is not enough to infer the existence of common regulatory mechanisms and instead additional information is required [108, 121], especially in higher organisms.