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Modeling Transcription Factor Target Promoters 131The drawback of these integrative approaches is that these programs tend tomiss many functional TFBSs that show very little sequence conservationeven across modestly distant species, because of single-nucleotide substitutionsand small indels within the regulatory regions.Novel high-throughput technologies, such as ChIP-chip, have enabledgenome-wide identification of the epigenetic mechanisms and protein–DNAinteractions that effect gene expression (29). In ChIP-chip experiments, chromatinimmunoprecipitation of specific protein/DNA complexes followed bymicroarray analysis is performed to probe a promoter microarray panel (e.g.,CpG-island microarray panel [30]). In recent years, the author (31–34) andothers (35) have successfully used ChIP-chip assays to find the target genesof TFs in mammalian systems. The major focus of this chapter is to introducedifferent bioinformatics tools that identify TFBS in a set of genomicsequences, and discuss the application of these methods in the high-levelanalysis of ChIP-chip experimental data.2. MaterialsThe user must have access to a computer with Internet access; for example,a PC running Microsoft Windows or Linux, an Apple Macintosh, or a UNIXworkstation. The user should be familiar with the use of Netscape Navigator orMicrosoft Internet Explorer, and the R statistical package http://www.r-project.org/.If the R programming package is not readily available the user can download theR base package from R-project website (through http://CRAN.R-project.org).The classification packages “rpart” and “randomForest” should be downloadedand installed in R. The user-friendly commercial CART software from Salfordsystems(http://www.salford-systems.com) and the professional version ofTRANSFAC from Genomatix (http://www.genomatix.de) would be helpful, butnot necessary. The list of commonly used TFBS prediction programs based onPWM and phylogenetic footprinting approaches are provided in Table 1.3. MethodsFirst an overview of the methodology is provided in Subheading 3.1., thena worked example is presented in Subheading 3.2.3.1. An Overview of In Silico Identification of TF Target PromotersQuite a few methods are available to scan for TFBSs in a candidate promotersequence. The simplest method of searching for a TFBS is by its consensussequence of preferred nucleotides at specific positions of the binding site (36).Perhaps the most widely used method is the PWM approach, wherein a candidateTFBS is represented by a matrix of nucleotide scores reflecting the likelihoodof each nucleotide at specific position (37). Although consensus sequence and