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Prediction Using PAINT 59In PAINT, the clustered data can be visualized as a matrix layout with the hierarchicaltree structure aligned to the rows and the columns of the Feasnet. Thezeros in the matrix are shown in black and the nonzero entries in the Feasnet arecolor based on the p-value of the corresponding TRE. The brightest shade of redrepresents low p-value (most significantly overrepresented in the Feasnet).Conversely, the brightest shades of cyan represent smaller p-values for underrepresentationin the observed Feasnet indicating more significantly underrepresentedTREs. This image can optionally represent the cluster index of each gene, whereinsuch cluster indices are generated from other sources such as expression or annotation-basedclustering. With such visualization, it is straightforward to explore therelationship between expression/annotation-based clusters and those based on cisregulatorypattern (i.e., Feasnet). The FeasNetViewer module can also generate anetwork layout diagram using the GraphViz libraries (available athttp://www.research.att.com/sw/tools/graphviz/). In the web-based PAINT, previousanalyses can be retrieved and/or continued using a job key provided for eachanalysis. The PAINT results are presented in a hyperlinked report and can also bedownloaded as a single compressed file for offline perusal.Nomenclature for this article includes bold italic for onscreen text, bold forbuttons, and courier font for files and folders.3. MethodsThe methods outlined next describe TRNA of biologically associated genesusing PAINT. Genes are typically associated by highly parallel experimentalapproaches such as microarray-based gene-expression analysis or proteomicanalyses. However, excellent results have been obtained by creating gene listsfrom extant literature by manual searches or computationally derived results from“knowledge database” searching.3.1. Identification of Overrepresented TF-Binding Sites Using PAINTA typical scenario of using PAINT is to study a group of genes identified orexpected to be coregulated under specific experimental conditions. PAINT isused to investigate whether these genes share any TF-binding sites in theirpromoters and if such a shared coincidence of binding sites is significantly higherthan random frequency as determined by Fisher’s exact test.3.1.1. Generation of PAINT-Compatible Input FileThe starting point to PAINT is a file containing the list of genes under investigation.The file should be a single column plaintext file, each row listing agene identifier. All the identifiers in the file need to be of the same type, forexample, Genbank accession number. An example gene list file (named