decided to systematically analyze both genome-wide transcription factor binding site predictions 
made in silico and comprehensive literaturederived information about target genes of selected TFs. First, we predicted which of the transcription factors have binding sites in the RefSeq gene promoters using the ProbTF tool combined with an empirical p-value computation. We LY3039478 site focused on genes that were identified by the previous LIGAP analysis and considered all transcription factors that had known binding specificities in TRANSFAC . We did not restrict our analysis only to those TFs whose transcripts are differentially expressed because, e.g., STAT6 is not differentially expressed ij et al. Recently, it was found out that most of the direct targets of STAT6, an important regulator of Th2 differentiation, were up-regulated in Th2 cells. Here we were interested in identifying TFs whose binding sites are enriched in the promoter regions of the genes which are differentially regulated in Th2 conditions, both among the up-regulated and down-regulated genes. Instead of looking at individual TF binding predictions that are prone to contain false positives, we used the Fisher’s exact test to search for enrichment of binding sites, in comparison to randomly selected gene set. The same analysis was carried out separately for all the differentially regulated gene sets and by taking into account the direction of regulation. Using a p-value cut-off of 0.01 for TF binding, we identified three hits from the enrichment analysis among Th2 specific up-regulated genes and three among the Th2 specific down-regulated genes. The results are depicted in motifs were combined and their targets were pooled. In accordance to our previously published results, the strongest hit within the Th2 up-regulated genes was STAT6, followed by NKX3A, and CDP. NKX3A is a member of the NKX family of homeobox genes that is expressed in prostate epithelium and functions as a potential prostate tumor suppressor. Recently, in a PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19796633 study focusing on Jurkat cells, a GATA3 binding site on the promoter of NKX3 gene was identified. Furthermore, in mouse increased expression of Nkx3a was observed to be regulated by IL-4 independently of STAT6. CDP is highly conserved homeodomain transcription factor involved in many cellular processes, including differentiation, development and proliferation.The three TF hits having enriched predicted binding sites among the Th2 down-regulated genes were the interferon regulatory factor family of TFs, IFN-stimulated genes factor 3 and STAT6. IRF family consists of IRF1 to IRF10 and has been shown to be essential in expression of type I interferon genes, IFN-stimulated genes and other pro-inflammatory response related cytokines. These genes are maintained down-regulated during Th2 proliferation and therefore, the results are in line with the Th2 effector cells characteristics. Moreover, IFN-induced expression of IRF1 and IRF2 has been shown to directly down-regulate IL-4 production by repressing IL-4 promoter sites. Opposing to other IRF family proteins, IRF4 has been shown to directly activate IL-4 promoter and IL-10 regulatory elements and be essential in Th2 cell differentiation by influencing the expression of GFI1, a transcriptional repressor in Th2 cells. However, the analysis relying on known TF binding specificities will not allow segregation of individual members of the IRF family. Further, an essential regulator of most ISGs is ISGF3 that is composed o