Hu and Gallo BMC Genomics 2010, 11:49 http://www.biomedcentral.com/1471-2164/11/49 Page 9 of 15 Figure 5 Overlapping tissue TF pairs and their functions between muscle tissues. (a) Venn diagram displaying the significant overlap for both tissue TF pairs and their functions in the group of skeletal muscle. (b) Significant overlap for both tissue TF pairs and their functions in the group of smooth muscle. Each circle indicates the number of tissue TF pairs. The number of overlapping tissue TF pairs and TF functions between two tissues is indicated in bold (# function/# TF). Also shown are their corresponding p-values from hypergeometric tests. networks were then compared to tissue-type TF-TF interaction networks to estimate the statistical signifi- cance for the distribution of liver-specific TFs. The results indicate that known liver-specific TFs were sig- nificantly enriched as internal TFs for these 7 tissue- type TF-TF interaction networks (bootstrap analysis; p < 10-20). By contrast, the total number of liver-specific TFs in these 7 tissue-type TF-TF interaction networks was not enriched (bootstrap analysis; p = 0.11). These results suggest that liver-specific TFs, other than initiating liver-specific transcriptional event, may play an impor- tant role in recruiting non-liver-specific TFs to the tis- sue-type TF-TF interaction network, thus offering the potential for coordinating and controlling gene expres- sion across a variety of conditions. Prediction of multiple interacting TFs As a first step to elucidate cis-regulatory modules involved in tissue gene regulation, we extended our ana- lysis to the interactions of 3 TFs (named as multiple interacting TFs). Using tissue TF pairs from each of the 79 tissues, we performed a two-step analysis of TFBS conservation and enrichment of overlapping orthologous genes between human and mouse (see Methods). Although it is likely that multiple interacting TFs may be under estimation by the use of tissue TF pairs instead of all predicted TF pairs, the predicted multiple interact- ing TFs are tissue-specific. Therefore, these predictions most likely represent cis-regulatory modules involved in tissue gene regulation. To this end, we identified 1735 unique interactions of 3 TFs for the 79 human tissues, ranging from 9 multiple interacting TFs for testis inter- stitial to 72 multiple interacting TFs for caudate nucleus (Additional File 5). The validity of these predicted multiple interacting TFs was assessed by using liver-specific single TFs from TRANSCFAC11.4 [25], as few known cis-regulatory modules were available. We performed analysis to see if known liver-specific TFs were statistically enriched in 30 predicted multiple interacting TFs from liver tissue. We found 4 of them (bootstrap analysis; p < 10-3) whose 3 TFs were all liver-specific, 18 (bootstrap analysis; p < 10-8) with at least 2 liver-specific TFs, and 28 (bootstrap analysis; p < 10-5) with at least 1 liver-specific TF. These results provide evidence for the enrichment of liver-spe- cific TFs in the predicted multiple interacting TFs, which in turn demonstrated the validity of the prediction. We next searched for all predicted multiple interacting TFs and their potential functions that are common between tissues. The results indicated that, although common multiple interacting TFs existed between most tissues, the highest overlap was within brain tissues and between brain and gland tissues. By contrast, there was little overlap for the functions of multiple interacting TFs, except within brain and cancer and between these 2 tissue types (Additional File 6). The latter is especially interesting to us, as cancer cells have a global effect on immune systems, which in turn control and shape developing cancer [31]. Six multiple interacting TFs were found to have common functions between immune systems and cancer tissues, including CEBPGAMMA: NKX25:PLZF, CEBPGAMMA:PAX4:PLZF, CP2:NFY: PAX4, FOXJ2:PAX4:POU3F2, CEBPGAMMA:PAX4: PLZF, and FOXJ2:HNF3:PAX4. These results revealed not only the common mechanisms for transcriptional regulation but also the common functional role of