Hu and Gallo BMC Genomics 2010, 11:49 http://www.biomedcentral.com/1471-2164/11/49 Page 3 of 15 Figure 1 Flowchart of analysis procedures. (a) Identification of tissue and tissue-type TF pairs. (b) Prediction of interactions of three TFs. (c) Reconstruction of tissue-type TF-TF interaction networks. HK: housekeeping genes; hs: human; mm: mouse. portion of the TF pairs performs ubiquitous roles across different tissues. The resulting tissue TF pairs range from 111 to 176 for different tissues. We also searched for TF pairs specific to one tissue (referred to tissue- unique TF pairs) and obtained from 2 to 20 such TF pairs for different tissues. The number of tissue TF pairs and tissue-unique TF pairs for each of the 79 human tissues are summarized in Table 1. The top 5 tissue TF pairs that have the most significant correlations between enriched TFBS pairs and enriched overlapping ortholo- gous genes are also listed for each tissue. Overall, we identified 2549 tissue and 803 tissue-unique TF pairs for the 79 human tissues. These results indicate that tissue gene expression is regulated by large sets of interacting TFs. Furthermore, the relative small number of tissue-unique TF pairs out of all tissue TF pairs suggests that identical tissue TF pairs in different tissues may play different functional roles, which prompted us to investi- gate their biological function. For this purpose, we used Gene2go http://www.ncbi.nlm.nih.gov/ to annotate human genes whose promoters contained the target TFBS pairs, as TFs control cellular biological processes via transcrip- tional regulation of groups of genes with similar functions. Significant (q-value < 0.1) biological processes for tissue TF pairs were obtained by comparing the number of TF target genes involved in a particular biological process to the number of genes for the same biological process in the whole human genome (Fisher’s exact test; p = 2.4 × 10-4 to 8 × 10-28). All tissue and tissue-unique TF pairs as well as their potential biological functions are listed in Addi- tional File 2. Evaluation by known interacting TFs Although the function conservation approach has been proven to be a successful means for predicting interacting TFs [22], we sought to assess the validity of the identified tissue TF pairs. We first used TRANS- Compel® 10.4 [25] to determine if known interacting TFs were statistically enriched in the predicted tissue TF pairs. The TRANSCompel® database contains 180 experimentally proven composite elements of two or more binding sites which were previously identified by individual wet lab studies from others. Of the180 com- posite elements, 105 were mapped to the 23,005 (214*215/2) possible combinations of 2 TFs from the 214 non-redundant position weight matrices (PWMs). We first investigated the statistical significance for the occurrence of known interacting TFs in both predicted TF pairs (before filtering) and tissue TF pairs in each of the 79 tissues. Figure 2 shows that known interacting TFs display enrichment more in the tissue TF pairs then in predicted TF pairs for both the number of tissues (37 vs. 9) and the degree of enrichment (Binomial test: p = 3.2 × 10-2 to 6.8 × 10-6 vs. p = 4 × 10-2 to 3.4 × 10-4). We also computed the occurrence of known interacting TFs in all predicted TFs pairs (before filtering) and all tissue TF pairs from the 79 tissues. We found that 40 (38.1%) of the 105 known interacting TFs were in both predicted TF pairs (Binomial test; p = 6.4 × 10-9) and tissue TF pairs (Binomial test; p = 5.4 × 10-11). To further verify our prediction, we next compared the tissue TF pairs to known tissue-specific TFs from liver, for which the cis-regulatory systems for both indi- vidual TF binding and synergistic actions have been thoroughly studied [3,4,25,26]. These studies found 40 liver-specific single TFs and 27 liver-specific interacting TFs. We first computed the tissue TF pairs whose two TFs were all liver-specific based on the 820 (40*41/2) possible combinations of 2 TFs from the 40 liver-