Hu and Gallo BMC Genomics 2010, 11:49 http://www.biomedcentral.com/1471-2164/11/49 those from housekeeping genes. The remaining TF pairs in each tissue were more tissue-specific, and therefore, were defined as tissue TF pairs. Similar results were obtained from using background sequences of either completely mixed nucleotides or keeping dinucleotides together or completely mixing nucleotides. The results from completely mixed nucleotides were used. Clustering analysis To group tissues based on their tissue TF pairs, a 2549 (tissue TF pairs) × 79 (tissues) matrix with binary num- bers was first built for all tissue TF pairs in the 79 human tissues. The presence of a tissue TF pair in the matrix was labeled with 1 and the absence was labeled with 0. A distance matrix was then built using the “bin- ary” method, and hierarchical clustering was subse- quently performed using the “complete” agglomeration method. All analysis was performed using the R statisti- cal package [40]. Predicting multiple interacting TFs A two-step analysis of TFBS conservation and enrich- ment of overlapping orthologous genes was performed to predict interactions of 3 TFs. For TFBS conservation, the identified tissue TFBS pairs were first used to con- struct all possible 3-TFBS combinations by searching paired tissue TFBS pairs with one shared TFBS between each other on exactly the same location of a gene’s pro- moter (Figure 1b) in a particular tissue. Orthologous gene pairs containing conserved 3-TFBS combination between human and mouse were then selected. Con- served 3-TFBS combinations are those whose 3 TFBSs have the same order and orientation on the promoter sequences between human and mouse orthologous genes. For enrichment of overlapping orthologous genes in a tissue, however, multiple interacting TFs from dif- ferent orthologous gene pairs were considered to be the same as long as they contained the same 3 TFs. Enriched multiple interacting TFs are those with 3- TFBS combinations occurring on at least 10 orthologous gene promoters and with their target orthologous genes displaying significant overlap between human and mouse (p = 3 × 10-2 to < 10-36 and q < 0.05). Statistical methods for enrichment analyses Two main statistical methods were employed for esti- mating the significance of enrichment in this study. For validating predicted TF pairs and tissue TF pairs by known interacting TFs, the binomial distribution prob- ability, as shown below, was used to determine if known interacting TFs were present more often in the pre- dicted TF pairs or tissue TF pairs than in a randomly selected group from a given list of TFs. Page 13 of 15 xNn  Nx p xf(1  p f )N x P    For example, in the case of estimating the statistical significance of known liver-specific interacting TFs in our predicted tissue TF pairs from liver tissue, the n is the number of known liver-specific interacting TFs in the predicted tissue TF pairs from this study; N the number of tissue TF pairs from liver tissue; and pf the background probability of liver-specific TF pairs in all possible combinations of 2 TFs from 214 PWMs. The statistical significance was computed using the hypergeometric distribution to estimate (1) the enrich- ment of overlapping tissue TF pairs and their overlap- ping functions between muscle tissues, and (2) overlapping orthologous genes in predicting multiple interacting TFs. P(X  c)   S1  N S1  min(s1,s2)   x  S2  x     N  xc    S2  In the case of overlapping orthologous genes in pre- dicting multiple interacting TFs, for example, c is the number of orthologous gene pairs containing conserved 3-TFBS combination between human and mouse; N the number of tissue-expressed genes for a particular tissue; S1 and S2 are the numbers of tissue-expressed genes with 3-TFBS combinations corresponding to those in c for human and mouse, respectively. The resulting p- value is the probability of observing c or more ortholo- gous gene pairs containing conserved 3-TFBS combina- tion from two sets of size S1 and S2 drawn from a set of N tissue-expressed genes. List of abbreviations TF: transcription factor; TFBS: transcription factor bind- ing site; PWM: position weight matrices. Additional file 1: Flowchart of analysis procedure for TF pair prediction. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 49-S1.pdf ] Additional file 2: Lists tissue and tissue-unique TF pairs and their potential functions for the 79 human tissues. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 49-S2.XLS ] Additional file 3: Lists tissue-type TF pairs for the 11 selected tissue groups. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 49-S3.XLS ]