significant correlation (Kendall’s tau = 0.03 p value = 0.34) be- tween expression similarity and DNA binding site motif similarity (Figure 1 in supplementary Data file S1). PCC only captures the pattern of expression across tissues and not the tissue-specific differences in expression, which is relevant for evolutionary selection. Any relationship between DNA binding site motif similarity and expression divergence is likely to be revealed in the context of individual tissues and is not evident if we consider overall gene profiles. To explore this hypothesis further, we directed our analysis towards tissue-specific expression divergence and its relationships to DNA binding site motif similarity. Figures 2a and 2b depict for all TF paralogous pairs, the relationship between the tissue-specific expression of the two TF genes and their DNA binding site motif similarity in adult Cerebellum and Heart tissues, respectively. These plots indicate that for paralogous pairs with similar DNA binding site motifs, in general, at most one of the paralogs is expressed at a high level. Conversely, if both paralogs are highly expressed then their DNA binding site motifs tend to be dissimilar. In other words, the points corresponding to high DNA binding site motif similarity are mostly found near the origin (both paralogs have low expression) and near the axes (exactly one of the paralogs is highly expressed), and these points become increasingly sparse as we move away from the origin (both paralogs highly expressed). We found that this trend is consistent in all 79 tissues in Novartis dataset (data not shown). This observed dependence of DNA binding site motif similarity on the expression values of the two paralogs can be approximated by the function LE~ 1=EX z1=EY , where EX and EY are the tissue-specific expression values of the two paralogs X and Y. Figures 3a and 3b show this theoretical dependence for the expression data in Cerebellum and Heart respectively. Thus, hE is ‘high’ if either both TF genes have low expression or if exactly one of the TFs has high expression i.e., the two paralogs have diverged in their expression. We next quantify the correlation between DNA binding site motif similarity and hE using the pipeline shown in Figure 4. Diversification of TF Paralogs Given the expression data for a tissue, for each paralogous TF pair (X, Y), we computed hE as defined above. We additionally computed the DNA binding site motif similarity §B between the PWMs corresponding to the two TFs using a previously benchmarked motif similarity measure based on the Pearson Correlation between PWM columns and Smith-Waterman un- gapped alignment of the PWMs[23]. Given the hE and §B values for each paralogous TF pair, we computed the Kendall’s tau rank correlation between hE and §B values and estimated its significance based on 1000 permutations of the expression data. Using other measures of correlation such as Pearson or Spearman does not change the results (see methods and discussion). We repeated the hE:§B correlation analysis for each of the 79 human tissues from the Novartis dataset[20]. We found that in 48 of the 79 (61%) tissues there was a significant positive correlation (p- value#0.05) between hE and §B. However, there is a high degree of similarity among the expression profiles of related tissues and thus, the 79 correlation tests are not independent. Therefore, we considered a subset of 23 tissues that were deemed to be non- redundant by the authors of the Novartis dataset[24]. We found that in 17 of the 23 (74%) tissues there was a significant correlation (p-value#0.05) between hE and §B. At a 0.01 p-value threshold, 14 (61%) of the tissues show significance, and 9 (39%) of the tissues show significance at a 0.001 p-value threshold. The mean and standard deviation of the correlations (tau value) in the significant cases were 0.21 and 0.05, respectively. As a control, we repeated the above experiment after randomizing the expression data and found a significant correlation only in 4.5% of the tissues. By random chance, we expect 5% of the tissues to show significance at p-value threshold of 0.05. We also repeated our analysis using paralogous TF-pairs obtained from the KOG data (www.ncbi. nlm.nih.gov/COG/), for which we obtained 242 paralogous pairs with 99 unique genes. This analysis yielded 67.1% tissues significant at the 0.05 p-value threshold and 54.4% tissues at a 0.01 threshold corresponding to a 13- to 54-fold enrichment. Thus, our overall conclusions are robust across different definitions Figure 2. Paralogous TFs with high expression tend to have dissimilar DNA binding site motifs and conversely, paralogs with similar DNA binding site motifs tend to diverge in their tissue-specific expression. The expression values for each TF gene in a paralogous pair are indicated on the x and y-axes, and the value of the corresponding DNA binding site motif similarity is depicted by the color of the point. Binding site motif similarity of paralogous TFs is depicted by the color (darker color indicates higher binding site motif similarity) at a co-ordinate determined by the expression values (depicted in log scale) of the two paralogs (x and y-axes) (a) Using gene expression in Cerebellum (b) Using gene expression in Heart. We used log scale for expression to accommodate for extreme expression values. doi:10.1371/journal.pone.0002345.g002 PLoS ONE | www.plosone.org 3 June 2008 | Volume 3 | Issue 6 | e2345