Functional Diversification of Paralogous Transcription Factors via Divergence in DNA Binding Site Motif and in Expression Larry N. Singh, Sridhar Hannenhalli* Penn Center for Bioinformatics, Department of Genetics, University of Pennsylvania, Philadephia, Pennsylvania, United States of America Abstract Background: Gene duplication is a major driver of evolutionary innovation as it allows for an organism to elaborate its existing biological functions via specialization or diversification of initially redundant gene paralogs. Gene function can diversify in several ways. Transcription factor gene paralogs in particular, can diversify either by changes in their tissue-specific expression pattern or by changes in the DNA binding site motif recognized by their protein product, which in turn alters their gene targets. The relationship between these two modes of functional diversification of transcription factor paralogs has not been previously investigated, and is essential for understanding adaptive evolution of transcription factor gene families. Findings: Based on a large set of human paralogous transcription factor pairs, we show that when the DNA binding site motifs of transcription factor paralogs are similar, the expressions of the genes that encode the paralogs have diverged, so in general, at most one of the paralogs is highly expressed in a tissue. Moreover, paralogs with diverged DNA binding site motifs tend to be diverged in their function. Conversely, two paralogs that are highly expressed in a tissue tend to have dissimilar DNA binding site motifs. We have also found that in general, within a paralogous family, tissue-specific decrease in gene expression is more frequent than what is expected by chance. Conclusions: While previous investigations of paralogous gene diversification have only considered coding sequence divergence, by explicitly quantifying divergence in DNA binding site motif, our work presents a new paradigm for investigating functional diversification. Consistent with evolutionary expectation, our quantitative analysis suggests that paralogous transcription factors have survived extinction in part, either through diversification of their DNA binding site motifs or through alterations in their tissue-specific expression levels. Citation: Singh LN, Hannenhalli S (2008) Functional Diversification of Paralogous Transcription Factors via Divergence in DNA Binding Site Motif and in Expression. PLoS ONE 3(6): e2345. doi:10.1371/journal.pone.0002345 Editor: Emmanouil T. Dermitzakis, The Wellcome Trust Sanger Institute, United Kingdom Received January 9, 2008; Accepted April 15, 2008; Published June 4, 2008 Copyright: 2008 Singh, Hannenhalli. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: NIH R21 (PI Hannenhalli) Competing Interests: The authors have declared that no competing interests exist. * E-mail: sridharh@pcbi.upenn.edu Introduction Gene or even whole genome duplication provides the essential spare parts for evolutionary innovation[1–4]. Relaxed purifying selection immediately after duplication allows an organism to elaborate its existing functional repertoire through specialization and diversification of individual gene functions[5–8]. In particular, expansion of transcription factor (TF) gene families has played a substantial role in the evolution of organismal complexity by elaborating transcriptional networks[9–11]. Paralogous genes (genes within a species evolutionarily-related by gene duplication events) that survive extinction often diversify by either assuming an entirely novel function, or specializing in some aspects of their original function while losing other functions[2–8]. However, in some cases an increased dosage is advantageous and the gene copies remain relatively conserved, partly mediated by gene conversion[12,13]. The initially identical gene copies may function- ally diversify via several distinct pathways. Figure 1 illustrates three possibilities that apply to TF genes in particular. First, the DNA binding domain may mutate, thereby altering the DNA binding site motif and hence the gene targets of the TF; this is common among paralogous TFs. Second, the interaction/activation domain may mutate thereby altering the TF’s interacting partners[14]. Two paralogous TFs having similar DNA binding domains but with different interaction domains may result in one TF acting as a suppressor of its paralog by interfering with the sibling’s binding; for example, Foxo1 and Foxa2 have highly similar DNA binding specificities but Foxo1 acts as a suppressor of Foxa2-mediated regulation of Pdx1 in pancreatic b-cells[15]. A third possible fate of duplicated TFs is the divergence of their gene expression[3]. Transcription factor Pax2, an essential regulator of nephrogenesis, regulates c-Ret specifically in kidney[16], while the paralog Pax3, with a similar DNA binding specificity, regulates the same gene in neural crest[17]. The relationship between these distinct pathways of functional diversification of paralogous TF genes is of fundamental interest from the perspective of understanding evolution of gene families, but has not been investigated previously. Although mutations directly alter the genome, the resulting functional change is what drives evolutionary selection. A comparison of various diversification pathways in terms of their PLoS ONE | www.plosone.org 1 June 2008 | Volume 3 | Issue 6 | e2345