Function and evolution of zinc finger transcription factors

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Unlike most transcription factors (TFs), the large C2H2 zinc finger family displays widespread duplication and diversifying selection in metazoans. Most C2H2 proteins are poorly characterized, and therefore it remains unknown what are their functional roles and what drives their evolutionary trajectories. In one case, however - that of the tetrapod-specific KRAB-C2H2 proteins, which represent roughly half of all human C2H2 proteins - recent evidence indicates that the family evolves to silence retroelements by recruiting the KAP1 corepressor. My lab recently showed that the genomic binding sites of C2H2 proteins are highly diverse and that each human KRAB-C2H2 protein binds a specific class of endogenous retroelements (EREs). Curiously, most of the EREs bound are ancient and have long been inactive, suggesting that the proteins that bind them, and/or the EREs themselves, have taken on additional roles. We have also found that C2H2 proteins, including KRAB-C2H2s, often have unique protein-protein interaction (PPI) profiles, suggesting that new host functions can be acquired by changing the effector function over relatively short evolutionary timescales. These observations suggest a genome-specific regulatory network model, distinct from the conserved regulatory models prevalent in the evolutionary development literature, as well as different forces that drive the gene expansions. Here, we propose two major lines of experimentation to explore C2H2 proteins in both tetrapod and non-tetrapod vertebrates, in order to gain a broader and more detailed view of their function and evolution. (1) We will map genomic binding sites to determine the rate at which DNA binding sites change for orthologous KRAB-C2H2 proteins across mammals, and whether EREs are bound at all by recently evolved C2H2 proteins in fish. (2) We will explore the mechanisms and evolution of PPIs with C2H2 proteins. We anticipate three different evolutionary “modes”, each of which we will explore in detail: (a) Diversification in structured protein-binding modules, (b) use of unstructured or non-domain polypeptide as a template for new PPIs, or (c) utilization of C2H2 (or other domains) for PPIs, which would represent an example of “moonlighting”. The project overall will make a major contribution to the study of vertebrate evolution, and to our understanding of regulatory networks and elements. **This is a renewal application for a Discovery Grant that was funded for one year. In the interim, my group has published two papers previously described as preliminary data, and submitted a third (see Appendices). I am grateful for the funding, which allowed generation of new preliminary data, and look forward to a longer funding interval on the basis of this extensively revised (and retitled) application.**