Synthetic protein–protein interaction domains created by shuffling Cys 2 His 2 zinc‐fingers

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Abstract
Cys2His2 zinc‐fingers (C2H2 ZFs) mediate a wide variety of protein–DNA and protein–protein interactions. DNA‐binding C2H2 ZFs can be shuffled to yield artificial proteins with different DNA‐binding specificities. Here we demonstrate that shuffling of C2H2 ZFs from transcription factor dimerization zinc‐finger (DZF) domains can also yield two‐finger DZFs with novel protein–protein interaction specificities. We show that these synthetic protein–protein interaction domains can be used to mediate activation of a single‐copy reporter gene in bacterial cells and of an endogenous gene in human cells. In addition, the synthetic two‐finger domains we constructed can also be linked together to create more extended, four‐finger interfaces. Our results demonstrate that shuffling of C2H2 ZFs can yield artificial protein‐interaction components that should be useful for applications in synthetic biology. ### Synopsis Construction of complex synthetic cellular networks will require access to a large toolbox of macromolecular ‘parts’ such as DNA‐binding proteins and protein multimerization domains. Natural gene regulatory networks in cells ranging from yeast to humans make extensive use of Cys2His2 zinc‐fingers (C2H2 ZFs) as such parts. C2H2 ZFs are compact molecular recognition domains found in 2–3% of all human genes ([Lander et al , 2001][1]; [Tupler et al , 2001][2]; [Venter et al , 2001][3]; [Muller et al , 2002][4]). These domains, identifiable as a sequence repeat containing pairs of conserved cysteines and histidines, are typically found in proteins as tandem arrays that can mediate specific recognition of different DNA ([Wolfe et al , 2000][5]), RNA ([Lu et al , 2003][6]), and protein sequences ([Mackay and Crossley, 1998][7]). The functional versatility and widespread prevalence of these domains in genomes ranging from flies to humans suggest that the C2H2 ZF fold is a useful scaffold for creating interactions with a variety of different macromolecules. DNA‐binding C2H2 ZFs derived from the Zif268, Sp1, and other naturally occurring transcription factors can be ‘mixed and matched’ to construct synthetic multifinger arrays possessing various novel DNA‐binding specificities ([Klug, 1999][8]; [Pabo et al , 2001][9]; [Falke and Juliano, 2003][10]; [Jamieson et al , 2003][11]; [Lee et al , 2003][12]; [Blancafort et al , 2004][13]; [Jantz et al , 2004][14]). These ‘designer’ DNA‐binding domains (DBDs) have been fused to transcriptional regulatory domains to create artificial transcription factors capable of regulating expression of specific endogenous genes in cell types ranging from yeast to human as well as in whole organisms ([Klug, 1999][8]; [Pabo et al , 2001][9]; [Falke and Juliano, 2003][10]; [Jamieson et al , 2003][11]; [Lee et al , 2003][12]; [Blancafort et al , 2004][13]; [Jantz et al , 2004][14]). In this report, we demonstrate that shuffling of C2H2 ZFs from dimerization zinc‐finger (DZF) domains found in the human Ikaros and other related transcription factors can yield synthetic multifinger domains with novel protein–protein interaction specificities. We were particularly interested in testing this idea because relatively little is currently understood about C2H2 ZF‐mediated protein–protein interactions. In addition, a finger shuffling strategy could provide a means to create a repertoire of different protein–protein interaction pairs. Such a collection of synthetic C2H2 ZF protein–protein interaction domains together with existing collections of designer C2H2 ZF DBDs would constitute a useful toolbox of parts for creating or modifying transcription and signaling networks in cells. To test whether protein‐interacting C2H2 ZFs could be ‘mixed and matched’ like some of their DNA‐binding counterparts, we created libraries of synthetic two‐finger DZFs by shuffling C2H2 ZFs obtained from DZF domains found in transcription factors ranging from Drosophila melanogaster to humans and then identified pairs of interacting two‐finger domains from these libraries using a bacterial two‐hybrid (B2H) system. DZFs consist of two C2H2 ZFs joined by a short linker ([Figure 1][15]) and have been identified in members of the mammalian Ikaros family of transcription factors as well as in the D. melanogaster Hunchback protein and the mammalian TRPS‐1 protein ([Figure 1][15]). DZFs mediate homo‐ and heterotypic interactions among these various transcription factors. Through database searches, we also identified potential DZFs in Hunchback homologs from grasshopper, leech, and Caenorhabditis elegans ([Figure 1][15]). We used eight of these naturally occurring DZFs as sources of C2H2 ZFs to create shuffled libraries of synthetic DZFs. Using the B2H system as a selection method, we identified interacting synthetic DZF pairs from our shuffled libraries ([Figure 3][16]). Four pairs of synthetic DZFs identified in our selections, in addition to mediating activation of a single‐copy test promoter in our bacterial cell‐based two‐hybrid system, mediated reconstitution of a synthetic bi‐partite activator in the nucleus of a human cell and transcriptional activation of the endogenous VEGF‐A gene. We also demonstrated that these four DZF pairs could mediate interaction in the cytoplasm of a human cell as judged by a co‐immunoprecipitation assay for DZF–DZF interactions. Previous studies have shown that DNA‐binding C2H2 ZFs can be linked together into tandem arrays capable of recognizing extended DNA sequences. For example, units composed of two C2H2 ZFs have been joined together by linkers to create four‐ and six‐finger proteins capable of binding 12 and 18 bp DNA sequences, respectively ([Moore et al , 2001][17]; [Tan et...