Synopsis

Subject Categories: Synthetic biology | Proteins

Molecular Systems Biology 2 Article number: 2006.0011  doi:10.1038/msb4100053
Published online: 21 March 2006
Citation: Molecular Systems Biology 2:2006.0011

Synthetic protein–protein interaction domains created by shuffling Cys2His2 zinc-fingers

Astrid V Giesecke1,2, Rui Fang1,3 & J Keith Joung1,3

  1. Molecular Pathology Unit, Department of Pathology, Massachusetts General Hospital, Charlestown, MA, USA
  2. Universität Regensburg, Institut für Zoologie, Regensburg, Germany
  3. Department of Pathology, Harvard Medical School, Boston, MA, USA

Correspondence to: J Keith Joung1,3 Molecular Pathology Unit, Department of Pathology, Massachusetts General Hospital, 149 13th Street, Room 7139, 7th floor, Charlestown, MA 02129, USA. Tel.: +1 6177269462; Fax: +1 6177265684; E-mail: Email: jjoung@partners.org

Received 20 June 2005; Accepted 20 January 2006; Published online 21 March 2006

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Article highlights

  • Shuffling Cys2His2 zinc-fingers from dimerization zinc finger (DZF) domains yields synthetic DZFs with novel protein-protein interaction specificities
  • Synthetic DZFs mediate protein-protein interactions in bacterial and human cells
  • Synthetic DZFs can be used to alter gene expression in bacterial and human cells
  • More extended protein-protein interaction interfaces can be created by linking together synthetic DZFs

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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; Tupler et al, 2001; Venter et al, 2001; Muller et al, 2002). 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), RNA (Lu et al, 2003), and protein sequences (Mackay and Crossley, 1998). 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; Pabo et al, 2001; Falke and Juliano, 2003; Jamieson et al, 2003; Lee et al, 2003; Blancafort et al, 2004; Jantz et al, 2004). 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; Pabo et al, 2001; Falke and Juliano, 2003; Jamieson et al, 2003; Lee et al, 2003; Blancafort et al, 2004; Jantz et al, 2004).

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) 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). 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). We used eight of these naturally occurring DZFs as sources of C2H2 ZFs to create shuffled libraries of synthetic DZFs.

Figure 1
Figure 1 : Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

DZF domains from various transcription factors. The top of the figure presents a schematic of a DZF domain with amino- (N) and carboxy- (C) terminal fingers represented as ovals and the interfinger linker as a connecting bar. The bottom portion of the figure shows an amino-acid sequence alignment of DZFs from human Ikaros, human Helios, human Aiolos, human Eos, human Pegasus, human TRPS-1, Drosophila melanogaster (D.m.) Hunchback, Locusta migratoria (L.m.) Hunchback, Helobdella triserialis (H.t.) Hunchback, and Caenorhabditis elegans (C.e.) Hunchback. Conserved cysteines and histidines are highlighted in blue. Positions showing 80% or greater conservation among the 10 DZF domains shown are highlighted in yellow.

Full figure and legend (171K)Figures & Tables index

Using the B2H system as a selection method, we identified interacting synthetic DZF pairs from our shuffled libraries (Figure 3). 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.

Figure 3
Figure 3 : Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

Schematic overview of shuffled DZF library construction and B2H selections to identify interacting synthetic DZFs. DZFs are represented as in Figure 1. Note that although two alpha-subunits are present in an RNAP complex, the DZF–DZF interaction illustrated can occur presumably because either the DZF domains on the RNAP alpha-fusion protein fail to dimerize (perhaps owing to a geometrical constraint) or because the RNAP complexes that are recruited to the promoter harbor one DZF domain alpha hybrid subunit and one wild-type alpha-subunit. Additional details are provided in the Results and Materials and methods section.

Full figure and legend (273K)Figures & Tables index

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; Tan et al, 2003; Urnov et al, 2005). We tested whether, by analogy, extended protein–protein interfaces might be constructed by linking together two synthetic DZFs to create 'double-DZFs' composed of four C2H2 ZFs. We found that our synthetic double-DZF pairs could mediate reconstitution of a synthetic bi-partite activator in human cells and that certain double-DZF pairs could robustly activate VEGF-A gene expression more strongly than the single DZF pairs from which they were constructed. This high level of activation was similar to that observed when the two parts of the artificial VEGF-A activator were covalently linked into a single molecule, suggesting that double-DZFs interact more strongly than single DZFs. Our results show that shuffling of DZF-derived C2H2 ZFs can yield synthetic DZFs with new specificities that can in turn be linked together to create more extended interaction interfaces. In future experiments, it will be interesting to determine whether additional protein-interacting C2H2 ZFs from other non-DZF domains described in the literature can also be shuffled to create novel protein interactions. In addition, we speculate that it may be possible to engineer synthetic, multi-DZF 'scaffolds' or 'adaptors' upon which various DZF-linked proteins might be assembled. Successful creation of such scaffolds could permit the creation of 'circuit components' in which various cellular pathway inputs (e.g., kinases, transcription factors) might be integrated into a single output.

The capability to engineer synthetic C2H2 ZFs with novel protein–protein interaction specificities, particularly when coupled with existing technologies for constructing designer C2H2 ZF DNA-binding proteins, will greatly expand the toolbox of 'parts' that can be used by synthetic biologists to construct artificial cellular networks or to disrupt and re-wire transcriptional and/or cell signaling circuits. Our experiments demonstrate that our artificial domains can mediate assembly of an active transcription complex at a single-copy gene in bacteria or at an endogenous gene in the nucleus of a human cell. Our results also show that our synthetic domains are active in the cytoplasm of a human cell, suggesting that they might be used to create nontranscriptional synthetic cellular networks (e.g., signaling pathways). Furthermore, because C2H2 ZFs are stably folded and active in a wide range of cells including bacteria, yeast, flies, worms, plants, and mammals, we expect that our synthetic protein-interacting C2H2 ZF domains will be useful in many cellular environments. Creation of large numbers of artificial DNA-binding and protein-interacting C2H2 ZF-based parts should facilitate the synthetic biologist's ability to create complex cellular networks in cells.

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Acknowledgements

We thank Andrew Hirsh for many helpful discussions and comments on the manuscript and Stacey Thibodeau for help with figures. AVG, RF, and JKJ were supported by grants from the NIH (K08 DK002883, R01 GM069906, and R01 GM072621) and by the MGH Department of Pathology. JKJ dedicates this paper to the memory of Robert L Burghoff, PhD, an outstanding scientist, patient teacher, and kind friend.

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