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A tunable zinc finger-based framework for Boolean logic computation in mammalian cells.

Lohmueller JJ, Armel TZ, Silver PA - Nucleic Acids Res. (2012)

Bottom Line: We describe 15 transcriptional activators that display 2- to 463-fold induction and 15 transcriptional repressors that show 1.3- to 16-fold repression.The split intein strategy is able to fully reconstitute the ZF-TFs, maintaining them as a uniform set of computing elements.Together, these components comprise a robust platform for building mammalian synthetic gene circuits capable of precisely modulating cellular behavior.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard University, Boston, MA 02115, USA.

ABSTRACT
The ability to perform molecular-level computation in mammalian cells has the potential to enable a new wave of sophisticated cell-based therapies and diagnostics. To this end, we developed a Boolean logic framework utilizing artificial Cys(2)-His(2) zinc finger transcription factors (ZF-TFs) as computing elements. Artificial ZFs can be designed to specifically bind different DNA sequences and thus comprise a diverse set of components ideal for the construction of scalable networks. We generate ZF-TF activators and repressors and demonstrate a novel, general method to tune ZF-TF response by fusing ZF-TFs to leucine zipper homodimerization domains. We describe 15 transcriptional activators that display 2- to 463-fold induction and 15 transcriptional repressors that show 1.3- to 16-fold repression. Using these ZF-TFs, we compute OR, NOR, AND and NAND logic, employing hybrid promoters and split intein-mediated protein splicing to integrate signals. The split intein strategy is able to fully reconstitute the ZF-TFs, maintaining them as a uniform set of computing elements. Together, these components comprise a robust platform for building mammalian synthetic gene circuits capable of precisely modulating cellular behavior.

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Determining the optimal ZF-TF split site. (A) Schematic representation of the plasmids used to test for split ZF activator function. Each ZF-N:int-N or Int-C:ZF-C fragment is expressed from a CMV expression plasmid. Activity is measured by the ability of each fragment pair to activate a cyan fluorescent protein reporter containing 6 copies of 9 bp ZF target sites versus activation by the Int-C:ZF-C fragment alone. (B) Schematic of the ZF-TF reconstitution process. After expression, the two split ZF-intein fragments bind together and undergo protein splicing to cleave away intein fragments and reconstitute the full ZF activator leading to activation of the BCR_ABL reporter. (C) BCR_ABL-1 amino acid sequence labeled with the 12 split sites assayed. (D) Characterization of the 12 ZF-intein activator pairs assayed by transient transfection in U-2 OS cells. CFP reporter expression was measured by flow cytometry and reported as total CFP expression. Fold changes in total CFP are listed for the activation of each split pair compared with activation by the corresponding Int-C:ZF-C fragment alone.
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gks142-F4: Determining the optimal ZF-TF split site. (A) Schematic representation of the plasmids used to test for split ZF activator function. Each ZF-N:int-N or Int-C:ZF-C fragment is expressed from a CMV expression plasmid. Activity is measured by the ability of each fragment pair to activate a cyan fluorescent protein reporter containing 6 copies of 9 bp ZF target sites versus activation by the Int-C:ZF-C fragment alone. (B) Schematic of the ZF-TF reconstitution process. After expression, the two split ZF-intein fragments bind together and undergo protein splicing to cleave away intein fragments and reconstitute the full ZF activator leading to activation of the BCR_ABL reporter. (C) BCR_ABL-1 amino acid sequence labeled with the 12 split sites assayed. (D) Characterization of the 12 ZF-intein activator pairs assayed by transient transfection in U-2 OS cells. CFP reporter expression was measured by flow cytometry and reported as total CFP expression. Fold changes in total CFP are listed for the activation of each split pair compared with activation by the corresponding Int-C:ZF-C fragment alone.

Mentions: We next sought to compute AND and NAND logic using our transcriptional activators and repressors. To use our split intein protein splicing strategy, we first set out to determine the optimal amino acid residue at which to split our ZF-TFs. We created twelve pairs of BCR_ABL-1:Jun activator split proteins. Each pair contained an amino- (N-) and carboxy-terminal (C-) fragment fused to the appropriate intein. These fragments were co-transfected, either together or separately, with the 6× BCR_ABL activator reporter (Figure 4A, B and C). Fold induction was calculated relative to CFP activation by the C-terminal fragment alone. Five out of twelve ZF split pairs displayed >3-fold signal output (Figure 4D and Supplementary Figure S14). The split site between residues 30 and 31 resulted in the highest activity, ∼19-fold, and was therefore chosen for further logic gate generation. Interestingly, all functional split sites are predicted to be located in protein loop regions of the ZF, suggesting the importance of the secondary structure on efficient protein splicing (Supplementary Figure S15). After performing this assay we discovered that a truncated form of the N-terminal intein fragment, intN*, lacking four C-terminal amino acids displayed higher splicing efficiency, and we used this intein fragment in all further experiments.Figure 4.


A tunable zinc finger-based framework for Boolean logic computation in mammalian cells.

Lohmueller JJ, Armel TZ, Silver PA - Nucleic Acids Res. (2012)

Determining the optimal ZF-TF split site. (A) Schematic representation of the plasmids used to test for split ZF activator function. Each ZF-N:int-N or Int-C:ZF-C fragment is expressed from a CMV expression plasmid. Activity is measured by the ability of each fragment pair to activate a cyan fluorescent protein reporter containing 6 copies of 9 bp ZF target sites versus activation by the Int-C:ZF-C fragment alone. (B) Schematic of the ZF-TF reconstitution process. After expression, the two split ZF-intein fragments bind together and undergo protein splicing to cleave away intein fragments and reconstitute the full ZF activator leading to activation of the BCR_ABL reporter. (C) BCR_ABL-1 amino acid sequence labeled with the 12 split sites assayed. (D) Characterization of the 12 ZF-intein activator pairs assayed by transient transfection in U-2 OS cells. CFP reporter expression was measured by flow cytometry and reported as total CFP expression. Fold changes in total CFP are listed for the activation of each split pair compared with activation by the corresponding Int-C:ZF-C fragment alone.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367183&req=5

gks142-F4: Determining the optimal ZF-TF split site. (A) Schematic representation of the plasmids used to test for split ZF activator function. Each ZF-N:int-N or Int-C:ZF-C fragment is expressed from a CMV expression plasmid. Activity is measured by the ability of each fragment pair to activate a cyan fluorescent protein reporter containing 6 copies of 9 bp ZF target sites versus activation by the Int-C:ZF-C fragment alone. (B) Schematic of the ZF-TF reconstitution process. After expression, the two split ZF-intein fragments bind together and undergo protein splicing to cleave away intein fragments and reconstitute the full ZF activator leading to activation of the BCR_ABL reporter. (C) BCR_ABL-1 amino acid sequence labeled with the 12 split sites assayed. (D) Characterization of the 12 ZF-intein activator pairs assayed by transient transfection in U-2 OS cells. CFP reporter expression was measured by flow cytometry and reported as total CFP expression. Fold changes in total CFP are listed for the activation of each split pair compared with activation by the corresponding Int-C:ZF-C fragment alone.
Mentions: We next sought to compute AND and NAND logic using our transcriptional activators and repressors. To use our split intein protein splicing strategy, we first set out to determine the optimal amino acid residue at which to split our ZF-TFs. We created twelve pairs of BCR_ABL-1:Jun activator split proteins. Each pair contained an amino- (N-) and carboxy-terminal (C-) fragment fused to the appropriate intein. These fragments were co-transfected, either together or separately, with the 6× BCR_ABL activator reporter (Figure 4A, B and C). Fold induction was calculated relative to CFP activation by the C-terminal fragment alone. Five out of twelve ZF split pairs displayed >3-fold signal output (Figure 4D and Supplementary Figure S14). The split site between residues 30 and 31 resulted in the highest activity, ∼19-fold, and was therefore chosen for further logic gate generation. Interestingly, all functional split sites are predicted to be located in protein loop regions of the ZF, suggesting the importance of the secondary structure on efficient protein splicing (Supplementary Figure S15). After performing this assay we discovered that a truncated form of the N-terminal intein fragment, intN*, lacking four C-terminal amino acids displayed higher splicing efficiency, and we used this intein fragment in all further experiments.Figure 4.

Bottom Line: We describe 15 transcriptional activators that display 2- to 463-fold induction and 15 transcriptional repressors that show 1.3- to 16-fold repression.The split intein strategy is able to fully reconstitute the ZF-TFs, maintaining them as a uniform set of computing elements.Together, these components comprise a robust platform for building mammalian synthetic gene circuits capable of precisely modulating cellular behavior.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard University, Boston, MA 02115, USA.

ABSTRACT
The ability to perform molecular-level computation in mammalian cells has the potential to enable a new wave of sophisticated cell-based therapies and diagnostics. To this end, we developed a Boolean logic framework utilizing artificial Cys(2)-His(2) zinc finger transcription factors (ZF-TFs) as computing elements. Artificial ZFs can be designed to specifically bind different DNA sequences and thus comprise a diverse set of components ideal for the construction of scalable networks. We generate ZF-TF activators and repressors and demonstrate a novel, general method to tune ZF-TF response by fusing ZF-TFs to leucine zipper homodimerization domains. We describe 15 transcriptional activators that display 2- to 463-fold induction and 15 transcriptional repressors that show 1.3- to 16-fold repression. Using these ZF-TFs, we compute OR, NOR, AND and NAND logic, employing hybrid promoters and split intein-mediated protein splicing to integrate signals. The split intein strategy is able to fully reconstitute the ZF-TFs, maintaining them as a uniform set of computing elements. Together, these components comprise a robust platform for building mammalian synthetic gene circuits capable of precisely modulating cellular behavior.

Show MeSH