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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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Engineering and characterization of ZF transcriptional activators. (A) Schematic representation of the assay used to test ZF activator function. Each transcriptional activator is expressed from the CMV promoter and tagged with a co-translationally cleaved t2a:mCherry fluorescent protein to monitor expression. ZF activator function is measured by the ability to activate the expression of 2 fused copies of cyan fluorescent protein from a reporter containing a minimal promoter and a variable number of 9 bp ZF target sites. (B) Characterization of the role of leucine zipper addition and target site copy number on ZF-TF transcriptional activation. BCR_ABL-1 activators fused to either no LZ (ZF-only), the c-Jun LZ (Jun), or the GCN4 LZ (GCN4) were co-transfected into U-2 OS cells along with reporter plasmids containing either 2, 4 or 6 copies of the corresponding 9 bp target site. CFP reporter expression as measured by flow cytometry and expressed as fold change over an off-target expression control. (C) Functional characterization of all ZF-activators co-transfected with reporter plasmids containing 6 copies of their 9 bp target sites.
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gks142-F1: Engineering and characterization of ZF transcriptional activators. (A) Schematic representation of the assay used to test ZF activator function. Each transcriptional activator is expressed from the CMV promoter and tagged with a co-translationally cleaved t2a:mCherry fluorescent protein to monitor expression. ZF activator function is measured by the ability to activate the expression of 2 fused copies of cyan fluorescent protein from a reporter containing a minimal promoter and a variable number of 9 bp ZF target sites. (B) Characterization of the role of leucine zipper addition and target site copy number on ZF-TF transcriptional activation. BCR_ABL-1 activators fused to either no LZ (ZF-only), the c-Jun LZ (Jun), or the GCN4 LZ (GCN4) were co-transfected into U-2 OS cells along with reporter plasmids containing either 2, 4 or 6 copies of the corresponding 9 bp target site. CFP reporter expression as measured by flow cytometry and expressed as fold change over an off-target expression control. (C) Functional characterization of all ZF-activators co-transfected with reporter plasmids containing 6 copies of their 9 bp target sites.

Mentions: We first generated a set of 15 ZF transcriptional activators and demonstrated the ability to increase their activity with increasing LZ strength. Each activator was comprised of a ZF DBD, either no LZ motif, a Jun LZ, or a GCN4 LZ, the transcriptional activation domain VP64 and the SV40 nuclear localization signal (NLS). Activators were expressed from the CMV promoter and tagged with co-translationally cleaved t2a:mCherry to monitor expression (Figure 1A) (21). We assayed activator function by co-transfection with reporter plasmids containing different numbers of ZF binding sites driving expression of AmCyan fluorescent protein CFP (Figure 1A). We first tested the BCR_ABL-1 activators and observed a wide range of signal output (2- to 163-fold) that increased with both the strength of the LZ binding domain and the number of ZF binding sites (Figure 1B and Supplementary Figure S2). We then compared the activity of ZF activators generated using different DBDs by co-transfection with the corresponding 6× Binding site (BS) reporter (Figure 1C and Supplementary Figure S3). A strong induction from all ZFs was observed (up to 463-fold), with HIV TFs displaying the strongest activation. No cross-reactivity was observed with activators co-transfected with off-target reporters, demonstrating the specificity of our ZF-TFs (Supplementary Figures S4 and S5).Figure 1.


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

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

Engineering and characterization of ZF transcriptional activators. (A) Schematic representation of the assay used to test ZF activator function. Each transcriptional activator is expressed from the CMV promoter and tagged with a co-translationally cleaved t2a:mCherry fluorescent protein to monitor expression. ZF activator function is measured by the ability to activate the expression of 2 fused copies of cyan fluorescent protein from a reporter containing a minimal promoter and a variable number of 9 bp ZF target sites. (B) Characterization of the role of leucine zipper addition and target site copy number on ZF-TF transcriptional activation. BCR_ABL-1 activators fused to either no LZ (ZF-only), the c-Jun LZ (Jun), or the GCN4 LZ (GCN4) were co-transfected into U-2 OS cells along with reporter plasmids containing either 2, 4 or 6 copies of the corresponding 9 bp target site. CFP reporter expression as measured by flow cytometry and expressed as fold change over an off-target expression control. (C) Functional characterization of all ZF-activators co-transfected with reporter plasmids containing 6 copies of their 9 bp target sites.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3367183&req=5

gks142-F1: Engineering and characterization of ZF transcriptional activators. (A) Schematic representation of the assay used to test ZF activator function. Each transcriptional activator is expressed from the CMV promoter and tagged with a co-translationally cleaved t2a:mCherry fluorescent protein to monitor expression. ZF activator function is measured by the ability to activate the expression of 2 fused copies of cyan fluorescent protein from a reporter containing a minimal promoter and a variable number of 9 bp ZF target sites. (B) Characterization of the role of leucine zipper addition and target site copy number on ZF-TF transcriptional activation. BCR_ABL-1 activators fused to either no LZ (ZF-only), the c-Jun LZ (Jun), or the GCN4 LZ (GCN4) were co-transfected into U-2 OS cells along with reporter plasmids containing either 2, 4 or 6 copies of the corresponding 9 bp target site. CFP reporter expression as measured by flow cytometry and expressed as fold change over an off-target expression control. (C) Functional characterization of all ZF-activators co-transfected with reporter plasmids containing 6 copies of their 9 bp target sites.
Mentions: We first generated a set of 15 ZF transcriptional activators and demonstrated the ability to increase their activity with increasing LZ strength. Each activator was comprised of a ZF DBD, either no LZ motif, a Jun LZ, or a GCN4 LZ, the transcriptional activation domain VP64 and the SV40 nuclear localization signal (NLS). Activators were expressed from the CMV promoter and tagged with co-translationally cleaved t2a:mCherry to monitor expression (Figure 1A) (21). We assayed activator function by co-transfection with reporter plasmids containing different numbers of ZF binding sites driving expression of AmCyan fluorescent protein CFP (Figure 1A). We first tested the BCR_ABL-1 activators and observed a wide range of signal output (2- to 163-fold) that increased with both the strength of the LZ binding domain and the number of ZF binding sites (Figure 1B and Supplementary Figure S2). We then compared the activity of ZF activators generated using different DBDs by co-transfection with the corresponding 6× Binding site (BS) reporter (Figure 1C and Supplementary Figure S3). A strong induction from all ZFs was observed (up to 463-fold), with HIV TFs displaying the strongest activation. No cross-reactivity was observed with activators co-transfected with off-target reporters, demonstrating the specificity of our ZF-TFs (Supplementary Figures S4 and S5).Figure 1.

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