A synthetic biology framework for programming eukaryotic transcription functions

Abstract

Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks.

Figure 1. Synthetic Construction of Eukaryotic Transcription Functions

Eukaryotic transcription factors (TFs) perform a variety of molecular functions to control promoters and facilitate the operation of genetic networks (top panel). Zinc fingers (ZFs) are modular domains found in many eukaryotic TFs that make sequence-specific contacts with DNA. Artificial ZF arrays were used as core building blocks for constructing synthetic TFs (sTFs) and gene circuitry in S. cerevisiae (bottom panel). The use of artificial ZF domains permits a fully decomposed design of a sTF, for which the molecular component properties are accessible, modular, and tunable (red italicized). The independent control of these component properties enables the systematic construction and modulation of transcriptional behavior. AD, transcriptional activation domain; GOI, gene of interest; REs, regulatory elements.

Figure 2. Artificial ZFs Can Be Used to Construct Synthetic Transcriptional Activators

(A) Circuit design for synthetic transcriptional cascade. Synthetic transcription factors (sTFs) are expressed from an ATc-inducible GAL1 promoter (pGAL1). sTF activators are composed of artificial ZF arrays fused to a herpes simplex VP16 activation domain (AD) and a nuclear localization sequence (NLS). Upon induction, the sTF operates on a cognate synthetic promoter—minimal CYC1 promoter engineered with ZF binding sequences directly upstream of the TATA box—to direct the expression of a yeast-enhanced green fluorescent protein (yEGFP) reporter. Circuits were chromosomally integrated into S. cerevisiae. (B) sTF activator circuits built from artificial ZF arrays activate transcription from cognate synthetic promoters in a dose-dependent fashion (ZF 37-12 shown here). Points represent mean values for three experiments ± SD. See also Figure S1.

Figure 3. Wiring a Library of Specific and Orthogonal Transcriptional Connections with Engineered ZF Arrays

(A) sTF-promoter pair library sequences. Amino acid residues of the recognition helices for 19 OPEN-engineered three-finger arrays, and corresponding DNA binding sequences (ZF binding sequences were inserted between EcoRI and BamHI sites within synthetic promoters). (B) sTFs activate transcription from cognate synthetic promoters. “Fold activation” values were calculated as the ratio of fluorescence values from induced cells (500 ng/ml ATc) to those from uninduced cells. Red stars denote the six sTF-promoter pairs chosen to test for orthogonality. (C) sTFs constructed from OPEN-engineered ZFs are orthogonal to one another. sTF_43-8 activated noncognate Promoter_21-16 due to the fortuitous creation of a sequence that is significantly similar to the binding sequence of 43-8, when the downstream BamHI restriction site is considered (A, blue boxes). (D) Fitness cost of sTF expression on host cell growth at 30 hr after circuit induction (“no ZF” = strain with synthetic promoter and sTF cassette lacking a ZF array). Error bars represent SD of three experiments. See also Figures S2 and S3.

Figure 4. Tuning Transcriptional Outputs by Rationally Adjusting Multiple Component Properties

(A) Tuning up output strength by increasing ZF operator number in synthetic promoter (sTF_43-8). (B) Integrating two distinct sTFs at a single synthetic promoter. sTF_43-8 and sTF_42-10 were expressed independently from ATc- and IPTG-inducible GAL1 promoters. (C) Schematic representation of the canonical Cys₂-His₂ ZF protein (top). Each finger is composed of two β strands and a recognition helix, which makes sequence-specific contacts to three DNA bps. Four arginine residues in the ZF framework that mediate nonspecific interactions with the phosphate backbone were targeted for mutation to alanine residues (gray boxes and highlighted in red) in order to alter the affinity of the ZF for its cognate binding sequence. (D) Tuning down activation output by engineering ZF affinity variants in sTF_42-10 (3x: R2A/R39A/R67A, 4x: R2A/R11A/R39A/R67A). Horizontal axis begins at basal (promoter-only) fluorescence level (B and D). (E) Phosphate backbone mutants of 42-10 rescue the fitness cost of sTF_42-10 on host cell growth. Error bars represent SD of three experiments. See also Figures S4 and S5.

Figure 5. Transcriptional Cooperative Systems Can Be Engineered from Weakly-Activating sTF “monomers” that Are Dimerized with a PDZ Interaction Domain

(A) The dimerization interaction promotes cooperative behavior in transcriptional activation. Syntrophin PDZ domain (dark gray) was fused to the C-terminal of ZF affinity mutant 43-8-4x, and the resulting AD-carrying sTF “monomer” was expressed from ATc-inducible pGAL1. The heterologous ligand (light gray) was fused to the C-terminal of 42-10-4x, and the resulting AD-less factor was expressed from IPTG-inducible pGAL1. The factors assemble at a synthetic “dimeric” promoter to cooperatively activate downstream transcription (“IPTG+” = full induction with 20 mM IPTG). (B) Disruption of the dimerization interaction abolishes cooperative behavior in transcriptional activation. A nonbinding ligand variant (GSGS-VKEAAA) was instead fused to 42-10-4x. Points represent mean values for three experiments ± SD.

Figure 6. Synthetic ZF-Based Transcription Framework Can Be Used to Engineer Diverse Two-Input Behaviors

(A) The transcriptional operation of a single sTF_43-8 (carrying a PDZ domain) at the proximal position of a two-input promoter. (B) Cooperative two-input synergy engineered with PDZ-carrying sTF_43-8-4x as the proximal activator and cognate ligand-carrying sTF_42-10-4x as the distal partner. (C) Cooperative two-input synergy further enhanced by the addition of an AD onto the distal partner to create a two-activator system. (D) A “null” two-input system engineered by abolishing the dimerization interaction with a PDZ nonbinding ligand on the distal partner, thus rendering it noncontributory. (E and F) Inhibitory two-input behavior engineered by reversing the activator location (from proximal to distal) and using either PDZ binding (E) or nonbinding ligands (F). (G and H) Inhibition by the proximal monomer can be further increased by increasing the proximal ZF affinity to DNA (43-8-4x to 43-8-3x) and decreasing the distal ZF affinity to DNA (42-10-3x to 42-10-4x) in both PDZ binding (G) and nonbinding cases (H). (I) By reversing the orientation of the operators, sTF_43-8-4x is converted from an inhibitor to a cooperative factor to, once again, obtain cooperative transcriptional synergy in the two-input behavior. All sTFs were expressed from either ATc- or IPTG-inducible pGAL1 (500 ng/ml ATc and/or 20 mM IPTG). Horizontal axes correspond to “mean fluorescence intensity per cell (AU)” and begin at basal (promoter-only) fluorescence level. Error bars represent SD of three experiments.