Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors

Abstract

The construction of increasingly sophisticated synthetic biological circuits is dependent on the development of extensible tools capable of providing specific control of gene expression in eukaryotic cells. Here, we describe a new class of synthetic transcription factors that activate gene expression in response to extracellular chemical stimuli. These inducible activators consist of customizable transcription activator-like effector (TALE) proteins combined with steroid hormone receptor ligand-binding domains. We demonstrate that these ligand-responsive TALE transcription factors allow for tunable and conditional control of gene activation and can be used to regulate the expression of endogenous genes in human cells. Since TALEs can be designed to recognize any contiguous DNA sequence, the conditional gene regulatory system described herein will enable the design of advanced synthetic gene networks.

Keywords: TALE; gene regulation; inducible gene expression; steroid hormone receptor; transcription factor.

Figure 1

Ligand-inducible TALE transcription factors. (a) TALE-TF proteins fused to ligand-binding domains (LBDs) from the estrogen receptor (ER) or progesterone receptor (PR) undergo intermolecular dimerization in response to 4-hydroxytamoxifen (4-OHT) or RU486, respectively, and up-regulate gene activation from DNA sequences that contain two direct or inverted repeat TALE binding sites. (b) TALE-TF proteins fused to the chimeric single-chain retinoid X-α/ecdysone (RXE) LBD undergo intramolecular rearrangement in response to ponasterone A (PonA) and up-regulate gene activation from target DNA that contains only a single TALE binding site. E.D. indicates effector domain.

Figure 2

Activation of gene expression by ligand-responsive TALE transcription factors. (a) Schematic illustration of the luciferase reporter system used to evaluate ligand-inducible TALE-TF gene activation. Avr15 target site repeats indicated. (b–d) Fold-activation of luciferase expression observed with the (b) PR/RU486, (c) ER/4-OHT, and (d) RXE/PonA regulatory systems. (e) Fold activation of luciferase expression observed by RXE-TFs with increasing concentrations of PonA. Error bars indicate standard deviation (n = 3; p-value < 0.0005; paired t-test).

Figure 3

Ligand-dependent gene activation from the ICAM-1 promoter by RXE-TFs. (a) (left) Location (red circles) of the TALE binding sites within the ICAM-1 promoter region, and (right) the sequence of each target site. 5′ thymidine nucleotides highlighted red. (b and c) Fold activation of luciferase expression after co-transfection of RXE-TF expression plasmids with luciferase reporter plasmids containing (b) four direct repeats of the ICAM-1 TF binding site or (c) the full-length ICAM-1 promoter upstream of a luciferase reporter gene. Error bars indicate standard deviation (n = 3; p-value < 0.0005; paired t-test).

Figure 4

Ligand-dependent gene activation from the Oct-4 promoter by RXE-TFs. (a) (Left) Location (red circles) of the TALE binding sites within the Oct-4 promoter region, and (right) the sequence composition of each target site. 5′ thymidine nucleotides highlighted red. (b and c) Fold activation of luciferase expression after co-transfection of RXE-TF expression plasmids with luciferase reporter plasmids containing (b) four direct repeats of the Oct-4 TF binding site or (c) the full-length Oct-4 promoter upstream of a luciferase reporter gene. Error bars indicate standard deviation (n = 3; p-value < 0.0005; paired t-test).

Figure 5

Regulation of endogenous ICAM-1 expression by RXE-TFs. (a) Location (red circles) of the TALE-binding sites within the ICAM-1 promoter region, and (right) the sequence composition of each target site. (b) Percentage of HeLa cells that showed an increase in ICAM-1 expression by flow cytometry after transfection with RXE-TFs targeting the endogenous ICAM-1 promoter. Data was normalized to mock-transfected cells. Error bars indicate standard deviation (n = 3; p-value < 0.002; paired t-test).