Light-inducible spatiotemporal control of gene activation by customizable zinc finger transcription factors

Abstract

Advanced gene regulatory systems are necessary for scientific research, synthetic biology, and gene-based medicine. An ideal system would allow facile spatiotemporal manipulation of gene expression within a cell population that is tunable, reversible, repeatable, and can be targeted to diverse DNA sequences. To meet these criteria, a gene regulation system was engineered that combines light-sensitive proteins and programmable zinc finger transcription factors. This system, light-inducible transcription using engineered zinc finger proteins (LITEZ), uses two light-inducible dimerizing proteins from Arabidopsis thaliana, GIGANTEA and the LOV domain of FKF1, to control synthetic zinc finger transcription factor activity in human cells. Activation of gene expression in human cells engineered with LITEZ was reversible and repeatable by modulating the duration of illumination. The level of gene expression could also be controlled by modulating light intensity. Finally, gene expression could be activated in a spatially defined pattern by illuminating the human cell culture through a photomask of arbitrary geometry. LITEZ enables new approaches for precisely regulating gene expression in biotechnology and medicine, as well as studying gene function, cell-cell interactions, and tissue morphogenesis.

Figure 1

The GI-ZFP fusion protein localizes to the ZFP DNA binding sequence upstream of the transgene. Blue light initiates heterodimerization between GI and LOV, which translocates VP16 to the gene of interest and activates transcription.

Figure 2

(a) Luciferase activity increases with blue-light illumination time in HeLa cells transfected with LOV-VP16, GI-ZFP2, and a luciferase reporter containing 9 copies of the ZFP2 binding site upstream of luciferase (*p < 0.0001 vs dark). (b) In cells transfected with LOV-VP16, a GI-ZFP, and a luciferase reporter, significant reporter activation was only observed when a GI-ZFP was paired with a luciferase reporter containing three copies (3x) of its corresponding ZFP binding site. Cells were illuminated for 30 h. A significant decrease in luciferase activity was observed in cells transfected with only a luciferase reporter and junk DNA (#p < 0.05).

Figure 3

(a) Light-induced luciferase activity increases with the number of upstream GI-ZFP binding sites. HeLa cells were transfected with the Seq2-Luc reporter with either 3, 6, 7, or 9 copies of the ZFP2 binding site and either junk DNA or LOV-VP16 and GI-ZFP2. Transfected cells were illuminated with pulsing blue light for 30 h or incubated in the dark. (*p < 0.0001 vs reporter only). (b) LITEZ is reversible and repeatable. HeLa cells were transfected with LOV-VP16, GI-ZFP2, and 9xSeq2-Luc. Cells were either incubated in the dark for the entire experiment (solid line) or illuminated with pulsing blue light (dotted line) for two separate 6-h periods (shaded areas) (p < 0.0001 vs dark).

Figure 4

LITEZ can be used to spatially control gene expression in human cells. (a) HEK 293T cells were co-transfected with LOV-VP16, GI-ZFP2, 9xSeq2-eGFP, and a constitutive dsRed expression plasmid and illuminated through a photomask (upper right) containing (a) rectangular slits with widths of 2, 1, 0.5, and 0.3 mm (left to right) or (b) happy face or spot array patterns. The 0.3 mm slit was too narrow to result in an observable pattern. eGFP intensity across the pattern was quantified by image analysis (a, top left). Inset (a, lower right) shows the sharp contrast between eGFP-positive and -negative cells at the border of the 2 mm slit. Scale bars = 2 mm.