Research ArticleSYNTHETIC BIOLOGY

Liquid-liquid phase separation of light-inducible transcription factors increases transcription activation in mammalian cells and mice

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Science Advances  01 Jan 2021:
Vol. 7, no. 1, eabd3568
DOI: 10.1126/sciadv.abd3568
  • Fig. 1 Design of droplet transcription factors (DropletTF).

    (A) Left: The conventional synthetic TF (TF-, TetR-eYFP-VP16) binds as a homodimer to tetO operators and recruits the preinitiation complex (PIC), initiating the expression of a downstream gene. Right: DropletTF (TF + FUS, TetR-eYFP-FUSn-VP16). FUS addition triggers the formation of coacervates at the tetO operator sites. The locally increased VP16 density increases promoter activity and downstream gene expression. (B) Distribution of TF- and TF + FUS. Human embryonic kidney (HEK)–293 cells were transfected with TF- or TF + FUS constructs and analyzed by fluorescence microscopy. (C) Effects of TF- and TF + FUS on transgene expression. tetOn-based SEAP reporters (n = 1 to 6, 26) were cotransfected with TF- and TF + FUS constructs. The stoichiometry of the expression vectors was adjusted to achieve approximately equal expression levels of TF- and TF + FUS. After 48 hours, SEAP production was quantified and TF expression levels were determined by flow cytometric detection of eYFP. (D) Temporal dynamics of SEAP production induced by TF- and TF + FUS and a tetO4 reporter. SEAP production was quantified at the indicated time points. (C and D) SEAP production was normalized to eYFP fluorescence. Curves represent the model fit of the data. Error bands are estimated with a model with a constant and relative Gaussian error. AU, arbitrary unit.

  • Fig. 2 Design of droplet optogenetic transcription factors (OptoTF).

    (A) Left: Conventional optogenetic TFs. CIBn-TetR is continuously bound to tetO. Upon blue light illumination, Cry2-eYFP-VP16 (OptoTF-) is recruited to the promoter via heterodimerization of Cry2 and CIBn. Homomultimerization of Cry2 leads to further accumulation of the TAD VP16, resulting in high expression of the reporter gene. Right: Droplet optogenetic TFs (Cry2-eYFP-IDR-VP16, OptoTF + IDR). Blue light triggers coacervate formation of OptoTF + IDR due to binding to CIBn-TetR and homo-oligomerization of Cry2. The resulting locally increased TAD concentration further increases reporter gene expression. (B) Light-responsive distribution of OptoTF- and different OptoTF + IDRs in HEK-293 cells. OptoTF- or OptoTF + IDR with three different IDRs (DDX4, FUS, and hnRNPA1) was cotransfected with a tetO13-based mCherry reporter and analyzed by fluorescence microscopy after 24-hour cultivation in the dark (left) or under blue light (465 nm, 5 μmol m−2 s−1). (C) Mobile fractions and half-recovery times of OptoTF + FUS droplets determined by fluorescence recovery after photobleaching (FRAP). OptoTF + FUS, CIBn-TetR, and a tetO7-based reporter were cotransfected into HEK-293 cells. Cells were illuminated with blue light (465 nm, 5 μmol m−2 s−1) for either 10 min or 24 hours before measurement. Both groups were compared by a two-tailed Welch’s t test.

  • Fig. 3 Effects of FUSn insertion into OptoTF constructs on gene expression levels.

    (A) Effect of the IDR integration site. FUSn was integrated into OptoTF- either between eYFP and VP16 or at the N terminus of Cry2. The constructs were cotransfected into HEK-293 cells together with a tetO7-based SEAP reporter and CIBn-TetR. Cells were cultivated in the dark or under blue light (465 nm, 5 μmol m−2 s−1) for 48 hours before quantifying SEAP production. (B) Gene expression mediated by OptoTF constructs. The experiment was performed as described in (A) except that reporters with different numbers of tetO repeats (1 to 6, 26) were used. The model fit to the data is represented by the curves, while the shaded error bands are estimated with an error model with a constant and relative Gaussian error. (C) OptoTF-mediated expression kinetics. OptoTF + FUS and OptoTF- were cotransfected together with a tetO4-based SEAP reporter and CIBn-TetR. Cells were cultivated in the dark or under blue light, and SEAP production was quantified at the indicated time points. The model fit to the data is represented by the curves, while the shaded error bands are estimated with an error model with a constant and relative Gaussian error.

  • Fig. 4 Difference in gene expression levels for transcription factors with and without FUS.

    (A) Ratio of the gene expression levels for TF + FUS/TF- (red) and OptoTF + FUS/OptoTF- (blue). Curves correspond to the ratio of the calibrated model trajectories for TF- and TF + FUS (Fig. 1C) and OptoTF- and OptoTF + FUS (Fig. 3B). (B) Light dose–dependent reporter gene expression. HEK-293 cells were cotransfected with OptoTF + FUS, CIBn-TetR, and a tetO7-based SEAP reporter and cultivated for 48 hours with the indicated blue light intensities. SEAP production was quantified. Curves represent the model prediction. Uncertainties (shaded bands) were calculated using the prediction profile likelihood method. (C) Differential reporter gene expression in mice. CIBn-TetR, OptoTF- or OptoTF + FUS, and a tetO7-Luciferase reporter were coadministered via hydrodynamic tail vein injection. Mice were either kept in darkness or exposed to blue light pulses for 11 hours (460 nm, 2-min pulses with the indicated intensity). For in vivo bioluminescence imaging, luciferin was injected intraperitoneally. Top: Mean bioluminescent radiance (p s−1 cm−2 sr−1) ± SEM, n = 4. P values were calculated with Student’s t test *P < 0.05, **P < 0.01. Bottom: Representative images for each condition (photo credit: Deqiang Kong, East China Normal University).

Supplementary Materials

  • Supplementary Materials

    Liquid-liquid phase separation of light-inducible transcription factors increases transcription activation in mammalian cells and mice

    Nils Schneider, Franz-Georg Wieland, Deqiang Kong, Alexandra A. M. Fischer, Maximilian Hörner, Jens Timmer, Haifeng Ye, Wilfried Weber

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