Fig. 3: Transcriptional regulation by nuclear condensates.
From: Programmable solid-state condensates for spatiotemporal control of mammalian gene expression
a, Schematics of transcriptional regulation by synthetic condensates. In the absence of grazoprevir, gene expression from episomal or genomic DNA is inhibited by ANR-mediated recruitment of NS3a(H1)-containing condensates to DBD-specific constitutive promoter regions. Grazoprevir disrupts the binding of ANR to NS3a(H1) and activates transcription by alleviating condensate-mediated steric hindrance. Using anchor proteins with ANR domains replaced by GNCR allows for grazoprevir-repressible gene expression. b, Grazoprevir-dependent derepression of SEAP transcription. Left, HEK-293 cells were cotransfected with a constitutive SEAP expression vector containing 14 TetR-specific tetO sites upstream of the constitutive PhCMV promoter (pYK262) and expression vectors for a TetR-based DNA anchor (NLS–TetR–3×ANR, pYK250) and nuclear condensate (NLS–PB1–AG–NS3a(H1), pYK164). Right, HEK-293 cells were cotransfected with a constitutive SEAP expression vector containing eight ZFP1-specific binding sites upstream of PhCMV (pYK479) and expression vectors for NLS–PB1–AG–NS3a(H1) and ZFP1–NLS–4×ANR (pYK164 and pYK521). Relative SEAP expression in cells cultivated in 1 µM grazoprevir or DMSO was profiled by RT–qPCR at 24 h after transfection. Transcript levels were normalized to GAPDH. c, On-demand derepression and silencing of episomal genes. Left, TetR-based systems. Cells cotransfected with SEAP reporter (pYK262) and (i) NLS–KRAB–NS3a(H1) (pYK582) and NLS–TetR–3×ANR (pYK250), (ii) NLS–PB1–AG–NS3a(H1) (pYK164) and NLS–TetR–3×ANR (pYK250), (iii) NLS–KRAB–NS3a(H1) (pYK582) and GNCR–NLS–TetR (pYK648) and (iv) NLS–PB1–AG–NS3a(H1) (pYK164) and GNCR–NLS–TetR (pYK648). Right, ZFP1-based systems. Cells cotransfected with SEAP reporter (pYK479) and (i) NLS–KRAB–NS3a(H1) (pYK582) and ZFP1–NLS–4×ANR (pYK521), (ii) NLS–PB1–AG–NS3a(H1) (pYK164) and ZFP1–NLS–4×ANR (pYK521), (iii) NLS–KRAB–NS3a(H1) (pYK582) and GNCR–ZFP1–NLS (pYK650) and (iv) NLS–PB1–AG–NS3a(H1) (pYK164) and GNCR–ZFP1–NLS (pYK650). SEAP expression in culture supernatants was profiled after 24 h treatment with 1 µM grazoprevir or DMSO. All data are shown as the mean ± s.d. of n = 3 independent experiments (b,c). Statistical significance was assessed using two-sided t-tests. ***P < 0.001 and ****P < 0.0001. Filled circles show individual results. Numbers above the bars represent the fold change between datasets.
