Fig. 5: Multivalent LLPS transcriptional activators potently enhance chemically regulated transcriptional activation.

a Schematic representation of the engineered LLPS-based transcriptional activation system. P4:NLS:6 × TIF2 binds to cccTag-dCas9:10×P3, and agonist-mediated recruitment of LBD-VPR activation domains undergoes LLPS, leading to gene activation of a reporter with a single DNA binding site. b Reporter transcriptional activation using cccTag-dCas9 fused to increasing P3 peptide repeats (1 × P3, 2 × P3, 4 × P3, and 10 × P3) measured 24 h after cortisol (COR, 50 nM) stimulation. c Estradiol (EST, 100 nM)-induced transcriptional activation in the ERβ-based system, demonstrating increased activation with higher dCas9-P3 peptide repeats. d Representative confocal image of HEK293T cells co-expressing dCas9:10P3, P4:NLS:BFP:6xTIF2, and GR2- or ERβ:NLS:VPR:YFP, along with [ab]nt sgRNA. The image shows nuclear localization of GR2- and ERβ-based LLPS puncta after 24 h stimulation with the ligands EST (100 nM) and COR (100 nM). Scale bar = 10 µm. Corresponding fluorescence intensity graphs for mCitrine and TagBFP are shown. Images are representative of two independent experiments. Arrowheads point to the representative condensates. e, f Dynamic regulation of GR2- and ERβ-based LLPS transcriptional activation through agonist and antagonist treatment. Cortisol-induced activation is inhibited by mifepristone (MIF) (e), while estradiol-induced activation is suppressed by 4-hydroxytamoxifen (4-OHT) (f). Agonist and antagonist ligand additions are indicated by filled circles beneath the graphs, with the corresponding expected outcomes marked by arrows. g Transcriptional activation of ASCL1 endogenous gene by GR2- (left) and ERβ- (right) based LLPS systems after stimulation of HEK293T cells with indicated ligands. In all experiments, HEK293T cells were co-transfected with 50 ng of the reporter plasmid [ab]:pMin:Fluc, 25 ng of the corresponding gRNA, 25 ng of the dCas9:n×P3, 50 ng of the P4:NLS:6(1)×TIF2, and 50 ng of either ERβ:NLS:VPR or GR2:NLS:VPR encoding plasmids. Results are presented as fold activation relative to non-stimulated mock cells transfected with an empty plasmid (pcDNA3) or reporter-only control. Data represent means ± s.d. from n = 8 biological replicates pooled from two independent experiments. Conditions were compared using a two-sided unpaired t-test with Welch’s correction (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns > 0.05). All schematics were created using Inkscape (ver. 1.2.1).