Fig. 4: SIMBA enables puncta formation and gene manipulation at the endogenous NFAT1 binding sites in the genome.
From: Engineering inducible biomolecular assemblies for genome imaging and manipulation in living cells
a nSIMBA consists of three components, NFAT1-8xSunTag, scFv-FKBP, and FRB-mCherry-HP1α. Upon ATP treatment, NFAT1-8xSunTag can translocate into nucleus and target NFAT1 binding motifs in the genome together with scFv-FKBP. Upon rapamycin stimulation, FRB-mCherry-HP1α can be further recruited to the genome sites targeted by NFAT1 and promote the puncta formation. b Time-lapse images show the labeling of NFAT1 binding sites through the nSIMBA system in HEK293T cells, upon the sequential treatments of 30 μM ATP and 100 nM rapamycin. Scale bar, 5 μm. c, d Diagram (c) and live cell images (d) showing the sequential recruitment of NFAT1-GFP and nSIMBA at the target genomic loci, upon ATP and rapamycin stimulation. Scale bar, 5 μm. e Heat-map shows differential gene expression between groups with (HP1α) or without HP1α (NC). f GO-term enrichment analysis of significantly altered genes from RNA-seq results by using DAVID. The bars show the functional gene groups that are downregulated by HP1α. The y axis shows the top seven enriched GO terms and the x axis shows the enrichment significance of Benjamini corrected P-values.
