Fig. 5: SIMBA recruits histone methyltransferase to regulate local transcription at target genomic sites.
From: Engineering inducible biomolecular assemblies for genome imaging and manipulation in living cells
a Volcano plot of HP1α-mediated transcriptomic changes at NFAT1 binding sites (Benjamini corrected P-value, Padj < 0.05). NYAP1, NGFR, and LRRC4B selected for following validation assays. Differentially regulated genes shown in blue (downregulated) and red (upregulated). b The CRISPR/dCas9 system targets the three endogenous genes in HEK293T cells. qPCR results show the relative expression (n = 3 biologically independent experiments, unpaired two-sided Student’s t test. NYAP1: p = 0.0039; LRRC4B: p = 0.0244; NGFR: p = 0.0191; No gRNA: p = 0.2662.) c Live cell imaging shows co-localization of HP1α and SUV39H1 N-terminal domain (SUV39H1ND) at NYAP1 locus before and after rapalog treatment (insets: enlarged images of the indicated BAs with enhanced clarity by applying more stringent brightness/contrast settings). Scale bar, 5 μm. d The puncta highlighted by the white box in (c) was quantified based on normalized intensities. e Dynamic fluorescence intensity change of HP1α (red) and SUV39H1ND (green) upon rapamycin (black arrow). Average intensity values of HP1α or SUV39H1ND at different time points were normalized to that of the time points before rapamycin. f NYAP1 gene expression determined by qPCR in response to chaetocin treatment (500 nM, 6 hrs), with or without HP1α BA formation. (n = 3 biologically independent experiments. One-way ANOVA with Tukey’s multiple comparison test; *p = 0.0202; **p = 0.0043; ns, p = 0.3535.). g, h Effects of H3.3K9M (g) and SUV39H1/2 knockdown (h) on SIMBA-induced gene suppression. Target gene: NYAP1. n = 3 biologically independent experiments, two-sided Fisher’s LSD test. p values are indicated in the figure. i The correlation between the distance of NFAT1 binding sites to TSS and fold change of the downregulated genes from nSIMBA RNA-seq data. Downregulated genes were divided into 3 categories based on the distance between nSIMBA binding sites to TSS. Standard boxplots, n = 145, 107, 97 genes, respectively. *** Two-sided Kruska–Wallis test, p = 4.9 × 10−7. Error bars, mean ± SD. ns, not significant. Source data are provided as a Source Data file.
