Fig. 1: Activation of the T-cell specific lncRNA Dreg1 in A20 B cells.
From: Activation of stably silenced genes by recruitment of a synthetic de-methylating module
a Fold activation of Dreg1 in A20 cells transduced with sgRNA targeting Dreg1 promoter together with SAM or SunTag-VP64 constructs. Fold change is calculated by ΔΔCt method. Data were analysed with unpaired two-sided Student’s t-test compared to control b DNA methylation (mCpG) profiles of naïve B and CD4 + T cells at the Dreg1 locus, plotted as population proportion of methylated cytosine in each CpG dinucleotide motif. c Schematics of TETact systems and corresponding construct designs—multiple copies of TET1CD are recruited to dCas9 via the GNC4 epitopes, whereas the activator domains (v1—VPR, v2—VP64-p65-hsf1, v3—p65-hsf1) are recruited via two MS2 aptamers. d Dreg1 lncRNA expression in A20 cells transduced with sgRNA targeting Dreg1 promoter in different activation systems as indicated. P = 0.0003 from one-way ANOVA with Dunnett’s post hoc test compared to SAM. e Bisulphite sequencing of Dreg1 TSS and promoter for A20-TETact-v3 cells transduced with either control or Dreg1-targeting sgRNA. Open lollipops represent non-methylated CpG whereas closed lollipops represent methylated CpG motif. Each row represents an individual clone. f Activation of Dreg1 lncRNA using different sgRNA targeting location. Expression level is relative to β-actin (Actb) level as 2−ΔCt. From left to right, P = 0.0248, 0.0124 from one-way ANOVA with Dunnett’s post hoc test compared to control. Data shown are mean ± s.e.m. from three independent transductions. n.s., non-significant, *P < 0.05, ***P < 0.001 Source data are provided as a Source Data file.
