Fig. 2
DNA methylation status and histone modifications in the conserved CNS of FOXP3 intron 1 during reactivation of FOXP3 in human CRISPRi/a cells. A heatmap of DNA methylation status of rich CpG sites in the conserved CNS of FOXP3 intron 1 during reactivation of FOXP3 in human HCC202 CRISPRi/a or HEK 293T CRISPRa cells determined by pyrosequencing. B quantitative expression analysis of FOXP3 before and after sgRNA transduction and 5-Aza-2'-deoxycytidine (5-Aza) treatment of CRISPRa HCC202 cells at days 0, 2, 4, and 7 as determined by qPCR. C quantitative expression analysis of FOXP3 before and after sgRNAs transduction and 5-Aza and Dox treatment of CRISPRi/a HCC202 cells at days 0, 2, 4, and 7 as determined by qPCR. D efficacy of transduction of FOXP3 sgRNA (mCherry) and transfection of SadCas9-TET1 (GFP) into CRISPRa HEK 293T cells at days 2 and 4 as determined by fluorescence microscopy. Scale bar, 1,000 μm. E quantitative expression analysis of FOXP3 before and after sgRNA transduction and SadCas9-TET1 transfection into CRISPRa HEK 293T cells at days 0, 2, and 4 as determined by qPCR. The fold change in expression was calculated using the 2-ΔΔ Ct method with GAPDH mRNA as an internal control. F IP with a specific anti-SadCas9 antibody (left panel: long exposure; right panel: short exposure) in CRISPRi/a HEK 293T cells. G SadCas9 ChIP-qPCR in the conserved CNS of FOXP3 intron 1 during CRISPRi/a-mediated activation of FOXP3 with or without XIST- and FOXP3-sgRNAs and Dox at day 4 in HEK 293T cells. The Y-axis represents % of input DNA. The values for the binding of SadCas9 to targeted locus were normalized to total input genomic DNA of each region. H-N various histone ChIP-qPCR analyses in the conserved CNS of FOXP3 intron 1 during CRISPRi/a-mediated activation of FOXP3 with or without XIST- and FOXP3-sgRNAs and Dox at day 4 in HEK 293T cells. Relative histone enrichment levels were normalized to total input genomic DNA of each region. The promoter loci of FOXP3 neighboring genes, PPP1R3F and CCDC22, were used as reference controls. Data are presented as the means ± standard deviation. p values were determined by a two-tailed t-test between two groups or ANOVA followed by Tukey’s post hoc test vs. a control group. All experiments were repeated three times