Supplementary Figure 7: Partial rescue of TKO phenotypes through PAX6 overexpression and targeted demethylation of the PAX6 P0 promoter.

a, Expression of neuroectoderm (endogenous PAX6:PAX6 (Endo), total PAX6:PAX6 (All), and FOXG1), neural crest (SOX10) and pluripotency (NANOG) markers at day 10 of neuroectoderm differentiation of WT cells, TKO cells without doxycycline treatment (TKO) and TKO cells treated with doxycycline during neuroectoderm differentiation (TKO + PAX6). n = 3 independent experiments. Data are presented as means ± s.d. For significance tests, black lines indicate comparison with WT. Statistical analysis was performed by one-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. b, Diagram of the dCas9–TET1 catalytic domain fusion protein (dCas9-TET1CD) and the dCas9–TET1 catalytic domain fusion protein in which the TET1 catalytic domain has been mutated (dCas9-TET1CD/Mut). c, Diagram of the gRNAs (blue arrows) designed to target the PAX6 P0 promoter in the region surrounding the 5hmC peak found in WT hESCs. The arrowhead indicates the 5′ end of the targeting gRNA. Regions previously analyzed for TET1 binding by ChIP–qPCR are enclosed by black rectangles. d, Heat map of MassARRAY analysis of 5mC in nontransfected TKO dCas9-TET1CD hESCs (NT) and TKO dCas9-TET1CD hESCs transiently transfected with gRNAs targeting the PAX6 P0 promoter (Cr6, Cr7, Cr9). The location of each row of CpGs with respect to the TSS is shown to the left of the heat map. The color key for percent methylation is shown to the right of the heat map. The graph on the bottom shows quantification of methylation in the region depicted in the heat map; n = 2 independent experiments. Data are presented as means ± s.d. For significance tests, all comparisons are to the nontransfected control (NT). Statistical analysis was performed by Student’s t test (two-sided): **P < 0.01, ***P < 0.001. e, Targeted demethylation at the bivalent promoters of PAX6 (left) and SOX10 (middle) and the enhancer of LEFTY2 (right). We used dCas9-TET1CD-targeted TKO hESCs expressing an HBB-targeting gRNA as a nontargeting (NT) control. Top, heat map of MassARRAY analysis of 5mC at the bivalent promoters of PAX6 and SOX10 and the enhancer of LEFTY2 after dCas9-TET1CD-mediated demethylation with either nontargeting or targeting gRNAs. – DOX indicates that the cell lines were not treated with doxycycline and thus do not express dCas9-TET1CD. The location of each row of CpGs with respect to the TSS is shown to the left of each heat map. The color key for percent methylation is shown to the right of the LEFTY2 heat maps. For each cell line and condition, three independent experiments are shown as three columns. Statistical analysis was performed by one-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Bottom, qPCR analysis of targeted gene expression either in hESCs (blue) or after neuroectoderm differentiation (red); n = 3 independent experiments. Data are presented as means ± s.d. Statistical analysis was performed by Student’s t test (two-sided): *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001