Extended Data Fig. 4: MEN1 and SUZ12 KO accelerates gene upregulation during neuroectoderm differentiation.
From: Genome-wide CRISPR screen identifies Menin and SUZ12 as regulators of human developmental timing
a, Normalized counts of additional representative markers of monotonic up cluster 1 (n = 3 independent differentiations). Data are the mean ± s.d. P values were calculated from two-way ANOVA test with post-hoc Tukey’s multiple comparisons test. b, Time course analysis of mRNA expression of representative markers from monotonic up cluster 1 and 4, relative to housekeeping gene, GAPDH, in NT and KO cells, assessed by qRT-PCR (n = 3 independent experiments). Data are the mean ± s.e.m. P values were calculated from two-way ANOVA test with post-hoc Dunnett’s multiple comparisons test. c, Day 4 expression levels of monotonic up and down genes, grouped by presence or absence of PAX6 binding at the promoter region. A list of PAX6-binding genes in hESCs41 (N = 3984) was intersected with each cluster; overlapping genes were classified as ‘PAX6 binding’, while the remaining genes were classified as ‘No PAX6 binding’. P values were calculated using two-tailed unpaired t-test. d, Time course analysis of neural markers PAX6, ZBTB16, and MAP2 in the PAX6 KO and its parental control lines during neuroectoderm differentiation (n = 4 for PAX6; n = 2 for ZBTB16 and MAP2), assessed by flow cytometry. Data are the mean ± range for PAX6. Data are the mean for ZBTB16 and MAP2. P values for PAX6 were calculated from two-way ANOVA test with post-hoc Šidák’s multiple comparisons test. e, Schematic of dox-inducible CRISPRa and CRISPRi of PAX6. H9 iCRISPRa or iCRISPRi hESCs were infected with NT or PAX6 gRNA and selected with antibiotics. PAX6 activation or inhibition was induced with 4-day doxycycline treatment (3 days at hESC stage and 1 day during the first day of differentiation). Cells were differentiated into neuroectoderm for 4 days and neural marker expression was assessed by flow cytometry. f, Representative histogram plots for neuroectoderm markers PAX6 and ZBTB16 in the H9 iCRISPRi lines. g, Representative dot plots of one gPAX6i line at day 4. Cell populations were colour-coded based on their PAX6 expression levels. h, Representative histogram plots for neuroectoderm markers PAX6 and ZBTB16 in the H9 iCRISPRa lines. i, mRNA expression of PAX6 relative to housekeeping gene, GAPDH, assessed by qRT-PCR, at day 0. ns, not significant (P > 0.05).
