Fig 2: Loss of UTX Results in Decreased Neurite Complexity and Electrophysiological Defects of hESC-Derived Neurons(A) Representative images of neurons derived from UTX WT and KO hESCs at day 40 of neural differentiation.(B) Sholl analysis showing that total neurite length reduced in UTX KO hESC-derived neurons (60 neurons in every group in 3 independent experiments).(C–E) Compared with WT group, UTX KO hESC-derived neurons exhibited decreased dendritic complexity as shown in reduced numbers of intersections (C), nodes (D), and ends (E).(F) Representative traces of membrane potential responding to step depolarization by current injection steps from -10 pA to +60 pA in 10-pA increments. Membrane potential was current-clamped at around -65 mV. Representative traces were displayed by WT neurons.(G) Quantification of the neuron maturity by recorded AP firing patterns at day 60 after differentiation (more than 15 neurons in every group have been recorded).(H) Representative traces of whole-cell currents in voltage-clamp mode. Cells were held at -70 mV. Step depolarization from -80 to +60 mV at 10-mV intervals was delivered. Inset shows Na+ currents.(I) Quantification of membrane capacitance in neurons at 60 days after differentiation. Error bars indicate ±SEM. *p < 0.05; a two-tailed t test was performed.(J) Quantification of membrane resistance in neurons at 60 days after differentiation. *p < 0.05.(K and L) Averaged current-voltage relationship (I-V curves) for Na+ currents, recorded from hESC-derived neurons. ns, not significant; *p < 0.05, **p < 0.01.(M and N) Averaged current-voltage relationship (I-V curves) for K+ currents, recorded from hESC-derived neurons. ns, not significant; *p < 0.05, **p < 0.01.

Fig 3: Gene expression profiling analysis of UTX-regulated genes using UTX-mutated and reconstituted cells. (A) UTX-mutated HCT116 cell lines (CR1 and CR2) were established by CRISPR gene editing and exogenous UTX was re-expressed using a lentivirus vector (vec: vector only; UTX: with exogenous UTX). UTX protein was detected by western blot analysis and the expression levels are shown under the lanes. Details of the targeted and mutated sequences are presented in Supplementary Fig. S3. (B) Gene expression profiling with microarray. Up- or down-regulated genes in the CR1 and CR2 clones are shown in the heatmap with a cut-off of 1.5-fold. Gene identities are shown on the right. (C) Expression levels of PCK2, CHOP, REDD1, CHAC1 and TRIB3 from the microarray analysis are shown in the bar charts. Data shows the mean of triplicates with S.E.M. and p-value (*< 0.05; **< 0.01; ***< 0.001). (D) RT-qPCR analysis. The same samples used for the microarray analysis were analyzed by RT-qPCR. n.s. indicates “not significant”. For microarray and RT-qPCR experiments, biological triplicates (N = 3) were processed.

Fig 4: UTX Loss-of-function Inhibits Neural Differentiation of hESCs(A) Immunostaining of neuron markers MAP2 and Tuj1 at day 40 of neural differentiation. Scale bar, 20 µm.(B) Quantitative analysis showing reduced percentages of MAP2+ and Tuj1+ neurons derived from UTX KO hESCs.(C) qRT-PCR analysis showing that the expression levels MAP2 and Tuj1 were significantly lower in UTX KO cells at day 40 of neural differentiation. Results are presented as mean ± SEM; n = 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: UTX Is Upregulated in Earlier Stages of Neural Differentiation of hESCs(A) Schematic illustration of neural differentiation of hESCs. hESCs, human embryonic stem cells; EB, embryoid bodies; hNSCs, human neural stem cells; hNPCs, human neural progenitor cells. Scale bar, 50 µm.(B) Real-time PCR analysis showing higher mRNA levels of UTX in earlier stages of neural differentiation of hESCs. GAPDH is used as an internal control for real-time PCR.(C) Western blotting indicating upregulated protein levels of UTX at earlier stages during neural differentiation of hESCs. ß-Tubulin is used as a loading control.(D) Representative images of H3K27me3 immunostaining at different stages of neural differentiation of hESCs. DAPI, a nuclei marker; Nestin, an NSC marker; DCX, an NPCs marker; MAP2, a mature neuron marker. Scale bar, 50 µm.(E) Western blotting showing protein levels of H3K27me3 and H3K27ac upon neural differentiation of hESCs. Histone 3 was used as a loading control.Results are presented as mean ± SEM; n = 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.