Extended Data Fig. 4: INTS10 is required for neural cell fate commitment.
From: The enhancer module of Integrator controls cell identity and early neural fate commitment
(a) Diagram of the INTS10 genomic locus and gRNA targeting strategy for generating haploinsufficient INTS10 clones. (b) CRISPR-Cas9 genome editing in iPSCs induced frameshift mutations that generate premature stop codons in INTS10. (c) Magnification of Fig. 4d to highlight the abnormal cellular features in INTS10 HET NPCs. Scale bar, 50 µm. (d) ICC analysis of neuronal markers TUJ1 and NEUN in WT and INTS10 HET neurons. INTS10 HET NPCs failed to initiate normal neuronal processes labeled by Tuj1. Scale bar, 50 µm. (e) Volcano plot shows that INTS10 depletion does not lead to transcriptomic changes in iPSCs as determined by 3′ mRNA Quant-seq (n = 3) (fold change >2, FDR < 0.1). (f) Volcano plot showing differentially expressed genes in INTS10 HET2 versus WT NPCs by 3′ mRNA Quant-seq (n = 3) (fold change >2, FDR < 0.1). (g) GO enrichment analysis of genes upregulated in INTS10 HET2 versus WT NPCs. Adjusted and exact p-values are listed in Supplementary Table 3. (h) GO enrichment analysis of genes downregulated in INTS10 HET2 versus WT NPCs. Adjusted and exact p-values are listed in Supplementary Table 3. (i) Immunoblot analysis of INTS10 expression (whole cell extracts) in haploinsufficient ReN cell clones. GAPDH was used as control. (j) ICC analysis of neural markers in WT and INTS10 HET ReN cells. INTS10 haploinsufficiency results in altered morphology and polarization of NPCs. Scale bar, 50 µm. (k) ICC analysis of neuronal markers in WT and INTS10 HET ReN cell-derived neurons at day 3 after differentiation. INTS10HET ReN cells developed less processes compared to WT ReN cells during differentiation. Scale bar, 50 µm. (l) Dot plot of GSEA analysis shows that INTS10 HET ReN cells start to lose neuronal signatures at 1 day after neural induction (3′ mRNA Quant-seq, n = 2). Adjusted and exact p-values are listed in Supplementary Table 3. (m) Immunoblot of INTS10 depletion in ReN cells upon lentiviral-transduced shRNAs. GAPDH was used as a control. (n) Dot plot of GSEA analysis shows that acute depletion of INTS10 leads to downregulation of neural differentiation pathways compared to shLUC control. Source numerical data and unprocessed blots are available in source data. Adjusted and exact p-values are listed in Supplementary Table 3.
