Extended Data Fig. 3: Genome-wide profiling of early neural commitment and neurogenesis using human iPSCs.
From: The enhancer module of Integrator controls cell identity and early neural fate commitment
(a) Nuclear extract of iPSCs was fractionated on a Superose 6 chromatography column. All Integrator subunits co-eluted at the high-molecular-weight fractions, corresponding to the fully assembled complex. INTS10, INTS13, and INTS14 also co-eluted at lower-molecular-weight fractions, suggesting the formation of a separate enhancer module similar to what we observed in SH-SY5Y cells. Two independent experiments produced consistent results. (b) Endogenous INTS13 nuclear interactomes in iPSC and NPC cells. IP was performed using commercially available antibodies, followed by MudPIT LC-MS/MS analysis. Log10 iBAQ protein scores of each interactor are plotted. (c-d) Endogenous INTS10 and ZYMND8 (c) and INTS13 and ZYMND8 interactomes (d) in NPC cells. IP was performed using commercially available antibodies, followed by MudPIT LC-MS/MS analysis. Log10 iBAQ protein scores of each interactor are plotted. (e) Heatmap of Log10 iBAQ protein scores from INTS13 IPs in SH-SY5Y, iPSC and NPCs cells for Zinc Finger Protein interactors. (f) Volcano plot showing differentially expressed genes in neurons versus NPCs, as determined by 3′ mRNA Quant-seq analysis (n = 3). Fold change >2, FDR < 0.1. (g) We plotted normalized read counts of INTS10, INTS13 and INTS14 during iPSC differentiation into neurons by 3′ mRNA Quant-seq analysis (n = 3). (h-j) Spearman’s correlation analysis of gene expression between previously a published neural differentiation model from iPSCs (Burke et al. 2020) and the datasets we generated in this study: iPSCs(h), NPCs (i) and cortical neurons (j). The horizontal black lines within the boxes denote median values (50th percentile), the black boxes contain the value from the 25th to the 75th percentile and the black whiskers denote values at the 5th and the 95th percentiles. (k) Average profiles and heatmaps of INTS10, INTS13 and ZMYND8 ChIP-seq at 3,369 RNAPII-bound enhancers in NPCs. INTS10 overlaps with INTS13 and ZMYND8 at these enhancers. (l) Screenshot of GLIS3 locus showing three active enhancers (yellow with star) that gained significant RNAPII binding in neurons. Increased signal for the Integrator enhancer module, H3K27ac, and H3K4me1 indicate robust activation. Source numerical data and unprocessed blots are available in source data.
