Abstract
Lineage-specific transcription factors operate as master orchestrators of developmental processes by activating select cis-regulatory enhancers and proximal promoters. Direct DNA binding of transcription factors ultimately drives context-specific recruitment of the basal transcriptional machinery that comprises RNA polymerase II (RNAPII) and a host of polymerase-associated multiprotein complexes, including the metazoan-specific Integrator complex. Integrator is primarily known to modulate RNAPII processivity and to surveil RNA integrity across coding genes. Here we describe an enhancer module of Integrator that directs cell fate specification by promoting epigenetic changes and transcription factor binding at neural enhancers. Depletion of Integrator’s INTS10 subunit upends neural traits and derails cells towards mesenchymal identity. Commissioning of neural enhancers relies on Integrator’s enhancer module, which stabilizes SOX2 binding at chromatin upon exit from pluripotency. We propose that Integrator is a functional bridge between enhancers and promoters and a main driver of early development, providing new insight into a growing family of neurodevelopmental syndromes.
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Data availability
Sequencing data that support the findings of this study have been deposited in the Gene Expression Omnibus under the accession codes GSE230928 (SuperSeries), GSE230894 (ChIP-seq data), GSE230917 (RNA-seq data), GSE230914 (Hi-C data), GSE230922 (scRNA-seq data), GSE230893 (ATAC-seq data) and GSE230889 (CUT&Tag data). Proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD054928. Source data are provided with this paper. All of the other data supporting the findings of this study are available from the corresponding author upon request.
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Acknowledgements
This study was supported by grants from the G. Harold and Leila Y. Mathers Charitable Foundation (to A.G.) and National Institutes of Health (NIH; R01 HL141326 and R01 CA252223). C.M.H., S.R.O. and A.Z. were supported by NIH training grant T32 CA09171. C.M.H. was also supported by a Ruth L. Kirschstein National Research Service Award (F31 CA265257). S.D. was supported by an NIH Supplement (HL141326-S1). We thank the Sarma Lab for reagents and useful discussions. We are grateful to the Genomics Core, Proteomics and Metabolomics Core and Imaging Facility of The Wistar Institute (P30-CA010815) for providing outstanding technical support. We also thank the iPSC Core Facility of the University of Pennsylvania for recommendations regarding iPSC protocols.
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Y.Z., C.M.H. and A.G. conceived of the study and experimental plan. Y.Z. and C.M.L. performed most of the experiments. K.A.L. and L.G. performed experiments for revisions, with help from S.R.O. M.G. and F.P. A.Z. provided support with the iPSC and NPC manipulation and differentiation protocols. C.M.H. performed most of the data analysis with help from S.D. and Y.Z. C.M.H. prepared the figures. A.G. wrote the manuscript with help from Y.Z. and C.M.H.
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Extended data
Extended Data Fig. 1 In vivo assembly of a ternary Integrator module in human neuronal cells.
(a) Heatmap clustering of all Integrator subunits (RNA expression levels=normalized transcripts per million bases, nTPM) across the full collection of non-neuronal human tissues and cell lines from the Human Protein Atlas project (proteinatlas.org). INTS10 expression is not increased across all tissues. (b) The number of INTS10, INTS13, and INTS14 peptides pulled down with GFP-FLAG, INTS10-FLAG, INTS13-FLAG, and INTS14-FLAG IPs in the nuclear extract of HEK293T cells stably overexpressing GFP-FLAG, INTS10-FLAG, INTS13-FLAG or INTS14-FLAG. (c) Immunoblot analysis of INTS10 IP from the nuclear extract of SH-SY5Y WT cells. Endogenous INTS10 co-precipitates along with other Integrator subunits (INTS1, INTS5 and INTS13). Two independent experiments yielded consistent results.(d) Endogenous INTS10 and INTS13 nuclear interactomes in parental SH-SY5Y cells. IP were performed using commercially available antibodies, followed by MudPIT LC-MS/MS analysis. Log10 iBAQ protein scores of each interactor are plotted. (e) Endogenous INTS5 and INTS10 nuclear interactomes in parental SH-SY5Y cells. IP were performed using commercially available antibodies, followed by MudPIT LC-MS/MS analysis. Log10 iBAQ protein scores of each interactor are plotted. Source numerical data and unprocessed blots are available in source data.
Extended Data Fig. 2 INTS10 is required for maintenance of neuronal cell identity.
(a) ICC of TUJ1 (green) and DAPI (blue) in WT and INTS10 KO2 (clone 24) and INTS10 KO3 (clone 37) SH-SY5Y cells. All clones show similar morphological changes. Scale bar, 50 µm. Three independent experiments produced consistent results. (b) Principal Component Analysis (PCA) of RNA-seq datasets for all INTS10 KO clones in SH-SY5Y cells. All KO clones cluster away from parental cells along the PC1 axis (74% of variance), suggesting that broad and consistent transcriptome changes ensue depletion of the enhancer module of Integrator. (c) Volcano plots showing the the differential gene expression profile in the three INTS10 KO clones (c10, c24, c37) versus SH-SY5Y WT cell as determined by 3′ mRNA Quant-seq (n = 2) (Fold change >2, FDR < 0.1). INTS10 knockout results in a similar scale of gene expression changes in each clone. (d-e) Average profiles and heatmaps of INTS10, INTS11, INTS13 and RNAPII ChIP-seq at 9,992 INTS10-bound promoters (d) and 12,687 INTS10-bound enhancers (e) in SH-SY5Y WT cells. INTS10 overlaps with INTS13, INTS11 and RNAPII at both proximal promoters and distal enhancers. (f-g) Volcano plots generated with Diffbind show genome-wide differences in H3K27ac (f) and H3K4me1 (g) deposition in INTS10 KO versus WT SH-SY5Y cells (CUT&TAG). We found increased H3K27ac at 12,619 sites and decreased H3K27ac at 10,128 sites. H3K4me1 was increased at 54,622 sites and decreased at 56,142 sites, suggesting broad epigenome changes due to loss of INTS10 (fold change >2, FDR < 0.1). (h-j) Average profile and heatmaps of H3K27ac (h), H3K4me1 (i) and H3K27me3 (j) in WT and INTS10 KO SH-SY5Y cells at 9,992 INTS10-bound promoters. Deletion of INTS10 did not result in broad epigenetic changes at proximal promoters. (k) Motif analysis of 12,687 INTS10-bound enhancers in SH-SY5Y WT cells shows that these cis-regulatory elements are enriched in TFs that regulate neuronal differentiation. Adjusted and exact p-values are listed in Supplementary Table 3. (l) Motif analysis of 11,923 enhancers with up-regulated histone acetylation in INTS10 KO SH-SY5Y cells shows that these H3K27ac-gained enhancers are enriched in TFs that modulate EMT programs. Source numerical data are available in source data. Adjusted and exact p-values are listed in Supplementary Table 3.
Extended Data Fig. 3 Genome-wide profiling of early neural commitment and neurogenesis using human iPSCs.
(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.
Extended Data Fig. 4 INTS10 is required for neural cell 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.
Extended Data Fig. 5 Single-cell analysis of iPSC-derived NPCs reveals diverging identity.
(a-b) Normalized expression of INTS10 (a) and SOX2 (b) in each cell on UMAP plots shows uniformly reduced INTS10 expression in HET2 NPCs, whereas changes in SOX2 expression between WT and HET2 NPCs are not significant. (c) SingleR annotations from the Human Primary Cell Atlas (which does not contain a NPC cell type) were used to build a heatmap of the 4 annotated clusters in Fig. 5b. The color scale reflects the percentage contained in each cluster for every cell type. Cluster 1 is primarily composed by neuron-like cells, whereas cluster 2 contains a variety of hematopoietic prognitors (likely mis-annotation of mesenchymal cells). Cluster 3 contains a higher proportion of iPS cells and cluster 4 is primarily composed by neuroepithelial cells. (d) Violin plots showing normalized expression of select marker genes in WT and HET2 cells.
Extended Data Fig. 6 Integrator’s enhancer module supports a SOX2 enhancer network.
(a) Violin plots show the expression of Integrator subunits computed from sc-RNAseq data. (b) GSEA analysis with the group of genes linked to the 602 enhancers that lose INTS10 in HET2 NPCs.The plot shows that genes linked to 602 INTS10 lost enhancers are significantly downregulated in HET2 cells. (c) Overlap of 602 enhancers that lose INTS10 in HET2 with histone marks ChIP-seq peaks from ENCODE in primary neuronal tissues. (d-e) Average profile and heatmap of INTS13 (d) and INTS11 (e) ChIP-seq in WT and HET2 NPCs at the 602 INTS10-lost enhancers (complement to Fig. 6c). (f) Violin plots showing the expression of SOX2, SOX3, SOX6 and SOX10 from scRNA-seq data. (g) GO analysis of genes linked to top 50 SOX2 enhancers by Hi-C. Adjusted and exact p-values are listed in Supplementary Table 3. (h) Snapshot of ChIP-seq and Hi-C tracks on a ~1 Mb region of chromosome 13 containing the SOX21 locus and several cis-regulatory elements (as highlighted with yellow bars and stars). Hi-C loops at 10 kb resolution were calculated with FitHiC. (i) Snapshot of the SOX1 locus in control and the SOX1 enhancer (E2) knockdown NPCs. Reduction of H3K27ac at the SOX1 promoter was observed (highlighted by the rectangle). Sox1 enhancers are highlighted with yellow bars and stars. (j) Average profiles and heatmaps of SOX2 and H3K27ac ChIP-seq at 1,341 enhancers with SOX2 localization in WT NPCs.(k) Immunoblot from whole cell extracts of WT and HET2 iPSCs and their respective NPC derivatives. GAPDH was used as a control. (l) Immunoblot from whole cell extracts of control or doxycycline treated NPCs with tet inducible SOX2. Histone H3 was used as a control. (m) Average profiles and heatmaps of SOX2 at 602 INTS10 lost enhancers in WT NPCs. (n) Average profiles and heatmaps of SOX2 (CUT&TAG) at 1,341 enhancers with SOX2 localization in WT NPCs. (o) Average profiles and heatmaps of SOX2 at 13,150 enhancers that gain SOX2 localization upon SOX2 overexpression in HET2 NPCs with tet inducible SOX2. (p) Motif analysis of 13,150 enhancers that gain significant SOX2 localization upon SOX2 overexression. Source unprocessed blots are available in source data. Adjusted and exact p-values are listed in Supplementary Table 3.
Extended Data Fig. 7 Depletion of SOX2 phenocopies reduction of INTS10 expression.
(a) GO analysis showing that the downregulated genes upon SOX2 depletion in NPCs are enriched for system developmental processes including nervous system development. Adjusted and exact p-values are listed in Supplementary Table 3. (b) GO analysis showing that the upregulated genes upon SOX2 depletion in NPCs are enriched for catabolic and autophagy pathways. Adjusted and exact p-values are listed in Supplementary Table 3. (c) Differential gene expression heatmap of 347 genes downregulated (yellow bar) and 44 upregulated (pink bar) caused by INTS10 (INTS10het) or SOX2 (shSOX2) depletion in NPCs and INTS10 (shINTS10) depletion in ReN cells, as determined by 3′ mRNA Quant-seq (n = 2). Depletion of SOX2 triggers similar gene expression changes to those seen upon depletion of INTS10 in ReN cells and in INTS10 HET2 NPCs. (d) Volcano plot showing few differentially expressed genes in shINTS10 versus shSOX2 NPCs. (Fold change >1, FDR < 0.1). (e) Volcano plot showing the differential binding profile of H3K27ac in shSOX2 versus shLUC-infected NPCs as revealed by CUT&TAG assays (Fold change >1, FDR < 0.1). (f) Motif analysis of 4,442 H3K27ac gained enhancers upon SOX2 depletion shows the enrichment of AP-1 motifs (black dots) but not SOX2 family motifs (red dots). Source numerical data are available in source data. Adjusted and exact p-values are listed in Supplementary Table 3.
Supplementary information
Supplementary Tables 1–3
Supplementary Table 1. List of SOX2-dependent enhancers. Supplementary Table 2. List of all reagents. Supplementary Table 3. Adjusted and exact P values used in the figure plots.
Source data
Source Data Figs. 1 and 4 and Extended Data Figs. 1, 3, 4 and 6
Unprocessed western blots.
Source Data Figs. 1–4, 6 and 7 and Extended Data Figs. 1–4 and 7
Statistical source data.
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Zhang, Y., Hill, C.M., Leach, K.A. et al. The enhancer module of Integrator controls cell identity and early neural fate commitment. Nat Cell Biol 27, 103–117 (2025). https://doi.org/10.1038/s41556-024-01556-y
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DOI: https://doi.org/10.1038/s41556-024-01556-y
