Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The enhancer module of Integrator controls cell identity and early neural fate commitment

Nature Cell Biology volume 27, pages 103–117 (2025)Cite this article

Subjects

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.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution
Fig. 1: In vivo assembly of a ternary Integrator module in human neural cells.
Fig. 2: INTS10 is required for maintenance of neural cell identity.
Fig. 3: Genome-wide profiling of early neural commitment and neurogenesis using human iPSCs.
Fig. 4: INTS10 is required for neural cell fate commitment.
Fig. 5: Single-cell analysis of iPSC-derived NPCs reveals diverging identity trajectories upon depletion of Integrator’s enhancer module.
Fig. 6: Integrator’s enhancer module supports a SOX2 enhancer network.
Fig. 7: Depletion of SOX2 phenocopies the reduction of INTS10 expression.

Similar content being viewed by others

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.

References

  1. Taverna, E., Gotz, M. & Huttner, W. B. The cell biology of neurogenesis: toward an understanding of the development and evolution of the neocortex. Annu. Rev. Cell Dev. Biol. 30, 465–502 (2014).

    CAS  PubMed  Google Scholar 

  2. Fish, J. L., Dehay, C., Kennedy, H. & Huttner, W. B. Making bigger brains—the evolution of neural-progenitor-cell division. J. Cell Sci. 121, 2783–2793 (2008).

    CAS  PubMed  Google Scholar 

  3. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

    CAS  PubMed  Google Scholar 

  4. Nehme, R. et al. Combining NGN2 programming with developmental patterning generates human excitatory neurons with NMDAR-mediated synaptic transmission. Cell Rep. 23, 2509–2523 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Qi, Y. et al. Combined small-molecule inhibition accelerates the derivation of functional cortical neurons from human pluripotent stem cells. Nat. Biotechnol. 35, 154–163 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Tchieu, J. et al. A modular platform for differentiation of human PSCs into all major ectodermal lineages. Cell Stem Cell 21, 399–410.e7 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Yao, B. et al. Epigenetic mechanisms in neurogenesis. Nat. Rev. Neurosci. 17, 537–549 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Cholewa-Waclaw, J. et al. The role of epigenetic mechanisms in the regulation of gene expression in the nervous system. J. Neurosci. 36, 11427–11434 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Bergsland, M. et al. Sequentially acting Sox transcription factors in neural lineage development. Genes Dev. 25, 2453–2464 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Wapinski, O. L. et al. Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons. Cell 155, 621–635 (2013).

    CAS  PubMed  Google Scholar 

  11. Singh, N. P. et al. Genome-wide binding analyses of HOXB1 revealed a novel DNA binding motif associated with gene repression. J. Dev. Biol. 9, 6 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Ortabozkoyun, H. et al. CRISPR and biochemical screens identify MAZ as a cofactor in CTCF-mediated insulation at Hox clusters. Nat. Genet. 54, 202–212 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Luo, Z. et al. Zic2 is an enhancer-binding factor required for embryonic stem cell specification. Mol. Cell 57, 685–694 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Hahn, M. A. et al. Reprogramming of DNA methylation at NEUROD2-bound sequences during cortical neuron differentiation. Sci. Adv. 5, eaax0080 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Welsh, S. A. & Gardini, A. Genomic regulation of transcription and RNA processing by the multitasking Integrator complex. Nat. Rev. Mol. Cell Biol. 24, 204–220 (2022).

    PubMed  PubMed Central  Google Scholar 

  16. Offley, S. R. et al. A combinatorial approach to uncover an additional Integrator subunit. Cell Rep. 42, 112244 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Lai, F., Gardini, A., Zhang, A. & Shiekhattar, R. Integrator mediates the biogenesis of enhancer RNAs. Nature 525, 399–403 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Elrod, N. D. et al. The Integrator complex attenuates promoter-proximal transcription at protein-coding genes. Mol. Cell 76, 738–752.e7 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Tatomer, D. C. et al. The Integrator complex cleaves nascent mRNAs to attenuate transcription. Genes Dev. 33, 1525–1538 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Beckedorff, F. et al. The human Integrator complex facilitates transcriptional elongation by endonucleolytic cleavage of nascent transcripts. Cell Rep. 32, 107917 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Fianu, I. et al. Structural basis of Integrator-dependent RNA polymerase II termination. Nature 629, 219–227 (2024).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Vervoort, S. J. et al. The PP2A–Integrator–CDK9 axis fine-tunes transcription and can be targeted therapeutically in cancer. Cell 184, 3143–3162.e32 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Zheng, H. et al. Identification of Integrator–PP2A complex (INTAC), an RNA polymerase II phosphatase. Science 370, eabb5872 (2020).

    CAS  PubMed  Google Scholar 

  24. Huang, K. L. et al. Integrator recruits protein phosphatase 2A to prevent pause release and facilitate transcription termination. Mol. Cell 80, 345–358.e9 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Barbieri, E. et al. Targeted enhancer activation by a subunit of the Integrator complex. Mol. Cell 71, 103–116.e7 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Xu, C. et al. R-loop-dependent promoter-proximal termination ensures genome stability. Nature 621, 610–619 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Oegema, R. et al. Human mutations in integrator complex subunits link transcriptome integrity to brain development. PLoS Genet. 13, e1006809 (2017).

    PubMed  PubMed Central  Google Scholar 

  28. Krall, M. et al. Biallelic sequence variants in INTS1 in patients with developmental delays, cataracts, and craniofacial anomalies. Eur. J. Hum. Genet. 27, 582–593 (2019).

    PubMed  PubMed Central  Google Scholar 

  29. Tepe, B. et al. Bi-allelic variants in INTS11 are associated with a complex neurological disorder. Am. J. Hum. Genet. 110, 774–789 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Mascibroda, L. G. et al. INTS13 variants causing a recessive developmental ciliopathy disrupt assembly of the Integrator complex. Nat. Commun. 13, 6054 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Graham, V., Khudyakov, J., Ellis, P. & Pevny, L. SOX2 functions to maintain neural progenitor identity. Neuron 39, 749–765 (2003).

    CAS  PubMed  Google Scholar 

  32. Favaro, R. et al. Hippocampal development and neural stem cell maintenance require Sox2-dependent regulation of Shh. Nat. Neurosci. 12, 1248–1256 (2009).

    CAS  PubMed  Google Scholar 

  33. Bertolini, J. A. et al. Mapping the global chromatin connectivity network for Sox2 function in neural stem cell maintenance. Cell Stem Cell 24, 462–476.e6 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Wei, C. L., Nicolis, S. K., Zhu, Y. & Pagin, M. Sox2-dependent 3D chromatin interactomes in transcription, neural stem cell proliferation and neurodevelopmental diseases. J. Exp. Neurosci. 13, 1179069519868224 (2019).

    PubMed  PubMed Central  Google Scholar 

  35. Zhang, X. et al. Biallelic INTS1 mutations cause a rare neurodevelopmental disorder in two chinese siblings. J. Mol. Neurosci. 70, 1–8 (2020).

    PubMed  Google Scholar 

  36. Sabath, K. et al. INTS10–INTS13–INTS14 form a functional module of Integrator that binds nucleic acids and the cleavage module. Nat. Commun. 11, 3422 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Kovalevich, J. & Langford, D. Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology. Methods Mol. Biol. 1078, 9–21 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Razew, M., Fraudeau, A., Pfleiderer, M. M., Linares, R. & Galej, W. P. Structural basis of the Integrator complex assembly and association with transcription factors. Mol. Cell 84, 2542–2552.e5 (2024).

    CAS  PubMed  Google Scholar 

  39. Fan, P. et al. Catalytic-independent functions of INTAC in conferring sensitivity to BET inhibition. Preprint at bioRxiv https://doi.org/10.1101/2024.02.07.579305 (2024).

  40. Sabath, K. et al. Basis of gene-specific transcription regulation by the Integrator complex. Mol. Cell 84, 2525–2541.e12 (2024).

    CAS  PubMed  Google Scholar 

  41. Zucco, A. J. et al. Neural progenitors derived from tuberous sclerosis complex patients exhibit attenuated PI3K/AKT signaling and delayed neuronal differentiation. Mol. Cell Neurosci. 92, 149–163 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Lee, J. E. Basic helix–loop–helix genes in neural development. Curr. Opin. Neurobiol. 7, 13–20 (1997).

    PubMed  Google Scholar 

  43. Suter, D. M., Tirefort, D., Julien, S. & Krause, K. H. A Sox1 to Pax6 switch drives neuroectoderm to radial glia progression during differentiation of mouse embryonic stem cells. Stem Cells 27, 49–58 (2009).

    CAS  PubMed  Google Scholar 

  44. Zuniga, A. et al. Mouse limb deformity mutations disrupt a global control region within the large regulatory landscape required for Gremlin expression. Genes Dev. 18, 1553–1564 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Conway, S. J. et al. The role of periostin in tissue remodeling across health and disease. Cell. Mol. Life Sci. 71, 1279–1288 (2014).

    CAS  PubMed  Google Scholar 

  46. Ricard-Blum, S. The collagen family. Cold Spring Harb. Perspect. Biol. 3, a004978 (2011).

    PubMed  PubMed Central  Google Scholar 

  47. Wei, S. C. et al. Matrix stiffness drives epithelial–mesenchymal transition and tumour metastasis through a TWIST1–G3BP2 mechanotransduction pathway. Nat. Cell Biol. 17, 678–688 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Cano, A. et al. The transcription factor snail controls epithelial–mesenchymal transitions by repressing E-cadherin expression. Nat. Cell Biol. 2, 76–83 (2000).

    CAS  PubMed  Google Scholar 

  49. Donato, R. et al. Differential development of neuronal physiological responsiveness in two human neural stem cell lines. BMC Neurosci. 8, 36 (2007).

    PubMed  PubMed Central  Google Scholar 

  50. Sarkar, A. & Hochedlinger, K. The Sox family of transcription factors: versatile regulators of stem and progenitor cell fate. Cell Stem Cell 12, 15–30 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Shaulian, E. & Karin, M. AP-1 as a regulator of cell life and death. Nat. Cell Biol. 4, E131–E136 (2002).

    CAS  PubMed  Google Scholar 

  52. Ameyar, M., Wisniewska, M. & Weitzman, J. B. A role for AP-1 in apoptosis: the case for and against. Biochimie 85, 747–752 (2003).

    CAS  PubMed  Google Scholar 

  53. Baillat, D. et al. Integrator, a multiprotein mediator of small nuclear RNA processing, associates with the C-terminal repeat of RNA polymerase II. Cell 123, 265–276 (2005).

    CAS  PubMed  Google Scholar 

  54. Fianu, I. et al. Structural basis of Integrator-mediated transcription regulation. Science 374, 883–887 (2021).

    CAS  PubMed  Google Scholar 

  55. Wang, H. et al. H3K4me3 regulates RNA polymerase II promoter-proximal pause-release. Nature 615, 339–348 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Stein, C. B. et al. Integrator endonuclease drives promoter-proximal termination at all RNA polymerase II-transcribed loci. Mol. Cell 82, 4232–4245.e11 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Hata, T. & Nakayama, M. Targeted disruption of the murine large nuclear KIAA1440/Ints1 protein causes growth arrest in early blastocyst stage embryos and eventual apoptotic cell death. Biochim. Biophys. Acta 1773, 1039–1051 (2007).

    CAS  PubMed  Google Scholar 

  58. Van den Berg, D. L. C. et al. Nipbl interacts with Zfp609 and the Integrator complex to regulate cortical neuron migration. Neuron 93, 348–361 (2017).

    PubMed  PubMed Central  Google Scholar 

  59. Zhang, Y. et al. The Integrator complex prevents dedifferentiation of intermediate neural progenitors back into neural stem cells. Cell Rep. 27, 987–996.e3 (2019).

    CAS  PubMed  Google Scholar 

  60. Miller, A. et al. Sall4 controls differentiation of pluripotent cells independently of the nucleosome remodelling and deacetylation (NuRD) complex. Development 143, 3074–3084 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Kosho, T., Okamoto, N. & Coffin-Siris Syndrome International, C. Genotype–phenotype correlation of Coffin–Siris syndrome caused by mutations in SMARCB1, SMARCA4, SMARCE1, and ARID1A. Am. J. Med. Genet. C Semin. Med. Genet. 166, 262–275 (2014).

    CAS  Google Scholar 

  62. Jung, Y. L., Hung, C., Choi, J., Lee, E. A. & Bodamer, O. Characterizing the molecular impact of KMT2D variants on the epigenetic and transcriptional landscapes in Kabuki syndrome. Hum. Mol. Genet. 32, 2251–2261 (2023).

    CAS  PubMed  Google Scholar 

  63. Gao, F. et al. Heterozygous mutations in SMARCA2 reprogram the enhancer landscape by global retargeting of SMARCA4. Mol. Cell 75, 891–904.e7 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Gabriele, M. et al. YY1 haploinsufficiency causes an intellectual disability syndrome featuring transcriptional and chromatin dysfunction. Am. J. Hum. Genet. 100, 907–925 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Zaugg, J. B. et al. Current challenges in understanding the role of enhancers in disease. Nat. Struct. Mol. Biol. 29, 1148–1158 (2022).

    CAS  PubMed  Google Scholar 

  66. Harada, T., Morooka, T., Ogawa, S. & Nishida, E. ERK induces p35, a neuron-specific activator of Cdk5, through induction of Egr1. Nat. Cell Biol. 3, 453–459 (2001).

    CAS  PubMed  Google Scholar 

  67. Knapska, E. & Kaczmarek, L. A gene for neuronal plasticity in the mammalian brain: Zif268/Egr-1/NGFI-A/Krox-24/TIS8/ZENK? Prog. Neurobiol. 74, 183–211 (2004).

    CAS  PubMed  Google Scholar 

  68. Sun, Z. et al. EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity. Nat. Commun. 10, 3892 (2019).

    PubMed  PubMed Central  Google Scholar 

  69. Jia, P. et al. ZMYND8 mediated liquid condensates spatiotemporally decommission the latent super-enhancers during macrophage polarization. Nat. Commun. 12, 6535 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Cimadamore, F., Amador-Arjona, A., Chen, C., Huang, C. T. & Terskikh, A. V.SOX2–LIN28/let-7 pathway regulates proliferation and neurogenesis in neural precursors. Proc. Natl Acad. Sci. USA 110, E3017–E3026 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Amador-Arjona, A. et al. SOX2 primes the epigenetic landscape in neural precursors enabling proper gene activation during hippocampal neurogenesis. Proc. Natl Acad. Sci. USA 112, E1936–E1945 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Soufi, A. et al. Pioneer transcription factors target partial DNA motifs on nucleosomes to initiate reprogramming. Cell 161, 555–568 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Soufi, A., Donahue, G. & Zaret, K. S. Facilitators and impediments of the pluripotency reprogramming factors’ initial engagement with the genome. Cell 151, 994–1004 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Chen, C. & Okayama, H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7, 2745–2752 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    CAS  PubMed  Google Scholar 

  76. Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).

    CAS  PubMed  Google Scholar 

  77. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    PubMed  PubMed Central  Google Scholar 

  78. Kolberg, L., Raudvere, U., Kuzmin, I., Vilo, J. & Peterson, H. gprofiler2—an R package for gene list functional enrichment analysis and namespace conversion toolset g:Profiler. F1000Res 9, ELIXIR-709 (2020).

    PubMed  PubMed Central  Google Scholar 

  79. Wu, T. et al. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. Innovation (Camb.) 2, 100141 (2021).

    CAS  PubMed  Google Scholar 

  80. Danecek, P. et al. Twelve years of SAMtools and BCFtools. GigaScience 10, giab008 (2021).

    PubMed  PubMed Central  Google Scholar 

  81. Ramirez, F. et al. deepTools2: A next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160–W165 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Zhang, Y. et al. Model-based analysis of ChIP-seq (MACS). Genome Biol. 9, R137 (2008).

    PubMed  PubMed Central  Google Scholar 

  83. Trapnell, C. et al. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat. Biotechnol. 32, 381–386 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. Aran, D. et al. Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage. Nat. Immunol. 20, 163–172 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. Corces, M. R. et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat. Methods 14, 959–962 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Burke, E. E. et al. Dissecting transcriptomic signatures of neuronal differentiation and maturation using iPSCs. Nat. Commun. 11, 462 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

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.

Author information

Author notes

  1. These authors contributed equally: Yingjie Zhang, Connor M. Hill.

Authors and Affiliations

  1. The Wistar Institute, Philadelphia, PA, USA

    Yingjie Zhang, Connor M. Hill, Kelsey A. Leach, Luca Grillini, Sandra Deliard, Sarah R. Offley, Martina Gatto, Francis Picone, Avery Zucco & Alessandro Gardini

  2. Cell and Molecular Biology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Connor M. Hill, Kelsey A. Leach & Sarah R. Offley

  3. Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy

    Luca Grillini & Martina Gatto

Authors
  1. Yingjie Zhang
    View author publications

    Search author on:PubMed Google Scholar

  2. Connor M. Hill
    View author publications

    Search author on:PubMed Google Scholar

  3. Kelsey A. Leach
    View author publications

    Search author on:PubMed Google Scholar

  4. Luca Grillini
    View author publications

    Search author on:PubMed Google Scholar

  5. Sandra Deliard
    View author publications

    Search author on:PubMed Google Scholar

  6. Sarah R. Offley
    View author publications

    Search author on:PubMed Google Scholar

  7. Martina Gatto
    View author publications

    Search author on:PubMed Google Scholar

  8. Francis Picone
    View author publications

    Search author on:PubMed Google Scholar

  9. Avery Zucco
    View author publications

    Search author on:PubMed Google Scholar

  10. Alessandro Gardini
    View author publications

    Search author on:PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Alessandro Gardini.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Cell Biology thanks Adrian Moore, Silvia Nicolis and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

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.

Source data

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.

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.

Source data

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.

Source data

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.

Source data

Supplementary information

Reporting Summary

Peer Review File

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

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-024-01556-y

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing