Bromodomain inhibition of the coactivators CBP/EP300 facilitate cellular reprogramming

Abstract

Silencing of the somatic cell type-specific genes is a critical yet poorly understood step in reprogramming. To uncover pathways that maintain cell identity, we performed a reprogramming screen using inhibitors of chromatin factors. Here, we identify acetyl-lysine competitive inhibitors targeting the bromodomains of coactivators CREB (cyclic-AMP response element binding protein) binding protein (CBP) and E1A binding protein of 300 kDa (EP300) as potent enhancers of reprogramming. These inhibitors accelerate reprogramming, are critical during its early stages and, when combined with DOT1L inhibition, enable efficient derivation of human induced pluripotent stem cells (iPSCs) with OCT4 and SOX2. In contrast, catalytic inhibition of CBP/EP300 prevents iPSC formation, suggesting distinct functions for different coactivator domains in reprogramming. CBP/EP300 bromodomain inhibition decreases somatic-specific gene expression, histone H3 lysine 27 acetylation (H3K27Ac) and chromatin accessibility at target promoters and enhancers. The master mesenchymal transcription factor PRRX1 is one such functionally important target of CBP/EP300 bromodomain inhibition. Collectively, these results show that CBP/EP300 bromodomains sustain cell-type-specific gene expression and maintain cell identity.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: A chromatin-focused chemical screen identifies CBP/EP300 bromodomain inhibitors as enhancers of reprogramming.
Fig. 2: CBP/EP300 bromodomain inhibition acts early to accelerate reprogramming.
Fig. 3: Efficient derivation of two-factor iPSCs with CBP/EP300 bromodomain inhibition.
Fig. 4: CBP/EP300 bromodomain inhibition downregulates fibroblast-specific genes.
Fig. 5: Genome-wide chromatin changes on CBP/EP300 bromodomain inhibition.
Fig. 6: Downregulation of PRRX1 by CBP/EP300 bromodomain inhibitors is necessary for reprogramming.

Data availability

RNA-sequencing, ChIP-sequencing and ATAC-sequencing data are deposited to the NCBI GEO database with the accession number GSE118220.

Code availability

The custom pipeline for ChIP-seq analysis can be found at: https://github.com/Acribbs/cribbslab/blob/master/Pipelines/pipeline_quantchip.py.

References

  1. 1.

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

    Article  CAS  Google Scholar 

  2. 2.

    Xu, Y. et al. Transcriptional control of somatic cell reprogramming. Trends Cell Biol. 26, 272–288 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Papp, B. & Plath, K. Epigenetics of reprogramming to induced pluripotency. Cell 152, 1324–1343 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Mikkelsen, T. S. et al. Dissecting direct reprogramming through integrative genomic analysis. Nature 454, 49–55 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Huangfu, D. et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat. Biotechnol. 26, 795–797 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Onder, T. T. et al. Chromatin-modifying enzymes as modulators of reprogramming. Nature 483, 598–602 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Cheloufi, S. et al. The histone chaperone CAF-1 safeguards somatic cell identity. Nature 528, 218–224 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Zhuang, Q. et al. NCoR/SMRT co-repressors cooperate with c-MYC to create an epigenetic barrier to somatic cell reprogramming. Nat. Cell Biol. 20, 400–412 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Chronis, C. et al. Cooperative binding of transcription factors orchestrates reprogramming. Cell 168, 442–459.e20 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Ichida, J. K. et al. Notch inhibition allows oncogene-independent generation of iPS cells. Nat. Chem. Biol. 10, 632–639 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Jackson, S. A., Olufs, Z. P. G., Tran, K. A., Zaidan, N. Z. & Sridharan, R. Alternative routes to induced pluripotent stem cells revealed by reprogramming of the neural lineage. Stem Cell Rep. 6, 302–311 (2016).

    Article  CAS  Google Scholar 

  13. 13.

    Zhao, Y. et al. A XEN-like state bridges somatic cells to pluripotency during chemical reprogramming. Cell 163, 1678–1691 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Ogryzko, V. V., Schiltz, R. L., Russanova, V., Howard, B. H. & Nakatani, Y. The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87, 953–959 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Delvecchio, M., Gaucher, J., Aguilar-Gurrieri, C., Ortega, E. & Panne, D. Structure of the p300 catalytic core and implications for chromatin targeting and HAT regulation. Nat. Struct. Mol. Biol. 20, 1040–1046 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Zeng, L., Zhang, Q., Gerona-Navarro, G., Moshkina, N. & Zhou, M.-M. Structural basis of site-specific histone recognition by the bromodomains of human coactivators PCAF and CBP/p300. Structure 16, 643–652 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Manning, E. T. T., Ikehara, T., Ito, T., Kadonaga, J. T. T. & Kraus, W. L. L. P300 forms a stable, template-committed complex with chromatin: role for the bromodomain. Mol. Cell. Biol. 21, 3876–3887 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Heintzman, N. D. et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat. Genet. 39, 311–318 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Visel, A. et al. ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457, 854–858 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Hnisz, D. et al. Super-enhancers in the control of cell identity and disease. Cell 155, 934–947 (2013).

    Article  CAS  Google Scholar 

  21. 21.

    Witte, S., Bradley, A., Enright, A. J. & Muljo, S. A. High-density P300 enhancers control cell state transitions. BMC Genomics 16, 903 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Iyer, N. G., Özdag, H. & Caldas, C. p300/CBP and cancer. Oncogene 23, 4225–4231 (2004).

    Article  CAS  Google Scholar 

  23. 23.

    Cribbs, A. et al. Inhibition of histone H3K27 demethylases selectively modulates inflammatory phenotypes of natural killer cells. J. Biol. Chem. 293, 2422–2437 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Arrowsmith, C. H. et al. The promise and peril of chemical probes. Nat. Chem. Biol. 11, 536–541 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Hay, D. A. et al. Discovery and optimization of small-molecule ligands for the CBP/p300 bromodomains. J. Am. Chem. Soc. 136, 9308–9319 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Picaud, S. et al. Generation of a selective small molecule inhibitor of the CBP/p300 bromodomain for leukemia therapy. Cancer Res. 75, 5106–5119 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Lasko, L. M. et al. Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours. Nature 550, 128–132 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Park, S. et al. Role of the CBP catalytic core in intramolecular SUMOylation and control of histone H3 acetylation. Proc. Natl Acad. Sci. USA 114, E5335–E5342 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Raisner, R. et al. Enhancer activity requires CBP/P300 bromodomain-dependent histone H3K27 acetylation. Cell Rep. 24, 1722–1729 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Weinert, B. T. et al. Time-resolved analysis reveals rapid dynamics and broad scope of the CBP/p300 acetylome. Cell 174, 231–244.e12 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Li, R. et al. A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell 7, 51–63 (2010).

    Article  CAS  Google Scholar 

  32. 32.

    Nohno, T. et al. A chicken homeobox gene related to drosophila paired is predominantly expressed in the developing limb. Dev. Biol. 158, 254–264 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Lu, M. F. et al. prx-1 functions cooperatively with another paired-related homeobox gene, prx-2, to maintain cell fates within the craniofacial mesenchyme. Development 126, 495–504 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Shao, Z. et al. Reprogramming by de-bookmarking the somatic transcriptional program through targeting of BET bromodomains. Cell Rep. 16, 3138–3145 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Yao, T. P. et al. Gene dosage-dependent embryonic development and proliferation defects in mice lacking the transcriptional integrator p300. Cell 93, 361–372 (1998).

    Article  CAS  Google Scholar 

  36. 36.

    Zhong, X. & Jin, Y. Critical roles of coactivator p300 in mouse embryonic stem cell differentiation and Nanog expression. J. Biol. Chem. 284, 9168–9175 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Fang, F. et al. Coactivators p300 and CBP maintain the identity of mouse embryonic stem cells by mediating long-range chromatin structure. Stem Cells 32, 1805–1816 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Cahan, P. et al. CellNet: network biology applied to stem cell engineering. Cell 158, 903–915 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Creyghton, M. P. et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl Acad. Sci. USA 107, 21931–21936 (2010).

    Article  Google Scholar 

  40. 40.

    Hammitzsch, A. et al. CBP30, a selective CBP/p300 bromodomain inhibitor, suppresses human Th17 responses. Proc. Natl Acad. Sci. USA 112, 10768–10773 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Conery, A. R. et al. Bromodomain inhibition of the transcriptional coactivators CBP/EP300 as a therapeutic strategy to target the IRF4 network in multiple myeloma. eLife 5, e10483 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Lynch, C. J. et al. The RNA polymerase II factor RPAP1 is critical for mediator-driven transcription and cell identity. Cell Rep. 22, 396–410 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Ortega, E. et al. Transcription factor dimerization activates the p300 acetyltransferase. Nature 562, 538–544 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Wang, W.-P. et al. The EP300, KDM5A, KDM6A and KDM6B chromatin regulators cooperate with klf4 in the transcriptional activation of POU5F1. PLoS One 7, e52556 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Merika, M., Williams, A. J., Chen, G., Collins, T. & Thanos, D. Recruitment of CBP/p300 by the IFN beta enhanceosome is required for synergistic activation of transcription. Mol. Cell 1, 277–287 (1998).

    Article  CAS  Google Scholar 

  46. 46.

    Simandi, Z. et al. OCT4 acts as an integrator of pluripotency and signal-induced differentiation. Mol. Cell 63, 647–661 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Ocaña, O. H. et al. Metastatic colonization requires the repression of the epithelial-mesenchymal transition inducer Prrx1. Cancer Cell 22, 709–724 (2012).

    Article  CAS  Google Scholar 

  48. 48.

    Du, B. et al. The transcription factor paired-related homeobox 1 (Prrx1) inhibits adipogenesis by activating transforming growth factor-β (TGFβ) signaling. J. Biol. Chem. 288, 3036–3047 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Yang, C. S., Lopez, C. G. & Rana, T. M. Discovery of nonsteroidal anti-inflammatory drug and anticancer drug enhancing reprogramming and induced pluripotent stem cell generation. Stem Cells 29, 1528–1536 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Park, I.-H. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146 (2008).

    Article  CAS  Google Scholar 

  51. 51.

    Seiler, C. Y. et al. DNASU plasmid and PSI:Biology-Materials repositories: resources to accelerate biological research. Nucleic Acids Res. 42, D1253–D1260 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Fidan, K. et al. Generation of integration-free induced pluripotent stem cells from a patient with familial Mediterranean fever (FMF). Stem Cell Res. 15, 694–696 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).

    Article  CAS  Google Scholar 

  54. 54.

    Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Sims, D. et al. CGAT: computational genomics analysis toolkit. Bioinformatics 30, 1290–1291 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We would like to thank A. Kocabay and A.C. Taşkın for help with mouse experiments, A. Ruacan (Koç University, School of Medicine, Department of Pathology) for examination of histological sections and E. Hookway as well as T. Seker, E. Kavak (Genomize Inc.) for help with the initial generation and analyses of the RNA-seq data. The authors gratefully acknowledge use of the services and facilities of the Koç University Research Center for Translational Medicine (KUTTAM), funded by the Republic of Turkey Ministry of Development. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Ministry of Development. K.S. was supported by a TUBITAK BIDEB Scholarship. Work in the U.O. laboratory was supported by Arthritis Research UK (program grant no. 20522), Cancer Research UK and the Rosetrees Foundation. The Structural Genomics Consortium is a registered charity (no. 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA) (ULTRA-DD grant no. 115766), Janssen, Merck KGaA Darmstadt Germany, MSD, Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome (106169/ZZ14/Z). The research has received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under REA grant agreement no. 609305. This work was supported by an EMBO Installation Grant (T.T.O.), a Newton Advanced Fellowship (U.O. and T.T.O.) and TUBITAK Project 213S182 (T.T.O.).

Author information

Affiliations

Authors

Contributions

A.E., K.S. and G.G.S. performed experiments and analyzed data. A.P.C. performed bioinformatics analyses. M.P. performed sequencing experiments. F.U. and T.M. analyzed RNA-seq data. J.E.D. provided materials. S.G. performed experiments. Ş.A. supervised research. U.O. designed the project, interpreted results and supervised the project. T.T.O. designed the project, interpreted results and wrote the manuscript.

Corresponding authors

Correspondence to Udo Oppermann or Tamer T. Önder.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Supplementary Figures 1–7

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ebrahimi, A., Sevinç, K., Gürhan Sevinç, G. et al. Bromodomain inhibition of the coactivators CBP/EP300 facilitate cellular reprogramming. Nat Chem Biol 15, 519–528 (2019). https://doi.org/10.1038/s41589-019-0264-z

Download citation

Further reading