Repurposing nuclear receptors for ligand-responsive liquid condensate formation and gene regulation

1. Bashor, C. J., Hilton, I. B., Bandukwala, H., Smith, D. M. & Veiseh, O. Engineering the next generation of cell-based therapeutics. Nat. Rev. Drug Discov. 21, 655–675 (2022).

2. Brayshaw, L. L. et al. The role of small molecules in cell and gene therapy. RSC Med. Chem. 12, 330 (2021).

   Google Scholar 

3. Fegan, A., White, B., Carlson, J. C. T. & Wagner, C. R. Chemically controlled protein assembly: Techniques and applications. Chem. Rev. 110, 3315–3336 (2010).

   Google Scholar 

4. Stanton, B. Z., Chory, E. J. & Crabtree, G. R. Chemically induced proximity in biology and medicine. Science 359. https://doi.org/10.1126/science.aao5902 (2018).

5. Derose, R., Miyamoto, T. & Inoue, T. Manipulating signaling at will: Chemically-inducible dimerization (CID) techniques resolve problems in cell biology. Pflugers Archiv Eur. J. Physiol. 465, 409–417 (2013).

6. Rihtar, E. et al. Chemically inducible split protein regulators for mammalian cells. Nat. Chem. Biol. 1–8 https://doi.org/10.1038/s41589-022-01136-x (2022).

7. Kang, S. et al. COMBINES-CID: An Efficient Method for de Novo Engineering of Highly Specific Chemically Induced Protein Dimerization Systems. J. Am. Chem. Soc. 141, 10948–10952 (2019).

   Google Scholar 

8. Glasgow, A. A. et al. Computational design of a modular protein sense-response system. Science 366, 1024–1028 (2019).

   Google Scholar 

9. Foight, G. W. et al. Multi-input chemical control of protein dimerization for programming graded cellular responses. Nat. Biotechnol. 37, 1209–1216 (2019).

   Google Scholar 

10. Martinko, A. J. et al. Switchable assembly and function of antibody complexes in vivo using a small molecule. Proc. Natl. Acad. Sci. Usa. 119, e2117402119 (2022).

    Google Scholar 

11. Miyamoto, T. et al. Rapid and orthogonal logic gating with a gibberellin-induced dimerization system. Nat. Chem. Biol. 8, 465–470 (2012).

    Google Scholar 

12. Liang, F.-S., Ho, W. Q. & Crabtree, G. R. Engineering the ABA Plant Stress Pathway for Regulation of Induced Proximity. Sci. Signal. 4, rs2 (2011).

    Google Scholar 

13. Spencer, D., Wandless, T., Schreiber, S. & Crabtree, G. Controlling signal transduction with synthetic ligands. Science 262, 1019–1024 (1993).

    Google Scholar 

14. Putyrski, M. & Schultz, C. Protein translocation as a tool: The current rapamycin story. FEBS Lett. 586, 2097–2105 (2012).

    Google Scholar 

15. Scholtes, C. & Giguère, V. Transcriptional control of energy metabolism by nuclear receptors. Nat. Rev. Mol. Cell Biol. 23, 750–770 (2022).

    Google Scholar 

16. Evans, R. M. & Mangelsdorf, D. J. Nuclear Receptors, RXR, and the Big Bang. Cell 157, 255–266 (2014).

    Google Scholar 

17. Sladek, F. M. What are nuclear receptor ligands?. Mol. Cell. Endocrinol. 334, 3–13 (2011).

    Google Scholar 

18. Rastinejad, F., Ollendorff, V. & Polikarpov, I. Nuclear receptor full-length architectures: confronting myth and illusion with high resolution. Trends Biochem. Sci. 40, 16–24 (2015).

    Google Scholar 

19. Weikum, E. R., Knuesel, M. T., Ortlund, E. A. & Yamamoto, K. R. Glucocorticoid receptor control of transcription: precision and plasticity via allostery. Nat. Rev. Mol. Cell Biol. 18, 159–174 (2017).

    Google Scholar 

20. Yu, X. et al. Structural Insights of Transcriptionally Active, Full-Length Androgen Receptor Coactivator Complexes. Mol. Cell 79, 812–823.e4 (2020).

    Google Scholar 

21. Fondell, J. D., Ge, H. & Roeder, R. G. Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex. Proc. Natl. Acad. Sci. USA. 93, 8329–8333 (1996).

    Google Scholar 

22. Lin, B. C., Hong, S. H., Krig, S., Yoh, S. M. & Privalsky, M. L. A conformational switch in nuclear hormone receptors is involved in coupling hormone binding to corepressor release. Mol. Cell. Biol. 17, 6131–6138 (1997).

    Google Scholar 

23. Santos, R. et al. A comprehensive map of molecular drug targets. Nat. Rev. Drug Discov. 16, 19–34 (2017).

    Google Scholar 

24. Ko, M. S., Takahashi, N., Sugiyama, N. & Takano, T. An auto-inducible vector conferring high glucocorticoid inducibility upon stable transformant cells. Gene 84, 383–389 (1989).

    Google Scholar 

25. Friedman, H. M. et al. Use of a glucocorticoid-inducible promoter for expression of herpes simplex virus type 1 glycoprotein gC1, a cytotoxic protein in mammalian cells. Mol. Cell. Biol. 9, 2303–2314 (1989).

    Google Scholar 

26. Gallinari, P. et al. A Functionally Orthogonal Estrogen Receptor-Based Transcription Switch Specifically Induced by a Nonsteroid Synthetic Ligand. Chem. Biol. 12, 883–893 (2005).

    Google Scholar 

27. Rössger, K., Charpin-El-Hamri, G. & Fussenegger, M. A closed-loop synthetic gene circuit for the treatment of diet-induced obesity in mice. Nat. Commun. 4, 2825 (2013).

    Google Scholar 

28. Braselmann, S., Graninger, P. & Busslinger, M. A selective transcriptional induction system for mammalian cells based on Gal4-estrogen receptor fusion proteins. Proc. Natl. Acad. Sci. 90, 1657–1661 (1993).

    Google Scholar 

29. Mata de Urquiza, A., Solomin, L. & Perlmann, T. Feedback-inducible nuclear-receptor-driven reporter gene expression in transgenic mice. Proc. Natl. Acad. Sci. 96, 13270–13275 (1999).

    Google Scholar 

30. Dirnberger, D., Unsin, G., Schlenker, S. & Reichel, C. A Small-Molecule–Protein Interaction System with Split-Ubiquitin as Sensor. ChemBioChem 7, 936–942 (2006).

    Google Scholar 

31. Choi, G. et al. Novel Estrogen Receptor Dimerization BRET-Based Biosensors for Screening Estrogenic Endocrine-Disrupting Chemicals. Biomater. Res. 28, 0010 (2024).

    Google Scholar 

32. Bledsoe, R. K. et al. Crystal Structure of the Glucocorticoid Receptor Ligand Binding Domain Reveals a Novel Mode of Receptor Dimerization and Coactivator Recognition. Cell 110, 93–105 (2002).

    Google Scholar 

33. Idevall-Hagren, O., Dickson, E. J., Hille, B., Toomre, D. K. & De Camilli, P. Optogenetic control of phosphoinositide metabolism. Proc. Natl. Acad. Sci. 109, E2316–E2323 (2012).

    Google Scholar 

34. Gao, Y. et al. Complex transcriptional modulation with orthogonal and inducible dCas9 regulators. Nat. Methods 13, 1043–1049 (2016).

    Google Scholar 

35. Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).

    Google Scholar 

36. Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583–588 (2015).

    Google Scholar 

37. Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging. Cell 159, 635–646 (2014).

    Google Scholar 

38. Scarsi, A., Pedone, D. & Paolo Pompa, P. A dual-color plasmonic immunosensor for salivary cortisol measurement. Nanoscale Adv. 5, 329–336 (2023).

    Google Scholar 

39. Watson, C. S., Jeng, Y.-J. & Kochukov, M. Y. Nongenomic actions of estradiol compared with estrone and estriol in pituitary tumor cell signaling and proliferation. FASEB J. 22, 3328–3336 (2008).

    Google Scholar 

40. Sabari, B. R. et al. Coactivator condensation at super-enhancers links phase separation and gene control. Science 361, eaar3958 (2018).

    Google Scholar 

41. Song, D. et al. ERα and ERβ Homodimers in the Same Cellular Context Regulate Distinct Transcriptomes and Functions. Front. Endocrinol. 13, 930227 (2022).

    Google Scholar 

42. Presman, D. M. et al. DNA binding triggers tetramerization of the glucocorticoid receptor in live cells. Proc. Natl. Acad. Sci. 113, 8236–8241 (2016).

    Google Scholar 

43. Chong, S. et al. Imaging dynamic and selective low-complexity domain interactions that control gene transcription. Science 361, eaar2555 (2018).

    Google Scholar 

44. Lebar, T., Lainšček, D., Merljak, E., Aupič, J. & Jerala, R. A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells. Nat. Chem. Biol. 16, 513–519 (2020).

    Google Scholar 

45. Rihtar, E. et al. Chemically inducible split protein regulators for mammalian cells. Nat. Chem. Biol. 19, 64–71 (2023).

    Google Scholar 

46. Fu, Y. et al. Dynamic properties of transcriptional condensates modulate CRISPRa-mediated gene activation. Nat. Commun. 16, 1640 (2025).

    Google Scholar 

47. Ziegler, M. J. et al. Mandipropamid as a chemical inducer of proximity for in vivo applications. Nat. Chem. Biol. 18, 64–69 (2022).

    Google Scholar 

48. Wang, T. et al. Repurposing salicylic acid as a versatile inducer of proximity. Nat. Chem. Biol. 1–13 https://doi.org/10.1038/s41589-025-01918-z (2025).

49. Chin, S. E. et al. A simeprevir-inducible molecular switch for the control of cell and gene therapies. Nat. Commun. 14, 7753 (2023).

    Google Scholar 

50. Beltrán, J. et al. Rapid biosensor development using plant hormone receptors as reprogrammable scaffolds. Nat. Biotechnol. 40, 1855–1861 (2022).

    Google Scholar 

51. Jan, M. et al. Reversible ON- and OFF-switch chimeric antigen receptors controlled by lenalidomide. Sci. Transl. Med. 13, eabb6295 (2021).

    Google Scholar 

52. Shui, S. et al. A rational blueprint for the design of chemically-controlled protein switches. Nat. Commun. 12, 5754 (2021).

    Google Scholar 

53. Giordano Attianese, G. M. P. et al. Dual ON/OFF-switch chimeric antigen receptor controlled by two clinically approved drugs. Proc. Natl. Acad. Sci. USA 121, e2405085121.

54. Feil, R., Wagner, J., Metzger, D. & Chambon, P. Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem. Biophys. Res. Commun. 237, 752–757 (1997).

    Google Scholar 

55. Lee, J. W., Cheong, J., Lee, Y. C., Na, S.-Y. & Lee, S.-K. Dissecting the molecular mechanism of nuclear receptor action: transcription coactivators and corepressors. Exp. Mol. Med. 32, 53–60 (2000).

    Google Scholar 

56. Watson, P. J., Fairall, L. & Schwabe, J. W. R. Nuclear hormone receptor co-repressors: Structure and function. Mol. Cell. Endocrinol. 348–135, 440–449 (2012).

    Google Scholar 

57. Bertschi, A., Wang, P., Galvan, S., Teixeira, A. P. & Fussenegger, M. Combinatorial protein dimerization enables precise multi-input synthetic computations. Nat. Chem. Biol. 19, 767–777 (2023).

    Google Scholar 

58. Gao, Y., Wang, L. & Wang, B. Customizing cellular signal processing by synthetic multi-level regulatory circuits. Nat. Commun. 14, 8415 (2023).

    Google Scholar 

59. Daniel, R., Rubens, J. R., Sarpeshkar, R. & Lu, T. K. Synthetic analog computation in living cells. Nature 497, 619–623 (2013).

    Google Scholar 

60. Wei, M.-T. et al. Nucleated transcriptional condensates amplify gene expression. Nat. Cell Biol. 22, 1187–1196 (2020).

    Google Scholar 

61. Chong, S. et al. Tuning levels of low-complexity domain interactions to modulate endogenous oncogenic transcription. Mol. Cell 82, 2084–2097.e5 (2022).

    Google Scholar 

62. Chen, R. et al. Specific multivalent molecules boost CRISPR-mediated transcriptional activation. Nat. Commun. 15, 7222 (2024).

    Google Scholar 

63. Schneider, N. et al. Liquid-liquid phase separation of light-inducible transcription factors increases transcription activation in mammalian cells and mice. Sci. Adv. 7, eabd3568 (2021).

    Google Scholar 

64. Yoshikawa, M., Yoshii, T., Ikuta, M. & Tsukiji, S. Synthetic Protein Condensates That Inducibly Recruit and Release Protein Activity in Living Cells. J. Am. Chem. Soc. 143, 6434–6446 (2021).

    Google Scholar 

65. Garabedian, M. V. et al. Designer membraneless organelles sequester native factors for control of cell behavior. Nat. Chem. Biol. 17, 998–1007 (2021).

    Google Scholar 

66. Wu, J. et al. Modulating gene regulation function by chemically controlled transcription factor clustering. Nat. Commun. 13, 2663 (2022).

    Google Scholar 

67. Qian, Z.-G., Huang, S.-C. & Xia, X.-X. Synthetic protein condensates for cellular and metabolic engineering. Nat. Chem. Biol. 18, 1330–1340 (2022).

    Google Scholar 

68. Wang, Y. et al. Programmable solid-state condensates for spatiotemporal control of mammalian gene expression. Nat. Chem. Biol. 1–10 https://doi.org/10.1038/s41589-025-01860-0 (2025).

69. Ramšak, M. et al. Programmable de novo designed coiled coil-mediated phase separation in mammalian cells. Nat. Commun. 14, 7973 (2023).

    Google Scholar 

70. Zhen, N. et al. Engineering bi-directional chemically-modulated synthetic condensates for cellular control. Nat. Commun. 16, 6587 (2025).

    Google Scholar 

71. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, (2009).

72. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25, 402–408 (2001).

    Google Scholar 

Download references