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A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling


Autoři: Khairunnisa Mentari Semesta aff001;  Ruilin Tian aff002;  Martin Kampmann aff002;  Mark von Zastrow aff005;  Nikoleta G. Tsvetanova aff001
Působiště autorů: Department of Pharmacology and Cancer Biology, Duke University, Durham, North Carolina, United States of America aff001;  Institute for Neurodegenerative Diseases, Department of Biochemistry and Biophysics, University of California, San Francisco, California, United States of America aff002;  Chen-Zuckerberg Biohub, San Francisco, California, United States of America aff003;  Biophysics Graduate Program, University of California, San Francisco, California, United States of America aff004;  Department of Psychiatry, University of California, San Francisco, California, United States of America aff005;  Department of Cellular & Molecular Pharmacology, University of California, San Francisco, California, United States of America aff006
Vyšlo v časopise: A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling. PLoS Genet 16(10): e32767. doi:10.1371/journal.pgen.1009103
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1009103

Souhrn

G protein-coupled receptors (GPCRs) allow cells to respond to chemical and sensory stimuli through generation of second messengers, such as cyclic AMP (cAMP), which in turn mediate a myriad of processes, including cell survival, proliferation, and differentiation. In order to gain deeper insights into the complex biology and physiology of these key cellular pathways, it is critical to be able to globally map the molecular factors that shape cascade function. Yet, to this date, efforts to systematically identify regulators of GPCR/cAMP signaling have been lacking. Here, we combined genome-wide screening based on CRISPR interference with a novel sortable transcriptional reporter that provides robust readout for cAMP signaling, and carried out a functional screen for regulators of the pathway. Due to the sortable nature of the platform, we were able to assay regulators with strong and moderate phenotypes by analyzing sgRNA distribution among three fractions with distinct reporter expression. We identified 45 regulators with strong and 50 regulators with moderate phenotypes not previously known to be involved in cAMP signaling. In follow-up experiments, we validated the functional effects of seven newly discovered mediators (NUP93, PRIM1, RUVBL1, PKMYT1, TP53, SF3A2, and HRAS), and showed that they control distinct steps of the pathway. Thus, our study provides proof of principle that the screening platform can be applied successfully to identify bona fide regulators of GPCR/second messenger cascades in an unbiased and high-throughput manner, and illuminates the remarkable functional diversity among GPCR regulators.

Klíčová slova:

Flow cytometry – G protein coupled receptors – Gene regulation – Genetic screens – Isoproterenol – Library screening – Regulator genes – cAMP signaling cascade


Zdroje

1. Hauser AS, Attwood MM, Rask-Andersen M, Schiöth HB, Gloriam DE. Trends in GPCR drug discovery: new agents, targets and indications. Nature reviews Drug discovery. 2017;16(12):829–42. Epub 2017/10/28. doi: 10.1038/nrd.2017.178 29075003.

2. Sharma S, Petsalaki E. Application of CRISPR-Cas9 Based Genome-Wide Screening Approaches to Study Cellular Signalling Mechanisms. Int J Mol Sci. 2018;19(4). doi: 10.3390/ijms19040933 29561791.

3. Lohse MJ, Engelhardt S, Eschenhagen T. What is the role of beta-adrenergic signaling in heart failure? Circ Res. 2003;93(10):896–906. doi: 10.1161/01.RES.0000102042.83024.CA 14615493.

4. Mutlu GM, Factor P. Alveolar epithelial beta2-adrenergic receptors. Am J Respir Cell Mol Biol. 2008;38(2):127–34. doi: 10.1165/rcmb.2007-0198TR 17709598.

5. Kampmann M. CRISPRi and CRISPRa Screens in Mammalian Cells for Precision Biology and Medicine. ACS Chem Biol. 2018;13(2):406–16. doi: 10.1021/acschembio.7b00657 29035510.

6. Maynard-Smith LA, Chen LC, Banaszynski LA, Ooi AG, Wandless TJ. A directed approach for engineering conditional protein stability using biologically silent small molecules. J Biol Chem. 2007;282(34):24866–72. doi: 10.1074/jbc.M703902200 17603093.

7. Tsvetanova NG, von Zastrow M. Spatial encoding of cyclic AMP signaling specificity by GPCR endocytosis. Nature chemical biology. 2014;10(12):1061–5. Epub 2014/11/05. doi: 10.1038/nchembio.1665 25362359.

8. Barlow CA, Kitiphongspattana K, Siddiqui N, Roe MW, Mossman BT, Lounsbury KM. Protein kinase A-mediated CREB phosphorylation is an oxidant-induced survival pathway in alveolar type II cells. Apoptosis. 2008;13(5):681–92. doi: 10.1007/s10495-008-0203-z 18392938.

9. Stern CM, Luoma JI, Meitzen J, Mermelstein PG. Corticotropin releasing factor-induced CREB activation in striatal neurons occurs via a novel Gbetagamma signaling pathway. PLoS One. 2011;6(3):e18114. doi: 10.1371/journal.pone.0018114 21448293.

10. Xie F, Li BX, Kassenbrock A, Xue C, Wang X, Qian DZ, et al. Identification of a Potent Inhibitor of CREB-Mediated Gene Transcription with Efficacious in Vivo Anticancer Activity. J Med Chem. 2015;58(12):5075–87. doi: 10.1021/acs.jmedchem.5b00468 26023867.

11. Tan Y, Liu W, Zhu Z, Lang L, Wang J, Huang M, et al. Selection and identification of transferrin receptor-specific peptides as recognition probes for cancer cells. Anal Bioanal Chem. 2018;410(3):1071–7. doi: 10.1007/s00216-017-0664-4 29046922.

12. Fredericks ZL, Pitcher JA, Lefkowitz RJ. Identification of the G protein-coupled receptor kinase phosphorylation sites in the human beta2-adrenergic receptor. J Biol Chem. 1996;271(23):13796–803. doi: 10.1074/jbc.271.23.13796 8662852.

13. Nobles KN, Xiao K, Ahn S, Shukla AK, Lam CM, Rajagopal S, et al. Distinct phosphorylation sites on the beta(2)-adrenergic receptor establish a barcode that encodes differential functions of beta-arrestin. Sci Signal. 2011;4(185):ra51. doi: 10.1126/scisignal.2001707 21868357.

14. Violin JD, DiPilato LM, Yildirim N, Elston TC, Zhang J, Lefkowitz RJ. beta2-adrenergic receptor signaling and desensitization elucidated by quantitative modeling of real time cAMP dynamics. The Journal of biological chemistry. 2008;283(5):2949–61. Epub 2007/11/30. doi: 10.1074/jbc.M707009200 18045878.

15. Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 2014;159(3):647–61. doi: 10.1016/j.cell.2014.09.029 25307932.

16. Thul PJ, Akesson L, Wiking M, Mahdessian D, Geladaki A, Ait Blal H, et al. A subcellular map of the human proteome. Science. 2017;356(6340). doi: 10.1126/science.aal3321 28495876.

17. Kampmann M, Bassik MC, Weissman JS. Functional genomics platform for pooled screening and generation of mammalian genetic interaction maps. Nat Protoc. 2014;9(8):1825–47. doi: 10.1038/nprot.2014.103 24992097.

18. Kim J, Ahn S, Ren XR, Whalen EJ, Reiter E, Wei H, et al. Functional antagonism of different G protein-coupled receptor kinases for beta-arrestin-mediated angiotensin II receptor signaling. Proc Natl Acad Sci U S A. 2005;102(5):1442–7. doi: 10.1073/pnas.0409532102 15671181.

19. Ren XR, Reiter E, Ahn S, Kim J, Chen W, Lefkowitz RJ. Different G protein-coupled receptor kinases govern G protein and beta-arrestin-mediated signaling of V2 vasopressin receptor. Proc Natl Acad Sci U S A. 2005;102(5):1448–53. doi: 10.1073/pnas.0409534102 15671180.

20. Chen JJ, Nathaniel DL, Raghavan P, Nelson M, Tian R, Tse E, et al. Compromised function of the ESCRT pathway promotes endolysosomal escape of tau seeds and propagation of tau aggregation. The Journal of biological chemistry. 2019;294(50):18952–66. Epub 2019/10/04. doi: 10.1074/jbc.RA119.009432 31578281.

21. Ramkumar P, Abarientos AB, Tian R, Seyler M, Leong JT, Chen M, et al. CRISPR-based screens uncover determinants of immunotherapy response in multiple myeloma. Blood advances. 2020;4(13):2899–911. Epub 2020/06/27. doi: 10.1182/bloodadvances.2019001346 32589729.

22. Tian R, Gachechiladze MA, Ludwig CH, Laurie MT, Hong JY, Nathaniel D, et al. CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons. Neuron. 2019;104(2):239–55.e12. Epub 2019/08/20. doi: 10.1016/j.neuron.2019.07.014 31422865.

23. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25(1):25–9. doi: 10.1038/75556 10802651.

24. Mi H, Huang X, Muruganujan A, Tang H, Mills C, Kang D, et al. PANTHER version 11: expanded annotation data from Gene Ontology and Reactome pathways, and data analysis tool enhancements. Nucleic Acids Res. 2017;45(D1):D183–D9. doi: 10.1093/nar/gkw1138 27899595.

25. The Gene Ontology C. The Gene Ontology Resource: 20 years and still GOing strong. Nucleic Acids Res. 2019;47(D1):D330–D8. doi: 10.1093/nar/gky1055 30395331.

26. Brown CR, Kennedy CJ, Delmar VA, Forbes DJ, Silver PA. Global histone acetylation induces functional genomic reorganization at mammalian nuclear pore complexes. Genes Dev. 2008;22(5):627–39. doi: 10.1101/gad.1632708 18316479.

27. Chen X, Xu L. Specific nucleoporin requirement for Smad nuclear translocation. Mol Cell Biol. 2010;30(16):4022–34. doi: 10.1128/MCB.00124-10 20547758.

28. Ibarra A, Benner C, Tyagi S, Cool J, Hetzer MW. Nucleoporin-mediated regulation of cell identity genes. Genes Dev. 2016;30(20):2253–8. doi: 10.1101/gad.287417.116 27807035.

29. Labade AS, Karmodiya K, Sengupta K. HOXA repression is mediated by nucleoporin Nup93 assisted by its interactors Nup188 and Nup205. Epigenetics Chromatin. 2016;9:54. doi: 10.1186/s13072-016-0106-0 27980680.

30. Liu F, Stanton JJ, Wu Z, Piwnica-Worms H. The human Myt1 kinase preferentially phosphorylates Cdc2 on threonine 14 and localizes to the endoplasmic reticulum and Golgi complex. Mol Cell Biol. 1997;17(2):571–83. doi: 10.1128/mcb.17.2.571 9001210.

31. Liu L, Wu J, Wang S, Luo X, Du Y, Huang D, et al. PKMYT1 promoted the growth and motility of hepatocellular carcinoma cells by activating beta-catenin/TCF signaling. Exp Cell Res. 2017;358(2):209–16. doi: 10.1016/j.yexcr.2017.06.014 28648520.

32. Hanyaloglu AC, von Zastrow M. Regulation of GPCRs by endocytic membrane trafficking and its potential implications. Annu Rev Pharmacol Toxicol. 2008;48:537–68. doi: 10.1146/annurev.pharmtox.48.113006.094830 18184106.

33. Nigg EA, Schafer G, Hilz H, Eppenberger HM. Cyclic-AMP-dependent protein kinase type II is associated with the Golgi complex and with centrosomes. Cell. 1985;41(3):1039–51. doi: 10.1016/s0092-8674(85)80084-2 2988780.

34. Godbole A, Lyga S, Lohse MJ, Calebiro D. Internalized TSH receptors en route to the TGN induce local Gs-protein signaling and gene transcription. Nat Commun. 2017;8(1):443. doi: 10.1038/s41467-017-00357-2 28874659.

35. Antoni FA, Wiegand UK, Black J, Simpson J. Cellular localisation of adenylyl cyclase: a post-genome perspective. Neurochem Res. 2006;31(2):287–95. doi: 10.1007/s11064-005-9019-1 16570209.

36. Calebiro D, Nikolaev VO, Gagliani MC, de Filippis T, Dees C, Tacchetti C, et al. Persistent cAMP-signals triggered by internalized G-protein-coupled receptors. PLoS Biol. 2009;7(8):e1000172. doi: 10.1371/journal.pbio.1000172 19688034.

37. Kotowski SJ, Hopf FW, Seif T, Bonci A, von Zastrow M. Endocytosis promotes rapid dopaminergic signaling. Neuron. 2011;71(2):278–90. doi: 10.1016/j.neuron.2011.05.036 21791287.

38. Lazar AM, Irannejad R, Baldwin TA, Sundaram AB, Gutkind JS, Inoue A, et al. G protein-regulated endocytic trafficking of adenylyl cyclase type 9. Elife. 2020;9. doi: 10.7554/eLife.58039 32515353.

39. Steinberg F, Gallon M, Winfield M, Thomas EC, Bell AJ, Heesom KJ, et al. A global analysis of SNX27-retromer assembly and cargo specificity reveals a function in glucose and metal ion transport. Nat Cell Biol. 2013;15(5):461–71. doi: 10.1038/ncb2721 23563491.

40. Horlbeck MA, Xu A, Wang M, Bennett NK, Park CY, Bogdanoff D, et al. Mapping the Genetic Landscape of Human Cells. Cell. 2018;174(4):953–67.e22. doi: 10.1016/j.cell.2018.06.010 30033366.

41. Tsvetanova NG, Trester-Zedlitz M, Newton BW, Riordan DP, Sundaram AB, Johnson JR, et al. G Protein-Coupled Receptor Endocytosis Confers Uniformity in Responses to Chemically Distinct Ligands. Mol Pharmacol. 2017;91(2):145–56. doi: 10.1124/mol.116.106369 27879340.

42. Bingham J, Sudarsanam S, Srinivasan S. Profiling human phosphodiesterase genes and splice isoforms. Biochem Biophys Res Commun. 2006;350(1):25–32. doi: 10.1016/j.bbrc.2006.08.180 16987497.

43. Stelzer G, Rosen N, Plaschkes I, Zimmerman S, Twik M, Fishilevich S, et al. The GeneCards Suite: From Gene Data Mining to Disease Genome Sequence Analyses. Curr Protoc Bioinformatics. 2016;54:1.30.1–1.3. doi: 10.1002/cpbi.5 27322403.

44. Dugan KA, Wood MA, Cole MD. TIP49, but not TRRAP, modulates c-Myc and E2F1 dependent apoptosis. Oncogene. 2002;21(38):5835–43. doi: 10.1038/sj.onc.1205763 12185582.

45. Feng Y, Lee N, Fearon ER. TIP49 regulates beta-catenin-mediated neoplastic transformation and T-cell factor target gene induction via effects on chromatin remodeling. Cancer Res. 2003;63(24):8726–34. 14695187.

46. Giebler HA, Lemasson I, Nyborg JK. p53 recruitment of CREB binding protein mediated through phosphorylated CREB: a novel pathway of tumor suppressor regulation. Mol Cell Biol. 2000;20(13):4849–58. doi: 10.1128/mcb.20.13.4849-4858.2000 10848610.

47. Van Orden K, Giebler HA, Lemasson I, Gonzales M, Nyborg JK. Binding of p53 to the KIX domain of CREB binding protein. A potential link to human T-cell leukemia virus, type I-associated leukemogenesis. J Biol Chem. 1999;274(37):26321–8. doi: 10.1074/jbc.274.37.26321 10473588.

48. Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013;154(2):442–51. doi: 10.1016/j.cell.2013.06.044 23849981.

49. Horlbeck MA, Gilbert LA, Villalta JE, Adamson B, Pak RA, Chen Y, et al. Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. Elife. 2016;5. doi: 10.7554/eLife.19760 27661255.

50. Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10(3):R25. doi: 10.1186/gb-2009-10-3-r25 19261174.


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