Identification of the transcription factor Miz1 as an essential regulator of diphthamide biosynthesis using a CRISPR-mediated genome-wide screen

Autoři: Jie Liu aff001;  Zehua Zuo aff001;  Meijuan Zou aff001;  Toren Finkel aff001;  Shihui Liu aff001
Působiště autorů: Aging Institute of University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, PA, United States of America aff001;  Division of Cardiology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States of America aff002;  Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States of America aff003
Vyšlo v časopise: Identification of the transcription factor Miz1 as an essential regulator of diphthamide biosynthesis using a CRISPR-mediated genome-wide screen. PLoS Genet 16(10): e32767. doi:10.1371/journal.pgen.1009068
Kategorie: Research Article
doi: 10.1371/journal.pgen.1009068


Diphthamide is a unique post-translationally modified histidine residue (His715 in all mammals) found only in eukaryotic elongation factor-2 (eEF-2). The biosynthesis of diphthamide represents one of the most complex modifications, executed by protein factors conserved from yeast to humans. Diphthamide is not only essential for normal physiology (such as ensuring fidelity of mRNA translation), but is also exploited by bacterial ADP-ribosylating toxins (e.g., diphtheria toxin) as their molecular target in pathogenesis. Taking advantage of the observation that cells defective in diphthamide biosynthesis are resistant to ADP-ribosylating toxins, in the past four decades, seven essential genes (Dph1 to Dph7) have been identified for diphthamide biosynthesis. These technically unsaturated screens raise the question as to whether additional genes are required for diphthamide biosynthesis. In this study, we performed two independent, saturating, genome-wide CRISPR knockout screens in human cells. These screens identified all previously known Dph genes, as well as further identifying the BTB/POZ domain-containing transcription factor Miz1. We found that Miz1 is absolutely required for diphthamide biosynthesis via its role in the transcriptional regulation of Dph1 expression. Mechanistically, Miz1 binds to the Dph1 proximal promoter via an evolutionarily conserved consensus binding site to activate Dph1 transcription. Therefore, this work demonstrates that Dph1-7, along with the newly identified Miz1 transcription factor, are likely to represent the essential protein factors required for diphthamide modification on eEF2.

Klíčová slova:

Biosynthesis – CRISPR – DNA methylation – Genetic screens – Library screening – Toxins – Transfection – HT1080 cells


1. Collier RJ. Understanding the mode of action of diphtheria toxin: a perspective on progress during the 20th century. Toxicon. 2001;39(11):1793–803. doi: 10.1016/s0041-0101(01)00165-9 11595641

2. Van Ness BG, Howard JB, Bodley JW. ADP-ribosylation of elongation factor 2 by diphtheria toxin. NMR spectra and proposed structures of ribosyl-diphthamide and its hydrolysis products. J Biol Chem. 1980;255(22):10710–6. 7430147

3. Van Ness BG, Howard JB, Bodley JW. ADP-ribosylation of elongation factor 2 by diphtheria toxin. Isolation and properties of the novel ribosyl-amino acid and its hydrolysis products. J Biol Chem. 1980;255(22):10717–20. 7000782

4. Jorgensen R, Purdy AE, Fieldhouse RJ, Kimber MS, Bartlett DH, Merrill AR. Cholix toxin, a novel ADP-ribosylating factor from Vibrio cholerae. J Biol Chem. 2008;283(16):10671–8. doi: 10.1074/jbc.M710008200 18276581

5. Liu S, Leppla SH. Retroviral insertional mutagenesis identifies a small protein required for synthesis of diphthamide, the target of bacterial ADP-ribosylating toxins. Mol Cell. 2003;12(3):603–13. doi: 10.1016/j.molcel.2003.08.003 14527407

6. Liu S, Milne GT, Kuremsky JG, Fink GR, Leppla SH. Identification of the proteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation factor 2. Mol Cell Biol. 2004;24(21):9487–97. doi: 10.1128/MCB.24.21.9487-9497.2004 15485916

7. Abdel-Fattah W, Scheidt V, Uthman S, Stark MJ, Schaffrath R. Insights into diphthamide, key diphtheria toxin effector. Toxins (Basel). 2013;5(5):958–68. doi: 10.3390/toxins5050958 23645155

8. Schaffrath R, Abdel-Fattah W, Klassen R, Stark MJ. The diphthamide modification pathway from Saccharomyces cerevisiae—revisited. Molecular microbiology. 2014;94(6):1213–26. doi: 10.1111/mmi.12845 25352115

9. Liu S, Wiggins JF, Sreenath T, Kulkarni AB, Ward JM, Leppla SH. Dph3, a small protein required for diphthamide biosynthesis, is essential in mouse development. Mol Cell Biol. 2006;26(10):3835–41. doi: 10.1128/MCB.26.10.3835-3841.2006 16648478

10. Chen CM, Behringer RR. Ovca1 regulates cell proliferation, embryonic development, and tumorigenesis. Genes Dev. 2004;18(3):320–32. doi: 10.1101/gad.1162204 14744934

11. Webb TR, Cross SH, McKie L, Edgar R, Vizor L, Harrison J, et al. Diphthamide modification of eEF2 requires a J-domain protein and is essential for normal development. J Cell Sci. 2008;121(Pt 19):3140–5. doi: 10.1242/jcs.035550 18765564

12. Liu S, Bachran C, Gupta P, Miller-Randolph S, Wang H, Crown D, et al. Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development. Proc Natl Acad Sci U S A. 2012;109(34):13817–22. doi: 10.1073/pnas.1206933109 22869748

13. Hawer H, Utkur K, Arend M, Mayer K, Adrian L, Brinkmann U, et al. Importance of diphthamide modified EF2 for translational accuracy and competitive cell growth in yeast. PloS one. 2018;13(10):e0205870. doi: 10.1371/journal.pone.0205870 30335802

14. Stahl S, da Silva Mateus Seidl AR, Ducret A, Kux van Geijtenbeek S, Michel S, Racek T, et al. Loss of diphthamide pre-activates NF-kappaB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor. Proc Natl Acad Sci U S A. 2015;112(34):10732–7. doi: 10.1073/pnas.1512863112 26261303

15. Su X, Lin Z, Lin H. The biosynthesis and biological function of diphthamide. Crit Rev Biochem Mol Biol. 2013;48(6):515–21. doi: 10.3109/10409238.2013.831023 23971743

16. Tsuda-Sakurai K, Miura M. The hidden nature of protein translational control by diphthamide: the secrets under the leather. J Biochem. 2019;165(1):1–8. doi: 10.1093/jb/mvy071 30204891

17. Mayer K, Mundigl O, Kettenberger H, Birzele F, Stahl S, Pastan I, et al. Diphthamide affects selenoprotein expression: Diphthamide deficiency reduces selenocysteine incorporation, decreases selenite sensitivity and pre-disposes to oxidative stress. Redox Biol. 2019;20:146–56. doi: 10.1016/j.redox.2018.09.015 30312900

18. Mattheakis LC, Shen WH, Collier RJ. DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae. Mol Cell Biol. 1992;12:4026–37. doi: 10.1128/mcb.12.9.4026 1508200

19. Mattheakis LC, Sor F, Collier RJ. Diphthamide synthesis in Saccharomyces cerevisiae: structure of the DPH2 gene. Gene. 1993;132(1):149–54. doi: 10.1016/0378-1119(93)90528-b 8406038

20. Nobukuni Y, Kohno K, Miyagawa K. Gene trap mutagenesis-based forward genetic approach reveals that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor 2. J Biol Chem. 2005;280(11):10572–7. doi: 10.1074/jbc.M413017200 15637051

21. Carette JE, Guimaraes CP, Varadarajan M, Park AS, Wuethrich I, Godarova A, et al. Haploid genetic screens in human cells identify host factors used by pathogens. Science. 2009;326(5957):1231–5. doi: 10.1126/science.1178955 19965467

22. Su X, Chen W, Lee W, Jiang H, Zhang S, Lin H. YBR246W is required for the third step of diphthamide biosynthesis. J Am Chem Soc. 2012;134(2):773–6. doi: 10.1021/ja208870a 22188241

23. Uthman S, Bar C, Scheidt V, Liu S, Ten HS, Giorgini F, et al. The amidation step of diphthamide biosynthesis in yeast requires DPH6, a gene identified through mining the DPH1-DPH5 interaction network. PLoS Genet. 2013;9(2):e1003334. doi: 10.1371/journal.pgen.1003334 23468660

24. Su X, Lin Z, Chen W, Jiang H, Zhang S, Lin H. Chemogenomic approach identified yeast YLR143W as diphthamide synthetase. Proc Natl Acad Sci U S A. 2012;109(49):19983–7. doi: 10.1073/pnas.1214346109 23169644

25. Duesbery NS, Webb CP, Leppla SH, Gordon VM, Klimpel KR, Copeland TD, et al. Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. Science. 1998;280(5364):734–7. doi: 10.1126/science.280.5364.734 9563949

26. Lee CS, Dykema KJ, Hawkins DM, Cherba DM, Webb CP, Furge KA, et al. MEK2 Is sufficient but not necessary for proliferation and anchorage-independent growth of SK-MEL-28 melanoma cells. PLoS One. 2011;6(2):e17165. doi: 10.1371/journal.pone.0017165 21365009

27. Vitale G, Pellizzari R, Recchi C, Napolitani G, Mock M, Montecucco C. Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages. Biochem Biophys Res Commun. 1998;248:706–11. doi: 10.1006/bbrc.1998.9040 9703991

28. Vitale G, Bernardi L, Napolitani G, Mock M, Montecucco C. Susceptibility of mitogen-activated protein kinase kinase family members to proteolysis by anthrax lethal factor. Biochem J. 2000;352 Pt 3:739–45.

29. Liu S, Leppla SH. Cell surface tumor endothelium marker 8 cytoplasmic tail-independent anthrax toxin binding, proteolytic processing, oligomer formation, and internalization. J Biol Chem. 2003;278:5227–34. doi: 10.1074/jbc.M210321200 12468536

30. Liu S, Crown D, Miller-Randolph S, Moayeri M, Wang H, Hu H, et al. Capillary morphogenesis protein-2 is the major receptor mediating lethality of anthrax toxin in vivo. Proc Natl Acad Sci U S A. 2009;106(30):12424–9. doi: 10.1073/pnas.0905409106 19617532

31. Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nature methods. 2014;11(8):783–4. doi: 10.1038/nmeth.3047 25075903

32. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014;343(6166):84–7. doi: 10.1126/science.1247005 24336571

33. Liu S, Moayeri M, Leppla SH. Anthrax lethal and edema toxins in anthrax pathogenesis. Trends Microbiol. 2014;22(6):317–25. doi: 10.1016/j.tim.2014.02.012 24684968

34. Barrilleaux BL, Burow D, Lockwood SH, Yu A, Segal DJ, Knoepfler PS. Miz-1 activates gene expression via a novel consensus DNA binding motif. PloS one. 2014;9(7):e101151. doi: 10.1371/journal.pone.0101151 24983942

35. Ashapkin VV, Kutueva LI, Vanyushin BF. Quantitative Analysis of DNA Methylation by Bisulfite Sequencing. Methods in molecular biology (Clifton, NJ). 2020;2138:297–312.

36. Liu J, Zuo Z, Sastalla I, Liu C, Jang JY, Sekine Y, et al. Sequential CRISPR-Based Screens Identify LITAF and CDIP1 as the Bacillus cereus Hemolysin BL Toxin Host Receptors. Cell host & microbe. 2020; 28: 402–410. e5. Epub 2020/06/17. doi: 10.1016/j.chom.2020.05.012 32544461.

37. Glatt S, Zabel R, Vonkova I, Kumar A, Netz DJ, Pierik AJ, et al. Structure of the Kti11/Kti13 heterodimer and its double role in modifications of tRNA and eukaryotic elongation factor 2. Structure. 2015;23(1):149–60. Epub 2014/12/30. doi: 10.1016/j.str.2014.11.008 25543256

38. Friedlander AM. Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process. J Biol Chem. 1986;261:7123–6. 3711080

39. Milne JC, Collier RJ. pH-dependent permeabilization of the plasma membrane of mammalian cells by anthrax protective antigen. Mol Microbiol. 1993;10:647–53. doi: 10.1111/j.1365-2958.1993.tb00936.x 7968541

40. Peukert K, Staller P, Schneider A, Carmichael G, Hanel F, Eilers M. An alternative pathway for gene regulation by Myc. EMBO J. 1997;16(18):5672–86. doi: 10.1093/emboj/16.18.5672 9312026

41. Dang CV. MYC on the path to cancer. Cell. 2012;149(1):22–35. doi: 10.1016/j.cell.2012.03.003 22464321

42. Stine ZE, Walton ZE, Altman BJ, Hsieh AL, Dang CV. MYC, Metabolism, and Cancer. Cancer Discov. 2015;5(10):1024–39. doi: 10.1158/2159-8290.CD-15-0507 26382145

43. Seoane J, Pouponnot C, Staller P, Schader M, Eilers M, Massague J. TGFbeta influences Myc, Miz-1 and Smad to control the CDK inhibitor p15INK4b. Nat Cell Biol. 2001;3(4):400–8. doi: 10.1038/35070086 11283614

44. Staller P, Peukert K, Kiermaier A, Seoane J, Lukas J, Karsunky H, et al. Repression of p15INK4b expression by Myc through association with Miz-1. Nat Cell Biol. 2001;3(4):392–9. doi: 10.1038/35070076 11283613

45. Seoane J, Le HV, Massague J. Myc suppression of the p21(Cip1) Cdk inhibitor influences the outcome of the p53 response to DNA damage. Nature. 2002;419(6908):729–34. doi: 10.1038/nature01119 12384701

46. Wanzel M, Russ AC, Kleine-Kohlbrecher D, Colombo E, Pelicci PG, Eilers M. A ribosomal protein L23-nucleophosmin circuit coordinates Mizl function with cell growth. Nat Cell Biol. 2008;10(9):1051–61. doi: 10.1038/ncb1764 19160485

47. Gebhardt A, Frye M, Herold S, Benitah SA, Braun K, Samans B, et al. Myc regulates keratinocyte adhesion and differentiation via complex formation with Miz1. The Journal of cell biology. 2006;172(1):139–49. doi: 10.1083/jcb.200506057 16391002

48. Wolf E, Gebhardt A, Kawauchi D, Walz S, von Eyss B, Wagner N, et al. Miz1 is required to maintain autophagic flux. Nat Commun. 2013;4:2535. doi: 10.1038/ncomms3535 24088869

49. Wiese KE, Walz S, von Eyss B, Wolf E, Athineos D, Sansom O, et al. The role of MIZ-1 in MYC-dependent tumorigenesis. Cold Spring Harb Perspect Med. 2013;3(12):a014290. doi: 10.1101/cshperspect.a014290 24296348

50. Adhikary S, Peukert K, Karsunky H, Beuger V, Lutz W, Elsasser HP, et al. Miz1 is required for early embryonic development during gastrulation. Molecular and cellular biology. 2003;23(21):7648–57. doi: 10.1128/mcb.23.21.7648-7657.2003 14560010

51. Gebhardt A, Kosan C, Herkert B, Moroy T, Lutz W, Eilers M, et al. Miz1 is required for hair follicle structure and hair morphogenesis. J Cell Sci. 2007;120(Pt 15):2586–93. doi: 10.1242/jcs.007104 17635993

52. Pomerantsev AP, Pomerantseva OM, Moayeri M, Fattah R, Tallant C, Leppla SH. A Bacillus anthracis strain deleted for six proteases serves as an effective host for production of recombinant proteins. Protein Expr Purif. 2011;80(1):80–90. doi: 10.1016/j.pep.2011.05.016 21827967

53. Li W, Xu H, Xiao T, Cong L, Love MI, Zhang F, et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 2014;15(12):554. doi: 10.1186/s13059-014-0554-4 25476604

Článek vyšel v časopise

PLOS Genetics

2020 Číslo 10

Nejčtenější v tomto čísle

Tomuto tématu se dále věnují…


Zvyšte si kvalifikaci online z pohodlí domova

Antiseptika a prevence ve stomatologii
nový kurz
Autoři: MUDr. Ladislav Korábek, CSc., MBA

Citikolin v neuroprotekci a neuroregeneraci: od výzkumu do klinické praxe nejen očních lékařů
Autoři: MUDr. Petr Výborný, CSc., FEBO

Zánětlivá bolest zad a axiální spondylartritida – Diagnostika a referenční strategie
Autoři: MUDr. Monika Gregová, Ph.D., MUDr. Kristýna Bubová

Diagnostika a léčba deprese pro ambulantní praxi
Autoři: MUDr. Jan Hubeňák, Ph.D

Význam nemocničního alert systému v době SARS-CoV-2
Autoři: doc. MUDr. Helena Lahoda Brodská, Ph.D., prim. MUDr. Václava Adámková

Všechny kurzy
Kurzy Doporučená témata Časopisy
Zapomenuté heslo

Nemáte účet?  Registrujte se

Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.


Nemáte účet?  Registrujte se