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Yeast two-hybrid screening reveals a dual function for the histone acetyltransferase GcnE by controlling glutamine synthesis and development in Aspergillus fumigatus

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Abstract

The acetyltransferase GcnE is part of the SAGA complex which regulates fungal gene expression through acetylation of chromatin. Target genes of the histone acetyltransferase GcnE include those involved in secondary metabolism and asexual development. Here, we show that the absence of GcnE not only abrogated conidiation, but also strongly impeded vegetative growth of hyphae in the human pathogenic fungus Aspergillus fumigatus. A yeast two-hybrid screen using a Saccharomyces cerevisiae strain whose tRNA molecules were specifically adapted to express A. fumigatus proteins identified two unprecedented proteins that directly interact with GcnE. Glutamine synthetase GlnA as well as a hypothetical protein located on chromosome 8 (GbpA) were identified as binding partners of GcnE and their interaction was confirmed in vivo via bimolecular fluorescence complementation. Phenotypic characterization of gbpA and glnA deletion mutants revealed a role for GbpA during conidiogenesis and confirmed the central role of GlnA in glutamine biosynthesis. The increase of glutamine synthetase activity in the absence of GcnE indicated that GcnE silences GlnA through binding. This finding suggests an expansion of the regulatory role of GcnE in A. fumigatus.

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References

  • Altwasser R, Baldin C, Weber J, Guthke R, Kniemeyer O, Brakhage AA, Linde J, Valiante V (2015) Network modeling reveals cross talk of MAP kinases during adaptation to caspofungin stress in Aspergillus fumigatus. PLoS One 10(9):e0136932

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Ashok Kumar T (2013) CFSSP: Chou and Fasman secondary structure prediction server. Wide Spectr 1(9):15–19

    Google Scholar 

  • Ballance DJ, Turner G (1985) Development of a high-frequency transforming vector for Aspergillus nidulans. Gene 36(3):321–331

    Article  CAS  PubMed  Google Scholar 

  • Bowyer P, Denning DW (2007) Genomic analysis of allergen genes in Aspergillus spp.: the relevance of genomics to everyday research. Med Mycol 45(1):17–26

    Article  CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Brakhage AA, Langfelder K (2002) Menacing mold: the molecular biology of Aspergillus fumigatus. Annu Rev Microbiol 56:433–455

    Article  CAS  PubMed  Google Scholar 

  • Brakhage AA, Van den Brulle J (1995) Use of reporter genes to identify recessive trans-acting mutations specifically involved in the regulation of Aspergillus nidulans penicillin biosynthesis genes. J Bacteriol 177(10):2781–2788

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Breeden L, Nasmyth K (1985) Regulation of the yeast HO gene. Cold Spring Harb Symp Quant Biol 50:643–650

    Article  CAS  PubMed  Google Scholar 

  • Brown GD, Denning DW, Gow NA, Levitz SM, Netea MG, White TC (2012) Hidden killers: human fungal infections. Sci Transl Med 4(165):165rv113

    Article  CAS  Google Scholar 

  • Canovas D, Marcos AT, Gacek A, Ramos MS, Gutierrez G, Reyes-Dominguez Y, Strauss J (2014) The histone acetyltransferase GcnE (GCN5) plays a central role in the regulation of Aspergillus asexual development. Genetics 197(4):1175–1189

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chang P, Fan X, Chen J (2015) Function and subcellular localization of Gcn5, a histone acetyltransferase in Candida albicans. Fungal Genet Biol 81:132–141

    Article  CAS  PubMed  Google Scholar 

  • Choudhary C, Weinert BT, Nishida Y, Verdin E, Mann M (2014) The growing landscape of lysine acetylation links metabolism and cell signalling. Nat Rev Mol Cell Biol 15(8):536–550

    Article  CAS  PubMed  Google Scholar 

  • da Silva Ferreira ME, Kress MR, Savoldi M, Goldman MH, Hartl A, Heinekamp T, Brakhage AA, Goldman GH (2006) The akuB(KU80) mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus. Eukaryot Cell 5(1):207–211

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Daniel JA, Grant PA (2007) Multi-tasking on chromatin with the SAGA coactivator complexes. Mutat Res 618(1–2):135–148

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dastidar RG, Hooda J, Shah A, Cao TM, Henke RM, Zhang L (2012) The nuclear localization of SWI/SNF proteins is subjected to oxygen regulation. Cell Biosci 2(1):30

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dunn-Coleman NS, Garrett RH (1980) The role fo glutamine synthetase and glutamine metabolism in nitrogen metabolite repression, a regulatory phenomenon in the lower eukaryote Neurospora crassa. Mol Gen Genet 179(1):25–32

    Article  CAS  PubMed  Google Scholar 

  • Dyda F, Klein DC, Hickman AB (2000) GCN5-related N-acetyltransferases: a structural overview. Annu Rev Biophys Biomol Struct 29:81–103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fedorova ND, Khaldi N, Joardar VS, Maiti R, Amedeo P, Anderson MJ, Crabtree J, Silva JC, Badger JH, Albarraq A, Angiuoli S, Bussey H, Bowyer P, Cotty PJ, Dyer PS, Egan A, Galens K, Fraser-Liggett CM, Haas BJ, Inman JM, Kent R, Lemieux S, Malavazi I, Orvis J, Roemer T, Ronning CM, Sundaram JP, Sutton G, Turner G, Venter JC, White OR, Whitty BR, Youngman P, Wolfe KH, Goldman GH, Wortman JR, Jiang B, Denning DW, Nierman WC (2008) Genomic islands in the pathogenic filamentous fungus Aspergillus fumigatus. PLoS Genet 4(4):e1000046

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Fields S, Song O (1989) A novel genetic system to detect protein–protein interactions. Nature 340(6230):245–246

    Article  CAS  PubMed  Google Scholar 

  • Georgakopoulos P, Lockington RA, Kelly JM (2013) The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex in Aspergillus nidulans. PLoS One 8(6):e65221

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gola S, Munder T, Casonato S, Manganelli R, Vicente M (2015) The essential role of SepF in mycobacterial division. Mol Microbiol 97(3):560–576

    Article  CAS  PubMed  Google Scholar 

  • Gsaller F, Hortschansky P, Beattie SR, Klammer V, Tuppatsch K, Lechner BE, Rietzschel N, Werner ER, Vogan AA, Chung D, Muhlenhoff U, Kato M, Cramer RA, Brakhage AA, Haas H (2014) The Janus transcription factor HapX controls fungal adaptation to both iron starvation and iron excess. EMBO J 33(19):2261–2276

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hassan AH, Prochasson P, Neely KE, Galasinski SC, Chandy M, Carrozza MJ, Workman JL (2002) Function and selectivity of bromodomains in anchoring chromatin-modifying complexes to promoter nucleosomes. Cell 111(3):369–379

    Article  CAS  PubMed  Google Scholar 

  • Hillmann F, Bagramyan K, Strassburger M, Heinekamp T, Hong TB, Bzymek KP, Williams JC, Brakhage AA, Kalkum M (2016) The crystal structure of peroxiredoxin Asp f3 provides mechanistic insight into oxidative stress resistance and virulence of Aspergillus fumigatus. Sci Rep 6:33396

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hoff B, Kück U (2005) Use of bimolecular fluorescence complementation to demonstrate transcription factor interaction in nuclei of living cells from the filamentous fungus Acremonium chrysogenum. Curr Genet 47(2):132–138

    Article  CAS  PubMed  Google Scholar 

  • Jöhnk B, Bayram O, Abelmann A, Heinekamp T, Mattern DJ, Brakhage AA, Jacobsen ID, Valerius O, Braus GH (2016) SCF ubiquitin ligase F-box protein Fbx15 controls nuclear co-repressor localization, stress response and virulence of the human pathogen Aspergillus fumigatus. PLoS Pathog 12(9):e1005899

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Jones P, Binns D, Chang HY, Fraser M, Li W, McAnulla C, McWilliam H, Maslen J, Mitchell A, Nuka G, Pesseat S, Quinn AF, Sangrador-Vegas A, Scheremetjew M, Yong SY, Lopez R, Hunter S (2014) InterProScan 5: genome-scale protein function classification. Bioinformatics 30(9):1236–1240

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kollath-Leiss K, Bonniger C, Sardar P, Kempken F (2014) BEM46 shows eisosomal localization and association with tryptophan-derived auxin pathway in Neurospora crassa. Eukaryot Cell 13(8):1051–1063

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kornberg A, Pricer WE Jr (1951) Enzymatic phosphorylation of adenosine and 2,6-diaminopurine riboside. J Biol Chem 193(2):481–495

    CAS  PubMed  Google Scholar 

  • Langfelder K, Jahn B, Gehringer H, Schmidt A, Wanner G, Brakhage AA (1998) Identification of a polyketide synthase gene (pksP) of Aspergillus fumigatus involved in conidial pigment biosynthesis and virulence. Med Microbiol Immunol 187(2):79–89

    Article  CAS  PubMed  Google Scholar 

  • Latge JP (1999) Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev 12(2):310–350

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lerin C, Rodgers JT, Kalume DE, Kim SH, Pandey A, Puigserver P (2006) GCN5 acetyltransferase complex controls glucose metabolism through transcriptional repression of PGC-1alpha. Cell Metab 3(6):429–438

    Article  CAS  PubMed  Google Scholar 

  • Linde J, Wilson D, Hube B, Guthke R (2010) Regulatory network modelling of iron acquisition by a fungal pathogen in contact with epithelial cells. BMC Syst Biol 4:148

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Lopez J, Mukhtar MS (2017) Mapping protein–protein interaction using high-throughput yeast 2-hybrid. Methods Mol Biol 1610:217–230

    Article  CAS  PubMed  Google Scholar 

  • Lundby A, Lage K, Weinert BT, Bekker-Jensen DB, Secher A, Skovgaard T, Kelstrup CD, Dmytriyev A, Choudhary C, Lundby C, Olsen JV (2012) Proteomic analysis of lysine acetylation sites in rat tissues reveals organ specificity and subcellular patterns. Cell Rep 2(2):419–431

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Magnani Dinamarco T, Brown NA, Couto de Almeida RS, Alves de Castro P, Savoldi M, de Souza Goldman MH, Goldman GH (2012) Aspergillus fumigatus calcineurin interacts with a nucleoside diphosphate kinase. Microb Infect 14(11):922–929

    Article  CAS  Google Scholar 

  • Marchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Geer LY, Bryant SH (2017) CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res 45(D1):D200–D203

    Article  CAS  PubMed  Google Scholar 

  • Munder T, Hinnen A (1999) Yeast cells as tools for target-oriented screening. Appl Microbiol Biotechnol 52(3):311–320

    Article  CAS  PubMed  Google Scholar 

  • Nossmann M, Pieper J, Hillmann F, Brakhage AA, Munder T (2017) Generation of an arginine-tRNA-adapted Saccharomyces cerevisiae strain for effective heterologous protein expression. Curr Genet 64(3):589–598

    Article  PubMed  CAS  Google Scholar 

  • Novy R, Drott D, Yaeger K, Mierendorf R (2001) Overcoming the codon bias of E. coli for enhanced protein expression. Innovations 12:1–3

    Google Scholar 

  • Nützmann HW, Fischer J, Scherlach K, Hertweck C, Brakhage AA (2013) Distinct amino acids of histone H3 control secondary metabolism in Aspergillus nidulans. Appl Environ Microbiol 79(19):6102–6109. https://doi.org/10.1128/AEM.01578-13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nützmann HW, Reyes-Dominguez Y, Scherlach K, Schroeckh V, Horn F, Gacek A, Schumann J, Hertweck C, Strauss J, Brakhage AA (2011) Bacteria-induced natural product formation in the fungus Aspergillus nidulans requires Saga/Ada-mediated histone acetylation. Proc Natl Acad Sci USA 108(34):14282–14287

    Article  PubMed  PubMed Central  Google Scholar 

  • O’Meara TR, Hay C, Price MS, Giles S, Alspaugh JA (2010) Cryptococcus neoformans histone acetyltransferase Gcn5 regulates fungal adaptation to the host. Eukaryot Cell 9(8):1193–1202

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Paolinelli R, Mendoza-Maldonado R, Cereseto A, Giacca M (2009) Acetylation by GCN5 regulates CDC6 phosphorylation in the S phase of the cell cycle. Nat Struct Mol Biol 16(4):412–420

    Article  CAS  PubMed  Google Scholar 

  • Pray-Grant MG, Schieltz D, McMahon SJ, Wood JM, Kennedy EL, Cook RG, Workman JL, Yates JR III, Grant PA (2002) The novel SLIK histone acetyltransferase complex functions in the yeast retrograde response pathway. Mol Cell Biol 22(24):8774–8786

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Quan J, Tian J (2009) Circular polymerase extension cloning of complex gene libraries and pathways. PLoS One 4(7):e6441

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Rice P, Longden I, Bleasby A (2000) EMBOSS: the European molecular biology open software suite. Trends Genet TIG 16(6):276–277

    Article  CAS  PubMed  Google Scholar 

  • Rösler SM, Kramer K, Finkemeier I, Humpf HU, Tudzynski B (2016) The SAGA complex in the rice pathogen Fusarium fujikuroi: structure and functional characterization. Mol Microbiol 102(6):951–974

    Article  PubMed  CAS  Google Scholar 

  • Salah Ud-Din AI, Tikhomirova A, Roujeinikova A (2016) Structure and functional diversity of GCN5-related N-acetyltransferases (GNAT). Int J Mol Sci 17(7):1018

    Article  PubMed Central  CAS  Google Scholar 

  • Samara NL, Wolberger C (2011) A new chapter in the transcription SAGA. Curr Opin Struct Biol 21(6):767–774

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sarikaya Bayram Ö, Latgé JP, Bayram Ö (2018) MybA, a new player driving survival of the conidium of the human pathogen Aspergillus fumigatus. Curr Genet 64(1):141–146. https://doi.org/10.1007/s00294-017-0740-5

    Article  CAS  PubMed  Google Scholar 

  • Sellam A, Askew C, Epp E, Lavoie H, Whiteway M, Nantel A (2009) Genome-wide mapping of the coactivator Ada2p yields insight into the functional roles of SAGA/ADA complex in Candida albicans. Mol Biol Cell 20(9):2389–2400

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Serebriiskii I, Estojak J, Berman M, Golemis EA (2000) Approaches to detecting false positives in yeast two-hybrid systems. Biotechniques 28(2):328–330, 332–326

    Article  CAS  PubMed  Google Scholar 

  • Soufi B, Soares NC, Ravikumar V, Macek B (2012) Proteomics reveals evidence of cross-talk between protein modifications in bacteria: focus on acetylation and phosphorylation. Curr Opin Microbiol 15(3):357–363

    Article  CAS  PubMed  Google Scholar 

  • Stajich JE, Harris T, Brunk BP, Brestelli J, Fischer S, Harb OS, Kissinger JC, Li W, Nayak V, Pinney DF, Stoeckert CJ Jr, Roos DS (2012) FungiDB: an integrated functional genomics database for fungi. Nucleic Acids Res 40(Database issue):D675–D681

    Article  CAS  PubMed  Google Scholar 

  • Tate JJ, Rai R, Cooper TG (2018) More than one way in: three Gln3 sequences required to telieve negative Ure2 regulation and support nuclear Gln3 import in Saccharomyces cerevisiae. Genetics 208(1):207–227

    Article  CAS  PubMed  Google Scholar 

  • Teichert S, Schonig B, Richter S, Tudzynski B (2004) Deletion of the Gibberella fujikuroi glutamine synthetase gene has significant impact on transcriptional control of primary and secondary metabolism. Mol Microbiol 53(6):1661–1675

    Article  CAS  PubMed  Google Scholar 

  • Thon M, Al Abdallah Q, Hortschansky P, Scharf DH, Eisendle M, Haas H, Brakhage AA (2010) The CCAAT-binding complex coordinates the oxidative stress response in eukaryotes. Nucleic Acids Res 38(4):1098–1113

    Article  PubMed  CAS  Google Scholar 

  • Vo TV, Das J, Meyer MJ, Cordero NA, Akturk N, Wei X, Fair BJ, Degatano AG, Fragoza R, Liu LG, Matsuyama A, Trickey M, Horibata S, Grimson A, Yamano H, Yoshida M, Roth FP, Pleiss JA, Xia Y, Yu H (2016) A proteome-wide fission yeast interactome reveals network evolution principles from yeasts to human. Cell 164(1–2):310–323

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wagner D, Wiemann P, Huss K, Brandt U, Fleissner A, Tudzynski B (2013) A sensing role of the glutamine synthetase in the nitrogen regulation network in Fusarium fujikuroi. PLoS One 8(11):e80740

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Wang Q, Zhang Y, Yang C, Xiong H, Lin Y, Yao J, Li H, Xie L, Zhao W, Yao Y, Ning ZB, Zeng R, Xiong Y, Guan KL, Zhao S, Zhao GP (2010) Acetylation of metabolic enzymes coordinates carbon source utilization and metabolic flux. Science 327(5968):1004–1007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wessel D, Flügge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138(1):141–143

    Article  CAS  PubMed  Google Scholar 

  • Wiemann P, Guo CJ, Palmer JM, Sekonyela R, Wang CC, Keller NP (2013) Prototype of an intertwined secondary-metabolite supercluster. Proc Natl Acad Sci USA 110(42):17065–17070

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • You D, Yin BC, Li ZH, Zhou Y, Yu WB, Zuo P, Ye BC (2016) Sirtuin-dependent reversible lysine acetylation of glutamine synthetases reveals an autofeedback loop in nitrogen metabolism. Proc Natl Acad Sci USA 113(24):6653–6658

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yu H, Braun P, Yildirim MA, Lemmens I, Venkatesan K, Sahalie J, Hirozane-Kishikawa T, Gebreab F, Li N, Simonis N, Hao T, Rual JF, Dricot A, Vazquez A, Murray RR, Simon C, Tardivo L, Tam S, Svrzikapa N, Fan C, de Smet AS, Motyl A, Hudson ME, Park J, Xin X, Cusick ME, Moore T, Boone C, Snyder M, Roth FP, Barabasi AL, Tavernier J, Hill DE, Vidal M (2008) High-quality binary protein interaction map of the yeast interactome network. Science 322(5898):104–110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

Lisa Reimer is acknowledged for excellent technical assistance. This study was supported by the Ernst-Abbe University of Applied Sciences Jena, by the Leibniz Institute for Natural Product Research and Infection Biology-Hans Knöll Institute Jena and the collaborative research center/transregio 124 FungiNet (Project A1 and Z2) funded by the Deutsche Forschungsgemeinschaft.

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Correspondence to Falk Hillmann, Thomas Munder or Axel A. Brakhage.

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Communicated by M. Kupiec.

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Nossmann, M., Boysen, J.M., Krüger, T. et al. Yeast two-hybrid screening reveals a dual function for the histone acetyltransferase GcnE by controlling glutamine synthesis and development in Aspergillus fumigatus. Curr Genet 65, 523–538 (2019). https://doi.org/10.1007/s00294-018-0891-z

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