The Gal4 protein is a well-known prototypic acidic activator that has multiple activation domains. We have previously identified a new activation domain called the nine amino acid transactivation domain (9aaTAD) in Gal4 protein. The family of the 9aaTAD activators currently comprises over 40 members including p53, MLL, E2A and other members of the Gal4 family; Oaf1, Pip2, Pdr1 and Pdr3. In this study, we revised function of all reported Gal4 activation domains. Surprisingly, we found that beside of the activation domain 9aaTAD none of the previously reported activation domains had considerable transactivation potential and were not involved in the activation of transcription. Our results demonstrated that the 9aaTAD domain is the only decisive activation domain in the Gal4 protein. We found that the artificial peptides included in the original Gal4 constructs were results of an unintended consequence of cloning that were responsible for the artificial transcriptional activity. Importantly, the activation domain 9aaTAD, which is the exclusive activation domain in Gal4, is also the central part of a conserved sequence recognized by the inhibitory protein Gal80. We propose a revision of the Gal4 regulation, in which the activation domain 9aaTAD is directly linked to both activation function and Gal80 mediated inhibition.

Gamma Delta T Cell Laboratory Department of Pathological Physiology Faculty of Medicine Masaryk University Brno Czech Republic

Laboratory of Cancer Biology and Genetics Department of Pathological Physiology Masaryk University Brno Czech Republic

Zobrazit více v PubMed

Hope IA, Struhl K. Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast. Cell. 1986;46: 885–894. PubMed

Ma J, Ptashne M. A new class of yeast transcriptional activators. Cell. 1987;51: 113–119. PubMed

Hope IA, Mahadevan S, Struhl K. Structural and functional characterization of the short acidic transcriptional activation region of yeast GCN4 protein. Nature. 1988;333: 635–640. 10.1038/333635a0 PubMed DOI

Johnston M, Dover J. Mutational analysis of the GAL4-encoded transcriptional activator protein of Saccharomyces cerevisiae. Genetics. 1988;120: 63–74. PubMed PMC

Peng G, Hopper JE. Gene activation by interaction of an inhibitor with a cytoplasmic signaling protein. Proc Natl Acad Sci USA. 2002;99: 8548–8553. 10.1073/pnas.142100099 PubMed DOI PMC

Johnston SA, Zavortink MJ, Debouck C, Hopper JE. Functional domains of the yeast regulatory protein GAL4. Proc Natl Acad Sci USA. 1986;83: 6553–6557. PubMed PMC

Englesberg E, Irr J, Power J, Lee N. Positive control of enzyme synthesis by gene C in the L-arabinose system. J Bacteriol. 1965;90: 946–957. PubMed PMC

Stargell LA, Struhl K. The TBP-TFIIA interaction in the response to acidic activators in vivo. Science. 1995;269: 75–78. PubMed

Chou S, Struhl K. Transcriptional activation by TFIIB mutants that are severely impaired in interaction with promoter DNA and acidic activation domains. Mol Cell Biol. 1997;17: 6794–6802. PubMed PMC

Dorris DR, Struhl K. Artificial Recruitment of TFIID, but Not RNA Polymerase II Holoenzyme, Activates Transcription in Mammalian Cells. Mol Cell Biol. 2000;20: 4350–4358. PubMed PMC

Thoden JB, Ryan LA, Reece RJ, Holden HM. The interaction between an acidic transcriptional activator and its inhibitor. The molecular basis of Gal4p recognition by Gal80p. J Biol Chem. 2008;283: 30266–30272. 10.1074/jbc.M805200200 PubMed DOI PMC

Drysdale CM, Dueñas E, Jackson BM, Reusser U, Braus GH, Hinnebusch AG. The transcriptional activator GCN4 contains multiple activation domains that are critically dependent on hydrophobic amino acids. Mol Cell Biol. 1995;15: 1220–1233. PubMed PMC

Jackson BM, Drysdale CM, Natarajan K, Hinnebusch AG. Identification of seven hydrophobic clusters in GCN4 making redundant contributions to transcriptional activation. Mol Cell Biol. 1996;16: 5557–5571. PubMed PMC

Natarajan K, Meyer MR, Jackson BM, Slade D, Roberts C, Hinnebusch AG, et al. Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast. Mol Cell Biol. 2001;21: 4347–4368. 10.1128/MCB.21.13.4347-4368.2001 PubMed DOI PMC

Jedidi I, Zhang F, Qiu H, Stahl SJ, Palmer I, Kaufman JD, et al. Activator Gcn4 employs multiple segments of Med15/Gal11, including the KIX domain, to recruit mediator to target genes in vivo. J Biol Chem. 2010;285: 2438–2455. 10.1074/jbc.M109.071589 PubMed DOI PMC

Krois AS, Ferreon JC, Martinez-Yamout MA, Dyson HJ, Wright PE. Recognition of the disordered p53 transactivation domain by the transcriptional adapter zinc finger domains of CREB-binding protein. Proc Natl Acad Sci USA. 2016;113: E1853–1862. 10.1073/pnas.1602487113 PubMed DOI PMC

Lee CW, Arai M, Martinez-Yamout MA, Dyson HJ, Wright PE. Mapping the interactions of the p53 transactivation domain with the KIX domain of CBP. Biochemistry. 2009;48: 2115–2124. 10.1021/bi802055v PubMed DOI PMC

Denis CM, Chitayat S, Plevin MJ, Wang F, Thompson P, Li S, et al. Structural basis of CBP/p300 recruitment in leukemia induction by E2A-PBX1. Blood. 2012; PubMed

Wang F, Marshall CB, Li G-Y, Yamamoto K, Mak TW, Ikura M. Synergistic interplay between promoter recognition and CBP/p300 coactivator recruitment by FOXO3a. ACS Chem Biol. 2009;4: 1017–1027. 10.1021/cb900190u PubMed DOI

Uesugi M, Verdine GL. The alpha-helical FXXPhiPhi motif in p53: TAF interaction and discrimination by MDM2. Proc Natl Acad Sci USA. 1999;96: 14801–14806. PubMed PMC

Radhakrishnan I, Pérez-Alvarado GC, Parker D, Dyson HJ, Montminy MR, Wright PE. Solution structure of the KIX domain of CBP bound to the transactivation domain of CREB: a model for activator:coactivator interactions. Cell. 1997;91: 741–752. PubMed

Lee CW, Martinez-Yamout MA, Dyson HJ, Wright PE. Structure of the p53 transactivation domain in complex with the nuclear receptor coactivator binding domain of CREB binding protein. Biochemistry. 2010;49: 9964–9971. 10.1021/bi1012996 PubMed DOI PMC

Wojciak JM, Martinez-Yamout MA, Dyson HJ, Wright PE. Structural basis for recruitment of CBP/p300 coactivators by STAT1 and STAT2 transactivation domains. EMBO J. 2009;28: 948–958. 10.1038/emboj.2009.30 PubMed DOI PMC

Ma J, Ptashne M. Deletion analysis of GAL4 defines two transcriptional activating segments. Cell. 1987;48: 847–853. PubMed

Ma J, Ptashne M. The carboxy-terminal 30 amino acids of GAL4 are recognized by GAL80. Cell. 1987;50: 137–142. PubMed

Melcher K, Johnston SA. GAL4 interacts with TATA-binding protein and coactivators. Mol Cell Biol. 1995;15: 2839–2848. PubMed PMC

Ding WV, Johnston SA. The DNA binding and activation domains of Gal4p are sufficient for conveying its regulatory signals. Mol Cell Biol. 1997;17: 2538–2549. PubMed PMC

Douglas HC, Hawthorne DC. ENZYMATIC EXPRESSION AND GENETIC LINKAGE OF GENES CONTROLLING GALACTOSE UTILIZATION IN SACCHAROMYCES. Genetics. 1964;49: 837–844. PubMed PMC

Douglas HC, Condie F. The genetic control of galactose utilization in Saccharomyces. J Bacteriol. 1954;68: 662–670. PubMed PMC

Matsumoto K, Adachi Y, Toh-e A, Oshima Y. Function of positive regulatory gene gal4 in the synthesis of galactose pathway enzymes in Saccharomyces cerevisiae: evidence that the GAL81 region codes for part of the gal4 protein. J Bacteriol. 1980;141: 508–527. PubMed PMC

Wu Y, Reece RJ, Ptashne M. Quantitation of putative activator-target affinities predicts transcriptional activating potentials. EMBO J. 1996;15: 3951–3963. PubMed PMC

Piskacek S, Gregor M, Nemethova M, Grabner M, Kovarik P, Piskacek M. Nine-amino-acid transactivation domain: establishment and prediction utilities. Genomics. 2007;89: 756–768. 10.1016/j.ygeno.2007.02.003 PubMed DOI

Piskacek M. 9aaTAD Prediction result (2006). Nature Precedings. 2009;

Piskacek M, Havelka M, Rezacova M, Knight A. The 9aaTAD Transactivation Domains: From Gal4 to p53. PLoS ONE. 2016;11: e0162842 10.1371/journal.pone.0162842 PubMed DOI PMC

Miller JH. Experiments in molecular genetics. Cold Spring Harbor Laboratory; 1972.

Baumgartner U, Hamilton B, Piskacek M, Ruis H, Rottensteiner H. Functional analysis of the Zn(2)Cys(6) transcription factors Oaf1p and Pip2p. Different roles in fatty acid induction of beta-oxidation in Saccharomyces cerevisiae. J Biol Chem. 1999;274: 22208–22216. PubMed

Warfield L, Tuttle LM, Pacheco D, Klevit RE, Hahn S. A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface. Proc Natl Acad Sci USA. 2014;111: E3506–3513. 10.1073/pnas.1412088111 PubMed DOI PMC

Hahn S. Structure(?) and function of acidic transcription activators. Cell. 1993;72: 481–483. PubMed

Himmelfarb HJ, Pearlberg J, Last DH, Ptashne M. GAL11P: a yeast mutation that potentiates the effect of weak GAL4-derived activators. Cell. 1990;63: 1299–1309. PubMed

Ptashne M. Two “what if” experiments. Cell. 2004;116: S71–72, 2 p following S76. PubMed

Ptashne M. The chemistry of regulation of genes and other things. J Biol Chem. 2014;289: 5417–5435. 10.1074/jbc.X114.547323 PubMed DOI PMC

Strubin M, Struhl K. Yeast and human TFIID with altered DNA-binding specificity for TATA elements. Cell. 1992;68: 721–730. PubMed

Erkina TY, Erkine AM. Nucleosome distortion as a possible mechanism of transcription activation domain function. Epigenetics Chromatin. 2016;9: 40 10.1186/s13072-016-0092-2 PubMed DOI PMC

Lu X, Ansari AZ, Ptashne M. An artificial transcriptional activating region with unusual properties. Proc Natl Acad Sci USA. 2000;97: 1988–1992. 10.1073/pnas.040573197 PubMed DOI PMC

Kumar PR, Yu Y, Sternglanz R, Johnston SA, Joshua-Tor L. NADP regulates the yeast GAL induction system. Science. 2008;319: 1090–1092. 10.1126/science.1151903 PubMed DOI PMC

Han Y, Kodadek T. Peptides selected to bind the Gal80 repressor are potent transcriptional activation domains in yeast. J Biol Chem. 2000;275: 14979–14984. PubMed

Klein J, Nolden M, Sanders SL, Kirchner J, Weil PA, Melcher K. Use of a genetically introduced cross-linker to identify interaction sites of acidic activators within native transcription factor IID and SAGA. J Biol Chem. 2003;278: 6779–6786. 10.1074/jbc.M212514200 PubMed DOI

Reeves WM, Hahn S. Targets of the Gal4 transcription activator in functional transcription complexes. Mol Cell Biol. 2005;25: 9092–9102. 10.1128/MCB.25.20.9092-9102.2005 PubMed DOI PMC

Majmudar CY, Labut AE, Mapp AK. Tra1 as a screening target for transcriptional activation domain discovery. Bioorg Med Chem Lett. 2009;19: 3733–3735. 10.1016/j.bmcl.2009.05.045 PubMed DOI PMC

Sakurai H, Hiraoka Y, Fukasawa T. Yeast GAL11 protein is a distinctive type transcription factor that enhances basal transcription in vitro. Proc Natl Acad Sci USA. 1993;90: 8382–8386. PubMed PMC

Suzuki Y, Nogi Y, Abe A, Fukasawa T. GAL11 protein, an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae. Mol Cell Biol. 1992;12: 4806 PubMed PMC

Nogi Y, Fukasawa T. A novel mutation that affects utilization of galactose in Saccharomyces cerevisiae. Curr Genet. 1980;2: 115–120. 10.1007/BF00420623 PubMed DOI

Hashimoto H, Kikuchi Y, Nogi Y, Fukasawa T. Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae. Isolation and characterization of the regulatory gene GAL4. Mol Gen Genet. 1983;191: 31–38. PubMed

Lin L, Chamberlain L, Zhu LJ, Green MR. Analysis of Gal4-directed transcription activation using Tra1 mutants selectively defective for interaction with Gal4. Proc Natl Acad Sci USA. 2012;109: 1997–2002. 10.1073/pnas.1116340109 PubMed DOI PMC

Larsson M, Uvell H, Sandström J, Rydén P, Selth LA, Björklund S. Functional studies of the yeast med5, med15 and med16 mediator tail subunits. PLoS ONE. 2013;8: e73137 10.1371/journal.pone.0073137 PubMed DOI PMC

Larschan E, Winston F. The Saccharomyces cerevisiae Srb8-Srb11 complex functions with the SAGA complex during Gal4-activated transcription. Mol Cell Biol. 2005;25: 114–123. 10.1128/MCB.25.1.114-123.2005 PubMed DOI PMC

Chang C, Gonzalez F, Rothermel B, Sun L, Johnston SA, Kodadek T. The Gal4 activation domain binds Sug2 protein, a proteasome component, in vivo and in vitro. J Biol Chem. 2001;276: 30956–30963. 10.1074/jbc.M102254200 PubMed DOI

Russell SJ, Johnston SA. Evidence that proteolysis of Gal4 cannot explain the transcriptional effects of proteasome ATPase mutations. J Biol Chem. 2001;276: 9825–9831. 10.1074/jbc.M010889200 PubMed DOI

Douglas HC, Hawthorne DC. Regulation of genes controlling synthesis of the galactose pathway enzymes in yeast. Genetics. 1966;54: 911–916. PubMed PMC

Douglas H.C. GP. A gene controlling inducibility of the galactose pathway enzymes in Saccharomyces. Biochimica et Biophysica Acta (BBA)—Specialized Section on Nucleic Acids and Related Subjects. 1963;68: 155–156.

Johnston SA, Salmeron JM, Dincher SS. Interaction of positive and negative regulatory proteins in the galactose regulon of yeast. Cell. 1987;50: 143–146. PubMed

Jiang F, Frey BR, Evans ML, Friel JC, Hopper JE. Gene activation by dissociation of an inhibitor from a transcriptional activation domain. Mol Cell Biol. 2009;29: 5604–5610. 10.1128/MCB.00632-09 PubMed DOI PMC

Egriboz O, Jiang F, Hopper JE. Rapid GAL gene switch of Saccharomyces cerevisiae depends on nuclear Gal3, not nucleocytoplasmic trafficking of Gal3 and Gal80. Genetics. 2011;189: 825–836. 10.1534/genetics.111.131839 PubMed DOI PMC

Carrozza MJ, John S, Sil AK, Hopper JE, Workman JL. Gal80 confers specificity on HAT complex interactions with activators. J Biol Chem. 2002;277: 24648–24652. 10.1074/jbc.M201965200 PubMed DOI

Weake VM, Workman JL. Inducible gene expression: diverse regulatory mechanisms. Nat Rev Genet. 2010;11: 426–437. 10.1038/nrg2781 PubMed DOI

Sandholzer J, Hoeth M, Piskacek M, Mayer H, de Martin R. A novel 9-amino-acid transactivation domain in the C-terminal part of Sox18. Biochem Biophys Res Commun. 2007;360: 370–374. 10.1016/j.bbrc.2007.06.095 PubMed DOI

Lindert U, Cramer M, Meuli M, Georgiev O, Schaffner W. Metal-responsive transcription factor 1 (MTF-1) activity is regulated by a nonconventional nuclear localization signal and a metal-responsive transactivation domain. Mol Cell Biol. 2009;29: 6283–6293. 10.1128/MCB.00847-09 PubMed DOI PMC

Piskacek M. Common Transactivation Motif 9aaTAD recruits multiple general co-activators TAF9, MED15, CBP and p300. Nature Precedings. 2009;

Piskacek M. 9aaTADs mimic DNA to interact with a pseudo-DNA Binding Domain KIX of Med15 (Molecular Chameleons). Nature Precedings. 2009;

Hong JY, Chae MJ, Lee IS, Lee YN, Nam MH, Kim DY, et al. Phosphorylation-mediated regulation of a rice ABA responsive element binding factor. Phytochemistry. 2011;72: 27–36. 10.1016/j.phytochem.2010.10.005 PubMed DOI

Shekhawat UKS, Ganapathi TR, Srinivas L. Cloning and characterization of a novel stress-responsive WRKY transcription factor gene (MusaWRKY71) from Musa spp. cv. Karibale Monthan (ABB group) using transformed banana cells. Mol Biol Rep. 2011;38: 4023–4035. 10.1007/s11033-010-0521-4 PubMed DOI

Lou S, Luo Y, Cheng F, Huang Q, Shen W, Kleiboeker S, et al. Human parvovirus B19 DNA replication induces a DNA damage response that is dispensable for cell cycle arrest at phase G2/M. J Virol. 2012;86: 10748–10758. 10.1128/JVI.01007-12 PubMed DOI PMC

Matsushita A, Inoue H, Goto S, Nakayama A, Sugano S, Hayashi N, et al. The nuclear ubiquitin proteasome degradation affects WRKY45 function in the rice defense program. Plant J. 2012; PubMed PMC

Aguilar X, Blomberg J, Brännström K, Olofsson A, Schleucher J, Björklund S. Interaction studies of the human and Arabidopsis thaliana Med25-ACID proteins with the herpes simplex virus VP16- and plant-specific Dreb2a transcription factors. PLoS ONE. 2014;9: e98575 10.1371/journal.pone.0098575 PubMed DOI PMC

Scharenberg MA, Pippenger BE, Sack R, Zingg D, Ferralli J, Schenk S, et al. TGF-β-induced differentiation into myofibroblasts involves specific regulation of two MKL1 isoforms. J Cell Sci. 2014;127: 1079–1091. 10.1242/jcs.142075 PubMed DOI

Piskacek M, Vasku A, Hajek R, Knight A. Shared structural features of the 9aaTAD family in complex with CBP. Mol Biosyst. 2015;11: 844–851. 10.1039/c4mb00672k PubMed DOI

Qiu Y, Li M, Pasoreck EK, Long L, Shi Y, Galvão RM, et al. HEMERA Couples the Proteolysis and Transcriptional Activity of PHYTOCHROME INTERACTING FACTORs in Arabidopsis Photomorphogenesis. Plant Cell. 2015; PubMed PMC

Lee K, Goh GYS, Wong MA, Klassen TL, Taubert S. Gain-of-Function Alleles in Caenorhabditis elegans Nuclear Hormone Receptor nhr-49 Are Functionally Distinct. PLoS ONE. 2016;11: e0162708 10.1371/journal.pone.0162708 PubMed DOI PMC

Farris TR, Dunphy PS, Zhu B, Kibler CE, McBride JW. Ehrlichia chaffeensis TRP32 is a Nucleomodulin that Directly Regulates Expression of Host Genes Governing Differentiation and Proliferation. Infect Immun. 2016; PubMed PMC

Presnell JS, Schnitzler CE, Browne WE. KLF/SP Transcription Factor Family Evolution: Expansion, Diversification, and Innovation in Eukaryotes. Genome Biol Evol. 2015;7: 2289–2309. 10.1093/gbe/evv141 PubMed DOI PMC

Myc 9aaTAD activation domain binds to mediator of transcription with superior high affinity

The evolution of the 9aaTAD domain in Sp2 proteins: inactivation with valines and intron reservoirs