Abstract
The Saccharomyces cerevisiae HEM13 gene codes for coproporphyrinogen oxidase (CPO), an oxygen-requiring enzyme catalysing the sixth step of heme biosynthesis. Its transcription is increased 40–50-fold in response to oxygen- or heme-deficiency. We have analyzed CPO activity and HEM13 mRNA levels in a set of isogenic strains carrying single or double deletions of the CYP1 (HAP1), ROX1, SSN6, or TUPI genes. The cells were grown in the presence or absence of oxygen and under heme-deficiency (hem1Δ background). Both Rox1p and Cyp1p partially repressed HEM13 in aerobic heme-sufficient cells, probably in an independent manner. In the absence of heme, Cyp1p activated HEM13 and strongly repressed ROX1, allowing de-repression of HEM13. Cyp1p had no effect on HEM13 expression in anaerobic cells. Deletions of SSN6 or TUP1 dramatically de-repressed HEM13 in aerobic cells. A series of deletions in the HEM13 promoter identified at least four regulatory regions that are required for HEM13 regulation. Two regions, containing motifs similar to the Rox1p consensus sequences, act as repression sites under aerobic growth. The two other sites act as activation sequences required for full induction under oxygen- or heme-deficiency. Taken together, these results suggest that induction of HEM13 occurs in part through relief of repression exerted by Rox1p and Cyp1p, and in part by activation mediated partly by Cyp1p under heme-deficiency and by unknown factors under oxygen-deficiency.
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References
Balasubramanian B, Lowry CV, Zitomer RS (1993) The Rox1 repressor of the Saccharomyces cerevisiae hypoxic genes is a specific DNA-binding protein with a high-mobility-group motif. Mol Cell Biol 13:6071–6078
Bennetzen JL, Hall BD (1982) Codon selection in yeast. J Biol Chem 257:3026–3031
Berben G, Dumont J, Gilliquet V, Bolle PA, Hilger F (1991) The YDp plasmids: a uniform set of vectors bearing versatile gene disruption cassettes for Saccharomyces cerevisiae. Yeast 7:475–477
Bonneaud N, Ozier-Kalogeropoulos O, Li G, Labouesse M, Minvielle-Sebastia L, Lacroute F (1991) A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors Yeast 7:609–615
Cooper JP, Roth SY, Simpson RT (1994) The global transcriptional regulators, SSN6 and TUP1, play distinct roles in the establishment of a repressive chromatin structure. Genes Dev 8:1400–1410
Creusot F, Verdière J, Gaisne M, Slonimski PP (1988) CYP1 (HAP1) regulator of oxygen-dependent gene expression in yeast. I. Overall organization of the protein sequence displays several novel structural domains. J Mol Biol 204:263–276
Defranoux N, Gaisne M, Verdière J (1994) Functional analysis of the zinc-cluster domain of the CYP1 (HAP1) complex regulator in heme-sufficient and heme-deficient yeast cells. Mol Gen Genet 242:699–707
Dorsman JC, Gozdzicka-Jozefiak A, Van Heeswijk WC, Grivell LA (1991) Multi-functional DNA proteins in yeast: the factors GFI and GFII are identical to the ARS-binding factor ABFI and the centromere-binding factor CPFI respectively. Yeast 7:401–412
Forsburg SL, Guarente L (1988) Mutational analysis of upstream activation sequence 2 of the CYC1 gene of Saccharomyces cerevisiae: a HAP2-HAP3-responsive site. Mol Cell Biol 8:647–654
Fujita A, Matsumoto S, Kuhara S, Misumi Y, Kobayashi H (1990) Cloning of the yeast SFL2 gene: its disruption results in pleiotropic phenotypes characteristic for tup1 mutants. Gene 89:93–99
Fytlovich S (1993) Analyse fonctionnelle du regulateur de transcription CYP1 implique dans la regulation par l'oxygene chez la levure Saccharomyces cerevisiae, PhD thesis, Paris 6 Universite, Paris, France
Gietz D, Jean ASt, Woods RA, Schiestl RH (1992) Improved method for high-efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425
Hodge MR, Kim G, Singh K, Cumsky MG (1989) Inverse regulation of the yeast COX5 genes by oxygen and heme. Mol Cell Biol 9:1958–1964
Keleher CA, Redd MJ, Schultz J, Carlson M, Johnson AD (1992) Ssn6-Tup1 is a general repressor of transcription in yeast. Cell 68:709–719
Keng T (1992) HAP1 and ROX1 form a regulatory pathway in the repression of HEM13 transcription in Saccharomyces cerevisiae. Mol Cell Biol 12:2616–2623
Kunkel TA, Roberts JD, Zakour RA (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol 154:367–382
Labbe P, Camadro JM, Chambon H (1985) Fluorometric assays for coproporphyrinogen oxidase and protoporphyrinogen oxidase. Anal Biochem 149:248–260
Labbe-Bois R, Camadro JM (1994) Ferrochelatase in Saccharomyces cerevisiae. In: Winkelman G, Winge DR (eds) Metal Ions in Fungi. Marcel Dekker Inc, New York, pp 413–453
Labbe-Bois R, Labbe P (1990) Tetrapyrrole and heme synthesis in the yeast Saccharomyces cerevisiae. In: Dailey HA (ed) Biosynthesis of heme and chlorophylls. McGraw-Hill, New York, pp 235–285
Lowry CV, Zitomer RS (1988) ROX1 encodes a heme-induced repression factor regulating ANB1 and CYC7 of Saccharomyces cerevisiae. Mol Cell Biol 8:4651–4658
Lowry CV, Cerdan ME, Zitomer RS (1990) A hypoxic consensus operator and a constitutive activation region regulate the ANB1 gene of Saccharomyces cerevisiae. Mol Cell Biol 10:5921–5926
Mehta KD, Smith M (1989) Identification of an upstream repressor site controlling the expression of an anaerobic gene (ANB1) in Saccharomyces cerevisiae. J Biol Chem 264:8670–8675
Myers AM, Tzagoloff A, Kinney DM, Lusty CJ (1986) Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions. Gene 45:299–310
Ohaniance L, Chaix P (1966) Effet inhibiteur de Zn2+ sur la biosynthèse induite par l'oxygène des enzymes respiratoires de la levure. Biochim Biophys Acta 128:228–238
Pfeifer K, Kim KS, Kogan S, Guarente L (1989) Functional dissection and sequence of yeast HAP1 activator. Cell 56:291–301
Pinkham JL, Keng T (1994) Heme-mediated gene regulation in Saccharomyces cerevisiae. In: Winkelman G, Winge DR (eds) Metal Ions in Fungi. Marcel Dekker Inc, New York, pp 455–501
Reece RJ, Ptashne M (1993) Determinants of binding-site specificity among yeast C6 zinc cluster proteins, Science 261:909–911
Rose MD, Winston F, Hieter P (1990) Methods in yeast genetics: a laboratory course manual, Cold Spring Harbour Laboratory, New York.
Rothstein R (1991) Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol 194:281–301
Sabova L, Zeman I, Supek F, Kolarov J (1993) Transcriptional control of the AAC3 gene encoding the mitochondrial ADP/ATP translocator in Sacchromyces cerevisiae by oxygen, heme and ROX1 factor. Eur J Biochem 213:547–553
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbour, New York
Schmitt ME, Brown TA, Trumpower BL (1990) A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res 18:3091–3092
Schneider JC, Guarente L (1991) Regulation of the yeast CYT1 gene encoding cytochrome c1 by HAP1 and HAP2/3/4. Mol Cell Biol 11:4934–4942
Schultz J, Carlson M (1987) Molecular analysis of SSN6, a gene functionally related to the SNF1 protein kinase of Saccharomyces cerevisiae. Mol Cell Biol 7:3637–3645
Trumbly RJ (1992) Glucose repression in the yeast Saccharomyces cerevisiae. Mol Microbiol 6:15–21
Turi TG, Loper JC (1992) Multiple regulatory elements control expression of the gene encoding the Saccharomyces cerevisiae cytochrome P45O, lanosterol 14alpha-demethylase (ERG11). J Biol Chem 267:2046–2056
Urban-Grimal D, Volland C, Garnier T, Dehoux P, Labbe-Bois R (1986) The nucleotide sequence of the HEM1 gene and evidence for a precursor form of the mitochondrial 5-aminolevulinate synthase in Saccharomyces cerevisiae. Eur J Biochem 156:511–519
Ushinsky SC, Keng T (1994) A novel allele of HAP1 causes uninducible expression of HEM13 in Saccharomyces cerevisiae. Genetics 136:819–831
Vashee S, Xu H, Johnston SA, Kodadek T (1993) How do “Zn2 Cys6” proteins distinguish between similar upstream activation sites? Comparison of the DNA-binding specificity of the GAL4 protein in vitro and in vivo. J Biol Chem 268:24699–24706
Verdière J, Gaisne M, Labbe-Bois R (1991) CYP1(HAP1) is a determinant effector of alternative expression of heme-dependent transcription in yeast. Mol Gen Genet 228:300–306
Williams FE, Trumbly RJ (1990) Characterization of TUP1 a mediator of glucose repression in Saccharomyces cerevisiae. Mol Cell Biol 10:6500–6511
Williams FE, Varanasi U, Trumbly RJ (1991) The CYC8 and TUP1 proteins involved in glucose repression in Saccharomyces cerevisiae are associated in a protein complex. Mol Cell Biol 11:3307–3316
Zagorec M, Labbe-Bois R (1986) Negative control of yeast coproporphyrinogen oxidase synthesis by heme and oxygen. J Biol Chem 261:2506–2509
Zagorec M, Buhler JM, Treich I, Keng T, Guarente L, Labbe-Bois R (1988) Isolation, sequence, and regulation by oxygen of the yeast HEM13 gene coding for coproporphyrinogen oxidase. J Biol Chem 263:9718–9724
Zhang M, Rosenblum-Vos LS, Lowry CV, Boakye KA, Zitomer RS (1991) A yeast protein with homology to the b-subunit of G proteins is involved in control of heme-regulated and cataboliterepressed genes. Gene 97:153–161
Zitomer RS, Lowry CV (1992) Regulation of gene expression by oxygen in Saccharomyces cerevisiae. Microbiol Rev 56:1–11
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Amillet, JM., Buisson, N. & Labbe-Bois, R. Positive and negative elements involved in the differential regulation by heme and oxygen of the HEM13 gene (coproporphyrinogen oxidase) in Saccharomyces cerevisiae . Curr Genet 28, 503–511 (1995). https://doi.org/10.1007/BF00518161
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DOI: https://doi.org/10.1007/BF00518161