Heme Levels Switch the Function of Hap1 of Saccharomyces cerevisiae between Transcriptional Activator and Transcriptional Repressor

ABSTRACT Changes in oxygen levels cause widespread changes in gene expression in organisms ranging from bacteria to humans. In Saccharomyces cerevisiae, this response is mediated in part by Hap1, originally identified as a heme-dependent transcriptional activator that functions during aerobic growth. We show here that Hap1 also plays a significant and direct role under hypoxic conditions, not as an activator, but as a repressor. The repressive activity of Hap1 controls several genes, including three ERG genes required for ergosterol biosynthesis. Chromatin immunoprecipitation experiments showed that Hap1 binds to the ERG gene promoters, while additional experiments showed that the corepressor Tup1/Ssn6 is recruited by Hap1 and is also required for repression. Furthermore, mutational analysis demonstrated that conserved Hap1 binding sites in the ERG5 5′ regulatory region are required for repression. The switch of Hap1 from acting as a hypoxic repressor to an aerobic activator is determined by heme, which is synthesized only in the presence of oxygen. The ability of Hap1 to function as a ligand-dependent repressor and activator is a property shared with mammalian nuclear hormone receptors and likely allows greater transcriptional control by Hap1 in response to changing oxygen levels.

[1]  W. Kaelin,et al.  Proline hydroxylation and gene expression. , 2005, Annual review of biochemistry.

[2]  A. Futcher,et al.  Use of polymerase chain reaction epitope tagging for protein tagging in Saccharomyces cerevisiae , 1995, Yeast.

[3]  J. Boeke,et al.  Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR‐mediated gene disruption and other applications , 1998, Yeast.

[4]  Li Zhang,et al.  The Molecular Chaperone Hsp90 Mediates Heme Activation of the Yeast Transcriptional Activator Hap1* , 2002, The Journal of Biological Chemistry.

[5]  P. Trocha,et al.  Location and regulation of early enzymes of sterol biosynthesis in yeast. , 1976, Archives of biochemistry and biophysics.

[6]  Li Zhang,et al.  A Novel Mode of Chaperone Action , 2004, Journal of Biological Chemistry.

[7]  B. Turcotte,et al.  Mutations in target DNA elements of yeast HAP1 modulate its transcriptional activity without affecting DNA binding. , 1996, Nucleic acids research.

[8]  G. Lauquin,et al.  Regulation by low temperatures and anaerobiosis of a yeast gene specifying a putative GPI‐anchored plasma membrane , 1996, Molecular microbiology.

[9]  C. Lowry,et al.  ROX1 encodes a heme-induced repression factor regulating ANB1 and CYC7 of Saccharomyces cerevisiae , 1988, Molecular and cellular biology.

[10]  J. Rine,et al.  Dual Activators of the Sterol Biosynthetic Pathway of Saccharomyces cerevisiae: Similar Activation/Regulatory Domains but Different Response Mechanisms , 2005, Molecular and Cellular Biology.

[11]  K. Kwast,et al.  Metabolic-State-Dependent Remodeling of the Transcriptome in Response to Anoxia and Subsequent Reoxygenation in Saccharomyces cerevisiae , 2006, Eukaryotic Cell.

[12]  H. Y. Steensma,et al.  A microarray‐assisted screen for potential Hap1 and Rox1 target genes in Saccharomyces cerevisiae , 2002, Yeast.

[13]  J. Buhler,et al.  Isolation, sequence, and regulation by oxygen of the yeast HEM13 gene coding for coproporphyrinogen oxidase. , 1988, The Journal of biological chemistry.

[14]  L. Grivell,et al.  Expression of the gene encoding subunit II of yeast QH2: Cytochrome c oxidoreductase is regulated by multiple factors , 1990, Current Genetics.

[15]  R. Tibshirani,et al.  Significance analysis of microarrays applied to the ionizing radiation response , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[16]  H. Shimoi,et al.  A hap1 mutation in a laboratory strain of Saccharomyces cerevisiae results in decreased expression of ergosterol-related genes and cellular ergosterol content compared to sake yeast. , 2004, Journal of bioscience and bioengineering.

[17]  L. Guarente,et al.  Functional dissection and sequence of yeast HAP1 activator , 1989, Cell.

[18]  Fred Winston,et al.  in Saccharomyces cerevisiae , 2005 .

[19]  S. Dent,et al.  Transcriptional repression by Tup1-Ssn6. , 2006, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[20]  Li Zhang,et al.  Heme: a versatile signaling molecule controlling the activities of diverse regulators ranging from transcription factors to MAP kinases , 2006, Cell Research.

[21]  John Quackenbush.  Microarray data normalization and transformation , 2002, Nature Genetics.

[22]  H. Erdjument-Bromage,et al.  The Hsp 70-Ydj 1 Molecular Chaperone Represses the Activity of the Heme Activator Protein Hap 1 in the Absence of Heme , 2008 .

[23]  L. Guarente,et al.  Structure of HAP1-18–DNA implicates direct allosteric effect of protein–DNA interactions on transcriptional activation , 1999, Nature Structural Biology.

[24]  J. Deckert,et al.  Multiple elements and auto-repression regulate Rox1, a repressor of hypoxic genes in Saccharomyces cerevisiae. , 1995, Genetics.

[25]  J C Loper,et al.  Multiple regulatory elements control expression of the gene encoding the Saccharomyces cerevisiae cytochrome P450, lanosterol 14 alpha-demethylase (ERG11). , 1992, The Journal of biological chemistry.

[26]  Ronald W. Davis,et al.  Genome-Wide Transcriptional Analysis of Aerobic and Anaerobic Chemostat Cultures of Saccharomyces cerevisiae , 1999, Journal of bacteriology.

[27]  A. Dudley,et al.  The Spt components of SAGA facilitate TBP binding to a promoter at a post-activator-binding step in vivo. , 1999, Genes & development.

[28]  Li Zhang,et al.  The Heme Activator Protein Hap1 Represses Transcription by a Heme-Independent Mechanism in Saccharomyces cerevisiae , 2005, Genetics.

[29]  J. Pinkham,et al.  Heme regulates SOD2 transcription by activation and repression in Saccharomyces cerevisiae , 1997, Current Genetics.

[30]  K. Davies,et al.  Regulatory mechanisms controlling expression of the DAN/TIR mannoprotein genes during anaerobic remodeling of the cell wall in Saccharomyces cerevisiae. , 2001, Genetics.

[31]  K. Kwast,et al.  Genomic Analyses of Anaerobically Induced Genes in Saccharomyces cerevisiae: Functional Roles of Rox1 and Other Factors in Mediating the Anoxic Response , 2002, Journal of bacteriology.

[32]  J. Rine,et al.  A Role for Sterol Levels in Oxygen Sensing in Saccharomyces cerevisiae , 2006, Genetics.

[33]  P. Philippsen,et al.  Additional modules for versatile and economical PCR‐based gene deletion and modification in Saccharomyces cerevisiae , 1998, Yeast.

[34]  M. Bard,et al.  Transcriptional regulation of the squalene synthase gene (ERG9) in the yeast Saccharomyces cerevisiae. , 1999, Biochimica et biophysica acta.

[35]  F. Foufelle,et al.  SREBP transcription factors: master regulators of lipid homeostasis. , 2004, Biochimie.

[36]  B. Guiard,et al.  Evidence for an interaction between the CYP1(HAP1) activator and a cellular factor during heme‐dependent transcriptional regulation in the yeast Saccharomyces cerevisiae. , 1993, The EMBO journal.

[37]  L. Guarente,et al.  Heme regulates transcription of the CYC1 gene of S. cerevisiae via an upstream activation site , 1983, Cell.

[38]  R. O. Poyton,et al.  A Mechanism of Oxygen Sensing in Yeast , 2003, Journal of Biological Chemistry.

[39]  S. Sturley,et al.  Conservation of eukaryotic sterol homeostasis: new insights from studies in budding yeast. , 2000, Biochimica et biophysica acta.

[40]  N. Defranoux,et al.  Functional analysis of the zinc cluster domain of the CYP1 (HAP1) complex regulator in heme-sufficient and heme-deficient yeast cells , 1994, Molecular and General Genetics MGG.

[41]  Nicola J. Rinaldi,et al.  Transcriptional regulatory code of a eukaryotic genome , 2004, Nature.

[42]  J. Hoheisel,et al.  The yeast transcriptome in aerobic and hypoxic conditions: effects of hap1, rox1, rox3 and srb10 deletions , 2002, Molecular microbiology.

[43]  E. M. Adkins,et al.  Recruitment of Tup1p and Cti6p regulates heme‐deficient expression of Aft1p target genes , 2006, The EMBO journal.

[44]  Á. Pascual,et al.  Nuclear hormone receptors and gene expression. , 2001, Physiological reviews.

[45]  H. Erdjument-Bromage,et al.  The Hsp70-Ydj1 Molecular Chaperone Represses the Activity of the Heme Activator Protein Hap1 in the Absence of Heme , 2001, Molecular and Cellular Biology.

[46]  M. Resnick,et al.  In vivo site-directed mutagenesis using oligonucleotides , 2001, Nature Biotechnology.

[47]  K. Kwast,et al.  Oxygen sensing and the transcriptional regulation of oxygen-responsive genes in yeast. , 1998, The Journal of experimental biology.

[48]  B Guiard,et al.  Some DNA targets of the yeast CYP1 transcriptional activator are functionally asymmetric--evidence of two half-sites with different affinities. , 1997, European journal of biochemistry.

[49]  C. Glass,et al.  Coactivator and corepressor complexes in nuclear receptor function. , 1999, Current opinion in genetics & development.

[50]  L. Guarente,et al.  Distinctly regulated tandem upstream activation sites mediate catabolite repression of the CYC1 gene of S. cerevisiae , 1984, Cell.

[51]  C. Herbert,et al.  A ‘natural’ mutation in Saccharomyces cerevisiae strains derived from S288c affects the complex regulatory gene HAP1 (CYP1) , 1999, Current Genetics.

[52]  R. Wenger.  Mammalian oxygen sensing, signalling and gene regulation. , 2000, The Journal of experimental biology.

[53]  L. W. Parks,et al.  Involvement of heme biosynthesis in control of sterol uptake by Saccharomyces cerevisiae , 1985, Journal of bacteriology.

[54]  D J Jamieson,et al.  Oxidative stress responses of the yeast Saccharomyces cerevisiae , 1998, Yeast.

[55]  Kara Dolinski,et al.  Saccharomyces Genome Database (SGD) provides tools to identify and analyze sequences from Saccharomyces cerevisiae and related sequences from other organisms , 2004, Nucleic Acids Res..

[56]  K. Kwast,et al.  Dynamical Remodeling of the Transcriptome during Short-Term Anaerobiosis in Saccharomyces cerevisiae: Differential Response and Role of Msn2 and/or Msn4 and Other Factors in Galactose and Glucose Media , 2005, Molecular and Cellular Biology.

[57]  John J. Wyrick,et al.  Genome-wide location and function of DNA binding proteins. , 2000, Science.

[58]  T. Keng,et al.  A novel allele of HAP1 causes uninducible expression of HEM13 in Saccharomyces cerevisiae. , 1994, Genetics.

[59]  M. Gaisne,et al.  CYP1 (HAP1) is a determinant effector of alternative expression of heme-dependent transcription in yeast , 1991, Molecular and General Genetics MGG.

[60]  Fred Winston,et al.  Construction of a set of convenient saccharomyces cerevisiae strains that are isogenic to S288C , 1995, Yeast.

[61]  K. Struhl,et al.  Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific Genomic Sequences In Vivo , 2004, Current protocols in molecular biology.

[62]  Jasper Rine,et al.  Upc2p and Ecm22p, Dual Regulators of Sterol Biosynthesis in Saccharomyces cerevisiae , 2001, Molecular and Cellular Biology.

[63]  L. Zhang,et al.  Molecular mechanism of heme signaling in yeast: the transcriptional activator Hap1 serves as the key mediator , 1999, Cellular and Molecular Life Sciences CMLS.

[64]  M. Brandriss,et al.  A regulatory region responsible for proline-specific induction of the yeast PUT2 gene is adjacent to its TATA box , 1988, Molecular and cellular biology.