ABSTRACT
Ixr1p from Saccharomyces cerevisiae has been previously studied because it binds to DNA containing intrastrand cross-links formed by the anticancer drug cisplatin. Ixr1p is also a transcriptional regulator of anaerobic/hypoxic genes, such as SRP1/TIR1, which encodes a stress-response cell wall manoprotein, and COX5B, which encodes the Vb subunit of the mitochondrial complex cytochrome c oxidase. However, factors controlling IXR1 expression remained unexplored. In the present study we show that IXR1 mRNA levels are controlled by oxygen availability and increase during hypoxia. In aerobiosis, low levels of IXR1 expression are maintained by Rox1p repression through the general co-repressor complex Tup1-Ssn6. Ixr1p itself is necessary for full IXR1 expression under hypoxic conditions. Deletion analyses have identified the region in the IXR1 promoter responsible for this positive auto-control (nucleotides -557 to -376). EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays show that Ixr1p binds to the IXR1 promoter both in vitro and in vivo. Ixr1p is also required for hypoxic repression of ROX1 and binds to its promoter. UPC2 deletion has opposite effects on IXR1 and ROX1 transcription during hypoxia. Ixr1p is also necessary for resistance to oxidative stress generated by H2O2. IXR1 expression is moderately activated by H2O2 and this induction is Yap1p-dependent. A model of IXR1 regulation as a relay for sensing different signals related to change in oxygen availability is proposed. In this model, transcriptional adaptation from aerobiosis to hypoxia depends on ROX1 and IXR1 cross-regulation.
Subject(s)
DNA-Binding Proteins/metabolism , High Mobility Group Proteins/metabolism , Oxygen/metabolism , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Adaptation, Physiological , Aerobiosis , Anaerobiosis , Chromatin Immunoprecipitation , DNA-Binding Proteins/genetics , Electrophoretic Mobility Shift Assay , Gene Expression Regulation, Fungal/drug effects , High Mobility Group Proteins/genetics , Models, Biological , Oxygen/pharmacology , Promoter Regions, Genetic/genetics , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Transformation, Genetic , alpha-Galactosidase/genetics , alpha-Galactosidase/metabolismABSTRACT
The function of KlSRB10 has been studied by diverse approaches. Primer extension analysis reveals several transcription start sites, position - 17 from ATG being predominant. Deletion of KlSRB10 diminishes growth in ethanol and decreases KlCYC1 transcript levels. A second phenotype associated with this deletion affects growth in galactose. These phenotypes are independent of the specific sequence connecting the ATP binding cassette and the kinase domain of Srb10p in yeasts. KlSrb10p is not necessary for LAC4 repression mediated by KlGal80p, as deduced by construction of a Klgal80Deltasrb10Delta double mutant. In the two-hybrid system, KlSrbp10p interacts with the protein encoded by KLLA0E08151g (KlSrbp11p).
Subject(s)
Cyclin-Dependent Kinases/physiology , Kluyveromyces/physiology , Amino Acid Sequence , Base Sequence , Blotting, Northern , Cyclin-Dependent Kinases/biosynthesis , Cyclin-Dependent Kinases/genetics , DNA, Fungal/chemistry , DNA, Fungal/genetics , Gene Expression Regulation, Fungal , Kluyveromyces/enzymology , Kluyveromyces/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Polymerase Chain Reaction , RNA, Fungal/chemistry , RNA, Fungal/genetics , Sequence Alignment , Transcription, Genetic , Two-Hybrid System TechniquesABSTRACT
Cloning, sequencing and functional analysis of the Kluyveromyces lactis KlHEM12 gene and its upstream region are reported. The gene encodes for a protein that is highly homologous to uroporphyrinogen decarboxylases from different organisms and complements its mutation in Saccharomyces cerevisiae. Secondary structure prediction allows outlining a topology diagram which is compatible with a (beta/alpha)8-barrel structure. A K. lactis haploid strain carrying a null allele of KlHEM12 showed decreased growth in media not supplemented with hemin (ferriprotoporphyrin IX) and red-fluorescent colonies due to the accumulation of porphyrins. KlHEM12 expression was analysed by Northern blot and promoter fusion to the reporter lacZ gene. Transcription of this gene is not under heme or glucose repression and it is slightly induced by non-fermentable carbon sources through the Hap2/3/4/5 complex.