PloS one, 2015, Vol.10 (12), p.e0144677-e0144677
In the fission yeast, two Tor isoforms, Tor1 and Tor2, oppositely regulate gene expression of amino acid permeases. To elucidate the transcriptional machinery for these regulations, here we have employed the cap analysis of gene expression (CAGE), a method of analyzing expression profiles and identifying transcriptional start sites (TSSs). The loss of Tor1 decreased, and Tor2 inhibition by its temperature sensitive mutation increased, mRNA expression of isp5+, per1+, put4+ and SPBPB2B2.01. In contrast, the loss of Tor1 increased, and Tor2 inhibition decreased, the expression of cat1+. These changes were confirmed by semi-quantitative RT-PCR. These opposite effects by the loss of Tor1 and Tor2 inhibition appeared to occur evenly across multiple TSSs for the respective genes. The motif discovery analysis based on the CAGE results identified the GATA motifs as a potential cis-regulatory element for Tor-mediated regulation. In the luciferase reporter assay, the loss of Tor1 reduced, and Tor2 inhibition and nitrogen depletion increased, the activity of isp5+ promoter as well as that of a GATAAG reporter. One of the GATAAG motifs in isp5+ promoter was critical for its transcriptional activity, and a GATA transcription factor Gaf1 was critical for the activities of isp5+ promoter and the GATAAG reporter. Furthermore, Tor2 inhibition and nitrogen depletion induced nuclear localization of Gaf1 from the cytosol and its dephosphorylation. These results suggest that Tor2 inhibition, which is known to be induced by nitrogen depletion, promotes nuclear localization of Gaf1, thereby inducing isp5+ transcription through Gaf1 binding to the GATAAG motif in its promoter. Since Gaf1 was also critical for transcription of per1+ and put4+, Tor-Gaf1 signaling may coordinate transcription of multiple amino acid permeases according to nutrient availability.
Acids ; Amino Acid Transport Systems - biosynthesis ; Amino Acid Transport Systems - genetics ; Amino acids ; Baking yeast ; Cages ; Cell growth ; Cell Nucleus - genetics ; Cell Nucleus - metabolism ; Cellular signal transduction ; Cytosol ; Dephosphorylation ; Depletion ; Gene expression ; Genetic aspects ; Genetic regulation ; Genomics ; Inhibition ; Isoforms ; Kinases ; Localization ; Machinery ; Machinery and equipment ; Mutation ; Nitrogen ; Nutrient availability ; Period 1 protein ; Phosphatidylinositol 3-Kinases - genetics ; Phosphatidylinositol 3-Kinases - metabolism ; Physiological aspects ; Polymerase chain reaction ; Protein Kinases - genetics ; Protein Kinases - metabolism ; Research ; Response Elements - physiology ; Schizosaccharomyces - genetics ; Schizosaccharomyces - metabolism ; Schizosaccharomyces pombe Proteins - genetics ; Schizosaccharomyces pombe Proteins - metabolism ; Signal Transduction - physiology ; Signaling ; Trans-Activators - genetics ; Trans-Activators - metabolism ; Transcription factors ; Transcription, Genetic - physiology ; Yeast
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