title: A novel CRE recombinase assay for quantification of GAL10-non coding RNA suppression on transcriptional leakage.
abstract: Eukaryotic promoters are tightly regulated and often securely repressed. However, recent reports indicated that transcripts originating from the strictly regulated GAL1-10 promoter can be detected by single-yeast cell imaging under repressive conditions. Such leaky, noisy transcription events were suppressed by a long non-coding RNA (GAL10-ncRNA) transcribed within the GAL1-10 locus. It was further suggested that GAL10-ncRNA repression of GAL1-10 promoter leakage tunes the bimodal expression pattern of the GAL network. Independent evidence has indicated that GAL10-ncRNA transcription establishes a repressive chromatin structure through the Set2 histone methyl-transferase and the Rpd3s histone deacetylase complex. In this report we set up a novel, simple genetic Cre recombinase assay in order to readily quantify transcriptional leakage from tightly repressed promoters. By applying this method we demonstrate that GAL10-ncRNA, Set2p and Rpd3p all suppress leaky GAL1-10 driven transcription. However, GAL10-ncRNA repression is not mediated by Set2p or Rpd3p. Moreover, as opposed to GAL10-ncRNA transcription, Set2 and Rpd3 do not influence the bimodal expression of GAL genes, despite their effect on GAL1-10 promoter leakage. We suggest that GAL10-ncRNA tunes the expression of GAL genes by additional mechanisms besides suppressing leaky transcription from the GAL1-10 promoter.
authors.affiliation: Biology Department, University of Crete, Heraklion, Crete, Greece.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Zacharioudakis
authors.affiliation: Biology Department, University of Crete, Heraklion, Crete, Greece. Electronic address: Tzamaria@imbb.forth.gr.
authors.forename: Dimitris
authors.initials: D
authors.lastname: Tzamarias
mesh_terms: D001483:Base Sequence; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D005812:Genes, Switch / Q000235:genetics*; D006655:Histone Deacetylases / Q000235:genetics; D019426:Integrases / Q000235:genetics*; D008969:Molecular Sequence Data; D022661:RNA, Untranslated / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D013489:Suppression, Genetic / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics; D059467:Transcriptome / Q000235:genetics
chemical_list: C518355:GAL10 protein, S cerevisiae; D022661:RNA, Untranslated; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; C045073:Cre recombinase; D019426:Integrases; C071665:RPD3 protein, S cerevisiae; C511735:Set3 protein, S cerevisiae; D006655:Histone Deacetylases
keywords: Cre; GAL10-ncRNA; Leaky transcription; Non-coding RNA