A novel CRE recombinase assay for quantification of GAL10-non coding RNA suppression on transcriptional leakage.

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.