Mating type switching and transcriptional silencing in Kluyveromyces lactis
Sammanfattning: To explore the similarities and differences of regulatory circuits among budding yeasts, we characterized the role of unscheduled meiotic gene expression 6 (UME6) and a novel mating type switching pathway in Kluyveromyces lactis. We found that Ume6 was required for transcriptional silencing of the cryptic mating-type loci HML? and HMRa. Ume6 acted directly at these loci by binding to the cis-regulatory silencers. Ume6 also served as a block to polyploidy and was required for repression of three meiotic genes, independently of the Rpd3 and Sin3 corepressors.Mating type switching from MAT? to MATa required the ?3 protein. The ?3 protein was similar to transposases of the mutator like elements (MULEs). Mutational analysis showed that the DDE-motif in ?3, which is conserved in MULEs was necessary for switching. During switching ?3 mobilizes from the genome in the form of a DNA circle. The sequences encompassing the ?3 gene circle junctions in the MAT? locus were essential for switching from MAT? to MATa. Switching also required a DNA binding protein, Mating type switch 1 (Mts1), whose binding sites in MAT? were important. Expression of Mts1 was repressed in MATa/MAT? diploids and by nutrients, limiting switching to haploids in low nutrient conditions.In a genetic selection for strains with increased switching rates we found a mutation in the RAS1 gene. By measuring the levels of the MTS1 mRNA and switching rates in ras1, pde2 and msn2 mutant strains we show that mating type switching in K. lactis was regulated by the RAS/cAMP pathway and the transcription factor Msn2. ras1 mutants contained 20-fold higher levels of MTS1 mRNA compared to wild type whereas pde2 and msn2 expressed less MTS1 mRNA and had decreased switching rates. Furthermore we found that MTS1 contained several potential Msn2 binding sites upstream of its ORF. We suggest that these observations explain the nutrient regulation of switching.
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