Functional diversity of silencers in budding yeasts

Area of Science:

• Molecular Biology
• Yeast Genetics
• Epigenetics

Background:

• Mating-type gene silencing is crucial for sexual reproduction in yeasts.
• Cryptic mating-type loci (HML and HMR) are transcriptionally repressed.
• Understanding silencing mechanisms provides insights into gene regulation and evolution.

Purpose of the Study:

• To investigate the molecular mechanisms of mating-type gene silencing in Kluyveromyces lactis.
• To identify specific DNA sequences and proteins involved in the HMLalpha and HMRa loci silencing.
• To compare silencing mechanisms between Kluyveromyces lactis and Saccharomyces cerevisiae.

Main Methods:

• Deletion and point mutagenesis of silencer regions.
• In vitro binding assays using recombinant proteins.
• Analysis of gene expression using temperature-sensitive mutants.

Main Results:

• A 102-bp minimal silencer fragment for HMLalpha was identified, containing essential regions A, B, and C.
• Recombinant Kluyveromyces lactis ribosomal DNA enhancer binding protein 1 (Reb1p) binds to the silencer's B box.
• Mutations in the B box abolished Reb1p binding and silencing function.
• Temperature-sensitive Reb1 gene mutants showed derepression of HMLalpha1 at non-permissive temperatures.
• A functional silencer element at the HMRa locus also contains Reb1p binding sites and A boxes.

Conclusions:

• Reb1p is a key protein involved in the silencing of cryptic mating-type loci in Kluyveromyces lactis.
• Distinct proteins bind to Kluyveromyces silencers compared to Saccharomyces silencers.
• Silencer evolution appears rapid in budding yeasts, with unique sequence elements and protein interactions.