The DNA End-Binding Protein Ku Regulates Silencing at the InternalHMLandHMRLoci inSaccharomyces cerevisiae

Abstract

Heterochromatin resides near yeast telomeres and at the cryptic mating-type loci, HML and HMR, where it silences transcription of the a-and a-mating-type genes, respectively. Ku is a conserved DNA end-binding protein that binds telomeres and regulates silencing in yeast. The role of Ku in silencing is thought to be limited to telomeric silencing. Here, we tested whether Ku contributes to silencing at HML or HMR . Mutant analysis revealed that yKu70 and Sir1 act collectively to silence the mating-type genes at HML and HMR. In addition, loss of yKu70 function leads to expression of different reporter genes inserted at HMR. Quantitative chromatin-immunoprecipitation experiments revealed that yKu70 binds to HML and HMR and that binding of Ku to these internal loci is dependent on Sir4. The interaction between yKu70 and Sir4 was characterized further and found to be dependent on Sir2 but not on Sir1, Sir3, or yKu80. These observations reveal that, in addition to its ability to bind telomeric DNA ends and aid in the silencing of genes at telomeres, Ku binds to internal silent loci via protein-protein interactions and contributes to the efficient silencing of these loci.

Mating-Type Genes andMATSwitching inSaccharomyces cerevisiae

“…There is a hierarchy of silencing, with HMR and HMR being more strongly silenced than telomeres. In general, telomeric silencing is less robust; several mutations that strongly affect telomeric silencing (e.g., yku70D (Moretti et al 1994;Wotton and Shore 1997;Vandre et al 2008) have either no effect on HM loci or have an effect only with a partially debilitated HMR-E sequence.…”

“…The idea that tethering HML or HMR to the nuclear periphery enhances silencing has been substantiated by the finding that the silencing that does occur in a sir1D is dependent on the yKu70 and YKu80 proteins (Patterson and Fox 2008;Vandre et al 2008). The Ku proteins, along with Esc1, redundantly tether telomeres and HM loci to the nuclear periphery (Taddei et al 2004(Taddei et al , 2005 and this sequestering may place the HM locus near a higher concentration of Sir2 and other silencing factors.…”

Donor Preference Meets Heterochromatin: Moonlighting Activities of a Recombinational Enhancer in Saccharomyces cerevisiae

“…Interestingly, measurements made by chromatin immunoprecipitation show that the RE promotes a-cell-specific enrichment of yKu70 at HML in the absence of HO induction (Bystricky et al 2009). Both yKu70 and yKu80, which together form a heterodimer involved in DNA repair, affect silencing and also contribute to donor preference (Ruan et al 2005;Coïc et al 2006b;Patterson and Fox 2008;Vandre et al 2008;Bystricky et al 2009). Given their connections to both silencing and RE activity, the Ku proteins could, in principle, contribute to the effect of the RE on silencing.…”

Mating-type Gene Switching in Saccharomyces cerevisiae

“…Telomeric silencing can extend for more than 10 kb (93), but strong silencing is confined to the first 1-2 kb (94). Several mutations that strongly affect telomeric silencing (e.g., yku70Δ) have either no effect on HM loci or have an effect only with a partially debilitated HMR-E sequence, suggesting that HM loci are more strongly silenced than telomeres (88,89,95).…”