Evidence for rapid evolution of silencers in budding yeasts.
Jimmy O. O. Sjöstrand, Andreas Kegel, Stefan Äström
Developmental Biology, Wenner-Grens Institut, Svante Arrheniusv 16,
Stockholm, 106 91, Stockholm
The cryptic mating type loci in Saccharomyces cerevisiae,
HMLalpha and HMRa, require flanking DNA sequences,
called silencers, to remain inactive. These are believed to act as a
nucleation point for the formation of heterochromatin. To learn more
about the architecture of a silencer we studied the silencing of
HMLalpha and HMRa in the related yeast
Kluyveromyces lactis. A 102bp minimal silencer fragment was
defined that was both necessary and sufficient for silencing of
HMLalpha. A saturation mutagenesis of the 102bp silencer revealed
three distinct regions (A, B, and C) that were important for silencing.
Recombinant K. lactis Reb1p could bind the silencer in vitro, and
point mutations in the B-box abolished both Reb1p binding and silencer
function. Furthermore, strains carrying temperature sensitive alleles of
the REB1 gene derepressed HMLalpha at the non permissive
temperature. The A-box was similar to a Ume6p binding site, which
recruits the Rpd3p histone deacetylase in S. cerevisiae. The
K. lactis RPD3 gene was identified and rpd3 strains had
better silencing than wild type strains. Currently, we are trying to
elucidate if the UME6 and ISW2 genes play any role in
HMLalpha silencing. Our data indicate that different proteins
bind to K. lactis silencers than to S. cerevisiae
silencers. We suggest that the evolution of silencers is rapid in
budding yeasts and discuss the similarities and differences between
silencers in Saccharomyces and Kluyveromyces.
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