Identification of a Hac1i-induced Factor that mediates transcriptional repression of URS1-controlled genes

The unfolded protein response (UPR) transcription factor Hac1i is best known for activating genes that restore endoplasmic reticulum proteostasis, but it also represses early meiotic genes (EMGs) in Saccharomyces cerevisiae via URS1 promoter elements. This repression depends on the Rpd3–Sin3 histone deacetylase complex (Rpd3L) yet does not involve direct Hac1i binding to URS1, implying the existence of Hac1i -induced effector proteins that act at these sites. The aim of this thesis is to define how Hac1i couples UPR activation to URS1-mediated repression and to establish experimental routes toward identifying such effectors.
Two complementary approaches were developed. First, a comparative proteomic strategy was used to probe Hac1i-dependent changes in Rpd3L composition. The canonical TAP tag was rebuilt into dominant drug-resistance backbones (natMX4, hphMX4, natNT2, hphNT1), enabling endogenous C-terminal tagging of Rpd3L subunits in prototrophic strains and supporting multi-locus engineering. TAP-tagged ASH1, DEP1, RXT2 and RPD3 strains were constructed and validated with URS1–lacZ reporters, confirming that TAP fusion preserved Hac1i-dependent repression. Tandem affinity purification of Rpd3L from wild-type, Hac1i-overexpressing and hac1Δ strains yielded partially enriched complexes and revealed a ~70 kDa species reduced in hac1Δ extracts, consistent with a Hac1i -dependent interactor, but low recovery and background contamination prevented definitive mass spectrometric identification.
Second, a genome-wide overexpression screen was established to identify dosage-sensitive repressors of an integrated URS1-containing IME2–lacZ reporter. Stable chromosomal reporters were generated in rme1Δ strains, an AB320/YEp13 genomic library was amplified and quality-controlled, and ~30,000 transformants were screened under sporulation conditions. Two independent clones reproducibly conferred a stably repressed reporter phenotype, indicating that the screen can recover candidate repressors, although plasmid instability and sequencing constraints precluded assignment of the underlying genes.
Overall, this work establishes a toolkit of genetic, biochemical and screening methods that links Hac1i to Rpd3L-mediated repression at URS1 and provides initial evidence for Hac1i-dependent changes in Rpd3L-associated factors and overexpression-sensitive repressors, laying the groundwork for future identification of the Hac1i -induced effector(s) that couple UPR signalling to chromatin-based control of meiotic entry