Figure 5.

Ubiquitylation at lysine 123 of H2B can be essential for viability. Plasmid shuffle was used to express WT H2B—and the K123R and K123+ mutants—as the sole source of histone H2B in strains FGY8, FGY9, and FGY10 (Table 1). (A) Lysine 123 of H2B is required for full GAL gene induction, but is not sufficient. Yeast were grown in raffinose and either uninduced (Raf) or induced with galactose (Gal) for 60 min. RNA was collected and reverse-transcribed into cDNA, and levels of GAL10 and 25S rRNA cDNAs were determined by quantitative PCR. “GAL10 cDNA” is expressed relative to the 25S rRNA control. (B) The K123+ and Δbre1 mutations are synthetically lethal. The indicated H2B proteins were expressed in either WT (BRE1; FGY8) or Δbre1 (FGY9) cells, together with a WT H2B protein expressed from a plasmid bearing the URA3 gene. Yeast were struck onto media containing 5-fluoroorotic acid (FOA) to eliminate cells expressing WT H2B. The K123+ H2B mutant supports viability in the BRE1, but not Δbre1, cells. (C) No genetic interaction between K123+ and Δubp8. Assay performed as in B but with UBP8 (FGY8) and Δubp8 (FGY10) cells. (D) No genetic interaction between K123+ and Δubp10. Assay performed as in B but with UBP10 (FGY8) and Δubp10 (FGY11) cells. (E) The K123+ and Δubp8/Δubp10 mutations are synthetically lethal. Assay performed as in B but with UBP8/10 (FGY8) and Δubp8/Δubp10 (FGY12) cells.