Figure 2.

Overexpression of Dot1 promotes H2BK123ub1 independent of deubiquitinases Ubp8 and Ubp10. (A) Immunoblot analysis showing monoubiquitination of H2B in WT, ubp8Δ and ubp10Δ strains with or without overexpression of Dot1 using an inducible GAL1 promoter on a multicopy (2 μ) plasmid. H2Bub1 can be detected by the slower migrating band using H2B antibodies. The asterisk indicates a non-specific band, shown as a loading control. The Dot1 antibody was used to show the Dot1 overexpression. (B) Quantification of the immunoblot shown in (A) (H2Bub1/H2B relative to WT) and biological replicates thereof (N = 3 +/- SD). Statistical significance as determined by an unpaired t-test is indicated by the asterisks (*P < 0.1, **P < 0.05). (C) Immunoblot analysis of strains harboring either WT H2B or H2B-K123R, with either overexpressed Dot1 or Dot1-G401R using an inducible GAL1 promoter on a multicopy (2 μ) plasmid. A site-specific antibody demonstrates that the site of ubiquitination is H2B-K123. Pgk1 was used as a loading control. (D) Immunoblot analysis of WT and bre1Δ cells with or without overexpression of Dot1 using an inducible GAL1 promoter on a multicopy (2 μ) plasmid. (E) Dot1 overexpression does not affect the expression level of Bre1, the main factor responsible for H2BK123ub1. An N-terminal FLAG tag was used to preserve the E3 activity. (F) Immunoblot analysis of strains expressing increasing amounts of Dot1 shows that Dot1 promotes H2BK123ub1 in a dose-dependent manner. (G) Quantification of the immunoblot shown in (F) (mean and individual data points of two biological replicates). (H) Suppression of synthetic sickness of Dot1-OE and ubp8Δ by an H2BK123R mutation. Dot1 was overexpressed in the strains indicated from a galactose-inducible GAL1 promoter on a multicopy (2 μ) plasmid (P_GAL1-DOT1) and an empty vector was used as a negative control (P_GAL1). Strains were spotted in a 10-fold dilution series and were pre-grown for 24 h in the carbon source indicated.