Figure 3
Derepression by Dot1 requires the histone acetyltransferase Gcn5. (A) Derepressor activity of Dot1 and Gcn5 in a wild-type strain (NKI5128 and NKI1088), a dot1 Δ strain (NKI5129 and NKI6020) or a gcn5 Δ strain (NKI5399 and NKI6018). Strains lacking endogenous Dot1 were grown at 37°C to suppress the silencing defect, (see Figure 2C). We found that Gcn5 had a more prominent role in the barrier than in the desilencing assay of Dot1. URA3 silencing in the dot1Δ strain at 37°C in the desilencing assay (NKI1088 background) was limited compared with URA3 silencing in the dot1Δ at 37°C in the barrier assay (NKI1084 background), which is the result of the telomeric context (see Additional file 2A). (B) URA3 expression (n = 2 +/- SEM) was determined by reverse transcriptase-PCR and normalized to SIR3 expression, which is expressed at similar levels as URA3 and at equal levels in wild-type cells and histone modifier mutants [24]. (C) WT, dot1 Δ and gcn5 Δ strains with a URA3 gene at its endogenous euchromatic location were grown on media with or without uracil (BY4702, NKI3006 and NKI1107). Growth on media lacking uracil requires activation of URA3 by Ppr1, which is not affected by the loss of Dot1 or Gcn5. (D) LexA, LexA-Dot1 and LexA-Dot1G401R were expressed from a high copy 2 μ plasmid (used for all experiments described here) in a wild-type strain (GCN5; NKI5128) or a gcn5 Δ strain (NKI5399), and compared with wild type strains expressing LexA fusion proteins from a single-copy CEN (centromere sequences) plasmid. Each LexA-tagged protein also contained a V5 tag, which was used for immunoblot detection. Pgk1 was used as loading control. (E) Barrier assay of the LexA-tagged proteins expressed from the plasmids described in (D).