Figure 2
Dot1 is a methyltransferase-dependent and -independent derepressor. (A) Outline of Dot1 deletion mutants showing the N terminus (white), the methyltransferase domain (black) and the H4 binding domain (grey). The G401R mutation (*) abolishes the catalytic activity of Dot1. All fusion proteins contained LexA and a V5 tag at the N terminus. (B) Protein and H3K79 methylation levels of Dot1 mutants described in (A) were determined in a dot1 Δ strain lacking LexA operators (NKI5070). H3K79 methylation levels were determined of a wild-type strain or a dot1Δ strain (NKI5376 and NKI5378) expressing LexA or LexA-Dot1. Protein expression and H3K79 methylation were determined by immunoblot analysis using a V5 antibody and antibodies specific for H3K79 mono-, di- and trimethylation or the histone H3 C terminus. (C) Barrier (NKI5128 and NKI5129; left) and desilencing assay (NKI5376 and NKI5378; right) in the presence and absence of DOT1. Deletion of DOT1 results in reduced silencing that could be bypassed at 37°C [24, 25]. Both LexA-Dot1 and LexA-Dot1G401R were still able to disrupt URA3 silencing in a dot1 Δ strain at 37°C, showing that derepressor activity does not require involvement of endogenous Dot1. Note that URA3 silencing is not completely lost in dot1 Δ at 30°C (NK5378). This is caused by enhancement of telomeric silencing by the TRP1 gene distal to URA3 (see Additional file 2A). (D) Derepressor activity of Dot1G401R and Dot1 deletion mutants at telomere VIIL (strains NKI5240, NKI5128 and NKI5376). Serial dilutions as presented before were quantified and plotted as bar graphs. (E) Derepressor activity of Dot1G401R and Dot1 deletion mutants at HML α (strains YXB85-n and YQY09). Growth on 5FOA observed for LexA-Dot1G401R, LexA-Dot1172-582 and LexA-Dot11-237 when targeted to LexA operators was caused by colonies that were uracil auxotrophs, which most likely represent URA3 mutants. (F) Derepressor activity of Dot1 and Dot1 mutants at the native chromosomes XIL, XVR and XVIL as described previously [82] (NKI2229, NKI2230 and NKI2231). (G) Dot1 and Dot1 mutants were expressed in dot1Δ strains with or without LexA operators, expressing wild-type histone H3 or histone H3 with K79 mutated to arginine (H3K79R), to determine whether K79 methylation is required for the methyltransferase-dependent derepressor activity (NKI6045, NKI6047, NKI6049 and NKI6051). Strains were grown at 37°C to enhance URA3 silencing.