Methylation of H3 K4 and K79 is not strictly dependent on H2B K123 ubiquitylation

Abstract

Covalent modifications of histone proteins have profound consequences on chromatin structure and function. Specific modification patterns constitute a code read by effector proteins. Studies from yeast found that H3 trimethylation at K4 and K79 is dependent on ubiquitylation of H2B K123, which is termed a "trans-tail pathway." In this study, we show that a strain unable to be ubiquitylated on H2B (K123R) is still proficient for H3 trimethylation at both K4 and K79, indicating that H3 methylation status is not solely dependent on H2B ubiquitylation. However, additional mutations in H2B result in loss of H3 methylation when combined with htb1-K123R. Consistent with this, we find that the original strain used to identify the trans-tail pathway has a genomic mutation that, when combined with H2B K123R, results in defective H3 methylation. Finally, we show that strains lacking the ubiquitin ligase Bre1 are defective for H3 methylation, suggesting that there is an additional Bre1 substrate that in combination with H2B K123 facilitates H3 methylation.

Figure 1.

The C-terminal tail of histone H2B contains a PIKK consensus site, which is not required for survival after DNA damage. (A) A cartoon of H2B showing the amino acid sequence of the C-terminal tail, which forms an α helix (αC; Luger et al., 1997). The putative PIKK consensus site is underlined, and the sequences of mutations used in this study are listed with the mutated residues highlighted in gray. (B) Serial dilutions of midlog cultures of wild type (WT; JDY183), htb1-S125stop (H2B S125*; JDY184), hta1-S129* (H2A S129*; JDY5), or cells lacking both tails (H2A S129*/H2B S125*; JDY6) were plated onto media lacking or containing phleomycin (phleo). (C) Serial dilutions of midlog cultures of htb1-S128* (H2B T128*; JDY7) or hta1-S129* (H2A S129*; JDY5) or lacking both tails (H2A S129*/H2B T128*; JDY8) were plated onto media in the absence or presence of phleomycin or methylmethanesulfonate (MMS).

Figure 2.

Genetic analysis of the C-terminal tail of histone H2B. (A) Serial dilutions of midlog cultures of wild-type (WT; JDY183), htb1-S125stop (S125*; JDY184), htb1-S125A/S126A (SS; JDY185), htb1-K123R (K; JDY186), and htb1-K123R/S125A/S126A (KSS; JDY187) mutant strains were plated onto media in the absence or the presence of phleomycin (phleo). (B) Western blot analysis of extracts from strains containing untagged H2B (WT; JDY183), Flag-tagged H2B (F-WT; JDY630), Flag-tagged htb1-S125A/S126A (F-SS; JDY206), Flag-tagged htb1-K123R (F-K; JDY251), and Flag-tagged htb1-K1233R/S125A/S126A (F-KSS; JDY253) using anti-Flag antiserum. (C) Serial dilutions of midlog cultures of FY406-based strains as in A and Y131-based strains; wild type (YM5037) and htb1-K123R in Y133 (YM5038) mutant strains were plated onto media in the absence or the presence of phleomycin. (D) Growth curve of wild type (JDY183) and the htb1-K123R/S125A/S126A (KSS; JDY187) mutant strain. (E) Serial dilutions of midlog cultures of untagged and Flag-tagged H2B-containing strains were plated onto media in the absence or presence of phleomycin. The strains used were untagged H2B (WT; JDY183), Flag-tagged H2B (F-WT; JDY630), untagged htb1-S125A/S126A (SS; JDY185), Flag-tagged htb1-S125A/S126A (F-SS; JDY206), untagged htb1-K123R (K; JDY186), Flag-tagged htb1-K123R (F-K; JDY251), untagged htb1-K123R/S125A/S126A (KSS; JDY187), and Flag-tagged htb1-K123R/S125A/S126A (F-KSS; JDY253). (F) Growth curve of untagged and tagged H2B-containing strains from E.

Figure 3.

Histone H3 trimethylation on K4 and K79 is not solely dependent on ubiquitylation of H2B K123. (A) Western blot analysis of the indicated strains using an antibody raised against trimethylated H3 K4 (H3K4me3), trimethylated H3 K79 (H3K79me3; the top band is a nonspecific cross-reacting species), or H3. FY406-based H2B mutant strains or Y131-based H2B mutant strains as in Fig. 2 C were compared. Strains lacking Dot1 (dot1Δ) or Set1 (set1Δ) were used as a control for antibody specificity. (B) Western blot analysis as in A of cell extracts from untagged (first, second, third, and fourth lanes) or Flag-tagged (fifth, sixth, seventh, and eighth lanes) H2B-expressing strains. (C) Western blot analysis of the strains in C using an antibody raised against trimethylated H3 K4 (H3K4me3), trimethylated H3 K79 (H3K79me3), or H3. (D) DNA damage hypersensitivity of Y131- or Y133-based strains in which the H2B-encoding plasmids were replaced with the constructs used in the FY406 strain background (Fig. 2 C). Serial dilutions of midlog cultures were plated onto media in the absence or the presence of phleomycin. (E) Serial dilutions of midlog cultures of wild-type (WT; W303α), bre1 (bre1Δ; JDY286), dot1 (dot1Δ; SKY2849), set1 (set1Δ; SKY2856), and dot1/set1 (dot1Δ/set1Δ; SKY2859) mutant strains were plated onto media in the absence or the presence of phleomycin.

Figure 4.

The Y133 strain carries a mutation in the genome that, in combination with htb1-K123R, results in hypersensitivity to phleomycin and loss of H3 trimethylation. (A) Serial dilutions of midlog cultures of Y131- or Y133-based strains and four independent segregants from the Y131 × Y131 cross or the Y131 × Y133 cross were plated onto media in the absence or the presence of phleomycin. All strains contain pJD173 (HTA1-htb1-K123R) as the sole source of H2B. (B) Western blot analysis of the strains in A using an antibody raised against trimethylated H3 K4 (H3K4me3), trimethylated H3 K79 (H3K79me3), or H3.

Figure 5.

The E3 ubiquitin ligase Bre1 is required for H3 K4 and K79 trimethylation. (A) Western blot analysis of wild-type (WT; JDY183), bre1 (bre1Δ; JDY193), dot1 (dot1Δ; SKY2849), and set1 (set1Δ; SKY2856) mutant yeast strains using an antibody raised against either trimethylated H3 K4 (H3K4me3), trimethylated H3 K79 (H3K79me3), or H3. (B) DNA damage hypersensitivity of strains lacking BRE1 combined with mutations in H2B. The strains used were wild type (WT; JDY183), bre1 (bre1Δ; JDY193), htb1-S125A/S126A (SS; JDY185), htb1-S125A/S126A/bre1 (SS bre1Δ; JDY195), htb1-K123R (K; JDY186), htb1-K123R/bre1 (K bre1Δ), and htb1-K123R/S125A/S126A/bre1 (KSS bre1Δ; JDY197).