Dot1 promotes H2B ubiquitination by a methyltransferase-independent mechanism

Abstract

The histone methyltransferase Dot1 is conserved from yeast to human and methylates lysine 79 of histone H3 (H3K79) on the core of the nucleosome. H3K79 methylation by Dot1 affects gene expression and the response to DNA damage, and is enhanced by monoubiquitination of the C-terminus of histone H2B (H2Bub1). To gain more insight into the functions of Dot1, we generated genetic interaction maps of increased-dosage alleles of DOT1. We identified a functional relationship between increased Dot1 dosage and loss of the DUB module of the SAGA co-activator complex, which deubiquitinates H2Bub1 and thereby negatively regulates H3K79 methylation. Increased Dot1 dosage was found to promote H2Bub1 in a dose-dependent manner and this was exacerbated by the loss of SAGA-DUB activity, which also caused a negative genetic interaction. The stimulatory effect on H2B ubiquitination was mediated by the N-terminus of Dot1, independent of methyltransferase activity. Our findings show that Dot1 and H2Bub1 are subject to bi-directional crosstalk and that Dot1 possesses chromatin regulatory functions that are independent of its methyltransferase activity.

Figure 1.

Genetic interactions between Dot1 overexpression and loss of SAGA-DUB suggest a common function of Dot1 and Ubp8. (A) Strains expressing endogenous Dot1 (WT), no Dot1 (dot1Δ), high levels of Dot1 (P_TDH3-DOT1) or high levels of catalytically inactive Dot1 (P_TDH3-DOT1-G401R) were crossed to an array of ∼1400 mutant strains and examined for fitness. (B) Fitness score (S) of mutants most affected by overexpression of Dot1 where S > 0 represents better growth than expected and S < 0 worse growth than expected. (C) Images of plates as an example of the colony fitness defect of the ubp8Δ strain. (D) Validation of the negative genetic interactions between ubp8Δ and overexpression of Dot1 from a galactose-inducible GAL1 promoter on a multicopy (2 μ) plasmid (+) or using an empty vector control (-).

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.

Figure 3.

Effects of Dot1 overexpression and loss of Ubp8 on H2BK123ub1 in chromatin. (A) ChIP-qPCR analysis of H2BK123ub1 relative to H2B in transcribed regions in bre1Δ, WT, ubp8Δ and Dot1-G401A overexpression under the control of the strong TDH3 promoter (mean and individual data points of two biological replicates). (B) Metagene plots of H2BK123ub1 ChIP-seq in WT, ubp8Δ and Dot1-G401A showing the average H2BK123ub1 pattern around the transcription start site. Colored lines represent five different groups based on gene expression level, from high (group 1) to low (group 5) expression. (C) Heatmaps of read-depth normalized H2BK123ub1 ChIP-seq counts showing the H2BK123ub1 signal in the five different gene expression groups indicated in B. Genes within each subgroup were ranked on gene length and centered on the gene midpoint.

Figure 4.

The N-terminus of Dot1 is necessary and sufficient to promote H2Bub1. (A) Schematic representation of the yeast Dot1 protein and different mutants used in this study. (B) Deletion of the Dot1 N-terminal part (DOT1-ΔN-1) abolished the effect of Dot1 on H2Bub1 without altering the expression level of constitutively overexpressed Dot1. (C) Inducible overexpression in galactose media of the N-terminus alone (Dot1-ΔC) was sufficient to promote H2Bub1. The lane with DOT1-ΔC-terminal originates from the same blot. The pan-Dot1 antibody was raised against full-length Dot1, but preferentially recognizes the N-terminus (Supplementary Figure S4). (D) Quantification of the immunoblot shown in (C) (mean and individual data points of two biological replicates). (E) A model for the mutual crosstalk between Dot1 and H2Bub. The catalysis of (de)modifying reactions is indicated by a black arrow or bar-headed line. Stimulation is visualized by white arrows. In short, H2BK123ub1 promotes H3K79 methylation by Dot1 and the N-terminus of Dot1 promotes ubiquitination of H2BK123. The latter stimulation is independent of deubiquitination by Upb8 and the recruitment of Bre1/Rad6 by the Paf complex, and is thus likely to directly act on ubiquitination by Bre1.