Histone H2BK123 monoubiquitination is the critical determinant for H3K4 and H3K79 trimethylation by COMPASS and Dot1

Abstract

Histone H2B monoubiquitination by Rad6/Bre1 is required for the trimethylation of both histone H3K4 and H3K79 by COMPASS and Dot1 methyltransferases, respectively. The dependency of methylation at H3K4 and H3K79 on the monoubiquitination of H2BK123 was recently challenged, and extragenic mutations in the strain background used for previous studies or epitope-tagged proteins were suggested to be the sources of this discrepancy. In this study, we show that H3K4 and H3K79 methylation is solely dependent on H2B monoubiquitination regardless of any additional alteration to the H2B sequence or genome. Furthermore, we report that Y131, one of the yeast histone H2A/H2B shuffle strains widely used for the last decade in the field of chromatin and transcription biology, carries a wild-type copy of each of the HTA2 and HTB2 genes under the GAL1/10 promoter on chromosome II. Therefore, we generated the entire histone H2A and H2B alanine-scanning mutant strains in another background, which does not express wild-type histones.

Figure 1.

Generation of antibodies specific to K123-monoubiquitinated H2B. (A) Development of polyclonal antibodies specific to monoubiquitinated H2B. Ubiquitinated H2B–specific antibodies generated in rabbit were affinity purified and characterized by Western blot analysis of whole cell extracts prepared from wild type (WT), H2BK123A, ubp8Δ, and rad6Δ. Both wild-type and ubp8Δ strains show the presence of monoubiquitinated H2B (24 kD and 23 kD, respectively); however, an increased amount of the ubiquitinated form of H2B was observed in the ubp8Δ strain as expected. No bands were detected in either H2BK123A or the rad6Δ strains, showing that our H2Bub antibody is capable of specifically recognizing ubiquitinated H2B in yeast. (B) Schematics of the plasmids used in this study. pSN888 was generated from pZS145 by deletion of the N-terminal Flag tag (Fg) from the HTB1 gene. Similarly, the Flag on H2B was removed in pRS315-HHT2-HHF2-HTA1-FlagHTB1 to generate H2B Flag-less strains.

Figure 2.

Di- and trimethylation of histone H3K4 and trimethylation of H3K79 are dependent solely on monoubiquitination of H2BK123. (A) Western blotting of whole cell extracts from strains transformed with a plasmid carrying wild-type (WT) H2B or mutant H2BK123 (K123A; K123R) either with or without an N-terminal Flag tag. Cell extracts were prepared from wild-type H2B or H2BK123 mutants in three different strain backgrounds and were subjected to SDS-PAGE and analyzed by Western blot analysis with antibodies to dimethyl H3K4, trimethyl H3K4, trimethyl H3K79 (αH3K4me2, αH3K4me3, and αH3K79me3), monoubiquitinated H2BK123 (αH2Bub), or Flag (αFLAG). An antibody against H3 (αH3) was used as a loading control. The calculated molecular masses of H2B, ubiquitinated H2B, and H3 are 14 kD, 23 kD, and 15 kD, respectively. The calculated molecular masses of Flag-tagged H2B and ubiquitinated, Flag-tagged H2B are 15 kD and 24 kD, respectively. White lines indicate that intervening lanes have been spliced out. (B) Western blotting of whole cell extracts from strains transformed with plasmid carrying wild-type or mutant H2BK123R in FY406 background.

Figure 3.

The H2A/H2B shuffle strain Y131 contains a galactose-regulated copy of HTA2-HTB2 genes on chromosome II. (A) Western blot analysis of whole cell extracts prepared from Flag-tagged wild-type H2B and Flag-H2BK123R grown under either glucose or galactose media. Cell extracts were subjected to SDS-PAGE and Western analysis with antibodies specific to H3K4me2, H3K4me3, H3K79me3, and Flag. An antibody against H3 (αH3) was used as a loading control. Triangles describe the increasing amounts of proteins loaded onto the gel. Letters D and G denote glucose and galactose, respectively. (B) Western analysis of whole cell extracts prepared from wild-type (WT) BY4742 (FM392) and its derivative rad6Δ, Flag-H2B (Y131 background), and Flag-H2BK123R (Y131 background). Cell extracts were subjected to SDS-PAGE and Western analysis with antibodies to trimethyl H3K4 (αH3K4me3), monoubiquitinated H2BK123 (αH2Bub), or Flag (αFLAG). An antibody against H3 was used as a loading control. The H2B monoubiquitination–specific antibody detected a faster migrating band, which is indicated by red arrows. This band represents an untagged version of H2B, which is only seen in Y131 when cells are grown in galactose-containing media. Blue arrows indicate the slower migrating Flag-tagged, monoubiquitinated H2B seen under both dextrose and galactose growth conditions only in wild-type cells and not H2BK123R. Black lines indicate that intervening lanes have been spliced out.

Figure 4.

In the Y131 strain, the GAL1/10 promoter is inserted between HTA2 and HTB2 on chromosome II. Schematic of the regions containing HTA2-HTB2 genes on chromosome II. Although this region was deleted and replaced with URA3 in the Y131 strain, a wild-type copy of HTA2-HTB2 controlled by the GAL1/10 promoter is present in this region.

Figure 5.

Generation of the entire H2A/H2B alanine-scanning collection in an FY406 background. (A) The H2A/H2B alanine-scanning library was generated as described in our previous study (Nakanishi et al., 2008). The complete removal of wild-type (WT) H2B was ensured by multiple rounds of 5-FOA selection and verified by sequencing. Each colony represents a strain expressing histones containing a single alanine substitution mutation of each of the residues of H2A and H2B. Red squares indicate the location of strains that are inviable in SD media containing 5-FOA (lethal mutants). For the key to the corresponding mutant strains within each plate, see Tables S2 and S3. (B) Western blot analysis of mutant strains in the FY406 background identified as defective for proper methylation of H3K4. Cell extracts prepared from each mutant strain were subjected to SDS-PAGE and analyzed by Western analysis with antibodies to dimethyl H3K4, trimethyl H3K4 (αH3K4me2 and αH3K4me3, respectively), monoubiquitinated H2BK123 (αH2Bub), or Flag (αFLAG). An antibody against H3 (αH3) was used as a loading control. White lines indicate that intervening lanes have been spliced out.