Polyubiquitylation of histone H2B

Abstract

Covalent modification of histones by ubiquitylation is a prominent epigenetic mark that features in a variety of chromatin-based events such as histone methylation, gene silencing, and repair of DNA damage. The prototypical example of histone ubiquitylation is that of histone H2B in Saccharomyces cerevisiae. In this case, attachment of ubiquitin to lysine 123 (K123) of H2B is important for regulation of both active and transcriptionally silent genes and participates in trans to signal methylation of histone H3. It is generally assumed that H2B is monoubiquitylated at K123 and that it is this single ubiquitin moiety that influences H2B function. To determine whether this assumption is correct, we have re-examined the ubiquitylation status of endogenous H2B in yeast. We find that, contrary to expectations, H2B is extensively polyubiquitylated. Polyubiquitylation of H2B appears to occur within the context of chromatin and is not associated with H2B destruction. There are at least two distinct modes of H2B polyubiquitylation: one that occurs at K123 and depends on the Rad6-Bre1 ubiquitylation machinery and another that occurs on multiple lysine residues and is catalyzed by an uncharacterized ubiquitin ligase(s). Interestingly, these ubiquitylation events are under the influence of different combinations of ubiquitin-specific proteases, suggesting that they have distinct biological functions. These results raise the possibility that some of the biological effects of ubiquitylation of H2B are exerted via ubiquitin chains, rather than a single ubiquitin group.

Figure 1.

Histone H2B is polyubiquitylated. Yeast cells were engineered to express either His-tagged WT or mutant (K48R or K63R) nonremovable (G76A) ubiquitin, together with either myc-tagged (FGY1; lane 1) or HA-epitope–tagged H2B (FGY2; lanes 2–5). Cells were lysed, and HA-tagged proteins present in the total lysate (input) or His-purified (Ni-NTA) material were detected by WB. The position of free H2B is indicated, as is the position of the H2B–Ub conjugates. Note that the monoubiquitylated form of H2B–HA appears as a doublet in the input; we presume that this corresponds to H2B–HA that is monoubiquitylated with either endogenous Ub (bottom panel) or the slightly larger His-Ub.

Figure 2.

There are at least two distinct pathways of H2B polyubiquitylation. Ubiquitylation of the indicated HA-tagged H2B mutants was measured as described in the legend to Figure 1, except that H2B–HA was expressed from a centromeric plasmid in strain BY4742. (A) The K123R H2B mutant is polyubiquitylated. (B) Ubiquitylation at lysine 123 depends exclusively on Rad6–Bre1. The ubiquitylation status of four different H2B mutants—NK+, coreK+, K123+, and K0 (described in the text)—was determined in either WT cells (lanes 1–4), or congenic strains in which RAD6 (FGY3; lanes 5–7) or BRE1 (FGY4; lanes 8–10) were deleted. (C) H2B proteins carrying single lysine residues are polyubiquitylated. The ubiquitylation status of K123+, K111+, and K3+ H2B mutants was examined in WT (lanes 1–3), Δrad6 (lanes 4–6), and Δbre1 (lanes 7–9) cells. Note that a more sensitive form of detection was used in the experiment in C than in B, because of the lower absolute signal from the single lysine K111+ and K3+ mutants. For this reason, monoubiquitylated K123+ H2B is visible in the input in C (cf. lanes in B with lane 1 in C).

Figure 3.

Deubiquitylating enzymes differentially affect Ub-chain length at particular H2B lysine residues. Ubiquitylation of the indicated HA-tagged H2B mutants was measured as described in the legend to Figure 1, except that H2B–HA was expressed from a centromeric plasmid in BY4742. Note also that to detect the effects of deleting UBP8 and/or UBP10, we used a cleavable form of His-Ub in which residue G76 of Ub is intact (Yaglom et al., 1995). Four congenic yeast strains were analyzed: WT (BY4742; lanes 1–3), Δubp8 (FGY5; lanes 4–6), Δubp10 (FGY6; lanes 7–9), and Δubp8/Δubp10 (FGY7; lanes 10–12).

Figure 4.

Ubiquitylated H2B is associated with chromatin. Wild-type yeast cells (JR5-2A) were engineered by plasmid shuffle to express the indicated H2B-HA mutants (from a centromeric plasmid) as well as His-tagged G76A ubiquitin. Chromatin fractionation was then performed as described (Liang and Stillman, 1997). Whole cell extracts (WCE; lanes 1, 6, and 11) were subject to a low-speed centrifugation (Lo-SP) and separated into soluble material (SN; lanes 2, 7, and 12) and an insoluble pellet containing chromatin (PL; lanes 3, 8, and 13). The insoluble material was then treated with micrococcal nuclease (MN) to release mono- and polynucleosomes into the soluble fraction. These extracts were then subject to a second round of centrifugation to separate remaining insoluble material (PL; lanes 5, 10, and 15) from the solubilized nucleosomes (SN; lanes 4, 9, and 14). Nickel-affinity chromatography was then used to isolate ubiquitylated proteins in each fraction. H2B–HA in the Ni-NTA and input material, as well as Orc3 (control) in the input, were detected by WB.

Figure 5.

Ubiquitylation at lysine 123 of H2B can be essential for viability. Plasmid shuffle was used to express WT H2B—and the K123R and K123+ mutants—as the sole source of histone H2B in strains FGY8, FGY9, and FGY10 (Table 1). (A) Lysine 123 of H2B is required for full GAL gene induction, but is not sufficient. Yeast were grown in raffinose and either uninduced (Raf) or induced with galactose (Gal) for 60 min. RNA was collected and reverse-transcribed into cDNA, and levels of GAL10 and 25S rRNA cDNAs were determined by quantitative PCR. “GAL10 cDNA” is expressed relative to the 25S rRNA control. (B) The K123+ and Δbre1 mutations are synthetically lethal. The indicated H2B proteins were expressed in either WT (BRE1; FGY8) or Δbre1 (FGY9) cells, together with a WT H2B protein expressed from a plasmid bearing the URA3 gene. Yeast were struck onto media containing 5-fluoroorotic acid (FOA) to eliminate cells expressing WT H2B. The K123+ H2B mutant supports viability in the BRE1, but not Δbre1, cells. (C) No genetic interaction between K123+ and Δubp8. Assay performed as in B but with UBP8 (FGY8) and Δubp8 (FGY10) cells. (D) No genetic interaction between K123+ and Δubp10. Assay performed as in B but with UBP10 (FGY8) and Δubp10 (FGY11) cells. (E) The K123+ and Δubp8/Δubp10 mutations are synthetically lethal. Assay performed as in B but with UBP8/10 (FGY8) and Δubp8/Δubp10 (FGY12) cells.