Analysis of H2B ubiquitylation in the genetically tractable yeast S. cerevisiae has been instrumental in revealing the extent to which this process can regulate gene activity. Exactly how ubiquitylation regulates H2B function is, however, unknown. To understand this mechanism, it is essential to understand the nature of how H2B is modified by ubiquitin. We used a directed method to examine the ubiquitylation status of H2B, and various mutants, in yeast cells. We found that, contrary to expectations, H2B is subject to extensive polyubiquitylation, both at the canonical K123 residue (which is assumed to be monoubiquitylated) and at multiple other sites within the protein. Ubiquitylation at K123 depends on the characterized Rad6–Bre1 pathway, whereas ubiquitylation at other sites occurs independent of Rad6 or Bre1. The existence of these multiple ubiquitylation events challenges the notion that a single Ub moiety is responsible for the biological activities of H2B–Ub and suggests that poly-Ub chains, perhaps attached to multiple sites on H2B, may influence its function.
Our studies have revealed two previously unrecognized types of H2B ubiquitylation: ubiquitylation at noncanonical lysine residues (i.e., not K123), and polyubiquitylation at lysine 123. Using “chain terminating” forms of Ub (), we have shown that most of these polyubiquitylation events involve K48-linked Ub chains and that there are multiple sites of Ub-chain formation within H2B. Analysis of single lysine H2B mutants reveals that most of the lysine residues within the protein can be subject to some ubiquitylation ( and data not shown), suggesting that there are either multiple pathways directing ubiquitylation at the various sites on H2B or that a smaller number of pathways can ubiquitylate H2B without targeting selected lysine residues. The latter scenario is particularly common in the Ub–proteasome system, where finding specific lysine residues required for Ub conjugation is the exception, rather than the rule (e.g., Crook et al., 1996
). It is important to note, however, that loss of Rad6 or Bre1 has little effect on the ubiquitylation status of NK+, coreK+, K111+, and K3+ mutants (), revealing that 1) Rad/Bre1 is fairly specific to K123 and 2) at least one other Ub-ligase must mediate ubiquitylation events at the other sites. It is also important to note that although the other Ub-ligase(s) may not have specificity toward particular lysine residues, the output of its activity could be very specific, because Ubp8 and Ubp10 appear to have different preferences for removing Ub from select lysine residues ().
What is the function of these noncanonical K48-linked polyubiquitin chains? Although K48-linked chains are typically implicated in proteolysis (Pickart and Fushman, 2004
), we think it is unlikely that these modifications direct H2B destruction. Histones, including H2B, are reported to be metabolically quite stable (Russev et al., 1975
). We have confirmed this result with bulk H2B–HA in our system (Supplemental Figure S2). This finding also suggests that the function of H2B polyubiquitylation is not linked to the degradation of excess histones (Gunjan and Verreault, 2003
). We favor the idea that K48-linked H2B polyubiquitylation plays a nonproteolytic, regulatory role in some aspect of chromatin function. This notion is supported by our finding that the ubiquitylated forms of the NK+ and coreK+ H2B mutants are associated with chromatin () and is analogous to what has been observed for the Met4 transcription factor, where limited K48-linked poly-Ub chains modulate Met4 function without altering its stability (Flick et al., 2004
; Flick et al., 2006
). It is possible that multiple Ub chains on H2B recruit Ub-dependent chaperones, such as Cdc48/p97 and the base of the 19S proteasome (Jentsch and Rumpf, 2007
), which can in turn alter chromatin structure. Recently, it was found that Cdc48/p49 can extract polyubiquitylated Aurora B kinase from chromatin (Ramadan et al., 2007
). By analogy, perhaps polyubiquitylation of H2B is important for mediating its eviction from chromatin as part of nonreplicative histone H2B exchange (Jamai et al., 2007
In addition to revealing ubiquitylation at noncanonical sites in H2B, our data also demonstrate that lysine 123 itself is subject to polyubiquitylation (). Intriguingly, both mono- and polyubiquitylation at K123 is dependent on RAD6/BRE1 (), revealing that the Rad6–Bre1 complex has selectivity for this residue and is the only ubiquitylation pathway that can target lysine 123. Given that many lysine residues on H2B can potentially serve as sites for Ub conjugation, the specificity between Rad6–Bre1 and K123 is striking and raises the issue of why other Ub-ligases fail to ubiquitylate K123. One possibility would be if the other Ub-ligases accessed a different pool of H2B than Rad6–Bre1, one in which K123 is not exposed in a context that is appropriate for ubiquitylation.
Regardless of how specificity between K123 and Rad6–Bre1 is established, these findings raise the distinct possibility that the previously described functions of H2B–K123 ubiquitylation are mediated by poly-Ub chains. This is an important realization, because poly-Ub chains may be able to interact with protein factors that cannot bind tightly to a single Ub moiety (Jamai et al., 2007
). As described above, poly-Ub chains could recruit Ub-dependent chaperones to chromatin, which in turn facilitate the known functions of K123 ubiquitylation. Indeed, we have previously found that H2B ubiquitylation is required for recruitment of 19S proteasomal ATPases to chromatin (Ezhkova and Tansey, 2004
) and that mutations in these ATPases result in loss of histone H3 (K4 and K79) methylation, which is itself a Rad6–Bre1/K123-dependent process. This finding has led us to propose that 19S proteins come to chromatin in a Ub-dependent manner and promote H3 methylation by altering local chromatin structure. One weakness in this model has been the fact that proteasome subunits have low affinity for mono-Ub (Deveraux et al., 1994
), making it difficult to imagine how monoubiquitylation of H2B at K123 could be linked to 19S recruitment. The demonstration that H2B is polyubiquitylated at this site makes a direct link between Rad6–Bre1-dependent ubiquitylation and 19S recruitment to chromatin more likely.
Our data also raise the possibility that transcriptional effects mediated by Ubp8 and Ubp10 could be a result of trimming poly-Ub chains, rather than deubiquitylating H2B entirely. It is difficult to dissect the relative contribution of mono- versus polyubiquitylation at lysine 123. Although the steady-state levels of polyubiquitylated H2B are considerably lower than the K123-monoubiquitylated form, this does not necessarily mean that H2B bearing a single Ub at K123 is the active species. Not only is ubiquitylation a highly dynamic process, but H2B polyubiquitylation may be spatially or temporally restricted, making it possible that H2B is extensively ubiquitylated at some sites in the genome where it the modification exerts its function. At this point, we do not know whether Rad6–Bre1 catalyzes K123-linked poly-Ub chains, or whether it primes H2B for polyubiquitylation by another ligase. We have found, however, that the E4, Ufd2, which is involved in Ub-chain elongation (Koegl et al., 1999
), is not required for polyubiquitylation at this site (data not shown), and we note that Rad6 can polyubiquitylate H2B in vitro (Sung et al., 1988
), suggesting that perhaps Rad6–Bre1 are sufficient for driving Ub-chain formation at K123.
Our genetic data () show that, in the presence of the K123+ form of H2B, disruption of either the Bre1 Ub-ligase or the Ubp8/Ubp10 deubiquitylating enzymes blocks yeast cell viability. This result strongly suggests that both H2B ubiquitylation and
deubiquitylation can be essential, supporting the notion that cycling of H2B ubiquitylation is a critical part of histone function (Emre and Berger, 2004
). Whether this phenomenon reflects cycling of total K123 ubiquitylation, or essential trimming of the poly-Ub chain on K123, however, remains to be determined.
Are histones polyubiquitylated in other contexts? Early studies identified polyubiquitylated forms of both H2A and H2B (Davie et al., 1987
), and found that both mono- and polyubiquitylated H2B are preferentially localized to transcriptionally active chromatin domains (Nickel et al., 1989
; Davie et al., 1991
). It is thus possible that poly-Ub chains broadly regulate histone function in eukaryotic cells. The demonstration of H2B polyubiquitylation in a genetically tractable model organism should permit future analyses of the role these polyubiquitylation events play in control of chromatin dynamics.