Triggering Gene Activity
Vikki M. Weake1and Jerry L. Workman1,*
1Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, USA
Recently, many of the enzymes responsible for the addition and removal of ubiquitin from the histones H2A
and H2B have been identified and characterized. From these studies, it has become clear that H2A and H2B
ubiquitination play critical roles in regulating many processes within the nucleus, including transcription ini-
tiation and elongation, silencing, and DNA repair. In this review, we present the enzymes involved in H2A and
H2B ubiquitination and discuss new evidence that links histone ubiquitination to other chromatin modifica-
tions, which has provided a model for the role of H2B ubiquitination, in particular, in transcription initiation
The Mechanism of Ubiquitination
The 76 amino acid protein ubiquitin is conjugated to substrate
proteins in a reaction involving three separate enzymatic activi-
ties (reviewed in Hochstrasser, 1996). Ubiquitin is first activated
by an ATP-dependent reaction involving a ubiquitin activating
enzyme (E1), followed by its conjugation via a thioester bond to
a cysteine residue in a ubiquitin-conjugating enzyme (E2). In
the final enzymatic step, ubiquitin is transferred from the E2 en-
a ubiquitin-protein isopeptide ligase (E3). E3 enzymes are often
characterized by the presence of a C3HC4 (RING) finger motif,
which binds zinc and is required for ubiquitin ligase activity. Sub-
strates can be mono- or polyubiquitinated; whereas polyubiqui-
tinationtargets proteins fordegradation viathe 26Sproteasome,
monoubiquitination generally acts as a tag that marks the sub-
strate protein to signal for a particular function. One well-charac-
terized example of this process is the monoubiquitination of his-
tones H2A and H2B. Early studies identified H2A as a target for
ubiquitination in higher eukaryotes, although this modification is
not detected in Saccharomyces cerevisiae (reviewed in Osley,
2006). Indeed, ubiquitinated H2A was originally considered
a unique histone-like chromosomal protein named A24. Around
the same time as the identification of H2A as a target for ubiqui-
tination, ubiquitinated H2B was also detected in mouse cells.
However, rather than being polyubiquitinated, it soon became
clear that only a single ubiquitin moiety is conjugated to both
H2A at Lys-119 (ubH2A) and H2B at Lys-120 (ubH2B) in mam-
mals (Osley, 2006). The ubiquitinated residue in H2B corre-
sponds to Lys-123 in S. cerevisiae, Lys-119 in Schizosaccharo-
myces pombe, and Lys-143 in Arabidopsis (Robzyk et al.,
2000; Sridhar et al., 2007; Tanny et al., 2007). Consistent with
a role in signaling, monoubiquitination is reversible; ubiquitin
can be removed from target substrates by a class of thiol prote-
ases known as ubiquitin-specific proteases (UBPs in yeast;
USPs in mammals) (reviewed in Nijman et al., 2005).
Rad6, the E2 ubiquitin conjugase that catalyzes H2B monoubi-
quitination, was first identified in S. cerevisiae (Robzyk et al.,
2000) (Table 1). Rad6 (Ubc2) was initially shown to catalyze
mono- and polyubiquitination of both H2A and H2B in vitro
(reviewed in Osley, 2006). However, studies in yeast using
a FLAG-tagged version of H2B coexpressed with an HA-tagged
ubiquitin (FLAG-H2B:HA-Ub) showed that Rad6 could monoubi-
quitinate only Lys-123 of H2B in vivo (Robzyk et al., 2000). Rad6
acts in conjunction with the E3 ubiquitin ligase Bre1 to monoubi-
quitinate H2B in vivo (Hwang et al., 2003; Wood et al., 2003a). In
an additional protein, Lge1, that is also required for H2B ubiqui-
taining the Rad6 homolog Rhp6, the Bre1 homologs Brl1 (Rfp2/
Spcc1919.15) and Brl2 (Rfp1/Spcc970.10c), and an additional
protein, Shf1, is required for H2B ubiquitination (Tanny et al.,
2007; Zofall and Grewal, 2007). Notably, Rad6 performs func-
tions distinct from H2B ubiquitination that are involved in the
DNA damage repair and protein degradation pathways and
require interactions with E3 enzymes other than Bre1, including
Rad18, Ubr1, and Rad5 (reviewed in Osley, 2006).
In mammals, there are two potential sequence homologs of
Rad6: HR6A and HR6B (Koken et al., 1991; Roest et al., 1996).
Lys-4 H3 methylation in rad6D yeast is rescued by expression
of either of the mouse Rad6 homologs (Sun and Allis, 2002).
However, HR6A and HR6B might act redundantly as Hrb6b-
knockout mice are viable, although male sterile, and have wild-
type levels of ubH2B (Baarends et al., 2007; Roest et al.,
1996). Another ubiquitin conjugase, UbcH6, can form a complex
in vitro with the human H2B E3 ubiquitin ligase that specifically
catalyzes monoubiquitination of nucleosomal H2B (Zhu et al.,
2005). However, the role of UbcH6 in H2B monoubiquitination
in vivo is less clear, and an interaction with the E3 ligase
(RNF20/40) in human cells has not been detected. Bre1 ho-
mologs have been identified in higher eukaryotes, including
Drosophila (Bray et al., 2005) and humans (Kim et al., 2005). In
humans, although both RNF20 and RNF40 share sequence
homology with Bre1, only RNF20 affects H2B ubiquitination in
cells (Kim et al., 2005; Zhu et al., 2005). There is evidence, how-
ever, that RNF20 and RNF40 form a complex in vivo (Zhu et al.,
Molecular Cell 29, March 28, 2008 ª2008 Elsevier Inc.
There are candidates for additional E3 enzymes mediating
H2B ubiquitination in human cells, for example Mdm2 (Minsky
and Oren, 2004). Mdm2 is a well-characterized negative regula-
tor of p53, and it ubiquitinates p53 and some of its cofactors (re-
viewed in Coutts and LaThangue, 2007). Supporting its potential
role in H2B ubiquitination, Mdm2 interacts with core histones
and ubiquitinates H2A and H2B in vitro, and Mdm2 overexpres-
sion increases the level of ubH2B in vivo. However, this ubiquiti-
nation does not seem to be specific for Lys-120 of H2B, as
mutation of both Lys-120 and Lys-125 of H2B is required to pre-
vent the increase in ubH2B caused by Mdm2 overexpression.
Notably, other E3ubiquitin ligases,suchasBRCA1,can catalyze
ubiquitination of both H2A and H2B in vitro, but the in vivo rele-
vance of these activities remains unclear (Chen et al., 2002; Mal-
lery et al., 2002; Xia et al., 2003).
UbH2B is a substrate for two ubiquitin-specific proteases in
S. cerevisiae: Ubp8 and Ubp10. The first of these, Ubp8, is a
component of the Spt-Ada-Gcn5-acetyltransferase (SAGA)
complex and deubiquitinates ubH2B in vitro and in vivo (Daniel
et al., 2004; Henry et al., 2003). SAGA integrity is required for
Ubp8 deubiquitination activity, as mutations disrupting the com-
plex, such as spt20D, or loss of Sgf11, which is required for the
association of Ubp8 with SAGA, increases the level of ubH2B
(Daniel et al., 2004; Henry et al., 2003; Ingvarsdottir et al.,
2005; Lee et al., 2005b; Powell et al., 2004). Recently, Ubp8
orthologs were identified in Drosophila and humans (Weake
et al., 2008; Zhang et al., 2008; Zhao et al., 2008). Drosophila
Nonstop and human USP22 reside within the SAGA and TFTC/
STAGA complexes, respectively, and require Sgf11 orthologs,
dSgf11/CG13379 and ATXN7L3, for their association with these
a component of the Sac3-Thp1 mRNA export complex (Rodri-
guez-Navarro et al., 2004), interacts with Ubp8 and Sgf11 and
is required for ubH2B deubiquitination (Kohler et al., 2006). The
Drosophila and human Sus1 orthologs E(y)2 and ENY2 are also
implicated in this process (Kurshakova et al., 2007; Zhao et al.,
The second ubH2B protease in yeast is Ubp10 (Dot4) (Emre
et al., 2005; Gardner et al., 2005). UBP10 deletion increases
the level of ubH2B similarly to ubp8D, but Ubp10 functions inde-
pendently of SAGA (Emre et al., 2005). Moreover, Ubp8 and
letion of both ubiquitin proteases results in a greater increase in
thelevelofubH2Bthaneither ofthesingle deletionsalone (Gard-
ner et al., 2005). Instead of functioning in transcription activation
like Ubp8, Ubp10 interacts with the silencing protein Sir4, is
enriched at silenced loci, and is important for Sir-mediated telo-
meric and rDNA silencing (reviewed in Osley, 2006). However,
Ubp10 function is not restricted to silenced regions, as muta-
tions in Ubp10 that specifically disrupt its silencing function,
but notits deubiquitination activity, still result in a global increase
in ubH2B (Gardner et al., 2005). Significantly, many more nonte-
lomeric genes show increased expression levels in the double
ubp10D; ubp8D deletion compared to either of the single dele-
tions, indicating that Ubp10 and Ubp8 might function redun-
dantly at some loci (Gardner et al., 2005). Phylogenetic analysis
suggests that the Drosophila homolog of Ubp10 might be
CG15817 (Weake et al., 2008). However, another ubiquitin-
specific protease in Drosophila, USP7, has been implicated in
Polycomb-mediated silencing and catalyzes deubiquitination
of ubH2B in vitro (van der Knaap et al., 2005). Loss of the Arabi-
dopsis H2B deubiquitinase SUP32/UBP26 increases global
levels of ubH2B and is associated with the loss of heterochro-
maticsilencing oftransgenes, areduction inthe levelof dimethy-
lated Lys-9 H3, and a reduction in promoter DNA methylation
(Sridhar et al., 2007). An additional ubiquitin protease, USP3,
has recently also been implicated in both ubH2B and ubH2A
deubiquitination in human cells (Nicassio et al., 2007).
Early Steps in Transcription Initiation and Elongation
Are Essential for H2B Ubiquitination
ingenetranscription activationandsilencing. Aninteresting con-
cept that has come out of these studies is crosstalk between
H2B ubiquitination and other histone modifications, including
histone H3 methylation. Some of the factors influencing H2B
ubiquitination have been identified, and they point to a series
of events involved in H2B ubiquitination during transcription
activation. In this model, Rad6 and Bre1 are initially recruited
to promoters through interactions with activators and then
Table 1. Enzymes Involved in H2A and H2B Ubiquitination/Deubiquitination in Different Organisms
H2B Ubiquitination H2B Deubiquitination H2A Ubiquitination H2A Deubiquitination
E2 E3 TranscriptionSilencingE2E3
S. cerevisiaeRad6 Bre1Ubp8 Ubp10
S. pombe Rhp6Brl1
Drosophila Dhr6Bre1 (CG10542)NonstopUSP7dRing (Sce)
USP21 USP3 ?
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associate with RNA polymerase II as it begins the process of
transcription. However, this promoter recruitment alone is not
sufficient for H2B ubiquitination, and stimulation of Rad6 ubiqui-
that associate with the elongating form of RNA polymerase II
Evidence supporting the activator-dependent recruitment of
Rad6 and Bre1 comes from studies showing that Bre1 interacts
with activators such as p53 in humans and Gal4 in yeast and is
recruited to promoters in an activator-dependent fashion inde-
pendently of Rad6 (Hwang et al., 2003; Kao et al., 2004; Wood
et al., 2003b) (Figure 1A). Rad6 requires Bre1 for its recruitment
to promoters, independently of ubH2B status (Kao et al., 2004;
Wood et al., 2003a). However, Rad6 recruitment is not sufficient
for H2B ubiquitination. Instead, many groups have shown that
components of the PAF complex are required for Rad6-medi-
ated H2B monoubiquitination (reviewed in Osley, 2006). PAF is
a complex comprising Paf1, Rtf1, Ctr9, Leo1, and Cdc73 that
associates with initiating and elongating RNA polymerase II
(reviewed in Shilatifard, 2006). Mutations in PAF components
such as Rtf1 and Paf1 cause a loss of ubH2B (Ng et al., 2003;
Wood et al., 2003b). In these mutants, Rad6 still localizes to
Figure 1. H2B Ubiquitination Requires Early Steps in Transcription Elongation
(A) The H2B ubiquitin ligase Bre1 interacts with acidic activators, such as Gal4, and recruits Rad6 and presumably its binding partner Lge1 to target promoters.
(B) Monoubiquitination of H2B by Rad6/Bre1 requires interactions with the PAF complex, the BUR complex, and the elongating form of RNA polymerase II that
has been phosphorylated on Ser-5 of the CTD by Kin28. The BUR complex phosphorylates Ser-120 of Rad6, which might stimulate its ubiquitin conjugase
(C) H2B ubiquitination is required for the recruitment of the Cps35 subunit of COMPASS, which activates di- and trimethylation of Lys-4 H3 by Set1. The 19S
proteasome and Ccr4-Not complex also link H2B ubiquitination to Lys-4 H3 methylation by mechanisms that are less clear.
Molecular Cell 29, March 28, 2008 ª2008 Elsevier Inc.
promoters, indicating that its recruitment is independent of the
PAF complex, although its catalytic activity requires the pres-
ence of PAF (Wood et al., 2003b; Xiao et al., 2005). Interestingly,
in vitro studies indicate that H2B ubiquitination requires tran-
scription itself as well as the presence of the PAF complex, be-
cause the appearance of ubH2B at the promoter on a chromatin
template in transcription studies required the addition of NTPs
(Pavri et al., 2006). Moreover, Rad6 associates with the elongat-
ing form of RNA polymerase II, and this association is disrupted
by loss of Bre1 or the PAF complex (Xiao et al., 2005). In the rtf1
mutant, although Rad6 is recruited to the promoter, it fails to
move into the open reading frame, indicating that the PAF com-
plex is required for Rad6 to travel with elongating RNA polymer-
ase II (Xiao et al., 2005).
Additional factors involved in transcription elongation are also
required for H2B ubiquitination (Figure 1B). For example, muta-
tions disrupting the Bur1/Bur2 cyclin-dependent protein kinase
complex (BUR complex) reduce H2B ubiquitination (Laribee
et al., 2005; Wood et al., 2005). This might result from a require-
ment of Bur1 and Bur2 for PAF complex recruitment (Laribee
et al., 2005; Wood et al., 2005). However, the BUR complex
also phosphorylates Rad6 at Ser-120, which might increase its
ubiquitination activity in vivo (Wood et al., 2005). Similarly, phos-
activity in vitro (Sarcevic et al., 2002). Thus, factors associated
with elongating RNA polymerase II appear to be required for
the activation of Rad6 ubiquitin conjugase activity. Furthermore,
transcription elongation itself might be required for H2B ubiquiti-
nation. During the early steps of transcription elongation, RNA
polymerase II is sequentially phosphorylated on its C-terminal
heptapeptide repeat sequences (CTD). The CTD is phosphory-
lated initially by Kin28 (CDK7 in humans) at Ser-5 and then by
Ctk1 (P-TEFb/CDK9 in humans) at Ser-2 (reviewed in Hartzog
and Tamkun, 2007). Kin28 loss, and the concomitant loss of
Ser-5 CTD phosphorylation, eliminates ubH2B, whereas loss of
Ctk1 has no effect (Xiao et al., 2005). This finding indicates that
some of the early steps that occur during transcription elonga-
tion are critical for H2B ubiquitination by Rad6 and Bre1.
H2B Monoubiquitination Is a Prerequisite for Lys-4 H3
and Lys-79 H3 Methylation
Many studies indicate thatRad6/Bre1-mediated H2B ubiquitina-
tion is required for Lys-4 H3 and Lys-79 H3 methylation in both
yeast and higher eukaryotes (reviewed in Osley, 2006). This his-
tone crosstalk appears to function unidirectionally: mutations af-
fecting H2B ubiquitination, including htbK123R, reduce the level
of these methylated H3 modifications; however, deletion of
either of the relevant methyltransferases, or mutation of the H3
methylation sites, has no reciprocal effect on H2B ubiquitination
in the components linking H2B ubiquitination to Lys-4 and Lys-
79 H3 methylation, although these downstream modifications
might ultimately result in different outcomes.
In yeast, Lys-79 H3 methylation is catalyzed by Dot1 (Ng et al.,
2002; van Leeuwen et al., 2002), and Lys-4 H3 methylation by
Set1, which functions as part of the COMPASS complex (MLL
complex in humans). COMPASS associates with the elongating
form of RNA polymerase II in a manner dependent on the Kin28
kinase and the PAF complex (reviewed in Shilatifard, 2006).
Although the interaction of COMPASS with RNA polymerase II
does not require the PAF component Rtf1, this PAF subunit is re-
quiredfor Rad6toassociatewithCOMPASS andfor subsequent
H2B monoubiquitination (Wood et al., 2003b).
H2B ubiquitination specifically affects di- and trimethylation of
(Dehe et al., 2005; Schneider et al., 2005; Shahbazian et al.,
2005). Furthermore, mutations in the BUR complex, which
reduce H2B ubiquitination, only affect trimethylation, and not di-
methylation, of Lys-4 H3 (Laribee et al., 2005). Thus, from these
studies, it is clear that the COMPASS methyltransferase com-
of Lys-4 H3. Recent work suggests a molecular mechanism
through which H2B ubiquitination might regulate COMPASS
methyltransferase activity (Lee et al., 2007). Lee and colleagues
showed that H2B ubiquitination controls the binding of the
Cps35 subunit of COMPASS, which is essential for methyltrans-
both the Cps35 subunit and the ability to di- and trimethylate
Lys-4 H3. In addition, Cps35 localization to the GAL1 promoter
is reduced significantly in rad6D or htbK123R strains. Notably,
the Lys-79 H3 methyltransferase Dot1 and Cps35 interact phys-
ically in coimmunoprecipitation studies, and methylated Lys-79
H3 is reduced in cps35D, indicating that Cps35, which is present
in multiple complexes inyeast, might also linkH2Bubiquitination
to Lys-79 H3 methylation.
The 19S regulatory complex of the proteasome also appears
to influence the events that occur between H2B ubiquitination
and Lys-4 and Lys-79 di- and trimethylation (Ezhkova and Tan-
sey, 2004). Specifically, two of the proteasomal ATPases, Rpt4
(Sug2) and Rpt6 (Sug1), are recruited to the promoters of genes
after activation, in a manner dependent on Rad6 and H2B ubiq-
uitination. Mutations affecting these ATPase subunits, such as
sug1-25, reduce global di- and trimethylated Lys-4 and Lys-79
H3. There is some evidence implicating the 19S regulatory
complex of the proteasome in transcription: specifically, the
19S complex is recruited independently of the 20S proteolytic
complex of the proteasome to the GAL1-10 SAGA-regulated
promoter by the Gal4 activator in yeast (Gonzalez et al., 2002).
Furthermore, the proteasomal ATPases Rpt4 and Rpt6 interact
genetically and physically with SAGA, and the loss of these
ATPases reduces global H3 acetylation and SAGA recruitment
to target promoters. In vitro studies show that the 19S regulatory
complex stimulates SAGA targeting by activators and its HAT
activity in an ATP-dependent manner (Lee et al., 2005a). How-
ever, SAGA recruitment and acetylation of Lys-9/14 H3 is inde-
pendent of H2B ubiquitination (Kao et al., 2004; Shukla et al.,
2006).Itisat presentunclear justhow the19Sproteasome might
be involved in linking H2B ubiquitination, Lys-4 H3 methyla-
tion, and ubH2B deubiquitination, and the effect of the 19S
proteasome on COMPASS recruitment and activity has yet to
Recent studies have also implicated the Ccr4-Not mRNA
production and processing complex in the steps between H2B
ubiquitination and Lys-4 H3 methylation (Laribee et al., 2007b;
Mulder et al., 2007). The Ccr4-Not complex contains at least
nine core subunits in yeast: Caf40, Caf130, Not 1–5, and the
Molecular Cell 29, March 28, 2008 ª2008 Elsevier Inc.
mRNA deadenylases Ccr4 and Caf1 (reviewed in Collart and
Timmers, 2004). This complex participates both in mRNA degra-
a repressive factor. Mutations in some of the components of this
Ccr4-Not complex, including not4, not5, and caf1, reduce trime-
thylated Lys-4 H3 but do not affect Lys-4 H3 mono- or dimethy-
lation or Lys-79 H3 methylation. Furthermore, components of
this complex show genetic and physical interactions with the
19S regulatory complex of the proteasome and the BUR com-
plex. Although some of the mutations in the Ccr4-Not complex
appear to reduce recruitment of the 19S regulatory complex to
and CTD phosphorylation of RNA polymerase II are unaffected.
There are conflicting data regarding the effect of mutations in
the Ccr4-Not complex on H2B ubiquitination and recruitment
of the PAF complex; currently it is unclear if this complex is
required for H2B ubiquitination (Laribee et al., 2007b; Mulder
et al., 2007). Intriguingly, the Not4 component of the complex
has a RING domain that is required for Lys-4 H3 trimethylation
and might potentially be an E3 ubiquitin ligase for as of yet
uncharacterized targets (Laribee et al., 2007b).
These data suggest a model linking H2B ubiquitination to Lys-
4 H3 methylation (Lee et al., 2007) (Figure 1C). In this model,
COMPASS associates withSer-5 phosphorylated RNA polymer-
ase II through interactions with the PAF complex. In the absence
of H2B ubiquitination, COMPASS lacks its Cps35 subunit and
can only monomethylate Lys-4 H3. Activation of Rad6 H2B ubiq-
uitination activity by the BUR complex and the association of
Rad6 with RNA polymerase II, the PAF complex, and COMPASS
follow this initial monomethylation. H2B ubiquitination enables
Cps35 recruitment, which activates COMPASS-mediated Lys-
4 H3 di- and trimethylation. This di/trimethylation requires com-
ponents of the 19S regulatory proteasome complex and the
Ccr4-Not mRNA processing complex. It is possible that these
components play some role in assisting recruitment or activation
of Cps35, or even of the SAGA HAT complex, which deubiquiti-
nates ubH2B via the ubiquitin-specific protease Ubp8 after
Lys-9/14 H3 acetylation. Notably, although Cps35 promoter
recruitment relies on H2B ubiquitination, it does not require the
COMPASS complex itself, indicating thatregulation of Cps35re-
cruitment might be an important step in linking H2B ubiquitina-
tion to Lys-4 H3 di- and trimethylation (Lee et al., 2007). A similar
pathway might be involved in linking Dot1-mediated Lys-79 H3
methylation to H2B ubiquitination, as many components re-
quired for Lys-4 H3 methylation also affect Lys-79 H3 methyla-
tion, with the exception of the Ccr4-Not complex (Laribee
et al., 2007b; Mulder et al., 2007).
Some data indicate that, in addition to its function in regulating
these initiation and early elongation steps in transcription, H2B
ubiquitination might be directly required for transcription by
RNA polymerase II. Evidence from in vitro transcription elonga-
tion experiments indicates that H2B ubiquitination might assist
the histone chaperone FACT in stimulating the passage of RNA
polymerase II through a nucleosomal template (Pavri et al.,
2006). The FACT histone chaperone complex can displace an
H2A/H2B dimer from a nucleosome core, enhancing transcrip-
tion elongation on chromatin templates (reviewed in Laribee
that the role of H2B ubiquitination in transcription elongation
might be independent of Lys-4 H3 methylation, asRNA polymer-
ase II occupancy is reduced at the 30end of coding sequences in
the htb1-K119R mutant (Tanny et al., 2007).
UbH2B Deubiquitination Is Required for Optimal
transcription elongation. Specifically Ubp8 is required for the re-
cruitment of Ctk1, the Ser-2 phosphorylated CTD form of RNA
polymerase II, and Set2 to the 50end of the open reading frame
sociated withthe lossof Ubp8, indicating that H2Bubiquitination
ase II. This interaction might be direct, as affinity-purified Ctk1
binds histones H2A and H2B but shows no interaction with
FLAG-H2B:HA-Ub.This finding suggeststhat H2Bubiquitination
might act as a checkpoint at which RNA polymerase II pauses
during early transcription elongation. In this model, H2B deubi-
quitination, mediated by Ubp8 within SAGA, is required for the
subsequent recruitment of the Ctk1 kinase, which phosphory-
gation (Figure 2). Furthermore, the authors showed that Ubp8
binds the elongating form of RNA polymerase II and travels into
the open reading frame with a subset of SAGA components dur-
gating RNA polymerase II indicates that there might be multiple
rounds of H2B ubiquitination and deubiquitination occurring dur-
ing transcriptionelongation. However,it isunclearwhetherthese
components associate with the same polymerase molecule
simultaneously. Are multiple rounds of H2B ubiquitination and
deubiquitination required as checkpoints to stall polymerase
and coordinate transcription with RNA processing events? In
higher eukaryotes, does H2A ubiquitination substitute for some
of the transcription checkpoint roles of H2B ubiquitination in
yeast? There are some indications that Ring1B and H2A ubiquiti-
tively transcribed in, ES cells (Stock et al., 2007). Studies on the
effect of Ubp8 and other deubiquitinases on RNA polymerase II
elongation through nucleosomal templates containing monoubi-
quitinated H2A or H2B are needed to clarify how these modifica-
tions function with regard to transcription elongation.
In humans, H2A ubiquitination is mediated by at least two differ-
ent E3 ubiquitin ligases, Ring1B and 2A-HUB, both of which are
associated with transcriptional silencing (Cao et al., 2005; Wang
et al., 2004; Zhou et al., 2008). Knockdown of Ring1B in human
cells largely reduces the level of ubH2A, indicating that this
enzyme is responsible for much of the H2A ubiquitination in
vivo (Wang et al., 2004). Ring1B (Ring2/Rnf2) associates with
three separate repressive complexes: Polycomb repressive
complex 1 (PRC1), E2F-6.com-1, and the FBXL10-BcoR com-
plex (Figure 3A). The first of these, PRC1, contains three RING
domain subunits, Ring1B (Ring2/Rnf2), Ring1A (RING1), and
Molecular Cell 29, March 28, 2008 ª2008 Elsevier Inc.
Bmi-1, and has E3 ubiquitin ligase activity specific to histone
H2A (Cao et al., 2005; Wang et al., 2004). PRC1 contains an ad-
ditional protein, human polyhomeotic 2 (HPH2) (Wang et al.,
2004). Although there are three potential RING domain E3 ubiq-
uitin ligases in PRC1, only Ring1B possesses in vitro E3 ligase
activity specific for H2A (Cao et al., 2005; Wang et al., 2004).
However, other subunits within PRC1, such as Bmi-1, strongly
stimulate the Ring1B E3 ubiquitin ligase activity (Cao et al.,
2005; Li et al., 2006; Wei et al., 2006). Furthermore, Ring1A
can substitute for Ring1B with regard to X inactivation in mice
(de Napoles et al., 2004). PRC1 and ubH2A colocalize on the
inactive X chromosome (Xi) in mouse, and RNAi-mediated
knockdown of Ring1B and Ring1A depletes ubH2A from Xi (de
Napoles et al., 2004; Fang et al., 2004). In flies and humans,
ubH2A localizes to the promoters of Polycomb-target genes,
including the Hox genes, in a Ring1B-dependent manner (Cao
et al., 2005; Wang et al., 2004; Wei et al., 2006).
Loss of H2A ubiquitination appears to have no effect on other
modifications involved in silencing such as Lys-27 H3 or Lys-9
H3 methylation, as knockdown of SUZ12, which reduces Lys-27
Figure 2. UbH2B Deubiquitination Is Required for Later Stages of Transcription Elongation
(A) In the absence of the ubH2B deubiquitinase Ubp8, persistent ubH2B inhibits recruitment of the Ctk1 kinase.
(B) Ubp8, within SAGA, deubiquitinates ubH2B, enabling Ctk1 recruitment and phosphorylation of Ser-2 within the CTD of RNA polymerase II.
(C) Deletion of the ubiquitin ligase Bre1 and the subsequent lack of ubH2B restores Ctk1 recruitment and Ser-2 phosphorylation even in yeast lacking Ubp8.
Molecular Cell 29, March 28, 2008 ª2008 Elsevier Inc.
and ubH2A localization at silenced promoters (Cao et al., 2005).
However, crosstalk between ubH2A and Lys-4 H3 methylation
was recently observed in studies on the H2A deubiquitinase
USP21 (Nakagawa et al., 2008). Specifically, ubH2A inhibits
MLL3-mediated di- and trimethylation of Lys-4 H3, repressing
transcription initiation, but not elongation, in vitro.
In addition to their role in the PRC1 complex, Ring1B and
Ring1A are also components of the E2F-6.com-1 repressive
complex that is involved in the silencing of E2F- and Myc-re-
sponsive genes in quiescent cells (Ogawa et al., 2002; Sanchez
et al., 2007). This complex contains factors including E2F-6,
Mga, Max, DP-1, HP1g, MBLR, h-l(3)mbt-like protein, YAF2,
and the Eu-HMTase1 (KMT1D) and NG36/G9a (KMT1C) Lys-9
H3 histone methyltransferases (Ogawa et al., 2002). It has not
yet been determined whether Ring1B within the E2F-6.com-1
repressive complex is able to ubiquitinate H2A.
Recently, a third complex containing Ring1B and Ring1A was
identified by using a proteomics approach (Gearhart et al., 2006;
Sanchez et al., 2007). The FBXL10-BcoR complex contains ad-
ditional proteins, including YAF2, CK2a, Skp1, HP1g, Cbx8,
tion of NSPC1, but not KDM2B, stimulates Ring1B-mediated
ubiquitination of H2A in HEK293 cells, indicating that the
FBXL10-BcoR complex might ubiquitinate H2A in vivo (Sanchez
et al., 2007). KDM2B is a nucleolar protein that demethylates
trimethylated Lys-4 H3 and represses the transcription of ribo-
somal RNA genes, but it is unclear yet whether H2A ubiquitina-
tion is involved in this repression (Frescas et al., 2007).
Figure 3. H2A Ubiquitin Ligases Are
Associated with Repressive Complexes
of three different repressive complexes: PRC1
Hox gene transcription; E2F6.com-1 methylates
Lys-9 H3 and binds E2F- and myc-response ele-
ments; and the FBXL10-BcoR complex contains
the Lys-4 H3 demethylase KDM2B and putative
additional proteins (dashed lines) and represses
(B) The 2A-HUB H2A ubiquitin ligase associates
with the NCoR/HDAC1/3 repressive complex and
inhibits FACT recruitment and transcription elon-
gation. Conversely, the 2A-DUB ubH2A deubiqui-
tinase associates with the PCAF/KAT2B coactiva-
tor and is required for gene activation at a subset
of chemokine gene promoters, potentially by
enhancing FACT recruitment.
Ring1B does not appear to be the sole
E3 ubiquitin ligase specific for H2A in
mammalian cells. Recent work identified
a novel H2A ubiquitin ligase: 2A-HUB/
hRUL138 (Zhou et al., 2008). 2A-HUB
associates with the N-CoR/HDAC1/3
complex at the promoters of a subset of
chemokine genes where it represses
transcription via inhibition of RNA poly-
merase II elongation (Figure 3B). Specifi-
cally, 2A-HUB-mediated H2A ubiquitina-
tion inhibits recruitment of the Spt16 subunit of FACT to these
genes. 2A-HUB knockdown, which reduces the level of ubH2A
at target promoters, enhances FACT recruitment and RNA poly-
merase II CTD phosphorylation, indicating that ubH2A represses
ing is intriguing, given that H2B ubiquitination appears to facili-
illustrating the significant differences between the roles of H2A
and H2B ubiquitination (Pavri et al., 2006).
Other potential ubiquitin ligases specific for H2A have been
uitination in vivo is unclear. A non-RING domain containing E3
ubiquitinligaseLASU1,whichpossesses analternative HECTdo-
main, was isolatedasa testis-specificubiquitin ligaseactivitythat
ubiquitinates H2A (Liu et al., 2005). LASU1 ubiquitinates H2A in
vitro in the presence of the UBC4-1 and UBC4-testis-specific E2
conjugases (Liu et al., 2005; Rajapurohitam et al., 1999). Despite
the identification of several E3 enzymes specific for ubiquitination
of H2A, the E2 enzyme responsible for H2A ubiquitination has yet
to be identified. Structural studies on the Ring1B/Bmi1 complex
in interactions between Ring1B and Bmi1 (Li et al., 2006). Several
E2 enzymes, including UbcH5a, b, c, or UbcH6, can all assist
Ring1B activity in vitro, and it is possible that these might act
redundantly in vivo (Buchwald et al., 2006; Li et al., 2006).
relate to the finding that ubH2A enhances the binding of the
linker histone H1 to reconstituted nucleosomes in vitro (Jason
Molecular Cell 29, March 28, 2008 ª2008 Elsevier Inc.
et al., 2005). Furthermore, mononucleosomes purified from
K119R H2Alack histone H1,indicating that H2Adeubiquitination
might cause the dissociation of linker histones from core nucle-
osomes (Zhu et al., 2007). This idea is consistent with the struc-
ture of the nucleosome in which the C terminus of H2A appears
to interact with linker histones (Luger et al., 1997). Thus, it is pos-
sible that ubH2A contributes directly to transcriptional repres-
sion by regulating higher-order chromatin structure, in addition
to inhibiting Lys-4 H3 methylation.
Monoubiquitination of the Histone Variant H2A.Z
Recent work in mammalian cells indicates that H2A.Z is monou-
biquitinated (ubH2A.Z) by the E3 ubiquitin ligase Ring1B at Lys-
120 or Lys-121 (Sarcinella et al., 2007). Furthermore, ubH2A.Z is
enriched on the inactive X chromosome (Xi) in female cells,
whereas unmodified H2A.Z is excluded from this chromosome
for its deposition on the inactive X chromosome but might rather
be a consequence of PRC1 activity on this chromosome.
Another H2A variant, macroH2A1.2, is subject to monoubiquiti-
nation at Lys-115, but the biological role of the ubiquitination is
not clear (Chu et al., 2006).
Intriguingly, in both S. cerevisiae and S. pombe, mutations di-
rectly affecting H2B ubiquitination are synthetically lethal with
mutations in the histone variant H2A.Z (pht1/htz1) (Hwang
et al., 2003; Tanny et al., 2007). It is not yet known whether
ubH2B affects H2A.Z deposition by the Swr1 complex and
how or if this might be involved in transcription activation.
Recently, three major deubiquitinases specific for ubH2A were
identified: Ubp-M, 2A-DUB, and USP21 (Joo et al., 2007; Naka-
gawa et al., 2008; Zhu et al., 2007). There is some evidence that
an addition deubiquitinase, USP3, also deubiquitinates ubH2A in
be involved in H2A deubiquitination came from earlier studies
showing that it possessed H2A deubiquitination activity in vitro
and that transient transfection of Ubp-M in human cells depleted
osome in vitro and that knockdown of Ubp-M increases ubH2A
levels in vivo. Consistent with the role of H2A ubiquitination in
Interestingly, H2A deubiquitination by Ubp-M appears to be
required for cell-cycle progression. Ubp-M is essential for Ser-10
H3 phosphorylation mediated by the Aurora B kinase and is
required for chromosome segregation during mitosis (Joo et al.,
2007). This finding fits with historical observations that Ser-10 H3
phosphorylation and ubH2A levels inversely correlate during the
phorylated and dephosphorylated, potentially by the cdc-2/cyclin
Furthermore, an enzymatically inactive form of Ubp-M fails to dis-
throughout metaphase and anaphase (Cai et al., 1999).
tion at a subset of promoters (Zhu et al., 2007). 2A-DUB contains
a JAMN/MPN+ domain, which can catalyze the hydrolysis of
ubiquitin chain isopeptide bonds. Knockdown of 2A-DUB by
siRNA in cell culture increases the global levels of ubH2A. 2A-
DUB interacts with the histone acetyltransferase p300/CBP-as-
sociated factor (PCAF/KAT2B) and preferentially deubiquitinates
hyperacetylated nucleosomes in vitro (Figure 3B).
USP21 is also involved in transcription initiation and can deu-
biquitinate ubH2A in vitro (Nakagawaet al.,2008).Thisdeubiqui-
tination relieves the repression of transcription initiation caused
by the ubH2A-mediated inhibition of Lys-4 H3 methylation.
Although recent studies implicate the H2B deubiquitinase
USP22 in deubiquitination of ubH2A in vitro (Zhao et al., 2008),
it has not been demonstrated that USP22 as part of SAGA has
any involvement in ubH2A deubiquitination in vivo.
Histone Ubiquitination and DNA Repair
tional nucleotide excision repair (Bergink et al., 2006). The ap-
histone H2AX (gH2AX), but phosphorylation is not required for
H2A ubiquitination. Similarly to H2AX phosphorylation, however,
H2A ubiquitination after DNA damage relies on the DNA damage
signaling kinase ATR (ATM and Rad3 related) (Bergink et al.,
2006). Several different E3 ubiquitin ligases, including Ring1B,
DDB1-CUL4ADDB2, and RNF8, have been implicated in DNA
damage-induced H2A ubiquitination (Bergink et al., 2006; Kape-
tanaki et al., 2006; Mailand et al., 2007). However, it remains
unclear which of these ubiquitin ligases is responsible for the
majority of H2A ubiquitination at sites of DNA damage. The
most recently characterized E3 enzyme, RNF8, is recruited to
sites of DNA damage through interactions with phosphorylated
MDC1, which itself binds directly to gH2AX. The accumulation
of conjugated ubiquitin and ubH2A at DNA lesions requires
RNF8, and recombinant RNF8 shows moderate polyubiquitina-
tion activity on H2A in vitro (Mailand et al., 2007). However,
a role for RNF8 in ubiquitination of H2AX at DNA lesions might
also be possible; RNF8 is functionally associated with an E2
ubiquitin conjugase, UBC13, and together these can catalyze
ubiquitination of gH2AX (Huen et al., 2007; Ikura et al., 2007;
Kolas et al., 2007; Wang and Elledge, 2007). Although the ap-
pearance of RNF8, UBC13, and ubiquitin conjugates are all re-
quired for the recruitment of proteins involved in later stages of
the response to DNA damage, it remains unclear how ubH2A
and ubH2AX are involved in the recruitment of these factors
(Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007).
Histones were first discovered to be ubiquitinated over a quarter
of a century ago. It is clear from the wealth of studies conducted
ification. Conjugation of a single ubiquitin moiety to histone H2A
results in significantly different outcomes when compared to the
addition of ubiquitin to H2B. Correspondingly, there are distinct
enzymes mediating the addition and removal of ubiquitin from
Molecular Cell 29, March 28, 2008 ª2008 Elsevier Inc.
the different histones. Whereas H2A ubiquitination may be con-
sidered as a repressive mark, it is clear that H2B ubiquitination
has roles both in transcription activation and in silencing. Al-
though H2A and H2B ubiquitination perform separate functions,
modifications and other chromatin effectors. One common
theme arising from the characterization of the enzymes involved
in H2A and H2B ubiquitination is the involvement of histone
crosstalk, particularly Lys-4 H3 di- and trimethylation, in mediat-
ing the effects of histone ubiquitination. In a broad sense, H2B
and H2A modifications often seem to have opposing effects:
H2B ubiquitination is required for Lys-4 H3 methylation, which
in turn is inhibited by H2A ubiquitination. Furthermore, whereas
H2B ubiquitination appears to assist FACT in stimulating RNA
polymerase II during transcription elongation, 2A-HUB-medi-
ated H2A ubiquitination inhibits recruitment of FACT and tran-
scription elongation at a subset of genes. Another common
thread in histone ubiquitination involves the association of en-
plexes involving multiplecatalytic activities.Itisclear thattheas-
sociation of these enzymes with additional proteins is essential
for their specificity and targeting, as demonstrated by the pro-
miscuity of many of these enzymes in vitro. Notably, both the
ubH2B and ubH2A deubiquitinases Ubp8 and 2A-DUB associ-
ate with histone acetyltransferase enzymes and are involved in
transcription activation. Might the mechanisms through which
these two complexes enhance transcription be more similar
than it might have originally seemed? Is there any relationship
or interaction between H2A and H2B ubiquitination during tran-
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that the lack of detectable H2A ubiquitination in S. cerevisiae
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higher eukaryotes. It will be of interest to determine whether
some of the silencing roles of ubH2B deubiquitination in particu-
lar are conserved in mammalian cells as they appear to be in
Drosophila. Despite a quarter of a century’s worth of studies on
histone ubiquitination, manyunanswered questions remain about
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