H2B Ubiquitylation Plays a Role in Nucleosome
Dynamics during Transcription Elongation
Alastair B. Fleming,1Cheng-Fu Kao,1,3Cory Hillyer,1Michael Pikaart,1,2and Mary Ann Osley1,*
1Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA
2Department of Chemistry, Hope College, Holland, MI 49423, USA
3Present address: Academia Sinica, Institute of Cellular and Organismic Biology, Taipai 115, Taiwan
The monoubiquitylation of histone H2B has been as-
sociated with transcription initiation and elongation,
but its role in these processes is poorly understood.
We report that H2B ubiquitylation is required for effi-
cient reassembly of nucleosomes during RNA poly-
merase II (Pol II)-mediated transcription elongation
in yeast. This role is carried out in cooperation with
the histone chaperone Spt16, and in the absence of
H2B ubiquitylation and functional Spt16, chromatin
structure is not properly restored in the wake of elon-
gating Pol II. Moreover, H2B ubiquitylation and Spt16
play a role in each other’s regulation. H2B ubiquityla-
tion is required for the stable accumulation of Spt16
at the GAL1 coding region, and Spt16 regulates the
formation of ubiquitylated H2B both globally and at
the GAL1 gene. These data provide a mechanism
linking H2B ubiquitylation to Spt16 in the regulation
of nucleosome dynamics during transcription elon-
A number of lysine modifications, including acetylation, methyl-
ation, and ubiquitylation, are placed on histones in defined pat-
(Kouzarides, 2007; Li et al., 2007a). In each case, the modifica-
tion machinery associates with the elongating form of Pol II,
and with the PAF complex mediating several of these interac-
tions (Krogan et al., 2003; Li et al., 2003; Ng et al., 2003; Squazzo
et al., 2002; Wood et al., 2003b; Xiao et al., 2005; Zhu et al.,
2005a, 2005b). Though the cotranscriptional modification of his-
tones has been linked to transcription elongation, the functions
of modified histones in this process are poorly understood.
One of the hallmarks of transcription elongation is the change
in nucleosome stability that occurs during the passage of Pol
II: nucleosomes are evicted in front of transcribing Pol II and rap-
idly reassembled in its wake (Bernstein et al., 2004; Dion et al.,
2007; Lee et al., 2004; Schwabish and Struhl, 2004). Eviction al-
lows Pol II to transcribe efficiently, and reassembly prevents the
useofcryptic transcription initiationsitesingenecodingregions.
Recent studies have implicated several histone modifications in
tion. Acetylation of H3 on lysines 9 and 14 by the Gcn5 histone
acetyltransferase has been connected to nucleosome eviction,
andmethylation ofH3onlysine 36bytheSet2methyltransferase
has a role in nucleosome reassembly (Carrozza et al., 2005;
Govind et al., 2007; Joshi and Struhl, 2005; Li et al., 2007b).
This raises the question of whether other histone modifications
also regulate nucleosome stability. One such modification is
the monoubiquitylation of the H2B C terminus (H2BK123ub1),
which is associated with active transcription both in yeast and
humans (Osley, 2006). H2B is transiently ubiquitylated at the
coding region of several highly expressed yeast genes though
the activities of the ubiquitin-conjugating enzyme Rad6 and the
ubiquitin ligase Bre1, which are tethered to elongating Pol II via
PAF (Henry et al., 2003; Hwang et al., 2003; Kao et al., 2004;
Robzyk et al., 2000; Wood et al., 2003a; Xiao et al., 2005). In the
absence of this modification, transcription of several inducible
genes is reduced, and cells become sensitive to 6-azauracil,
exhibiting slow growth or lethal phenotypes when combined
(Xiao et al., 2003). These latter data implicate H2BK123ub1 in
transcription elongation, although they do not indicate how this
modification functions during this process.
During transcription elongation, nucleosome dynamics are
also regulated by the activity of histone-binding chaperones,
which include Asf1, Spt6, and Spt16 (Belotserkovskaya et al.,
2003; Kaplan et al., 2003; Mason and Struhl, 2003; Schwabish
and Struhl, 2006). These chaperones tether specific histones
during nucleosome eviction or promote histone deposition after
the passage of transcribing Pol II. Like several histone-modifying
enzymes, the histone chaperones also track with elongating Pol
II and have been implicated in control of the elongation process
(Belotserkovskaya et al., 2003; Endoh et al., 2004; Hartzog et al.,
1998; Kaplan et al., 2000; Mason and Struhl, 2003; Saunders
et al., 2003; Schwabish and Struhl, 2006). This raises the possi-
tone chaperones cooperate to regulate nucleosome dynamics
during transcription elongation. A functional interaction between
H2BK123ub1 and the histone chaperone Spt16 was recently de-
of the evolutionarily conserved FACT complex, which stimulates
transcription elongation on nucleosomal templates both in vitro
and in vivo (Belotserkovskaya et al., 2003; Formosa et al.,
2001; Mason and Struhl, 2003; Orphanides et al., 1999; Witt-
meyer et al., 1999). H2BK123ub1 was found to cooperate with
Molecular Cell 31, 57–66, July 11, 2008 ª2008 Elsevier Inc. 57
human FACT to promote efficient Pol II elongation on a reconsti-
tuted chromatin-transcription template (Pavri et al., 2006).
Although FACT promotes disassembly of H2A-H2B dimers
bly during transcription elongation in vivo, the possible interplay
between Spt16 and H2BK123ub1 in the control of nucleosome
dynamics was not investigated (Belotserkovskaya et al., 2003;
Kaplan et al., 2003; Schwabish and Struhl, 2004). We asked if
H2BK123ub1 and yeast Spt16 also interact during transcription
elongation in vivo, and if these two factors affect nucleosome
stability during Pol II transcription. We found that H2BK123ub1
and Spt16 regulate histone deposition at the GAL1 gene in the
wake of elongating Pol II and suppress cryptic transcription initi-
ation sites in selected gene-coding regions. In addition, these
factors influence the kinetic properties of elongating Pol II.
Collectively, the data support a role for H2BK123ub1 and
Spt16 in the restoration of chromatin structure during the tran-
scription elongation process to promote accurate and efficient
Pol II transcription.
H2BK123ub1 Affects GAL-Regulated Transcription
Independently of Its Regulation of H3 Methylation
H2BK123ub1 mediates a transhistone regulatory pathway lead-
2002; Shahbazian et al., 2005; Sun and Allis, 2002). Because
both methylation marks have also been associated with active
transcription, we asked if the transcriptional phenotypes associ-
ated with the absence of H2B ubiquitylation resulted from the
failure to methylate these H3residues (Bernstein et al.,2002; Po-
kholok et al., 2005; Santos-Rosa et al., 2002; Schneider et al.,
2004). By comparing the phenotypes of htb-K123R to hht-K4A
or hhtK79A mutants, which have lost all forms of methylation
(mono, di, tri) on lysine 4 or 79, we conclude this is not necessar-
ily the case. First, an htb-K123R mutant, but not an hht-K4A
mutant, exhibits sensitivity to 6-azauracil (6-AU), a drug that
causes transcription elongation stress (Xiao et al., 2005). Sec-
ond, genome-wide transcription studies have revealed little
overlap in the gene expression profiles among htb-K123R, hht-
K4A, and hht-K79A strains (Mutiu et al., 2007; C.F.K. and
upon analysis of global gene expression patterns in mutants de-
fective for H2B ubiquitylation or H3K4 methylation in the fission
yeast S. pombe (Tanny et al., 2007). Third, we noted differences
between htb-K123R and hht-K4A strains in induction of the
mulated more slowly and to lower levels in htb-K123R and more
rapidly and to higher levels in hht-K4A compared with wild-type
(Figure 1A). Pol II occupancy followed a similar pattern, support-
ing the view that H2BK123ub1 has a role in transcription that
is independent of its role in controlling H3K4 methylation (Fig-
by an hht-K79A mutation, and GAL1 transcripts accumulated
to higher levels than wild-type in an hht-K4A hht-K79A double
mutant (see Figure S1 available online). Thus, H2BK123ub1
has specific functions in GAL-regulated transcription that are
independent of its regulation of H3 methylation.
Effect of H2BK123ub1 on Histone Occupancy at GAL1
During Pol II transcription, nucleosomes are removed in front of
elongating Pol II and rapidly reassembled in its wake (Schwabish
plays a role in nucleosome dynamics during transcription elon-
gation because the ubiquitin mark is also dynamically regulated,
such that it is formed and then turned over along the GAL1 open
reading frame (ORF) (Xiao et al., 2005). Moreover, the discovery
that H2B ubiquitylation and the histone chaperone Spt16 pro-
mote efficient Pol II elongation on a chromatin template in vitro
raised the possibility that these factors cooperate to control
nucleosome eviction in front of elongating Pol II (Pavri et al.,
in the wake of transcribing Pol II, suggesting that H2BK123ub1
might alsoactwith Spt16in thisaspect of nucleosome dynamics
(Mason and Struhl, 2003).
To investigate if H2BK123ub1 affects either nucleosome evic-
tion or reassembly during transcription in vivo and if it interacts
with Spt16 in this regard, we analyzed the phenotypes of single-
and double-htb-K123R and spt16 mutants. The double mutant
was viable but grew more slowly than either parent strain at
the permissive temperature for spt16. To examine the effects
of the mutations on nucleosome stability during transcription,
Figure 1. H2B Ubiquitylation and H3 Lysine 4 Methylation Differen-
tially Affect GAL-Regulated Transcription
Wild-type,htb-K123R,orhht-K4A cellscontainingaGAL-YLR454w genewere
grown at 30?in YP + 2% raffinose and then shifted to YP + 2% galactose.
(A) GAL-YLR454w transcript levels were measured by RT-qPCR and normal-
ized to ACT1 mRNA levels.
(B) Two hours after galactose induction, Pol II occupancy was measured by
ChIP at the promoter and 2 kb intervals across the GAL-YLR454w ORF. The
results represent the means from two to three independent experiments,
with bars depicting SEM.
H2B Ubiquitylation Regulates Nucleosome Dynamics
58 Molecular Cell 31, 57–66, July 11, 2008 ª2008 Elsevier Inc.
wild-type and mutant strains were induced with galactose and
shifted to 37?to inactivate the essential Spt16 protein, and his-
tone occupancy was measured across the GAL1 gene by ChIP
(Figure 2A). A genome-wide analysis found that transcription
rate inversely correlated with histone occupancy, with highly
transcribed yeast genes having lower levels of histones than
poorly transcribed genes (Bernstein et al., 2004; Dion et al.,
2007). Mutations that affected histone eviction led to higher
levels of histones in gene-coding regions, whereas those that al-
tered histone deposition resulted in low levels of both Pol II and
We expected that if H2BK123ub1 were required for eviction of
histones, then higher levels of H2B or H3 would be present at
the GAL1 ORF in htb-K123R relative to wild-type. However, in
the absence of H2BK123ub1, H2B occupancy was similar to
wild-type, whereas H3 occupancy was reproducibly, but not
significantly, higher (Figure 2A). Thus, during transcription elon-
gation at GAL1, H2BK123ub1 is dispensable for eviction of
histones that constitute both the H2A-H2B dimer and H3-H4
tetramer. Similar results were obtained when histone occupancy
was measured at the GAL1 promoter (Figure 2A), indicating that
scription initiation; however, in this study, we focus on the role of
H2B ubiquitylation in nucleosome dynamics during transcription
elongation. In spt16, GAL1 histone levels were also similar to
wild-type (Figure 2A), consistent with a previous report that
Spt16 does not regulate nucleosome eviction at the GAL1 ORF
(Schwabish and Struhl, 2004). However, the levels of H2B and
H3 were significantly lower at the ORF in the spt16 htb-K123R
double mutant compared with each single mutant (Figure 2A).
In contrast, there was no further reduction in histone levels at
the GAL1 promoter in the double mutant. Moreover, all of the ef-
fects on histone occupancy were observed only when the GAL1
gene was induced with galactose; under conditions of glucose
repression, the levels of H2B and H3 at the ORF were not signif-
icantly different between the wild-type and mutant strains
(Figure S2). Together, the data suggest that H2BK123ub1 and
Spt16 functionally interact during transcription elongation at
GAL1 to control the levels of histones associated with this
gene. As shown here, this interaction regulates nucleosome
reassembly in the wake of elongating Pol II.
H2BK123ub1 and Spt16 Functionally Interact
to Repress Cryptic Transcription Initiation
When histone and Pol II occupancies were compared at the
GAL1 ORF, we noted that the inverse histone-Pol II relationship
was lost in htb1-K123R and spt16, with low levels of H2B and
H3 associated with low levels of Pol II (Figures 2A and 2B). This
phenotype was consistent with the reported defect in nucleo-
some reassembly in spt16, and suggested that H2BK123ub1
Pol II (Schwabish and Struhl, 2004). Moreover, because even
lower levels of histones were present at GAL1 in the htb-K123R
spt16 doublemutant, H2BK123ub1 andSpt16 could functionally
interact to control histone deposition. To address this possibility,
weanalyzedthepatternof mRNAproduction orPolIIoccupancy
at several genes that contain cryptic transcription initiation sites
in their coding regions. These sites are normally occluded by nu-
cleosomes and not recognized by the Pol II initiation machinery.
However, when nucleosome reassembly is defective, the sites
can be revealed and Pol II will initiate transcription, leading to in-
creased Pol II occupancy in the 30coding region and elevated
production of 30transcripts. We examined the levels of internal
plan et al., 2003). In htb-K123R, increased levels of 30transcripts
were detected at two of three genes examined in cells grown at
30?(Figure 3A). Although not as dramatic, this effect was similar
to that observed in strains that lack H3K36 methylation (Fig-
ure 3A), which has been linked to the prevention of cryptic tran-
scription (Carrozza et al., 2005; Joshi and Struhl, 2005; Li et al.,
2007b). In support of the RNA data, Pol II occupancy was
Figure 2. H2B Ubiquitylation and Spt16 Functionally Interact to Con-
trol Histone and Pol II Occupancy at GAL1
Wild-type, htb-K123R, spt16, and spt16 htb-K123R cells were grown at 30?in
YP+2%raffinosefor2hr,shifted toYP+2%galactose for2hr,followed byan
additional 1 hr at 37?.
(A) Histone occupancy at the GAL1 promoter and 50ORF.
(B) Pol II occupancy at the GAL1 promoter, 50and 30ORF. Pol II occupancy
was normalized to wild-type, and histone occupancy was normalized to the
ORF-free region, INT-V. The results represent the means from three to four
tically significant difference between the double and single mutants as deter-
mined by Student’s t test (p < 0.05).
H2B Ubiquitylation Regulates Nucleosome Dynamics
Molecular Cell 31, 57–66, July 11, 2008 ª2008 Elsevier Inc. 59
prevents the utilization of internal initiation sites at a subset of
yeast genes, supporting a role for this histone modification in
the regulation of nucleosome reassembly.
Next, we monitored internal transcription at these same genes
plus the STE11 gene after strains were grown for 1 hr at 37?to
inactivate Spt16 (Figure 3C and Figure S3). In spt16, 30transcript
levels were elevated at all four genes, consistent with a broad
role for Spt16 in nucleosome reassembly (Kaplan et al., 2003).
Cryptic transcripts were also produced from YLR454w, FLO8,
and VPS72 in htb-K123R, although their levels were consistently
lower than those in spt16 (Figure 3C). In the spt16 htb-K123R
double mutant the levels of 30transcripts from three genes
that H2B ubiquitylation and Spt16 functionally interact to reas-
semble histones during transcription elongation, again at a sub-
set of genes (Figure 3C and Figure S3).
Chromatin Structure Is Altered in the Absence
of H2BK123ub1 and Spt16
Our findings suggested that H2BK123ub1 and Spt16 cooperate
to restore normal chromatin structure in the wake of elongating
Pol II, and in their absence, chromatin will be disrupted. To
address this possibility, we examined the micrococcal nuclease
(MNase) sensitivity of chromatin extracted from wild-type, htb-
K123R, spt16, and spt16 htb-K123R cells grown in glucose for
1 hr at 37?(Figure 4). The MNase patterns revealed that the nu-
cleosomal ladder ofbulkchromatinwasslightlylesspronounced
in spt16 compared with chromatin from wild-type or htb-K123R,
double mutant. These effects were more apparent when we ex-
amined the MNase sensitivity of two genes (FLO8 and STE11)
that exhibited enhanced cryptic transcription in the absence of
both Spt16 and H2BK123ub1. At each gene, the absence of
H2BK123ub1 further disrupted the significant loss of the nucleo-
someladder seeninspt16.To determineifthiseffectwasrelated
to transcription, we also investigated the MNase accessibility of
atranscriptionally silentlocus, HMLa, whoseexpression was not
derepressed in any of the mutant strains (data not shown). Over-
all, the nucleosome ladder at HMLa was similar among the four
strains, although chromatin from spt16 exhibited an increased
sensitivity to MNase that resulted in the recovery of less DNA.
However, this increased sensitivity was not further enhanced
by the additional loss of H2BK123ub1. This suggests that
Spt16, independent of H2BK123ub1, which is not enriched at
HMLa (Kao et al., 2004), could play a role in establishing or main-
taining HMLa chromatin structure. Together, the data support
the view that H2B ubiquitylation and Spt16 combine to maintain
chromatin structure primarily at transcriptionally active genes.
H2BK123ub1 and Spt16 Regulate the Rate
of Histone Deposition
To directly test the hypothesis that H2BK123ub1 and Spt16 reg-
ulate nucleosome reassembly during transcription elongation,
we measured the kinetics of histone deposition at two positions
on the GAL1 ORF following a galactose-to-glucose shift at 37?
(Figure 5). Glucose represses GAL1 transcription, and this assay
measures the rate of histone deposition during the last wave of
Pol II transcription that occurs after initiation has been blocked.
In wild-type cells, H2B and H3 deposition was complete by
16 min after glucose addition. However, in htb-K123R, the
deposition of both histones occurred more slowly and was not
complete until 30 min. In this strain, the final levels of restored
histones were similar to those in wild-type cells, although less
H2B was deposited at the 30end of the coding region, suggest-
ing a more important role for H2BK123ub1 in assembly of the
H2A-H2B dimer. These findings provide direct evidence that
H2BK123ub1 regulates the rate of nucleosome reassembly dur-
ing transcription elongation.
The deposition of H2B and H3 was severely compromised in
spt16, consistent with the enhanced internal initiation phenotype
of this mutant. However, measurement of histone deposition in
the htb-K123R spt16 double mutant yielded a surprising result.
Figure 3. H2B Ubiquitylation and Spt16 Functionally
Interact to Repress Internal Transcription Initiation
(A) Total RNA was extracted from wild-type, htb-K123R, or
hht-K36A cells grown at 30?in YPD. RT-qPCR reactions
were performed with primers corresponding to the 30and 50
coding regions of the YLR454w, FLO8, and VPS72 genes,
and the ratio of 30/50ORF products was calculated.
gene after 2 hr of galactose induction at 30?.
(C) Wild-type, htb-K123R, spt16, and spt16 htb-K123R cells
were grown in YPD for 1 hr at 37?. RT-qPCR reactions were
performed as described in (A). The data represent the means
from three to six independent experiments after normalization
to wild-type, with bars depicting SEM. Black asterisks high-
light a significant difference between the single mutants and
wild-type (*p < 0.05; ** p < 0.005), and the gray asterisk indi-
cates a significant difference between spt16 htb-K123R and
spt16 (p < 0.05) as determined by Student’s t test.
H2B Ubiquitylation Regulates Nucleosome Dynamics
60 Molecular Cell 31, 57–66, July 11, 2008 ª2008 Elsevier Inc.
In this strain, the rate of histone deposition was intermediate be-
indicating that the defect in nucleosome reassembly in spt16
could be partially suppressed by the absence of H2BK123ub1.
As shown in Figure 2A, H2B and H3 occupancy at GAL1 under
conditions of steady-state transcription was significantly lower
in the htb-K123R spt16 double mutant compared with each sin-
gle mutant. Thus, although the rate of histone deposition was in-
and Spt16 is inherently unstable or functionally aberrant, a con-
clusion supported by the enhanced accessibility of spt16 htb-
K123R chromatin to MNase digestion (Figure 4). Collectively,
chromatin structure during transcription elongation at GAL1.
H2BK123ub1 and Spt16 Regulate the Properties
of Elongating Pol II
We next asked if the nucleosome reassembly defects in htb-
K123R and spt16 single and double mutants affected Pol II elon-
gation rate orprocessivity. The rate of PolII elongation was mea-
sured in the same samples used to follow histone deposition,
and the data represent the release of Pol II from the GAL1
ORF, again during the last wave of Pol II transcription
(Figure 6A). The results suggest that Pol II was released from
the 50ORF slightly more slowly in htb-K123R and spt16 single
mutants than in wild-type, indicative of a possible defect in the
rate of Pol II elongation. However, an unexpected phenotype
again occurred in the htb-K123R spt16 double mutant. In this
strain, Pol II was released from the GAL1 template significantly
faster than in wild-type, an effect that was more pronounced at
the 50end of the coding region (Figure S4A). This effect could
be due to either decreased stability of Pol II or increased Pol II
elongation rate. We favor the latter mechanism based on mea-
strains under conditions of galactose activation (Figure 6B). In
htb-K123R or spt16, less Pol II was present at the 30ORF relative
Figure 4. H2B Ubiquitylation and Spt16 Interact to
Regulate Chromatin Structure
Wild-type, htb-K123R, spt16, and spt16 htb-K123R cells were
grown in YPD for 1 hr at 37?. Nuclei were incubated with
MNase over time, and the extracted DNA was analyzed by se-
quential Southern blot analysis using labeled probes corre-
sponding to the Ya region of the silent HMLa locus and the
transcriptionally active FLO8 and STE11 genes. The MNase
digestion pattern of total genomic DNA was visualized by
ethidium bromide staining. Exposure times for the HMLa
tween wild-type and htb-K123R, and spt16 and spt16 htb-
to the 50ORF, indicative of a processivity defect.
This is consistent with a slightly decreased rate of
Pol II elongation, which leads to a higher probability
that Pol IIwill fall offthe template. Nonetheless, full-
length GAL1 transcripts were still produced (Figure S4B), per-
haps due to a cellular quality-control system that removes non-
level of Pol II increased at the GAL1 30ORF in the htb-K123R
spt16 double mutant. Because the GAL1 gene does not harbor
an intenal transcription initiation site, we attribute this latter phe-
notype to an increased rate of Pol II elongation as the result of
a more ‘‘open’’ chromatin structure in this strain. Although the
data support this mechanism, we cannot eliminate other, more
complex explanations for the Pol II phenotype in the double
Regulatory Interplay between Spt16 and H2BK123ub1
In a reconstituted chromatin-transcription system, the monoubi-
quitylation of H2B depended on the Spt16-containing FACT
complex, and H2B ubiquitylation, in turn, facilitated the activity
of FACT to stimulate transcription elongation (Pavri et al., 2006).
To test if this regulatory interplay also occurs in vivo, we first ex-
amined the effect of spt16 on the global levels of H2BK123ub1.
Upon Spt16 inactivation, H2BK123ub1 levels were reduced
?5-fold, whereas the amount of unmodified H2B was only mod-
estly decreased (Figure 7A). A chromatin double-immunoprecip-
itation assay was then used to monitor the accumulation of
H2BK123ub1 at the GAL1 ORF after galactose induction at 37?
(Figure 7B). In wild-type cells, H2BK123ub1 accumulated tran-
siently at the GAL1 50and 30ORF, as previously reported (Kao
et al., 2004; Xiao et al., 2005). In spt16, H2BK123ub1 levels
were significantly reduced across the coding region. Spt16 was
also found to be required for accumulation of H2BK123ub1 at
the ORFs of three constitutively transcribed genes (Figure S5).
ing H2B ubiquitylation levels at a subset of yeast genes.
Next, we determined if H2BK123ub1 regulated the properties
of Spt16. Spt16 is recruited to gene coding regions in vivo with
the same pattern as elongating Pol II (Mason and Struhl, 2003).
We examined the role of H2BK123ub1 in Spt16 recruitment
by measuring the kinetics of Spt16 association with GAL1 in
wild-type and htb-K123R after galactose induction at 37?
(Figure 7C). Spt16 stably accumulated across the GAL1 ORF in
H2B Ubiquitylation Regulates Nucleosome Dynamics
Molecular Cell 31, 57–66, July 11, 2008 ª2008 Elsevier Inc. 61
wild-type but was not present at the promoter (data not shown).
Spt16 was recruited to the GAL1 ORF with the same kinetics in
htb-K123R, and it accumulated to higher levels at the 30end of
the ORF before and after galactose induction (Figure S6). How-
ever, in this mutant, Spt16 accumulated for only 60 min before
its levels declined (Figure 7C and Figure S6). This indicates that
H2BK123ub1 is dispensable for the initial recruitment of Spt16,
but is required for Spt16 to stably associate with the coding
region. The htb-K123R mutation also reduced the amount
of Spt16 associated with the ORFs of three constitutive genes
tein levels, neither of which decreased in the absence of
H2BK123ub1 (data not shown). Thus, the data indicate that
in vivo, H2BK123ub1 regulates the stability of Spt16 at gene-
coding regions, and Spt16 in turn regulates the level of H2B
The findings in this study suggest that H2B ubiquitylation plays
several roles during transcription elongation in vivo. First,
H2BK123ub1 regulates nucleosome reassembly at the GAL1
tionally interacts with the histone chaperone Spt16 to restore
proper chromatin structure during transcription elongation and
and Spt16, with H2BK123ub1 leading to stable accumulation of
ubiquitylation and Spt16 during activated transcription.
H2B Ubiquitylation and Spt16 Regulate Nucleosome
Reassembly Coupled to Elongating Pol II to Restore
Proper Chromatin Structure
Nucleosome eviction and reassembly are integral features of Pol
II transcription elongation, but how they are signaled during the
Figure 5. H2B Ubiquitylation and Spt16 Functionally Interact
during Histone Reassembly at GAL1
Wild-type, htb-K123R, spt16, and spt16 htb-K123R cells were grown at
30?in YP + 2% raffinose for 2 hr, shifted to YP + 2% galactose for 2 hr,
followed by an additional 1 hr at 37?. Glucose was added to 2%, and
H2B or H3 occupancy was measured at the GAL1 50and 30ORF. Occu-
pancies were normalized to the levels present in galactose-induced cells
(set as 1.0 for each strain). The results represent the average of three to
four independent experiments, with bars depicting SEM.
elongation process is just beginning to be revealed. Some of
thesesignalsinvolveparticular histonemodifications thatare
also regulated by the passage of Pol II, and they include H3/
H4 acetylation and H3 methylation. Acetylation of H3 was re-
cently shown to regulate eviction of the H3/H4 tetramer, and
H3K36 methylation has emerged as a key signal for nucleo-
some reassembly through its recruitment of the Rpd3S his-
tone deacetylase complex (Carrozza et al., 2005; Govind
et al., 2007; Joshi and Struhl, 2005; Li et al., 2007b). Our re-
search suggests that H2B ubiquitylation is also linked to his-
tone deposition during Pol II transcription elongation, with this
modification regulating the kinetics of nucleosome reassembly
Although H3K36 methylation and H2BK123ub1 are both con-
nected to nucleosome reassembly, these two modifications are
likely to signal different reassembly pathways. First, we have
shown that H2BK123ub1 functionally interacts with the histone
chaperone Spt16 to restore proper chromatin structure during
Pol II elongation, whereas H3K36 methylation has been re-
ported to act in conjunction with another histone chaperone,
Spt6 (Carrozza et al., 2005). Second, the internal transcription
initiation defect that occurs in the absence of H3K36 methyla-
tion is more pronounced and involves more genes than the de-
fect associated with the absence of H2BK123ub1 (Carrozza
et al., 2005; Li et al., 2007b). The reassembly pathway con-
trolled by H3K36me functions predominantly at long genes
that are infrequently transcribed (Li et al., 2007b). Though the
spectrum of genes subject to the H2BK123ub1/Spt16 reassem-
bly pathway is not known, the H2B modification is present
across the coding region of highly transcribed genes in yeast
and humans (Minsky et al., 2008; and C.F.K., M.A.O., and
S.B., unpublished data), and Spt16 has been reported to func-
tion at genes with strongly positioned nucleosomes (Jimeno-
Gonzalez et al., 2006). Together, the data suggest that H2B
ubiquitylation, along with Spt16, regulates nucleosome reas-
sembly at a subset of genes that might have a distinctive chro-
The findings also indicate that the role of H2BK123ub1 and
Spt16 in nucleosome reassembly is to restore proper chromatin
structure in the wake of elongating Pol II. This conclusion is sup-
ported by the phenotypes associated with the absence of both
factors. First, there was an enhanced disruption of chromatin at
genes where H2BK123ub1 and Spt16 cooperatively repress
cryptic transcription initiation. Second, Pol II elongation rate
was apparently accelerated at GAL1 in the double mutant, sug-
though the data cannot entirely eliminate a role for Spt16 and
H2B Ubiquitylation Regulates Nucleosome Dynamics
62 Molecular Cell 31, 57–66, July 11, 2008 ª2008 Elsevier Inc.
H2BK123ub1 in transcription-independent chromatin stability,
these factors appear to affect chromatin structure primarily
through transcription-coupled effects.
Regulatory Interplay between H2B Ubiquitylation
In addition to functional interactions, a regulatory interplay oc-
curs between H2BK123ub1 and Spt16, with Spt16 promoting
H2B ubiquitylation and H2BK123ub1 stabilizing Spt16 at
ORFs. H2B ubiquitylation is controlled by several factors that lo-
calize the Rad6/Bre1 enzymatic machinery to gene-coding re-
gions. A key factor in this localization is PAF, which tethers
Rad6/Bre1 to elongating Pol II through its own association with
polymerase (Krogan et al., 2002, 2003; Laribee et al., 2005;
Wood et al., 2003b; Xiao et al., 2005). As the spt16 mutation
does not alter the transcription of genes known to regulate
H2B ubiquitylation (unpublished observations), PAF recruitment
or stability is also a likely mechanism for the role of Spt16 in es-
tablishing H2BK123ub1 levels. This would also be consistent
with the reported physical interaction between Spt16 and PAF
(Krogan et al., 2002; Pavri et al., 2006; Squazzo et al., 2002). Al-
ternatively, the role of Spt16 could be indirect: in the absence of
Spt16, Pol II accumulates slowly in the GAL1 coding region be-
cause transcription initiation is inhibited (Mason and Struhl,
2003). Thus, the levels of H2BK123ub1, which are coupled to
Pol II, would correspondingly be reduced.
In the absence of H2BK123ub1, Spt16 was recruited to the
GAL1 coding region withnormal kinetics but did notstably accu-
mulate there (Figure 7C). This suggests that chromatin assem-
bled in the absence of H2BK123ub1 leads to the eventual disso-
ciation of Spt16. How could H2B ubiqitylation stabilize Spt16
accumulation? It might alter chromatin structure to enhance
Spt16 interaction with other components of the nucleosome,
or the ubiquitin moiety itself might directly interact with Spt16
anism, the lowering of Spt16 levels in gene-coding regions might
partially account for the presence of internally initiated tran-
scripts in htb-K123R. The Spt16-containing FACT complex is
also involved in the mechanism by which H2A ubiquitylation re-
the H2A modification blocks recruitment of FACT to a subset of
signals from both H2A and H2B ubiquitylation.
We interpret the combined regulatory and functional interac-
tions between H2BK123ub1 and Spt16 in the context of tran-
scription elongation at GAL1 (Figure S7). Upon association of
the H2B ubiquitylation machinery with elongating Pol II, the ma-
Ubp8, a protease that deubiquitylates H2B (Wyce et al., 2007;
Xiao et al., 2005). Together, the two sets of factors promote
the transient formation of H2BK123ub1 along the ORF. Spt16
also associates with the ORF and tracks with Pol II, perhaps
through its association with PAF. The presence of H2BK123ub1
in chromatin maintains high levels of Spt16, and in turn, high
levels of Spt16 promote full levels of H2B ubiquitylation. We
propose that this relationship represents a positive feedback
loop that contributes to the timely and efficient deposition of
nucleosomes as Pol II elongates.
Other chromatin assembly factors have also been reported to
regulate nucleosome reassembly at the GAL1 ORF, notably
Asf1, which plays a role in H3-H4 deposition (Kim et al., 2007;
Schwabish and Struhl, 2006). However, the more severe defect
in histone deposition in spt16 suggests that Spt16 plays a major
role in this process at GAL1. What, then, is the role of H2B ubiq-
uitylation in nucleosome reassembly and how is this role cou-
pled to the Spt16 pathway? H2BK123ub1 could in principle ac-
tivate another reassembly pathway, such as one regulated by
Asf1. However, we favor the model that H2BK123ub1 regulates
nucleosome reassembly at GAL1 primarily by promoting the
Spt16 deposition pathway, and that it has both positive and
negative roles in this process. H2BK123ub1’s positive role
would be to stabilize Spt16 association with the GAL1 ORF,
while its negative role would be to block other nucleosome as-
sembly pathways from operating. This model could account for
the phenotypes of both the htb-K123R single mutant and htb-
K123R spt16 double mutant. Histone deposition would occur
more slowly in the absence of H2BK123ub1, not only because
H2B ubiquitylation normally stabilizes Spt16 association with
the ORF, but through the derepression of alternate and less
Figure 6. H2B Ubiquitylation and Spt16 Regulate the Kinetic Proper-
ties of Elongating Pol II
(A) Pol II occupancy was measured at the GAL1 50ORF in the samples de-
scribed in Figure 5, and normalized to the level present in galactose-induced
cells (set as 1.0 for each strain). The results represent the average of three
to four independent experiments, with bars depicting SEM. Asterisks indicate
a statistically significant difference between wild-type and spt16 K123R dou-
ble mutant (p < 0.05) as determined by Student’s t test.
(B) Data from Figure 2B were used to determine the ratio of Pol II occupancy at
the GAL1 30ORF relative to the 50ORF. The results were further normalized to
Pol II occupancy in the wild-type strain. The asterisk represents a statistically
significant difference between spt16 htb-K123R and spt16 (p < 0.05). There
was no significant difference between wild-type and spt16 htb-K123R.
H2B Ubiquitylation Regulates Nucleosome Dynamics
Molecular Cell 31, 57–66, July 11, 2008 ª2008 Elsevier Inc. 63
efficient reassembly factors that compete with Spt16. When
both Spt16 and H2BK123ub1 are absent, these derepressed
reassembly pathways partially compensate for the absence of
Spt16 to deposit histones but are unable to assemble stable
chromatin structure, leading to decreased histone occupancy
at GAL1 and enhanced internal transcription initiation at other
genes. In this model, H2B ubiquitylation provides specificity
to the nucleosome reassembly process at GAL1 by ensuring
that chromatin structure is restored by the dominant histone
deposition factor, Spt16.
tion to nucleosome reassembly raises fundamental questions
about the origins of reassembled nucleosomes: Are these nucle-
osomes formed from a pool of evicted and modified histones or
from a de novo pool of unmodified (or premodified) histones?
Both the ubiquitylation and acetylation marks are formed and
then turned over during transcription elongation (Henry et al.,
2003; Li et al., 2007a; Xiao et al., 2005). The turnover of
H2BK123ub1 is dependent on the Ubp8 ubiquitin protease,
which also associates with elongating Pol II (Wyce et al., 2007).
modified with ubiquitin, then Ubp8 might remove the mark once
H2BK123ub1 is assembled into a nucleosome (Figure S7). The
presence of the ubiquitin modification on evicted H2B would
promote histone deposition, while the subsequent removal of
ubiquitin might signal that nucleosome reassembly has been
Figure 7. Regulatory Interplay between H2BK123ub1 and Spt16
YPD at 30?or shifted to 37?for 1 hr. Whole-cell lysates were separated on
15% acrylamide gels, and blots were probed with the indicated anti-
bodies. Representative results from three to four independent experi-
ments are shown.
(B) Wild-type, htb-K123R, and spt16 cells were grown at 30?in YP + 2%
raffinose for 1.5 hr and transferred to 37?for 30 min; galactose was then
added to 2%, and cells were kept at 37?. H2BK123ub1 levels were
measured by sequential ChIP. The data represent the average of two
(C) Spt16 ChIP was performed on the samples described in (B). The data
represent the means from two to three independent experiments, with
bars depicting SEM.
Strains and Growth Conditions
S. cerevisiae strains were in the W303 background (Table S1). Quadruple
histone knockout strains were obtained from David Allis and M. Mitchell
Smith. Strains were grown at 30?, except for those containing the temper-
ature-sensitive spt16-197 mutation, which were shifted from 30?to 37?for
1 hr to inactivate Spt16.
Chromatin Immunoprecipitation Analysis
ChIP was performed as described (Henry et al., 2003; Kao et al., 2004;
Xiao et al., 2005) using the following antibodies: Pol II (8WG16; Covance,
Inc., Princeton, NJ), H3 (ab1791; Abcam, Cambridge, England), Flag
CA), and Spt16 (generous gift of T. Formosa). With the exception of Pol II
antibody, antibodies were prebound to protein A or protein G sepharose
beads. DNAs were analyzed by real-time quantitative PCR (qPCR) using
a SYBR Green Master Mix (ABI, Foster City, CA) and ABI 9700 PCR ma-
chine. The IP/input ratio for GAL1 sequences was normalized to the IP/
input ratio of TEL-V(PolII,Spt16, H2BK123ub1)or INT-V(H2B,H3) sequences
as described previously (Xiao et al., 2005).
RT-qPCR and Protein Analyses
For RT-qPCR analysis, RNA was was extracted from cells by the hot phenol
method (Xiao et al., 2005), treated with DNase I (Promega, Fitchburg, WI),
and used to generate cDNA with a poly(dT) primer and SuperScript III Reverse
Transcriptase kit (Invitrogen). PCR reactions were performed in triplicate using
a SYBR Green Master Mix (ABI) and ABI 9700 PCR machine.
Protein lysates were prepared using a urea-SDS lysis buffer (Gardner et al.,
2005). Lysates (0.125–0.25 OD600) were electrophoresed on 15% acrylamide-
Tris HCl gels (Bio-Rad, Hercules, CA), and proteins were transferred to an Im-
mobilon filter (Millipore; Billerica, MA). The filters were hybridized with H3
(ab1791; Abcam), Flag (M2-F3165; Sigma-Aldrich), or G6PDH (A9521;
Sigma-Aldrich) antibodies, and developed with Immun-Star chemilumines-
cence reagent (Bio-Rad).
One liter of cells was grown in YPD at 30?to OD600of ?0.8, shifted to pre-
warmed YPD, and incubated for 1 hr at 37?. Nuclei were isolated and aliquots
described (Tsukuda et al., 2005). Extracted DNA was separated on 1.2% aga-
rose gels in TBE buffer and transferred to aGeneScreen filter (Millipore). Filters
The Supplemental Data include seven figures and one table and are available
H2B Ubiquitylation Regulates Nucleosome Dynamics
64 Molecular Cell 31, 57–66, July 11, 2008 ª2008 Elsevier Inc.
We thank Kevin Struhl, Fred Winston, David Allis, and M. Mitchell Smith for
strains and plasmids. Tim Formosa is gratefully acknowledged for his gift of
Spt16 antibody. Supported by NIH grant GM40118 to M.A.O. and a Leukemia
and Lymphoma Society Special Fellowship to C.F.K.
Received: July 6, 2007
Revised: January 15, 2008
Accepted: April 25, 2008
Published: July 10, 2008
Belotserkovskaya, R., Oh, S., Bondarenko, V.A., Orphanides, G., Studitsky,
V.M.,and Reinberg, D.(2003).FACT facilitates transcription-dependent nucle-
osome alteration. Science 301, 1090–1093.
Bernstein, B.E., Humphrey, E.L., Erlich, R.L., Schneider, R., Bouman, P., Liu,
J.S., Kouzarides, T., and Schreiber, S.L. (2002). Methylation of histone H3
Lys 4 in coding regions of active genes. Proc. Natl. Acad. Sci. USA 99,
Bernstein, B.E., Liu, C.L., Humphrey, E.L., Perlstein, E.O., and Schreiber, S.L.
(2004). Global nucleosome occupancy in yeast. Genome Biol. 5, R62.
Carrozza, M.J., Li, B., Florens, L., Suganuma, T., Swanson, S.K., Lee, K.K.,
Shia, W.J., Anderson, S., Yates, J., Washburn, M.P., et al. (2005). Histone
H3 methylation by Set2 directs deacetylation of coding regions by Rpd3S to
suppress spurious intragenic transcription. Cell 123, 581–592.
Dion, M.F., Kaplan, T., Kim, M., Buratowski, S., Friedman, N., and Rando, O.J.
(2007). Dynamics of replication-independent histone turnover in budding
yeast. Science 315, 1405–1408.
M.,and Shilatifard, A. (2002). Methylation of histone H3 byCOMPASS requires
ubiquitination of histone H2B by Rad6. J. Biol. Chem. 277, 28368–28371.
Duina, A.A., Rufiange, A., Bracey, J., Hall, J., Nourani, A., and Winston, F.
(2007). Evidence that the localization of the elongation factor Spt16 across
transcribed genes is dependent upon histone H3 integrity in Saccharomyces
cerevisiae. Genetics 177, 101–112.
Endoh, M., Zhu, W., Hasegawa, J., Watanabe, H., Kim, D.K., Aida, M., Inukai,
N., Narita, T., Yamada, T., Furuya, A., et al. (2004). Human Spt6 stimulates
transcription elongation by RNA polymerase II in vitro. Mol. Cell. Biol. 24,
Formosa, T., Eriksson, P., Wittmeyer, J., Ginn, J., Yu, Y., and Stillman, D.J.
(2001). Spt16-Pob3 and the HMG protein Nhp6 combine to form the nucleo-
some-binding factor SPN. EMBO J. 20, 3506–3517.
Gardner, R.G., Nelson, Z.W., and Gottschling, D.E. (2005). Ubp10/Dot4p reg-
ulates the persistence of ubiquitinated histone H2B: distinct roles in telomeric
silencing and general chromatin. Mol. Cell. Biol. 25, 6123–6139.
Govind, C.K., Zhang, F., Qiu, H., Hofmeyer, K., and Hinnebusch, A.G. (2007).
Gcn5 promotes acetylation, eviction, and methylation of nucleosomes in tran-
scribed coding regions. Mol. Cell 25, 31–42.
Hartzog, G.A., Wada, T., Handa, H., and Winston, F. (1998). Evidence that
Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II
in Saccharomyces cerevisiae. Genes Dev. 12, 357–369.
Henry,K.W.,Wyce,A.,Lo,W.S.,Duggan,L.J.,Emre,N.C., Kao, C.F., Pillus,L.,
Shilatifard, A., Osley, M.A., and Berger, S.L. (2003). Transcriptional activation
via sequential histone H2B ubiquitylation and deubiquitylation, mediated by
SAGA-associated Ubp8. Genes Dev. 17, 2648–2663.
Hwang, W.W., Venkatasubrahmanyam, S., Ianculescu, A.G., Tong, A., Boone,
C., and Madhani, H.D. (2003). A conserved RING finger protein required for
histone H2B monoubiquitination and cell size control. Mol. Cell 11, 261–266.
Jimeno-Gonzalez, S., Gomez-Herreros, F., Alepuz, P.M., and Chavez, S.
(2006). A gene-specific requirement for FACT during transcription is related
to the chromatin organization of the transcribed region. Mol. Cell. Biol. 26,
Joshi, A.A., and Struhl, K. (2005). Eaf3 chromodomain interaction with methyl-
ated H3–K36 links histone deacetylation to Pol II elongation. Mol. Cell 20, 971–
Kao, C.F., Hillyer, C., Tsukuda, T., Henry, K., Berger, S., and Osley, M.A.
(2004). Rad6 plays a role in transcriptional activation through ubiquitylation
of histone H2B. Genes Dev. 18, 184–195.
Kaplan, C.D., Morris, J.R., Wu, C., and Winston, F. (2000). Spt5 and Spt6 are
associated with active transcription and have characteristics of general elon-
gation factors in D. melanogaster. Genes Dev. 14, 2623–2634.
Kaplan, C.D., Laprade, L., and Winston, F. (2003). Transcription elongation
factors repress transcription initiation from cryptic sites. Science 301, 1096–
Kim, H.J., Seol, J.H., Han, J.W., Youn, H.D., and Cho, E.J. (2007). Histone
chaperones regulate histone exchange during transcription. EMBO J. 26,
Kouzarides, T. (2007). Chromatin modifications and their function. Cell 128,
Shilatifard, A., Buratowski, S., and Greenblatt, J.F. (2002). RNA polymerase II
elongation factors of Saccharomyces cerevisiae: a targeted proteomics ap-
proach. Mol. Cell. Biol. 22, 6979–6992.
Krogan, N.J., Dover, J., Wood, A., Schneider, J., Heidt, J., Boateng, M.A.,
Dean, K., Ryan, O.W., Golshani, A., Johnston, M., et al. (2003). The Paf1 com-
plex is required for histone H3 methylation by COMPASS and Dot1p: linking
transcriptional elongation to histone methylation. Mol. Cell 11, 721–729.
Laribee, R.N., Krogan, N.J., Xiao, T., Shibata, Y., Hughes, T.R., Greenblatt,
J.F., and Strahl, B.D. (2005). BUR kinase selectively regulates H3 K4 trimethy-
lation and H2B ubiquitylation through recruitment of the PAF elongation com-
plex. Curr. Biol. 15, 1487–1493.
Lee, C.K., Shibata, Y., Rao, B., Strahl, B.D., and Lieb, J.D. (2004). Evidence for
nucleosome depletion at active regulatory regions genome-wide. Nat. Genet.
Li, B., Howe, L., Anderson, S., Yates, J.R., III, and Workman, J.L. (2003). The
Set2 histone methyltransferase functions through the phosphorylated car-
boxyl-terminal domain of RNA polymerase II. J. Biol. Chem. 278, 8897–8903.
Li,B.,Carey,M.,andWorkman, J.L. (2007a).Theroleofchromatinduringtran-
scription. Cell 128, 707–719.
Li, B., Gogol, M., Carey, M., Pattenden, S.G., Seidel, C., and Workman, J.L.
(2007b). Infrequently transcribed long genes depend on the Set2/Rpd3S path-
way for accurate transcription. Genes Dev. 21, 1422–1430.
Mason, P.B., and Struhl, K. (2003). The FACT complex travels with elongating
RNA polymerase II and is important for the fidelity of transcriptional initiation
in vivo. Mol. Cell. Biol. 23, 8323–8333.
Minsky, N., Shema, E., Field, Y., Schuster, M., Segal, E., and Oren, M. (2008).
Monoubiquitinated H2B is associated with the transcribed region of highly
expressed genes in human cells. Nat. Cell Biol. 16 10.1038/ncb1712.
Mutiu, A.I., Hoke, S.M., Genereaux, J., Liang, G., and Brandl, C.J. (2007). The
role of histone ubiquitylation and deubiquitylation in gene expression as deter-
mined by the analysis of an HTB1(K123R) Saccharomyces cerevisiae strain.
Mol. Genet. Genomics 277, 491–506.
Ng, H.H., Dole, S., and Struhl, K. (2003). The Rtf1 component of the Paf1 tran-
scriptional elongation complex is required for ubiquitination of histone H2B. J.
Biol. Chem. 278, 33625–33628.
Orphanides, G., Wu, W.H., Lane, W.S., Hampsey, M., and Reinberg, D. (1999).
The chromatin-specific transcription elongation factor FACT comprises
human SPT16 and SSRP1 proteins. Nature 400, 284–288.
Osley, M.A. (2006). Regulation of histone H2A and H2B ubiquitylation. Brief.
Funct. Genomic Proteomic 4, 179–189.
Pavri, R., Zhu, B., Li, G., Trojer, P., Mandal, S., Shilatifard, A., and Reinberg, D.
(2006). Histone H2B monoubiquitination functions cooperatively with FACT to
regulate elongation by RNA polymerase II. Cell 125, 703–717.
H2B Ubiquitylation Regulates Nucleosome Dynamics
Molecular Cell 31, 57–66, July 11, 2008 ª2008 Elsevier Inc. 65
Pokholok, D.K., Harbison, C.T., Levine, S., Cole, M., Hannett, N.M., Lee, T.I.,
Bell, G.W., Walker, K., Rolfe, P.A., Herbolsheimer, E., et al. (2005). Genome-
wide map of nucleosome acetylation and methylation in yeast. Cell 122,
Robzyk, K., Recht, J., and Osley, M.A. (2000). Rad6-dependent ubiquitination
of histone H2B in yeast. Science 287, 501–504.
Santos-Rosa, H., Schneider, R., Bannister, A.J., Sherriff, J., Bernstein, B.E.,
Emre, N.C., Schreiber, S.L., Mellor, J., and Kouzarides, T. (2002). Active genes
are tri-methylated at K4 of histone H3. Nature 419, 407–411.
Saunders, A., Werner, J., Andrulis, E.D., Nakayama, T., Hirose, S., Reinberg,
D., and Lis, J.T. (2003). Tracking FACT and the RNA polymerase II elongation
complex through chromatin in vivo. Science 301, 1094–1096.
Schneider, R., Bannister, A.J., Myers, F.A., Thorne, A.W., Crane-Robinson, C.,
and Kouzarides, T. (2004). Histone H3 lysine 4 methylation patterns in higher
eukaryotic genes. Nat. Cell Biol. 6, 73–77.
Schwabish, M.A., and Struhl, K. (2004). Evidence for eviction and rapid depo-
sition of histones upon transcriptional elongation by RNA polymerase II. Mol.
Cell. Biol. 24, 10111–10117.
Schwabish,M.A.,and Struhl, K.(2006).Asf1mediates histone eviction and de-
position during elongation by RNA polymerase II. Mol. Cell 22, 415–422.
Shahbazian, M.D., Zhang, K., and Grunstein, M. (2005). Histone H2B ubiquity-
lation controls processive methylation but not monomethylation by Dot1 and
Set1. Mol. Cell 19, 271–277.
Squazzo, S.L., Costa, P.J., Lindstrom, D.L., Kumer, K.E., Simic, R., Jennings,
J.L., Link, A.J., Arndt, K.M., and Hartzog, G.A. (2002). The Paf1 complex phys-
ically and functionally associates with transcription elongation factors in vivo.
EMBO J. 21, 1764–1774.
Sun, Z.W., and Allis, C.D. (2002). Ubiquitination of histone H2B regulates H3
methylation and gene silencing in yeast. Nature 418, 104–108.
Tanny, J.C., Erdjument-Bromage, H., Tempst, P., and Allis, C.D. (2007). Ubiq-
uitylation of histone H2B controls RNA polymerase II transcription elongation
independently of histone H3 methylation. Genes Dev. 21, 835–847.
Tsukuda, T., Fleming, A.B., Nickoloff, J.A., and Osley, M.A. (2005). Chromatin
remodelling at a DNA double-strand break site in Saccharomyces cerevisiae.
Nature 438, 379–383.
VanDemark, A.P., Xin, H., McCullough, L., Rawlins, R., Bentley, S., Heroux, A.,
Stillman, D., Hill, C.P., and Formosa, T. (2008). Structural and functional anal-
ysis of the Spt16p N-terminal domain reveals overlapping roles of yFACT sub-
units. J. Biol. Chem. 283, 5058–5068.
Wittmeyer, J., Joss, L., and Formosa, T. (1999). Spt16 and Pob3 of Saccharo-
myces cerevisiae form an essential, abundant heterodimer that is nuclear,
chromatin-associated, and copurifies with DNA polymerase alpha. Biochem-
istry 38, 8961–8971.
Wood, A., Krogan, N.J., Dover, J., Schneider, J., Heidt, J., Boateng, M.A.,
Dean, K., Golshani, A., Zhang, Y., Greenblatt, J.F., et al. (2003a). Bre1, an
E3 ubiquitin ligase required for recruitment and substrate selection of Rad6
at a promoter. Mol. Cell 11, 267–274.
Wood, A., Schneider, J., Dover, J., Johnston, M., and Shilatifard, A. (2003b).
The Paf1 complex is essential for histone monoubiquitination by the Rad6-
Bre1 complex, which signals for histone methylation by COMPASS and
Dot1p. J. Biol. Chem. 278, 34739–34742.
Wyce, A., Xiao, T., Whelan, K.A., Kosman, C., Walter, W., Eick, D., Hughes,
T.R., Krogan, N.J., Strahl, B.D., and Berger, S.L. (2007). H2B ubiquitylation
acts as a barrier to Ctk1 nucleosomal recruitment prior to removal by Ubp8
within a SAGA-related complex. Mol. Cell 27, 275–288.
Wyers, F., Rougemaille, M., Badis, G., Rousselle, J.C., Dufour, M.E., Boulay,
J., Regnault, B., Devaux, F., Namane, A., Seraphin, B., et al. (2005). Cryptic
pol II transcripts are degraded by a nuclear quality control pathway involving
a new poly(A) polymerase. Cell 121, 725–737.
Xiao, T., Hall, H., Kizer, K.O., Shibata, Y., Hall, M.C., Borchers, C.H., and
Strahl, B.D. (2003). Phosphorylation of RNA polymerase II CTD regulates H3
methylation in yeast. Genes Dev. 17, 654–663.
Xiao, T., Kao, C.F., Krogan, N.J., Sun, Z.W., Greenblatt, J.F., Osley, M.A., and
Strahl, B.D. (2005). Histone H2B ubiquitylation is associated with elongating
RNA polymerase II. Mol. Cell. Biol. 25, 637–651.
Zhou, W., Zhu, P., Wang, J., Pascual, G., Ohgi, K.A., Lozach, J., Glass, C.K.,
and Rosenfeld, M.G. (2008). Histone H2A monoubiquitination represses tran-
scription by inhibiting RNA polymerase II transcriptional elongation. Mol. Cell
Zhu, B., Mandal, S.S., Pham, A.D., Zheng, Y., Erdjument-Bromage, H., Batra,
S.K., Tempst, P., and Reinberg, D. (2005a). The human PAF complex coordi-
nates transcription with events downstream of RNA synthesis. Genes Dev. 19,
Zhu, B., Zheng, Y., Pham, A.D., Mandal, S.S., Erdjument-Bromage, H.,
Tempst, P., and Reinberg, D. (2005b). Monoubiquitination of human histone
H2B: the factors involved and their roles in HOX gene regulation. Mol. Cell
H2B Ubiquitylation Regulates Nucleosome Dynamics
66 Molecular Cell 31, 57–66, July 11, 2008 ª2008 Elsevier Inc.