Current Biology 22, 56–63, January 10, 2012 ª2012 Elsevier Ltd All rights reservedDOI 10.1016/j.cub.2011.11.042
The Rpd3 Core Complex Is
a Chromatin Stabilization Module
Xiao-Fen Chen,1Benjamin Kuryan,1Tasuku Kitada,1
Nancy Tran,1Jing-Yu Li,1Siavash Kurdistani,1
Michael Grunstein,1Bing Li,2and Michael Carey1,*
1Department of Biological Chemistry, 351A Biomedical
University of California, Los Angeles, Los Angeles,
CA 90095-1737, USA
2Department of Molecular Biology, University of Texas
Dallas, TX 75390, USA
The S. cerevisiae Rpd3 large (Rpd3L) and small (Rpd3S)
histone deacetylase (HDAC) complexes are prototypes for
understanding transcriptional repression in eukaryotes .
The current view is that they function by deacetylating
chromatin, thereby limiting accessibility of transcriptional
factors to the underlying DNA. However, an Rpd3 catalytic
mutant retains substantial repression capability when tar-
geted to a promoter as a LexA fusion protein . We investi-
gated the HDAC-independent properties of the Rpd3
complexes biochemically and discovered a chaperone func-
tion, which promotes histone deposition onto DNA, and
a novel activity, which prevents nucleosome eviction but
not remodeling mediated by the ATP-dependent RSC
complex. These HDAC-independent activities inhibit Pol II
transcription on a nucleosomal template. The functions of
the endogenous Rpd3 complexes can be recapitulated with
recombinant Rpd3 core complex comprising Sin3, Rpd3,
and Ume1. To test the hypothesis that Rpd3 contributes to
chromatin stabilization in vivo, we measured histone H3
density genomewide and found that it was reduced at
promoters in an Rpd3 deletion mutant but partially restored
in a catalytic mutant. Importantly, the effects on H3 density
are most apparent on RSC-enriched genes . Our data
suggest that the Rpd3 core complex could contribute to
repression via a novel nucleosome stabilization function.
Results and Discussion
Rpd3S Contains H3K36me3-Independent Histone
Chaperone and Nucleosome Stabilization Functions
The Rpd3 HDAC is the prototype for understanding gene
repression on chromatin . HDACs function by removing
acetyl marks placed on histone tails by histone acetyl-
transferases such as SAGA and NuA4 . Acetylated histones
decondense chromatin directly  and/or serve as targets for
ATP-dependent remodeling enzymes including SWI/SNF 
and RSC . Bromodomains within these enzymes recruit
them to acetylated chromatin and enhance their remodeling
function . In yeast, Rpd3L and Rpd3S share three subunits:
Rpd3, Sin3, and Ume1 [10, 11]. Rpd3L contains numerous
additional subunits  and is targeted to promoters by
sequence-specific DNA binding proteins like Ume6 [13, 14].
Importantly, the Rpd3 HDAC activity contributes to but is not
essential for repression on promoters when targeted as
a LexA fusion . The Rpd3S complex contains two additional
subunits, Rco1 and Eaf3, which target it to H3K36 trimethy-
H3K36me3 and associates with Pol II . Rpd3S maintains
a hypoacetylated state in the ORF and suppresses cryptic
transcription [10, 11, 17]. Recently, Rpd3S was reported to
interact with elongating Pol II, and its recruitment to tran-
scribed regions was dependent on phosphorylation of the
carboxy-terminal domain of Rpb1 .
Several aspects of Rpd3 function were of interest to us.
First, the in vivo roles of both Rpd3S and Rpd3L were consis-
tent with a nucleosome stability function. Second, in addition
to Rpd3, the two complexes share two other subunits, Ume1
of correlating with repression of transcription on chromatin
.Finally, the observation thatRpd3 catalyticmutants retain
some repression capabilities when targeted via LexA fusions
suggested that some other aspect of the protein was contrib-
unessential, only that other aspects of Rpd3 complexes may
cooperate with the HDAC to ensure full repression. To explore
the HDAC-independent functions of Rpd3, we considered
the possibility that it might affect nucleosome remodeling.
For example, a previous study by Kingston and colleagues
revealed that human SWI/SNF ATPases copurified with a
Sin3/HDAC complex and that their remodeling activities were
compromised by the HDAC .
interest in the mechanism of Pol II elongation on nucleosomal
templates, which in our system requires nucleosome remodel-
ing and octamer eviction by RSC. Because we began with
Rpd3S, we also asked whether H3K36me3 would affect
nucleosome remodeling. Tandem affinity purification (TAP)
was employed to purify the RSC and Rpd3S proteins from
S. cerevisiae (Figure 1A) . H3K36me3 histones were gener-
ated with the methyl-lysine analog (MLA) technology .
H3K36 was first mutated to cysteine (H3K36C) and then
alkylated with (2-bromoethyl) trimethylammonium bromide
to form a methyl-lysine analog or MLA (H3K36C-me3). We
will refer to the MLA as H3K36me3 for convenience. The
MLA is recognized in a western blotting experiment by an
H3K36me3 antibody (Figure S1A available online). Subse-
quently, unmethylated (naive) or H3K36me3 mononucleo-
somes were reconstituted on a
containing the 601 nucleosome positioning sequence [23,
24]. Rpd3S bound to both nucleosomes in an EMSA assay
and displayed a higher affinity for H3K36me3 nucleosomes
as shown previously  (Figure S1B).
The assembled nucleosomes were incubated with RSC in
the presence or absence of Rpd3S and analyzed by native
gel electrophoresis. RSC mobilized the histone octamer as
indicated by the faster migration of the 601 nucleosome on
a native gel. However, Rpd3S did not significantly inhibit
this activity (Figure 1B). Similar effects were observed on
H3K36me3 nucleosomes (Figure S1C). We conclude that
32P-labeled DNA fragment
RSC - + + + +
Rpd3S - - -
Octamer - + + + + +
FACT - - + ---
1 2 3 4 5 6
Rpd3S - -
RSC - + + + +
Acceptor DNA - + + + +
1 2 3 4 5
Relative amount of
0 13 39 78 nM
0 6 12 18 nM
Figure 1. Rpd3S Inhibits RSC-Dependent Nucleosome Eviction and Promotes Nucleosome Assembly In Vitro
(A) Silver stain gel of TAP-purified RSC2, Rpd3S, and FACT complexes.
(B) The effect of Rpd3S on RSC-dependent nucleosome remodeling. 2 nM RSC was incubated with 0.3 nM32P-labeled mononucleosome and 0, 13, 39, or
78 nM Rpd3S. The remodeling products were fractionated by native PAGE. A phosphorimage of the gel is shown. See also Figures S1A–S1C for the effect of
H3K36me3 on Rpd3S in binding and RSC-mediated nucleosome remodeling reactions.
in the presence of 10 ng of pGEM3Z601R acceptor DNA. Bar graph on the right represents quantitation by ImageQuant TL (GE) of the three independent
experiments. The relative amounts of free DNA generated by eviction were plotted as a bar graph normalized to that generated by 6 nM RSC alone, which
wasassignedavalueof 100.The errorbarsshow6standarddeviation (SD). The pvalueis calculatedbyStudent’s ttest.See alsoFigure S1Dforthe effect of
H3K36me3 on Rpd3S in RSC-mediated nucleosome eviction.
(D) Rpd3S-mediated chromatin assembly assay. Left, the reaction contained 18 nM FACT, or 6, 12, 18 nM of Rpd3S, respectively, with recombinant
Xenopus octamers and a32P-labeled 601 DNA fragment. A phosphorimage of a native gel is shown. Graph on the right represents quantitation of the
amounts of assembled nucleosomes by Rpd3S relative to no Rpd3S control (i.e., octamers alone). The free DNA and assembled nucleosome are indicated.
The error bars show 6standard deviation (SD). The p value is calculated with Student’s t test. For chaperone assays see also Figure S1E for the effect of
H3K36me3 on Rpd3S, Figure S1F for the effect of Rpd3S mutants, and Figure S1G for the effect of trichostatin.
The Rpd3 Chromatin Stabilization Module
Rpd3S does not inhibit nucleosome remodeling under the
experimental conditions tested in our assays.
As reported previously , RSC has the ability to transfer
a histone octamer from one DNA molecule to another, often
termed an acceptor. This octamer transfer capability, referred
to as eviction, is important for RSC’s function . Upon
addition of an unlabeled supercoiled acceptor DNA to our
remodeling reactions, RSC transferred the majority of
octamers from the labeled DNA probe to the unlabeled DNA,
thereby generating substantial amounts of free
(Figure 1C, lane 2). Importantly, the amount of eviction, as
measured by accumulation of free DNA, decreased signifi-
cantly with increasing doses of Rpd3S (Figure 1C, lanes 3–5
and accompanying bar graph), while the amount of remodeled
nucleosome increased. Similar effects on remodeling and
eviction were observed at similar Rpd3S concentrations on
H3K36me3 chromatin (Figures S1C and S1D). We conclude
that Rpd3S inhibits RSC-mediated octamer eviction in an
H3K36me3-independent manner in vitro.
Inhibition of ATP-dependent octamer eviction is an activity
that may act to maintain nucleosome stability and histone
density over a regulatory region and gene. Another activity
that could serve a similar role would be the ability of Rpd3S
to act as a chaperone by assembling histones onto DNA.
Histone chaperones such as FACT, Nap1, Asf1, and Spt6
play important roles in transcription regulation in yeast [27,
28]. To address this possibility, Rpd3S was first incubated
with unmodified, naive octamers to allow protein-protein
interactions, followed by incubation with a32P-labeled DNA
bearing the 601 positioning sequence. Nucleosome formation
was analyzed on a native gel (Figure 1D). To verify the
efficiency of our in vitro system, we compared Rpd3S with
the well-studied histone chaperone FACT (Figure 1A) ,
which is known to specifically load histones onto DNA. As
reported previously , FACT strongly stimulated nucleo-
some formation (Figure 1D, lane 3). Importantly, Rpd3S also
promoted nucleosome assembly in adose-dependent manner
(Figure 1D, lanes 4–6). Surprisingly, the concentration of
Rpd3S necessary to assemble nucleosomes was measurably
lower than that required to inhibit eviction. H3K36me3 did
not enhance and even inhibited the chaperone function of
Rpd3S (Figure S1E). Moreover, an Rpd3S mutant lacking the
PHD domain of Rco1 and the chromo domain (CHD) of Eaf3,
both of which target Rpd3S to H3K36me3 , displayed
similar relative nucleosome assembly activity as did the wild-
type protein. H3K36me3 negatively affected the mutant’s
chaperone function similar to its effect on wild-type Rpd3S
(Figure S1F). We conclude that the Rpd3S complex acts as
a histone chaperone to promote histone deposition onto
DNA in vitro in an H3K36me3-independent manner.
The inhibition of chaperone function by H3K36me3 was
quite interesting given that Rpd3S normally targets this modi-
fication for binding within an ORF. However, the effect was
also observed in a mutant of Rpd3S lacking the targeting
domains suggesting that H3K36me3 inhibits a specific aspect
of the chaperone function. For example, H3K36me3 also
negatively affected the chaperone activity of FACT (data not
shown). Collectively, the chaperone and eviction data suggest
that Rpd3S possesses a chromatin stabilization function,
which is independent of the specific H3K36me3 targeting
function. Although our assays utilized unacetylated histones,
it remained a remote possibility that the HDAC function of
Rpd3 might somehow contribute. However, Rpd3S promoted
nucleosome formation at the same efficiency in the absence
or presence of trichostatin concentrations sufficient to inhibit
90% of Rpd3’s HDAC activity (Figure S1G).
The 3-Subunit Core of Rpd3L and Rpd3S Mediates the
Nucleosome Stabilization Function
We next asked whether the Rpd3L complex exhibited similar
properties as Rpd3S because the two enzymes share a set
of three core subunits [10, 11]. To address this question, we
TAP purified the Rpd3L complex (Figure 2A) and tested its
effect on RSC-dependent remodeling (data not shown) and
octamer eviction (Figure 2B). Like Rpd3S, Rpd3L had little
effect on remodeling but significantly inhibited octamer evic-
tion as indicated by the reduced amounts of free DNA
over, like Rpd3S, Rpd3L stimulated nucleosome assembly
with naive octamers (Figure 2C).
Because Rpd3L shares a 3-subunit core complex (3-core)
with Rpd3S, we next asked whether this module contributes
to the nucleosome stabilization function. The 3-subunit core
complex was reconstituted by coexpression of S. cerevisiae
Ume1, Rpd3, and Sin3 in Sf9 cells via a baculovirus system.
Sin3 was tagged with the FLAG epitope and the complex
was purified with a two-step procedure involving an anti-
FLAG immuno-affinity column followed by gel filtration chro-
matography. The final products were relatively pure except
for an unknown protein that copurified (Figure 2D). We
observed a significant and dose-dependent inhibition of
RSC-mediated octamer eviction by the 3-subunit core
complex (Figure 2E). The core complex also enhanced nucle-
osome formation in a chaperone assay, similar to Rpd3S and
Rpd3L (Figure 2C).
In an attempt to identify the subunit responsible for nucle-
osome stabilization, we used FLAG-affinity chromatography
to purify each of the individual subunits (Figure S2A).
In side-by-side purifications of similar scale and yield, only
Ume1 purified to near homogeneity as a single species.
Sin3 was degraded slightly and Rpd3 copurified with several
higher molecular weight bands. Nevertheless, the amounts of
full-length subunits were sufficient for testing. Surprisingly,
no individualsubunit inhibited
eviction (Figure S2B) or assembled nucleosomes to any
significant extent (Figure S2C). We conclude that the entire
Rpd3 core complex (Sin3, Ume1, and Rpd3) is necessary
and sufficient for the chromatin stabilization function in vitro.
The Nucleosome Stabilization Function Inhibits
Nucleosomal Transcription In Vitro
To further study the nucleosome stabilization function of
Rpd3 HDACs, we performed in vitro transcription. Previously,
we established a system to study Pol II transcription through
a nucleosome by using a ‘‘C-tail’’ template bearing a single-
stranded stretch of dC ligated to a DNA fragment encompass-
ing the 601 positioning sequence (Figure 3A) . Pol II
employs the C-tail as a promoter and elongates into the 601
nucleosome. RSC was shown to stimulate Pol II elongation
through the nucleosomal barrier . Our current view is that
RSC stimulates transcription by evicting the nucleosome. We
wished to determine whether the nucleosome stabilization
transcription. Consistent with our previous study, transcrip-
tion with TAP-purified yeast Pol II generated only small
amounts of full-length (FL) transcripts (Figure 3B, lane 1).
Pol II arrested at discrete locations and short transcripts
were produced. In the presence of ATP and acceptor DNA,
Current Biology Vol 22 No 1
RSC strongly stimulated transcription as reflected by the
decrease in arrested transcripts and by increased production
of full-length (FL) transcripts (Figure 3B, lane 2). The addition
of the Rpd3 HDACs, in the form of either Rpd3S, Rpd3L, or
the 3-subunit core complex, all diminished transcription in
a dose-dependent manner on naive chromatin (Figure 3B,
lanes 3–8). However, the presence of the chaperone FACT,
which has no effect on RSC eviction (data not shown), did
not affect the production of full-length transcripts (Figure 3B,
lanes 9–10). The inhibition of transcription by Rpd3 HDACs
was specific to a nucleosomal template as shown by the fact
Because Rpd3S displayed a higher affinity for H3K36me3
nucleosomes , we compared the inhibitory effect of
Rpd3S and 3-subunit core complex on transcription with
naive or H3K36me3 nucleosomal template. The H3K36me3
nucleosomes enhanced repression by Rpd3S but not by the
3-subunit core complex (Figure 3D). The results are consistent
with the higher affinity of Rpd3S for H3K36me3 nucleosomes.
These data indicate that the Rpd3 complexes antagonize
RSC-mediated stimulation of Pol II transcription elongation
through a nucleosome in vitro. The inhibition of transcription
elongation by Rpd3S is consistent with an in vivo study
showing that deletion of Rpd3 bypasses the requirement of
positive elongation factor Bur1/Bur2 .
Our approach does not indicate whether Rpd3S, for
example, can block an elongating Pol II molecule in vivo. It is
not known how Pol II elongation occurs in living cells and
whether there are situations where it would encounter
Rpd3S-bound nucleosomes. Indeed, the current model, for
which there is little experimental support, suggests that
Rpd3S-bound nucleosomes accumulate behind Pol II .
Nevertheless, our assay provides a measure of the stability
of the nucleosome conferred by Rpd3 in the presence of the
strong ATP-dependent remodeling activity of RSC and the
potent NTP-dependent DNA translocase activity of Pol II.
Rpd3 Affects H3 Density Preferentially at RSC-Bound
Genes in Vivo
The ability of the two Rpd3 complexes to stabilize nucleo-
somes independent of HDAC activity suggested that they
might play similar roles in vivo. To address this hypothesis,
we prepared strains of yeast with the endogenous Rpd3
gene deleted but bearing an empty vector or vectors express-
ing either the wild-type or H150A catalytically inactive Rpd3
.Themutant andwild-type Rpd3pwereexpressed atsimilar
levels (Figure S3A). The H150A mutation appears to be
previously via an in vitro assay. However, it still interacts
with Sin3 and partially retains the transcription repression
0 15 45 90 nM
Relative amount of
Relative amount of
0 15 45 90 nM
Figure 2. Rpd3L and 3-Subunit Core Complex Prevent RSC-Dependent Nucleosome Eviction and Promote Nucleosome Assembly In Vitro
(A) Silver stain gel of TAP-purified Rpd3L.
(B) The effect of Rpd3L on RSC-dependent nucleosome eviction. 6 nM RSC was incubated with 0, 15, 45, 90 nM Rpd3L, respectively, and analyzed as
described in Figure 1C legend.
(C) Nucleosome assembly with Xenopus octamers and 18 nM Rpd3L or 3-subunit core complex, respectively, as in Figure 1D legend.
(D) Silver stain gel of recombinant 3-subunit core complex. The asterisk indicates an unknown protein that copurified with the 3-subunit core complex.
(E) The effect of 3-subunit core complex on RSC-dependent nucleosome eviction. 6 nM RSC was incubated with 601 nucleosome and 0, 15, 45, 90 nM
of recombinant core complex, respectively, and analyzed as in Figure 1C legend. See also Figure S2A for silver-stained gels of the individual subunits,
Figure S2B for their effect on RSC-mediated nucleosome eviction, and Figure S2C for chaperone assays.
The Rpd3 Chromatin Stabilization Module
function . To test the effect in vivo, we chose two known
targets of Rpd3, histone H3K18ac and H4K5ac . The data
demonstrate that the H150A and null Rpd3 mutants lead to
that the point mutant is largely inactive in vivo (Figure S3B).
Finally, we measured histone H3 density genome-wide in
each of these three strains by using Agilent tiling arrays.
Upon deletion of Rpd3, we observed a significant decrease
in H3 density at intergenic/promoter regions genome-wide,
while the coding regions/ORFs were less affected (Figure 4A)
although still significant in some regions (data not shown).
Importantly, however, the H150A derivative maintained signif-
icantly higher H3 density than in Rpd3-deletion cells (empty
vector), although not as high as in Rpd3 wild-type cells. The
data suggest a global role of Rpd3 in affecting histone density
although the effect is more apparent in promoter regions.
Our biochemical studies showed that Rpd3 complexes
prevent RSC from evicting histones on chromatin (Figures 1
and 2). We hypothesized that the observed changes in H3
density upon Rpd3 mutation might be more evident on
genes that are bound by RSC in vivo. To test this hypothesis,
we compared our data with that of the published genome-
wide distribution of five subunits of the RSC1 and RSC2
complexes in S. cerevisiae . We analyzed intergenic/
promoter regions scored for high RSC binding by the authors
(as measured via all five subunits; p < 0.001 for each region)
and compared them with a similar number of targets display-
ing the least RSC binding (Figure 4B). We then analyzed the
H3 density changes observed upon Rpd3 mutation in these
same two subsets of targets. The deletion or mutation of
Rpd3 minimally impaired H3 occupancy at low RSC-bound
targets, while a more significant decrease was observed at
+ + + + + + + + +
1 2 3 4 5 6 7 8 9 10
+ + + + + + + + +
RSC - + + + + + + + - + + + + + + +
Figure 3. Rpd3 HDACs Inhibit RSC-Mediated Activation of Nucleosome Transcription
(A) Schematic of the C-tail template. The template contains the 601 positioning sequence and a 20-nucleotide single-stranded C-tail with an intervening
polylinker from pGEM3Z601R. Pol II initiates from the C-tail.
(B) The template was assembled into a mononucleosome with naive recombinant Xenopus octamers and then preincubated with 3 nM RSC in the presence
or absence of 30 or 60 nM Rpd3S, Rpd3L, 3-subunit core complex, or FACT for 1 hr at 30?C. Pol II, a-[32P]CTP, NTPs, and RNase H were then added for
15 min at 30?C. The32P-labeled RNA products were fractionated on a 10% polyacrylamide/urea gel. A phosphorimage of the gel is shown.
(C) Same assay as performed in (B), except that naked C-tail DNA template was used instead of nucleosomal template.
(D) In vitro transcription was performed on naive or H3K36me3 nucleosomes. 3 nM RSC was incubated with 0, 5, 15, 45 nM Rpd3S or recombinant 3-subunit
core complex, respectively.
Current Biology Vol 22 No 1
the RSC-enriched targets (Figures 4C and 4D). Importantly,
the largest difference in H3 density between wild-type and
either the null or H150A Rpd3 mutant was observed at RSC-
enriched regions. The data suggest the possibility that the
Rpd3 complex can somehow influence nucleosome stability
at RSC-enriched promoter regions in vivo. It is unclear how
this would affect gene expression because recent findings
suggest that the nucleosome occupancy of Rpd3 targets is
not always correlated with transcription frequency [32–34].
Additionally, Rpd3 affects transcription of full length and
the true effect.
Conclusions and Perspectives
We have demonstrated that Rpd3S and Rpd3L possess
a previously unrecognized capacity to promote nucleosome
assembly like a histone chaperone. Additionally, both Rpd3
HDAC complexes prevent RSC-dependent histone eviction
from nucleosomes, possibly through their histone chaperone
activity. However, we note that the eviction and chaperone
functions displayed different concentration dependence. We
were also able to establish that the three common subunits
totheshared activitiesof thesmallandlarge Rpd3HDACs. We
speculate that the combination of a histone chaperone activity
and inhibition of RSC eviction facilitate chromatin stability and
complement the transcriptional repression function of the
The diminished density of H3 in strains upon Rpd3 deletion
or mutation was significant mainly on the promoter/intergenic
regions. Despite the observation that transcription level is
inversely correlated with H3 density , the levels of H3 in
transcribed regions significantly exceed those in promoter
regions. It remains a possibility that although Rpd3S is found
primarily in ORFs, the normally high histone density masks
its stabilization function. Histone density and chromatin stabi-
lization in the ORF is known to involve numerous proteins,
including chaperones such as Spt6 and FACT, many of which
generate a cryptic transcription phenotype when mutated
[36–38]. Hence the role of Rpd3’s stabilization function in the
ORF regions may be less apparent and require more sensitive
assays or combinatorial mutations of other chaperones to
become more evident. Alternately, the ability of H3K36me3 to
inhibit Rpd3’s chaperone function may disable that function
A prediction of the role of Rpd3, as either a chaperone or
in chromatin stabilization, is that it should copurify with
substantial amounts of H3. Indeed, we observed that Rpd3
Low Rsc2 genes High Rsc2 genes
p (WT-Vector) = 0.03
p (H150A-Vector) = 0.25
p (WT-Vector) = 2.00-e8
p (H150A-Vector) = 9.76-e4
Rsc2 promoter enrichment
p (WT-Vector) = 1.13-e14
p (H150A-Vector) = 7.50-e7
Figure 4. Rpd3 Complexes Stabilize Chromatin In Vivo
(A) H3 levels were measured by ChIP in wild-type (WT), rpd3 H150A (H150A), and rpd3D (Vector) cells. ChIP DNA of Histone H3 and inputs were amplified,
labeled, and hybridized to Agilent Tiling arrays. The average binding of 6,572 annotated genes and their upstream 500 bp regions are shown. Enrichment
of H3 ChIP DNA is shown as the log2 ratios of ChIP versus input DNA. p values for the promoters were calculated with the Mann-Whitney test. See also
Figure S3A for Rpd3 native versus H150A protein levels, Figure S3B for global acetylation levels of H3K18 and H4K5 in Rpd3 native, H150A, and null back-
grounds, and Figures S3C and S3D for Rpd3 association with Pol II and H3.
(B) Box and whisker plot for two subsets of targets that have high or low Rsc2 enrichment. Each subset has 495 and 506 targets, respectively. The p values
are calculated by Student’s t test.
(C and D) H3 levels of the low (C) and high (D) Rsc2 targets were measured in wild-type, rpd3 H150A, and rpd3D cells.p values for the promoters were calcu-
lated by the Mann-Whitney test.
The Rpd3 Chromatin Stabilization Module
complexes, purified with an Rpd3-TAP strain, contained
a significant amount of H3 and less, but still detectable,
amounts of Pol II. Stoichiometry measurements revealed that
the ratio of Rpd3:H3:Pol II is 10:4.5:1 (Figures S3C and S3D).
This result was obtained with concentrations of heparin and
ethidium bromide, which are known to disfavor protein-DNA
interactions. In the case of H3, these data are consistent with
a chaperone function. In the case of Pol II, these data are
consistent with the direct interaction of Pol II and Rpd3S
proposed by Hinnebusch and colleagues .
It should be pointed out that our unpublished microarray
data show that deletion of Rpd3 causes upregulation and
downregulation of many genes as reported  (data not
shown). Although it would be easy to dismiss the downregu-
lated genes as indirect effects, the result belies a complex
role of Rpd3 in transcription. For example,little isknown about
the mechanisms underlying global histone deacetylation and
what role it plays. Additionally, Rpd3 has been shown to be
directly required for activation of stress-inducible genes,
and in some cases, the effect requires the catalytic activity
[32–34]. In such scenarios, nucleosome density and the chro-
matin stabilization function may not always correlate with
Rpd3 occupancy. Therefore, although our results reveal an
additional function for Rpd3, which may have implications
for the function of these proteins in higher eukaryotes, much
remains to be learned of how this protein functions in genomic
database (accession # GSE33829).
Supplemental Information includes Supplemental Experimental Proce-
dures and three figures and can be found with this article online at
This work was supported by the National Institute of General Medical
Sciences grant (1R01GM085002) to M.C. B.L. is a W.A. ‘‘Tex’’ Moncrief, Jr.,
Scholar in Medical Research and is supported by grants from the National
Institutes of Health (R01GM090077), the Welch Foundation (I-1713), the
March of Dimes Foundation, and the American Heart Association. We are
grateful to K. Struhl at Harvard Medical School for providing the Rpd3
wild-type and H150A plasmids. We thank Y. Guo for her initial experimental
contributions to this work.
Received: September 29, 2011
Revised: November 21, 2011
Accepted: November 21, 2011
Published online: December 15, 2011
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The Rpd3 Chromatin Stabilization Module