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
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The Rpd3 Chromatin Stabilization Module