MOLECULAR AND CELLULAR BIOLOGY, Mar. 2009, p. 1401–1410
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 29, No. 6
The H3K4 Demethylase Lid Associates with and Inhibits Histone
Nara Lee,1,2Hediye Erdjument-Bromage,3Paul Tempst,3Richard S. Jones,4and Yi Zhang1,2*
Howard Hughes Medical Institute1and Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center,2
University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295; Molecular Biology Program,
Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 100213; and Department of
Biological Sciences, Southern Methodist University, Dallas, Texas 752754
Received 21 October 2008/Returned for modification 17 November 2008/Accepted 19 December 2008
JmjC domain-containing proteins have been shown to possess histone demethylase activity. One of these
proteins is the Drosophila histone H3 lysine 4 demethylase Little imaginal discs (Lid), which has been
genetically classified as a Trithorax group protein. However, contrary to the supposed function of Lid in
gene activation, the biochemical activity of this protein entails the removal of a histone mark that is
correlated with active transcription. To understand the molecular mechanism behind the function of Lid,
we have purified a Lid-containing protein complex from Drosophila embryo nuclear extracts. In addition
to Lid, the complex contains Rpd3, CG3815/Drosophila Pf1, CG13367, and Mrg15. Rpd3 is a histone
deacetylase, and along with Polycomb group proteins, which antagonize the function of Trithorax group
proteins, it negatively regulates transcription. By reconstituting the Lid complex, we demonstrated that
the demethylase activity of Lid is not affected by its association with other proteins. However, the
deacetylase activity of Rpd3 is greatly diminished upon incorporation into the Lid complex. Thus, our
finding that Lid antagonizes Rpd3 function provides an explanation for the genetic classification of Lid as
a positive transcription regulator.
Gene transcription is regulated in part by modulating the
conformation of chromatin, which consists of DNA wrapped
around histone proteins. Chromatin conformation can be
altered by posttranslational modifications of histone tails,
such as acetylation and methylation (12). The acetylation of
lysine residues on histone tails likely regulates transcription
in a positive manner through one of two mechanisms. First,
the acetylation of histone tails may inhibit chromatin com-
paction, as exemplified by acetylated lysine 16 of histone H4
(31). Second, acetylated histone tails may serve as docking
sites for effector proteins that promote gene transcription
(19). The steady state of histone acetylation is regulated by
a balance between histone acetyltransferases that add acetyl
groups and histone deacetylases (HDACs) that remove
these moieties from histone tails (43). One of the most
prominent deacetylases is Rpd3 (reduced potassium depen-
dency 3), which was initially identified in yeast as a tran-
scriptional repressor with homologues in higher eukaryotes
that belong to the class I HDACs (26, 42). The repressive
function of Rpd3 is further underscored by the fact that it
can act in concert with some Polycomb group (PcG) protein
complexes to efficiently silence target genes by virtue of
histone deacetylation (22, 35, 40).
The methylation of histone lysines is associated with ei-
ther active or repressed gene transcription, depending on
the modified lysine residues. In general, the methylation of
lysines 4, 36, and 79 of histone H3 is associated with active
transcription, whereas methylation on lysines 9 and 27 of
histone H3, as well as lysine 20 of histone H4, correlates
with gene repression (4). Although histone methylation was
believed previously to be a static modification, recent stud-
ies indicated that this histone mark can also be actively
removed by histone demethylases (30, 37). The majority of
histone demethylases identified so far contain the evolution-
arily conserved JmjC domain as a signature motif (10).
Based on sequence homology in the JmjC domain and the
overall architecture of associated motifs, JmjC domain-con-
taining proteins have been classified into seven groups (10),
and members of five groups have been demonstrated to
possess histone lysine demethylase activity (1). The Dro-
sophila protein Little imaginal discs (Lid) has been shown to
possess demethylase activity toward trimethylated histone
H3 lysine 4 (H3K4me3) (7, 15, 29), a mark that is enriched
in promoters and that correlates with active transcription
(28). Genetically, the gene lid has been classified as a mem-
ber of the Trithorax group (TrxG) of genes (9), which act as
transcriptional activators and antagonize PcG protein func-
tion (25). TrxG proteins, in particular Trithorax and Ash1
(absent, small, or homeotic discs 1), methylate H3K4 and
thus contribute to active transcription (3, 5, 33). Unexpect-
edly, the enzymatic activity of Lid is to remove H3K4 meth-
ylation, a mark set by other members of its genetic class.
This raises the question of what may be the molecular basis
for the genetic classification of lid as a TrxG gene.
by Lid, we have purified a Lid-containing protein complex from
Drosophila embryonic nuclear extracts (NE). Interestingly, one
component of this complex is Rpd3, a well-known HDAC. En-
* Corresponding author. Mailing address: Department of Biochem-
istry and Biophysics, Lineberger Comprehensive Cancer Center, Uni-
versity of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295.
Phone: (919) 843-8225. Fax: (919) 966-4330. E-mail: yi_zhang@med
?Published ahead of print on 29 December 2008.
zymatic studies with a reconstituted complex show that the
HDAC activity of Rpd3 is greatly diminished in the context of
the Lid complex. This finding can be recapitulated in Drosoph-
ila S2 cells, suggesting that Lid may function as a transcription
activator by inhibiting histone deacetylation.
MATERIALS AND METHODS
Purification of a Lid-containing complex. Drosophila embryonic NE was pre-
pared from 0- to 24-h-old embryos as described previously (8). The scheme for
conventional chromatography purification is outlined in Fig. 1A. Peak fractions
of Lid were identified using a previously described rabbit polyclonal antibody
FIG. 1. Rpd3 copurifies with a Lid-containing protein complex. (A) The purification scheme for isolating a Lid-containing complex from
Drosophila embryonic NE is shown. Molar concentrations refer to KCl in fractions. FT, flowthrough; DE-5PW, DEAE-5PW; mass spec, mass
spectrometry. (B) Following anti-Lid affinity purification, proteins were resolved by SDS gel electrophoresis. Coomassie blue-stained bands were
identified by mass spectrometry. Asterisks show nonspecific bands. aa, amino acids; chromo, chromodomain. (C) Rpd3 and Mrg15 coelute with
Lid following gel filtration chromatography on a Superose 6 column. The elution profile of protein standards for this column and the estimated
molecular mass of the Lid complex are indicated at the top. (D) Coimmunoprecipitation using embryo NE demonstrates the association of Lid
with Rpd3 and Mrg15. As a control for antibody specificity, anti-Pcl antibody was included in the coimmunoprecipitation experiments. IP,
immunoprecipitation; WB, Western blotting.
1402LEE ET AL.MOL. CELL. BIOL.
against the N-terminal region of Lid (15). Approximately 900 mg of NE was
fractionated on a phosphocellulose P11 (Sigma) column by stepwise elution
using elution buffer (40 mM HEPES [pH 7.9], 0.2 mM EDTA, 10% glycerol, 1
mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride) containing the KCl
concentrations indicated in Fig. 1A. The 0.4 M fraction was loaded onto a
DEAE-5PW column (TosoHaas), and the bound proteins were eluted with a
linear gradient from 50 mM (BD50) to 600 mM (BD600) ammonium sulfate in
elution buffer. Fractions containing the Lid complex eluting at BD160 to BD190
were pooled and further separated by gel filtration chromatography on a Super-
ose 6 column (Amersham) using elution buffer containing 300 mM KCl. For
affinity purification of Lid-associated proteins, anti-Lid antibody was coupled to
protein A-agarose beads (RepliGen). The resin was incubated overnight at 4°C
with fractions 11 to 16 from the Superose 6 fractionation (see Fig. 1C) and
washed extensively. The immunoadsorbed complex was separated by sodium
dodecyl sulfate (SDS) gel electrophoresis, and the resulting protein bands were
subjected to mass spectrometry analysis. For complex verification, rabbit anti-
Rpd3 and anti-Mrg15 antibodies (kind gifts from James Kadonaga, University of
California, San Diego, and Thomas Kusch, Rutgers, respectively) were used at a
1:5,000 dilution for Western blot analysis.
Reconstitution of the Lid complex. Clones of the Lid complex component
cDNAs (Lid clone no. LD40310, Drosophila Pf1 [dPf1] clone no. GH06635,
CG13367 clone no. RH61522, Rpd3 clone no. GM14158, and Mrg15 clone no.
LD22902) were obtained from the Drosophila Genomics Resource Center. The
cDNAs were cloned into the pFastBac-HTB vector (Invitrogen) expressing an
N-terminal His tag or into a modified version of this vector expressing an
N-terminal Flag tag to generate baculoviruses for protein expression in Sf9 cells.
The optimal viral titer for each component was empirically determined and used
for purifying the reconstituted complex by Flag immunoprecipitation (Flag-IP)
using anti-Flag M2 affinity beads (Sigma) and gel filtration chromatography. To
purify Rpd3-containing subcomplexes, Sf9 cells were infected with baculovirus
expressing Flag-Rpd3, along with baculovirus expressing His-Mrg15, His-
CG13367, or His-Lid. Cell lysates were subjected to nickel-nitrilotriacetic acid
(Ni-NTA) affinity purification according to the instructions of the Ni-NTA man-
ufacturer (Qiagen), followed by Flag-IP. For Western blot analysis, anti-Flag
(Sigma) and anti-His (Santa Cruz) antibodies were used at a 1:1,000 dilution.
Histone demethylase reaction. The histone demethylase reaction was carried
out in demethylase buffer [50 mM HEPES (pH 7.9), 25 ?M Fe(NH4)2(SO4)2, 1
mM ?-ketoglutarate, 2 mM ascorbate) using synthetically methylated histone
substrates generated as described by Simon et al. (32). The reaction mixtures
were incubated for 3 h at 30°C, and the reactions were stopped by the addition
of SDS loading buffer and subsequently analyzed by Western blotting using
rabbit anti-dimethylated H3K4 (anti-H3K4me2) antibody (product no. ab7766
[Abcam]; 1:500 dilution) and mouse anti-H3 antibody (product no. ab10799
[Abcam]; 1:2,000 dilution).
HDAC reaction. Histone deacetylation was carried out in deacetylation buffer
(75 mM Tris [pH 7.5], 150 mM NaCl, 2 mM dithiothreitol, 0.1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride) using HeLa cell core histones, which were pre-
pared as described by Zhang et al. (47), as substrates. For the deacetylation
reactions involving the mixing of Rpd3 with other proteins (see Fig. 3E), Flag-
Rpd3 enzyme was preincubated for 30 min at 30°C with the other proteins prior
to the addition of core histones. The deacetylation reaction mixtures were incu-
bated at 30°C for 1 h and analyzed by Western blotting using rabbit anti-
acetylated histone H3 (anti-AcH3) antibody (product no. 06-599 [Upstate];
1:1,000 dilution). Alternatively, differently acetylated histone forms were re-
solved by Triton-acetic acid-urea (TAU) gel electrophoresis followed by Coo-
massie blue staining as described previously (16). To determine the level of
histone H4 acetylation by Western blotting, anti-acetylated histone H4 (anti-
AcH4) antibody (product no. 06-866; Upstate) was used at a 1:2,000 dilution.
Cell culture and immunostaining. S2 cells (obtained from Greg Rogers, Uni-
versity of North Carolina) were grown in Sf-900 II serum-free medium (Gibco)
at room temperature. Mrg15, CG13367, wild-type Lid, and H637A mutant Lid
(LidH637A) cDNAs including sequences encoding N-terminal Flag tags were
cloned into the pAc5.1 vector (Invitrogen), and cells were transfected using
Effectene according to the instructions of the manufacturer (Qiagen). For im-
munostaining, cells were fixed in 2% paraformaldehyde in Brower buffer (50 mM
PIPES [pH 6.9], 1 mM MgSO4, 0.5 mM EGTA, 0.5% NP-40) for 2 h at 4°C.
Afterward, cells were washed in PBNT (0.5 M NaCl, 10 mM NaPO4[pH 7.0], 1%
bovine serum albumin [BSA], 0.1% Triton X-100) and blocked in PBNT con-
taining 10% goat serum for 30 min. Mouse anti-Flag antibody was used at a
1:2,000 dilution, and rabbit anti-Odd antibody (a kind gift from James Skeath,
Washington University in St. Louis) was used at a 1:100 dilution.
RNA isolation and RT-PCR. RNA from S2 cells was isolated using Trizol
(Invitrogen), and cDNA for reverse transcriptase PCR (RT-PCR) was generated
using SuperScript III according to the instructions of the manufacturer (Invitro-
gen). Primers used for RT-PCR were as follows: for ftz, 5?-CGAGGAGACTT
TGGCATCAG-3? and 5?-ACGCCGGGTGATGTATCTATT-3?; for eve, 5?-CC
CTGGTTGTGGACCTCTT-3? and 5?-ACTGGATAGGCATTCATTTGG-3?;
for odd, 5?-AGCAACATAACCGTGGATGAC-3? and 5?-AGCATGGGGGAG
AGACTTG-3?; for lid, 5?-ACGGCCTTTATCGGGTATTT-3? and 5?-TTGAA
CAGCAACACCACCAG-3?; and for rp49, 5?-TGCACCAGGAACTTCTTGA
AT-3? and 5?-ATACAGGCCCAAGATCGTGAA-3?.
ChIP. Chromatin immunoprecipitation (ChIP) was carried out using a dual
cross-linking approach with DSG [di(N-succinimidyl) glutarate] followed by
formaldehyde as described online at www.epigenome-noe.net (PROT29). The
following primers were used to quantify the immunoprecipitated material by
real-time PCR: for ftz, 5?-TGCACATCGCAGAGTTAGAGA-3? and 5?-ATGT
TGTCGGCGTAGCTGTAG-3?; for eve, 5?-AGAGCGCAGCGGTATAAAA
G-3? and 5?-GGCAGTTAGTTGTTGACTGTGC-3?; and for odd, 5?-AAAGC
AAAAGCAAAAGCAACA-3? and 5?-ACGCTTGAGAATCGAAGTGAA-3?.
Immunostaining of polytene chromosomes. The yw strain of Drosophila mela-
nogaster was used as the wild type. The salivary gland-specific AB1-Gal4 line was
obtained from the Bloomington stock collection (stock no. 1824). To generate
transgenic flies that overexpress Lid, Lid cDNA including a sequence encoding
an N-terminal Flag tag was cloned into the pUAST vector. For Gal4-driven
overexpression, flies were reared at 27°C. Polytene chromosomes were immu-
nostained as described previously (23). In brief, salivary glands of mid-third-
instar larvae were dissected and fixed in a solution of 45% acetic acid and
4% paraformaldehyde for 8 min on poly-L-lysine-coated slides before being
squashed. The slides were washed twice in phosphate-buffered saline (PBS),
incubated in PBS–1% Triton X-100 for 10 min, and blocked in PBS–5% nonfat
dried milk for 1 h. Primary antibodies (rabbit anti-Rpd3, 1:200; mouse anti-Flag,
1:1,000; and rabbit anti-AcH3, 1:250) were diluted in PBS–1% BSA and incu-
bated for 2 h at room temperature. Secondary antibodies (anti-rabbit immuno-
globulin G [IgG]-Cy3, 1:400, and anti-mouse IgG-AF488, 1:400) were diluted in
PBS containing 2% normal donkey serum and incubated for 1 h at room tem-
perature. The slides were washed in washing buffer (PBS containing 0.2% Tween
20 and 0.2% NP-40) with NaCl concentrations of 300, 400, and 500 mM. Chro-
mosomes were stained in DAPI (4?,6-diamidino-2-phenylindole; 100 ng/ml) and
mounted with fluorescent mounting medium (DakoCytomation).
Purification of a Lid-containing complex. Lid has been ge-
netically identified as a member of the TrxG of proteins (9),
which act as transcriptional activators. Contrary to the genetic
classification of Lid, the biochemical activity of the protein is to
demethylate H3K4, thereby removing a mark of active tran-
scription. To resolve this contradiction, we sought to identify
functional partners of Lid. Using conventional chromatogra-
phy, we purified a Lid-containing protein complex from Dro-
sophila embryonic NE (Fig. 1A). Mass spectrometry analysis
identified Lid-associated proteins as CG3815, Rpd3, CG13367,
and Mrg15 (Fig. 1B). CG3815 (hereinafter referred to as dPf1)
is homologous to mammalian Pf1 (PHD zinc finger protein 1),
which has been reported to associate with HDACs such as
Rpd3 (44). CG13367 is an uncharacterized protein distantly
related to mammalian ocular development-associated gene/
GATA zinc finger domain-containing protein 1 (ODAG/
Gatad1) (38). Mrg15 contains an N-terminal chromodomain
whose mammalian homologue has been shown to bind to
H3K36me3 (46). Additionally, Mrg15 is part of the Drosoph-
ila Tip60 complex that functions in histone exchange (13),
and Mrg15 is also found in a mammalian HDAC-containing
complex together with Pf1 (45).
To verify that these proteins form a complex, we compared
the elution profile of Lid with those of Rpd3 and Mrg15 by
using a gel filtration column. Western blot analysis confirmed
that Rpd3 and Mrg15 coelute with Lid (Fig. 1C). We could not
examine the elution patterns of dPf1 and CG13367 because
VOL. 29, 2009 Lid INHIBITS Rpd3 ACTIVITY1403
antibodies against these proteins were not available. To further
verify the association of these proteins, we carried out coim-
munoprecipitation studies of Lid with Rpd3 and Mrg15. An-
tibodies against both Rpd3 and Mrg15 coimmunoprecipitated
Lid from embryonic NE (Fig. 1D, lanes 3 and 4). Reciprocal
immunoprecipitation using a Lid antibody confirmed the asso-
ciation of Lid with Rpd3 (Fig. 1D, lane 8). The coimmunopre-
cipitation of Mrg15 was not evaluated due to the similarity of
its molecular weight to those of antibody heavy chains. These
associations are specific as the Lid antibody did not immu-
noprecipitate Polycomb-like (Pcl) protein, which is a com-
ponent of a PcG complex, and vice versa (Fig. 1D, lanes 5
and 11) (21, 27).
Reconstitution and characterization of the demethylase ac-
tivity. To characterize the enzymatic activities of the Lid com-
plex, we attempted to reconstitute the complex in vitro. To this
end, we generated baculoviruses that expressed Flag-Rpd3
along with the remaining components, each harboring a His
tag. After the coinfection of Sf9 cells, the complex was first
affinity purified by Flag-IP and then subjected to gel filtration
chromatography (Fig. 2A). The overexpressed proteins copu-
rified as a protein complex (Fig. 2B), demonstrating that
complex formation with the identified subunits can be recapit-
Previous studies have shown that the activities of histone-
modifying enzymes can be modulated through association with
FIG. 2. The incorporation of Rpd3 into the Lid complex reduces the deacetylase activity of Rpd3. (A) Reconstitution scheme for the Lid
complex. Sf9 cells were infected with baculoviruses expressing His-tagged Lid, His-dPf1, His-CG13367, Flag-Rpd3, and His-Mrg15. The complex
was immunoprecipitated from cell lysate by using Flag antibodies and further purified with a Superose 6 column. (B) Silver-stained gel with the
reconstituted Lid complex obtained as depicted in panel A. (C) Histone demethylase assay with the Lid complex (Lid-com) and recombinant Lid.
The amount of Lid in the complex was determined (top), and the complex was subsequently subjected to histone demethylation reactions using
synthetic H3K’4me3 (middle) or H3K’4me3K’36me3 (bottom) as a substrate. Western blotting (WB) using anti-H3K4me2 antibody was carried
out to detect the reaction product. Western blotting with anti-H3 antibody served as a loading control. ?, no Lid. (D) The HDAC activity of Rpd3
is inhibited by its incorporation into the complex. The amount of Rpd3 in the complex was determined (top), and HDAC reactions were carried
out by incubating the Lid complex and recombinant Rpd3 with core histones and then performing Western blot analysis using anti-AcH3 antibody
(middle). Western blotting with anti-H3 antibody served as a loading control (bottom). (E) Core histones were incubated with the Lid complex
or with increasing amounts of recombinant Rpd3. The reaction products were analyzed by electrophoresis on a TAU gel to visualize differentially
acetylated histone forms. While as little as 0.1 ?g of recombinant Rpd3 is sufficient to show deacetylase activity, a larger amount of Rpd3
incorporated into the Lid complex is unable to deacetylate core histones.
1404LEE ET AL.MOL. CELL. BIOL.
other proteins (6, 14). To determine whether the enzymatic
activity of Lid is modulated by its association with other com-
ponents, we first quantified the amount of Lid in the complex
by comparing the complex to given amounts of recombinant
Lid by Western blot analysis (Fig. 2C, top panel). We then
compared the demethylase activity of the complex to that of
recombinant Lid alone. For the demethylase assays, we em-
ployed chemically methylated histones as a substrate (32) and
monitored the demethylation reaction by Western blot anal-
ysis using methylation state-specific histone antibodies. We
adopted this approach because the use of homogeneously
methylated histone substrates proved to offer the most sen-
sitive readout for the demethylase activity. We generated
chemically trimethylated lysine 4 analogues of histone H3
(H3K’4me3, where the apostrophe denotes the lysine ana-
logue) and monitored the generation of H3K4me2 by Western
blot analysis using an H3K4me2-specific antibody. Results
shown in Fig. 2C (middle panels) indicate that the Lid complex
and Lid alone have similar enzymatic activities when equal
amounts of Lid are compared. Thus, we conclude that the
incorporation of Lid into the complex does not significantly
alter its demethylase activity under our assay conditions.
Previous studies have demonstrated that Mrg15 binds to
H3K36me3 through the Mrg15 chromodomain (46). This find-
ing prompted us to test whether methylation on H3K36 might
help the recruitment of the Lid complex to its substrate and
consequently lead to enhanced H3K4 demethylase activity. To
this end, synthetic histones that were methylated on both lysine
4 and lysine 36 were generated and used as substrates. Results
shown in Fig. 2C (bottom panels) demonstrate that doubly
methylated histones are no better substrates for the Lid com-
plex. However, we cannot rule out the possibility that under
physiological conditions, methylation at H3K36 might stimu-
late the demethylase activity through the interaction of Mrg15
with methylated histones. Furthermore, the lack of enhanced
enzymatic activity of the Lid complex in vitro may be due to the
lack of other as yet unidentified factors in our assay system.
The HDAC activity of Rpd3 is inhibited by the association of
Rpd3 with the Lid complex. Next, we analyzed whether the
deacetylase activity of Rpd3 is affected by the incorporation of
Rpd3 into the Lid complex. To this end, the reconstituted Lid
complex and increasing amounts of recombinant Flag-Rpd3
were incubated with core histones and the reaction products
were analyzed by Western blotting using an antibody against
AcH3. Results shown in Fig. 2D indicate that the incorpora-
tion of Rpd3 into the Lid complex greatly inhibited the
deacetylase activity of Rpd3 (cf. lanes 2 and 5). We indepen-
dently confirmed this finding by TAU gel electrophoresis, as
this technique allows the visualization of differentially acety-
lated histone forms. While as little as 0.1 ?g of recombinant
Rpd3 showed detectable enzymatic activity, the Lid complex
containing fivefold the amount of Rpd3 was unable to deacety-
late core histones (Fig. 2E, cf. lanes 2 and 3).
Next, we attempted to resolve which subunit of the Lid
complex is responsible for the inhibition of Rpd3. To this end,
we first determined which subunit interacts with Rpd3 by coin-
fecting Sf9 cells with baculoviruses expressing Flag-tagged
Rpd3 and a His-tagged version of each component of the Lid
complex. Proteins that associate with Rpd3 were coimmuno-
precipitated using anti-Flag antibody and detected by Western
blotting using anti-His antibody. Rpd3 was found to interact
with Mrg15, CG13367, and Lid, while it did not interact with
dPf1 under the same conditions (Fig. 3A). Next, we reconsti-
tuted Rpd3-containing subcomplexes that contained Rpd3 in
association with each of its interacting partners by using Ni-
NTA affinity followed by Flag-IP (Fig. 3B) and compared the
deacetylase activities of these two-component subcomplexes
with that of Rpd3 alone. Unexpectedly, we found that the
association of Rpd3 with any of the three components of the
Lid complex inhibited its HDAC activity (Fig. 3C).
To rule out that the observed inhibition of Rpd3 was caused
by the purification procedure involving Ni-NTA affinity and
Flag-IP, we purified each component bearing a Flag tag from
Sf9 cells (Fig. 3D, left panel) and added the components indi-
vidually to the deacetylation reaction mixtures. Results shown
in Fig. 3E (left panel) demonstrate that the addition of each of
the Rpd3 binding partners to the reaction mixtures inhibited
the deacetylase activity of Rpd3. To show that the inhibitory
nature of these proteins toward Rpd3 is specific, we added
unrelated proteins, such as BSA and other JmjC domain-con-
taining proteins (Flag-JHDM3A and His-JHDM1A) (Fig. 3D,
right panel) (11, 37) to the deacetylase assay mixtures. Results
shown in Fig. 3E (middle panel) demonstrate that the addition
of the above-mentioned proteins did not affect the deacetylase
activity. Moreover, to show that the inhibition of Rpd3 is
caused by direct interaction with its binding partner and is not
due to aggregation, we carried out deacetylation reactions with
increasing amounts of Lid (Fig. 3E, right panel). Taken to-
gether, these results indicate that the inhibitory effects of
Mrg15, CG13367, and Lid on Rpd3 are specific.
Lid inhibits the activity of Rpd3 at an Rpd3 target gene. As
Mrg15, CG13367, and Lid inhibit the activity of Rpd3 in vitro,
we examined whether this inhibition is also observed in vivo.
To this end, we transfected Drosophila S2 cells with constructs
expressing Flag-tagged Mrg15, CG13367, and Lid and ana-
lyzed whether the overexpression of these proteins results in
the inhibition of Rpd3 activity by monitoring the global AcH3
level. We found that none of these proteins when overex-
pressed are able to elicit an obvious increase in the global H3
acetylation level (data not shown). One possible explanation
for the lack of global HDAC inhibition is the inability of Rpd3
proteins, which are preassembled into existing Rpd3-contain-
ing complexes, to associate with the overexpressed proteins.
Nevertheless, we asked whether the inhibition of Rpd3 could
occur in a gene-specific manner. Previous studies have shown
that Rpd3 regulates segmentation genes, such as fushi tarazu
(ftz), even-skipped (eve), and odd-skipped (odd), during embry-
ogenesis (18, 34). To determine whether any of these genes are
regulated by Rpd3 in S2 cells in an HDAC activity-dependent
manner, we treated S2 cells with the HDAC inhibitor tricho-
statin A (TSA) and checked whether these segmentation genes
become upregulated upon TSA treatment. RT-PCR analysis
showed that only odd was derepressed after TSA treatment
(Fig. 4A), indicating that odd may be an Rpd3-responsive
target gene in S2 cells. We then examined whether the over-
expression of Mrg15, CG13367, or Lid is able to upregulate
odd expression and consequently result in an enhanced protein
level. Immunostaining with antibodies against Odd (41) dem-
onstrated that the levels of Odd protein were increased in cells
overexpressing Lid (34 of 55 cells; 62%) but not in cells over-
VOL. 29, 2009 Lid INHIBITS Rpd3 ACTIVITY1405
FIG. 3. Association with Mrg15, CG13367, and Lid inhibits Rpd3 activity in vitro. (A) Rpd3 interacts with Mrg15, CG13367, and Lid. Sf9 cells
were coinfected with baculoviruses expressing Flag-Rpd3 (F-Rpd3) in combination with His-tagged proteins of the Lid complex. Cell lysates were
subjected to immunoprecipitation (IP) with Flag antibody, and interaction partners were detected by Western blot (WB) analysis using anti-His
antibody. ctrl, control (no Flag-Rpd3); ?, absent; ?, present. (B) Reconstitution of Rpd3-containing subcomplexes. Sf9 cells were coinfected with
baculoviruses expressing the indicated proteins, and Rpd3-containing subcomplexes were purified from cell lysates using Ni-NTA affinity followed
by Flag-IP. A Coomassie blue-stained gel (top) and a Western blot with Rpd3 subcomplexes detected by anti-His antibody (bottom) are shown.
H, His; F, Flag. (C) Core histones were incubated with Rpd3 subcomplexes, and HDAC activity was analyzed by Western blotting using anti-AcH3
antibody. Comparable amounts of Rpd3 were used in all reactions, as shown by Western blotting with anti-Rpd3 (top). Association with Mrg15,
CG13367, and Lid diminishes the deacetylase activity of Rpd3 in vitro (middle). Anti-H3 Western blotting served as a loading control (bottom).
(D) Coomassie blue-stained SDS gels with the indicated Flag-tagged proteins purified from baculovirus-infected Sf9 cells, BSA, and His-JHDM1A
purified from E. coli are shown. (E) The inhibition of Rpd3 activity can be achieved by mixing individual interaction partners of Rpd3 in vitro.
Flag-Rpd3 (0.2 ?g) and equimolar amounts of Mrg15, CG13367, and Lid were added to the deacetylation reaction mixtures prior to the addition
of core histones. Western blot analysis using anti-AcH3 antibody showed that the addition of each of the interacting partners could inhibit the
deacetylase activity of Rpd3 (left panel) but that unrelated proteins did not show any inhibition (middle panel). Increasing amounts of Lid as
indicated were added to 0.2 ?g of Rpd3 to show dose-dependent inhibition of HDAC activity (right panel). Western blotting with anti-H3 antibody
served as a loading control.
1406 LEE ET AL.MOL. CELL. BIOL.
expressing Mrg15 or CG13367 (Fig. 4B). To rule out the pos-
sibility that the derepression of odd was due to the histone
demethylase activity of Lid and thus independent of the
HDAC activity of Rpd3, we overexpressed a catalytically inac-
tive form of Lid (LidH637A) (15). Results shown in Fig. 4B
(bottom panels) demonstrate that the overexpression of the
mutant Lid also led to increased levels of Odd at a comparable
frequency (37 of 67 cells; 58%), indicating that the derepres-
sion of odd was independent of the demethylase activity of Lid.
To evaluate whether the upregulation of odd was due to en-
hanced transcription and not caused solely by protein stabili-
zation, we cotransfected S2 cells with constructs expressing Lid
and green fluorescent protein (GFP) at a ratio of 3:1 and
isolated Lid-overexpressing cells by fluorescence-activated cell
sorter (FACS) analysis based on GFP fluorescence. We found
that the FACS step was necessary, as the transfection efficiency
was less than 15%. RT-PCR analysis showed that odd tran-
scripts were upregulated in cells overexpressing Lid (Fig. 4C).
Considering the facts that odd is a known Rpd3 target (18, 34)
and that TSA treatment can upregulate the expression of odd
in S2 cells (Fig. 4A), in combination with the fact that Lid can
inhibit the activity of Rpd3, the upregulation of odd mediated
by the overexpression of Lid is likely to be due to the inhibition
of the HDAC activity of Rpd3.
To further examine whether the upregulation of odd is
caused by Lid-mediated inhibition of Rpd3, we performed
ChIP assays analyzing the localization of Rpd3, AcH3, and Lid
at the odd locus. As a control, we included the promoter
regions of ftz and eve. Expectedly, we observed Rpd3 localiza-
tion at the odd promoter region and its absence at the ftz and
FIG. 4. Lid inhibits the HDAC activity of Rpd3 in vivo. (A) Identification of Rpd3 targets in S2 cells. S2 cells were treated with 100 ng/ml TSA
for 48 h to repress deacetylation by Rpd3. RNA was isolated from dimethyl sulfoxide-treated control cells (ctrl) or TSA-treated cells (TSA) and
examined for the reactivation of Rpd3 target genes by RT-PCR. Of the three tested segmentation genes, only odd was derepressed by TSA
treatment. rp49 served as a loading control. ?, with; ?, without. (B) S2 cells were transfected with constructs expressing Flag-tagged Mrg15,
CG13367, wild-type Lid (Flag-LidWT), or catalytically inactive Lid (Flag-LidH637A). Cells were stained with anti-Flag antibody, anti-Odd antibody,
and DAPI. Only cells overexpressing wild-type Lid (34 of 55 cells; 62%) and LidH637A(37 of 67 cells; 58%) showed ectopic staining of Odd at
comparable frequencies. (C) The levels of odd mRNA in Lid-overexpressing cells are increased. S2 cells were transfected with constructs expressing
Lid and GFP at a ratio of 3:1. Lid-overexpressing cells were isolated by FACS analysis using GFP fluorescence. RNA was isolated from control
cells (GFP negative) and Lid-overexpressing cells (GFP positive) and subjected to RT-PCR to detect transcripts of lid and odd. rp49 served as a
loading control. (D) ChIP analysis for the promoter regions of the ftz, eve, and odd gene locus. ChIP was carried out with anti-Rpd3, anti-AcH3,
and anti-Lid antibodies and anti-IgG antibody (data not shown) by using chromatin from wild-type (WT) and Lid-overexpressing (Lid) cells.
Real-time PCR was employed to quantify the immunoprecipitated material relative to the input.
VOL. 29, 2009 Lid INHIBITS Rpd3 ACTIVITY1407
eve promoters in wild-type S2 cells (Fig. 4D, top panel), con-
sistent with the transcriptional responsiveness of the odd gene
to TSA treatment. Interestingly, we also found Lid to be
present at the odd promoter but absent at the promoters of ftz
and eve (Fig. 4D, bottom panel). Following Lid overexpression,
the level of AcH3 at the odd promoter increased significantly,
consistent with the transcriptional reactivation of odd (Fig. 4D,
middle panel). The levels of AcH3 at the ftz and eve promoters
were unaffected. Intriguingly, we observed that the occupancy
of Lid at the odd promoter was unchanged upon Lid overex-
pression and that the amount of Rpd3 binding was decreased
moderately but reproducibly. These results suggest that an
excessive nuclear pool of Lid may sequester Rpd3 from its
target gene. The merely moderate decrease in Rpd3 occupancy
may reflect the fact that a heterogeneous population of cells
was analyzed by ChIP, as only approximately 60% of cells
overexpressing Lid displayed ectopic odd expression (Fig. 4B).
The unchanged level of Lid at the odd locus despite its over-
expression may be explained by the fact that the recruitment of
Lid requires additional limiting factors. Taken together, these
results indicate that the reduced binding of Rpd3 is sufficient to
elicit the observed increase in the level of acetylated histones
and, thus, elevated transcription.
Lid antagonizes the function of Rpd3 in a transgenic fly line.
A recent study has shown that the levels of acetylated histones
on larval polytene chromosomes in lid mutants are severely
reduced (17). Based on our finding that Lid can inhibit the
HDAC activity of Rpd3 in vitro, the reduced levels of acety-
lated histones may be caused by overactive Rpd3 in the ab-
sence of Lid in these mutants. To further substantiate a link
between Lid and histone acetylation, we generated a trans-
genic fly line in which Flag-tagged Lid was under the control of
a Gal4-inducible upstream activation sequence promoter. Us-
ing the salivary gland-specific AB1-Gal4 driver, we observed
robust overexpression of Lid in salivary gland tissue, while the
level of Rpd3 remained unaffected (Fig. 5A, upper panels).
Immunostaining of polytene chromosomes of wild-type larvae
with anti-Rpd3 antibody showed a distribution of Rpd3 on less
condensed interbands throughout all chromosome arms and its
absence from the chromocenter, as described previously (Fig.
5B, left panels) (24). Notably, the overexpression of Lid re-
sulted in a marked decrease in Rpd3 binding on polytene
chromosomes, suggesting that excessive Lid is able to displace
Rpd3 from chromatin (Fig. 5B, right panels). To rule out that
the reduced binding of Rpd3 was caused by compromised
chromosome integrity upon Lid induction, we coimmunos-
tained the polytene chromosomes with anti-Flag antibody. The
distribution of the ectopic Flag-tagged Lid did not differ from
the previously reported distribution of endogenous Lid (15), as
it localized to interbands and was absent from the chromo-
center. The decreased binding of Rpd3 to polytene chromo-
somes, however, was not accompanied by elevated levels of
AcH3, as analyzed by Western blotting and immunostaining of
polytene chromosomes (Fig. 5A, lower panels, and data not
shown). In addition, we examined whether the global level of
AcH4 was altered by Lid overexpression and concomitant re-
duction in Rpd3 localization. As the acetylation of lysine 16 of
histone H4 is enriched on the male X chromosome as a con-
sequence of dosage compensation (39), we analyzed histones
of salivary glands from male and female larvae separately.
However, in neither case did we observe an elevated level of
AcH4 (Fig. 5A). These results suggest that the temporary
removal of Rpd3 from chromatin alone in this tissue is not
sufficient to increase the level of acetylated histones.
To shed light on the molecular mechanism of how the his-
tone demethylase Lid regulates transcription, we have purified
a Lid-containing protein complex, which includes dPf1, Rpd3,
CG13367, and Mrg15, from Drosophila embryonic NE. Previ-
ous studies have shown that the activities of chromatin-modi-
FIG. 5. The overexpression of Lid reduces the binding of Rpd3 to polytene chromosomes. (A) Western blot analysis of protein extracts derived
from wild-type (WT) and Lid-overexpressing salivary glands. Ten pairs of salivary glands from mid-third-instar larvae were used in the analysis to
detect the levels of Lid, Rpd3, AcH3, and AcH4. The arrow indicates cross-reacting acetylated H2B. Western blotting using anti-H3 antibody
served as a loading control. UAS, upstream activation sequence. (B) Immunostaining of polytene chromosomes from wild-type and Lid-
overexpressing larvae. Polytene chromosomes were immunostained using anti-Rpd3 and anti-Flag antibodies to detect ectopically expressed Lid.
1408 LEE ET AL.MOL. CELL. BIOL.
fying enzymes can be modulated through association with
other proteins in a complex. Although we did not observe an
alteration in histone demethylase activity upon the formation
of the Lid complex compared to the activity of recombinant
Lid alone, we cannot rule out the possibility that additional
factors are required to mediate this stimulatory effect. As we
could not use nucleosomes as a substrate for reasons of sensi-
tivity, it is possible that the Lid complex is irresponsive to
enhanced demethylase activity on methylated histones. On the
other hand, a different Lid-containing complex that is primarily
responsible for histone demethylation may exist. Previously,
Lid has been reported to interact with dMyc and another TrxG
protein, Ash2, in larval eye imaginal discs (29), implying that
other, tissue- and developmental stage-specific Lid-containing
complexes may exist.
Intriguingly, we observed inhibition of the HDAC activity of
Rpd3 in the Lid complex. In this respect, the major function of
Lid in this particular complex may be to counteract the tran-
scriptional repression mediated by the deacetylase activity of
Rpd3. Notably, Rpd3 has been shown previously to interact
with the PRC2 (Polycomb repressive complex 2) complex and
to enhance PcG-mediated gene silencing through histone
deacetylation (36, 40). As the H3K4 demethylase activity of
Lid is not required for odd gene activation (Fig. 4B), it is
tempting to speculate that the genetic characterization of lid as
a TrxG gene is due in part to its inhibitory effect on Rpd3. By
inhibiting the HDAC activity of Rpd3, Lid may counteract the
full extent of PcG-mediated suppression of gene expression,
providing an explanation for the contradictory genetic classi-
fication of lid as a TrxG gene and the enzymatic activity of Lid
to remove an active histone mark. From this point of view, it
appears possible that the histone demethylase activity of Lid is
developmentally dispensable. However, we have observed that
lid homozygous mutant flies can be rescued only by a transgene
encoding wild-type Lid and not by a transgene encoding a
catalytically inactive mutant form of Lid (N. Lee and Y. Zhang,
unpublished observations), indicating that H3K4 demethyla-
tion is developmentally important. Thus, Lid appears to fulfill
two possibly distinct functions during development, and these
functions may act independently of each other. One function is
to demethylate H3K4, whereas the other is to antagonize
HDAC activity to promote transcription.
The findings of a recent study substantiate the antagonistic
behavior of Lid toward Rpd3. Lloret-Llinares et al. reported
that lid mutant alleles act as an enhancer of position effect
variegation (17), whereas some mutations in Rpd3 have been
found to confer suppressor-of-variegation phenotypes (20).
Moreover, polytene chromosomes of lid mutants have been
shown to have reduced levels of AcH3 (17), which is consistent
with our finding that the overexpression of Lid is able to reduce
the binding of Rpd3 to polytene chromosomes. Thus, in the
absence of Lid, the balance between Lid and Rpd3 would be
tilted toward Rpd3, resulting in reduced levels of AcH3.
A similar HDAC complex containing Pf1 and Mrg15 in
mammals has been described previously (45). We can envisage
that Lid is recruited to a core HDAC complex consisting of
Rpd3, dPf1, and Mrg15 (and possibly including additional fac-
tors that are part of the HDAC complex) and thereby inhibits
the HDAC activity. The recruitment of Lid to the sites of the
HDAC complex may act as a switch to turn on the expression
of target genes during development. We have shown on a
gene-specific level for the odd gene in S2 cells by ChIP analysis
and on a global level by the immunostaining of polytene chro-
mosomes that the overexpression of Lid results in a marked
decrease in Rpd3 binding, suggesting that excessive Lid is able
to interact with and displace Rpd3 from its target sites (Fig. 4D
and Fig. 5B). We have to point out, however, that our findings
are based on conditions of robust overexpression of Lid and
that our observations need to be confirmed for target genes of
the Lid complex in the context of development.
It is surprising that we find Mrg15 to negatively regulate the
HDAC activity of Rpd3 in vitro, because Mrg15 has been
shown previously to contribute to transcription repression (45).
In this regard, it is possible that the interaction solely between
Rpd3 and Mrg15 results in enzymatic inhibition and that in-
teraction with additional factors, such as Sin3, may be required
to restore the HDAC activity. Provided that the Lid complex
identified in this study does play a role in regulating dynamic
histone methylation, another role for Mrg15 is conceivable.
The chromodomain of Mrg15 may potentially be involved in
recruiting the Lid complex to target genes. The trimethylation
of H3K4 peaks in the promoter region, whereas the trimeth-
ylation of H3K36 is enriched in the 3? region of genes (2, 28).
During the process of transcription, the chromodomain of
Mrg15 may target the Lid complex to the bodies of genes
through its interaction with H3K36me3 and induce the re-
moval of H3K4 trimethylation, resulting in the enrichment of
the 5? region of genes with this modification. In the absence of
Lid, this distinct border of the different methyl marks would
not be sustained and transcription efficiency would deteriorate,
thus offering an explanation for the function of Lid in active
Future genome-wide location studies of Lid and the other
components of the complex will reveal which target genes are
controlled by this complex. Furthermore, it will be interesting
to find out where within target genes the complex is located.
Does the complex bind to the bodies of genes to demethylate
H3K4, or does the binding take place at promoter regions to
regulate dynamic histone deacetylation? The identification of
Lid-associated proteins has set the stage for these detailed
studies, which will reveal insight into the mechanism underly-
ing transcription regulation by Lid.
We thank James Kadonaga, Thomas Kusch, James Skeath, and
Greg Rogers for reagents and Robert Klose and Kathryn Gardner for
comments and discussion.
This work was supported by NIH grants GM68804 (to Y.Z.),
GM46567 (to R.S.J.), and P30 CA08748 (to P.T.). Y.Z. is an investi-
gator of the Howard Hughes Medical Institute. N.L. is funded by the
International Human Frontier Science Program Organization.
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