Ligand-Dependent Nuclear Receptor Corepressor LCoR Functions by Histone Deacetylase-Dependent and -Independent Mechanisms
LCoR (ligand-dependent corepressor) is a transcriptional corepressor widely expressed in fetal and adult tissues that is recruited to agonist-bound nuclear receptors through a single LXXLL motif. LCoR binding to estrogen receptor alpha depends in part on residues in the coactivator binding pocket distinct from those bound by TIF-2. Repression by LCoR is abolished by histone deacetylase inhibitor trichostatin A in a receptor-dependent fashion, indicating HDAC-dependent and -independent modes of action. LCoR binds directly to specific HDACs in vitro and in vivo. Moreover, LCoR functions by recruiting C-terminal binding protein corepressors through two consensus binding motifs and colocalizes with CtBPs in the nucleus. LCoR represents a class of corepressor that attenuates agonist-activated nuclear receptor signaling by multiple mechanisms.
Molecular Cell, Vol. 11, 139–150, January, 2003, Copyright 2003 by Cell Press
Ligand-Dependent Nuclear Receptor Corepressor
LCoR Functions by Histone Deacetylase-Dependent
and -Independent Mechanisms
al., 2000). Crystal structures of agonist- and antagonist-
bound LBDs have revealed conserved ␣ helical struc-
tures (Bourget et al., 1995; Renaud et al., 1995; Wagner
et al., 1995; Brzozowski et al., 1997). Agonist binding
induces conformational changes that reorient the C-ter-
Han S. Lee,
Nicholas R. Bertos,
Victor K.M. Han,
and John H. White
* minal AF-2 helix (helix 12) to create a binding pocket
recognized by coactivators.
Department of Physiology
Department of Medicine Several coregulatory proteins control nuclear receptor
function (Robyr et al., 2000; Glass and Rosenfeld, 2000;
Department of Oncology
McGill University Dilworth and Chambon, 2001; Rosenfeld and Glass,
2001). Their diversity suggests that transcriptional acti-Montreal, Quebec, H3G 1Y6
Department of Biochemistry vation by receptors occurs through recruitment of multi-
ple factors acting sequentially or combinatorially. Co-University of Montreal
Montreal, Quebec, H3C 3J7 activators of the p160 family, SRC1/NCoA1, TIF-2/
GRIP-1, and pCIP/AIB1/RAC3/ACTR/TRAM-1 (Onate et
Department of Paediatrics
Department of Obstetrics and Gynecology al., 1995; Chakravarti et al., 1996; Hong et al., 1996;
Voegel et al., 1996; Anzick et al., 1997; Chen et al., 1997),Department of Biochemistry and Anatomy
MRC Group in Fetal and Neonatal Health which interact with ligand-bound receptors through
LXXLL motifs or NR boxes (Voegel et al., 1996; Heeryand Development
Lawson Research Institute et al., 1997). Cocrystallographic studies of ligand-bound
receptors revealed ␣-helical NR boxes oriented within aUniversity of Western Ontario
London, Ontario, N6A 4V2 hydrophobic pocket containing helix 12 held by a charge
clamp composed of conserved residues in helices 3 andCanada
12 (Darimont et al., 1998; Feng et al., 1998; Nolte et al.,
1998; Shiau et al., 1998). P160 coactivators recruit other
proteins essential for transactivation, including CREBSummary
binding protein (CBP) and its homolog p300 (Kamei et
al., 1996; Chen et al., 1997; Torchia et al., 1997). SeveralLCoR (ligand-dependent corepressor) is a transcrip-
tional corepressor widely expressed in fetal and adult coactivators including CBP/p300 and associated factor
p/CAF possess histone acetyltransferase activity, re-tissues that is recruited to agonist-bound nuclear re-
ceptors through a single LXXLL motif. LCoR binding quired for chromatin remodeling (Ogryzko et al., 1996;
Yang et al., 1996; Chen et al., 1997; Kurokawa et al.,to estrogen receptor ␣ depends in part on residues
in the coactivator binding pocket distinct from those 1998) and subsequent access of the transcriptional ma-
chinery to promoters.bound by TIF-2. Repression by LCoR is abolished by
histone deacetylase inhibitor trichostatin A in a recep- Corepressors NCoR and SMRT mediate ligand-inde-
pendent repression by thyroid and retinoic acid recep-tor-dependent fashion, indicating HDAC-dependent
and -independent modes of action. LCoR binds di- tors (Horlein et al., 1995; Chen and Evans, 1995; Perissi
et al., 1999) and recruit multiprotein complexes impli-rectly to specific HDACs in vitro and in vivo. Moreover,
LCoR functions by recruiting C-terminal binding pro- cated in transcriptional repression and histone deacety-
lation (Alland et al., 1997; Hassig et al., 1997; Heinzel ettein corepressors through two consensus binding mo-
tifs and colocalizes with CtBPs in the nucleus. LCoR al., 1997; Kadosh and Struhl, 1997; Laherty et al., 1997;
Nagy et al., 1997; Pazin and Kadonaga, 1997). Histonerepresents a class of corepressor that attenuates ago-
nist-activated nuclear receptor signaling by multiple deacetylases (HDACs) fall into three classes based on
homology, domain structure, subcellular localization,mechanisms.
and catalytic properties (Khochbin et al., 2001; Ng and
Bird, 2001; Wade, 2001). NCoR and SMRT are compo-
nents of several different complexes containing distinct
Nuclear receptors are ligand-regulated transcription
combinations of ancillary proteins and class I or class
factors whose activities are controlled by a range of
II HDACs (Rosenfeld and Glass, 2001), suggesting that
lipophilic extracellular signals. They directly regulate
their function depends on cell type, combinations of
transcription of genes whose products control many
transcription factors bound to specific promoters, and
aspects of physiology and metabolism (Chawla et al.,
phase of the cell cycle.
2001). Receptors are composed of a series of conserved
Here, we have identified a ligand-dependent core-
domains, A–F. Many N-terminal A/B regions contain
pressor (LCoR) that interacts with ER␣ and other class
transactivation domains (activating function-1; AF-1),
I and class II nuclear receptors through a single NR box.
which can cooperate with AF-2, located in the C-terminal
LCoR, which is expressed from the earliest stages of
ligand binding domain (LBD) (Tora et al., 1989; Robyr et
mammalian development, functions in an HDAC-depen-
dent and -independent manner through interactions with
multiple cofactors. LCoR represents a distinct class of
Figure 1. LCoR Gene, Transcript, and Pro-
(A) The LCoR two-hybrid cDNA clone (top)
and clones isolated from a prostate cDNA
library (below) are shown. LCoR ESTs are
shown below the composite 4813 bp cDNA
sequence (white bar). The open reading frame
of LCoR is indicated by the start codon and
the downstream stop codon. The first up-
stream in-frame stop codons are also indi-
cated. Human ESTs were identified using the
INFOBIOGEN site (http://www.infobiogen.fr/
BF761899, BF677797, AU132324, AK023248,
and BI029242/B1029025 are from adult co-
lon, adult prostate, NT2 teratocarcinoma cell
line, and adult marrow cDNA libraries, re-
spectively. A 4747 bp cDNA (AB058698) iden-
tified from a human brain library (Nagase et
al., 2001) containing an extra 5⬘UTR exon is
indicated at the bottom.
(B) Structure of the LCoR gene deduced using
the Human Genome Browser (http://genome.
ucsc.edu/cgi-bin/hggateway). The extra 5⬘UTR
exons present in the human brain cDNA
AB058698 are indicated as white bars. Intron
sizes are indicated where known.
(C) Schematic representation of LCoR pro-
tein. The NR box LSKLL, nuclear localization
signal (NLS), and putative helix-loop-helix
(HLH) domain are indicated. The homologies
of the HLH with other proteins are shown,
with asterisks indicating positions of amino
acid similarity. Existence of the HLH was pre-
dicted using Psired (http://bioinf.cs.ucl.
ac.uk) and Network Protein Sequence Analy-
nuclear receptor corepressor that acts to attenuate sig- 339 that is homologous to a simple nuclear localization
signal (NLS) of the SV40 large T antigen-type. The NLSnaling by agonist-bound receptors.
lies at the N terminus of a putative helix-loop-helix do-
main (Figure 1C and see Supplemental Figure S1 at
DC1 for LCoR sequence), which is 48%, 48%, and 43%
Identification of LCoR
homologous to motifs encoded by the Eip93F, T01C1.3,
LCoR was isolated from a yeast two-hybrid library as a
and MBLK-1 genes of Drosophila, C. elegans, and Hon-
cDNA containing a 1299 bp open reading frame (433
eybee (Apis mellifera; Takeuchi et al., 2001), respectively
amino acids; 47,006 kDa; Figures 1A and 1D) encoding a
(Figure 1C). The domain also bears 35% homology to
protein that interacted with the ER␣ LBD in an estradiol-
the pipsqueak motif (PSQ) repeated four times in the
dependent manner. Additional cDNAs were obtained from
DNA binding domain of the Drosophila transcription fac-
a human prostate cDNA library, and several expressed
tor pipsqueak (Lehmann et al., 1998).
sequence tags (ESTs; Figure 1A). Human sequences
were also highly homologous (ⵑ95%) to several mouse
ESTs, including multiple clones from a two-cell embryo LCoR Is Widely Expressed in Fetal and Adult Tissues
LCoR transcripts are broadly expressed at varying levelslibrary (data not shown), indicating that LCoR is ex-
pressed from the earliest stages of mammalian develop- in human adult and fetal tissues, with highest expression
observed in placenta, the cerebellum, and corpus callo-ment. The 4.8 kb of cDNA sequence encompasses seven
exons on chromosome 10q24.1, including four short sum of the brain, adult kidney and a number of fetal
tissues (see Supplemental Figure S2 at http://www.5⬘UTR exons that contain several in-frame stop codons
(Figure 1B and data not shown). A human brain EST molecule.org/cgi/content/full/11/1/139/DC1). LCoR
transcripts were also detected in a wide variety of human(Nagase et al., 2001) contains a single exon insert that
lengthens the 5⬘UTR without extending the open reading cell lines (Figure 2A), with highest levels of expression
observed in intestinal Caco-2 cells and embryonicframe and contains an upstream stop codon (Figures
1A and 1B). The initiator ATG of LCoR lies within a con- HEK293 kidney cells. While LCoR transcripts were abun-
dant in MDA-MB361 breast carcinoma cells, expressionsensus Kozak sequence RNNatgY (Kozak, 1996).
LCoR bears only limited resemblance to known co- was weaker in MDA-MB231 and MCF-7 breast cancer
lines (Figure 2A). Along with the EST data cited above,regulators. There is a single LXXLL motif (NR box) at
amino acid 53 and a PRKKRGR motif at amino acid these results indicate that LCoR transcripts are widely
Nuclear Receptor Corepressor LCoR
Figure 2. LCoR Transcripts Are Widely Ex-
(A) Northern blot of 15 g of total RNA iso-
lated from the cell lines indicated with LCoR
or ubiquitin probes. SCC4, SCC9, SCC15, and
SCC25 are human head and neck squamous
carcinoma lines; MDA-MB231, MDA-MB361,
and MCF-7 are human breast carcinoma cell
lines; HeLa, LNCaP, and CaCo-2 are human
cervical, prostate, and colon carcinoma lines,
respectively. HEK293 cells are derived from
human embryonic kidney and COS-7 from
(B) In situ hybridization analysis of LCoR ex-
pression in human placenta. (i and ii) Bright
and dark field photomicrographs of the cho-
rionic villi (CV) of a near term placenta (36
weeks) probed with a 443 b
antisense probe. Magnification, 20⫻. (ii) (in-
set) Dark field photomicrograph of a section
probed with a control LCoR sense probe. (iii
and iv) As in (i) and (ii), except at 40⫻ magnifi-
cation. Syn, syncytiotrophoblast; cm, chori-
expressed throughout fetal development and in the mone-treated MCF-7 cells (Wijayaratne and McDonnell,
Given the robust expression of LCoR transcripts in Interaction of ER␣ and LCoR in vivo was further tested
by bioluminescence resonance energy transfer (BRET)placenta and the complex placental steroid physiology,
LCoR expression was investigated further by in situ hy- in living COS-7 cells transiently cotransfected with plas-
mids expressing ER␣-EYFP and LCoR-rluc fusion pro-bridization analysis of a section of human placenta (Fig-
ure 2B). The results reveal that LCoR is predominantly teins. BRET and its variant fluorescence resonance en-
ergy transfer (FRET) have been used in the past to studyexpressed in the syncytiotrophoblast layer of terminally
differentiated cells, which acts as a barrier between ma- receptor-coregulator interactions (Llopis et al., 2000).
Treatment with estradiol or diethylstilbestrol (DES) en-ternal circulation and the fetus whose function is critical
for controlling maternal hormonal signals that modulate hanced BRET ratios 2.5- to 3-fold (Figure 3C), consistent
with agonist-dependent interaction of LCoR and ER␣,fetal metabolism and development (Pepe and Albrecht,
1995). whereas treatment with antiestrogens 4-hydroxytamoxi-
fen (OHT) or raloxifene had no significant effect. More-
over, mutation of the NR box of LCoR to LSKAA largely
Agonist-Dependent Interaction of LCoR
disrupted hormone-dependent interaction and reduced
and ER␣ In Vivo
hormone-independent interaction of the two proteins
An affinity-purified antibody developed against an LCoR
by approximately 2-fold (Figure 3C), indicating that the
peptide detected a protein of approximately 50 kDa in
LCoR LXXLL motif is essential for ligand-dependent in-
MCF-7, HEK293, and COS-7 cell extracts (Figure 3A),
teraction with ER␣.
in excellent agreement with cDNA cloning data. The
antibody also specifically detected several LCoR fusion
proteins and deletion mutants (data not shown). Immu- Interaction of LCoR with Nuclear Receptor
Ligand Binding Domains In Vitronocytochemical studies with the antibody in all three
lines revealed a nuclear protein (data not shown and In vitro translated LCoR selectively bound to the ER␣
LBD fused to GST (GST-ER␣-LBD) in a partially estro-see below). Consistent with two-hybrid cloning, endoge-
nous LCoR coimmunoprecipitated with endogenous gen-dependent manner (Figure 4A). Consistent with
BRET analyses, antiestrogens OHT, raloxifene, or ICIER␣ in an estradiol-dependent manner from MCF-7 cell
extracts (Figure 3B). No immunoprecipitation of ER␣ 164,384 did not induce interaction of LCoR with ER␣
(Figure 4A), and hormone-dependent binding of ER␣or LCoR was observed when anti-ER␣ antibody was
replaced by control IgG (Figure 3B). Note that reduced was abolished by mutation of the LCoR NR box (LSKAA;
Figure 4B). Similar results were obtained with GST-ER␣ER␣ expression after estradiol treatment is consistent
with enhanced turnover of the receptor observed in hor- fusions and in vitro translated LCoR-LSKAA (data not
Figure 3. Interaction of LCoR and ER␣ In Vivo
(A) Western analysis of LCoR in 20, 50, or 100 g of extract from
MCF-7, HEK293, and COS-7 cells using a rabbit polyclonal antipep-
(B) Coimmunoprecipitation of LCoR with ER␣. Western blots (WB) of
ER␣ (left) and LCoR (right) in immunoprecipitates of ER␣ with control
Figure 4. Characterization of LCoR Interaction In Vitro with ER␣,
mouse IgG or mouse monoclonal anti-ER␣ antibody from extracts of
ER␤, and VDR by GST Pull-Down Assay
MCF-7 cells treated for 4 hr with vehicle (⫺) or estradiol (E2).
Estradiol (E2), hydroxytamoxifen (OHT), raloxifene (Ral), and
(C) Bioluminescence resonance energy transfer (BRET) assays on
ICI164,384 (ICI), vitamin D3 (D3) were added to 10
M as indicated.
COS-7 cells transiently cotransfected with plasmids expressing
Inputs (lanes 1) represent 10% of the amount of labeled protein
EYFP-ER␣ and rluc-LCoR or rluc-LCoR-LSKAA fusion proteins and
used in assays.
treated with 10
M ␤-estradiol (E2), hydroxytamoxifen (OHT), raloxi-
(A) Ligand-dependent interaction of in vitro translated LCoR with
fene, diethylstilbestrol (DES), or ethanol (⫺). BRET ratios were calcu-
lated as described in the Experimental Procedures. The data shown
(B and D) Interaction of in vitro translated ER␣ (HEG0; [B]) or ER378
represent the mean ⫾ SEM of three experiments.
(D) with GST fused to LCoR, LCoR-LSKAA, or TIF2.1 as indicated.
(C) Interaction of LCoR with GST-ER␣ or a helix 12 mutant
shown). Double point mutation of ER␣ helix 12 (L539A,
(E and F) Interaction of GST fusions of wild-type ER␣ LBD or LBD
L540A; mAF-2) abolished ligand-dependent binding of
mutants T347A, H356R, N359S, and K362A with LCoR (E) or TIF-
LCoR (Figure 4C), demonstrating the importance of the
2.1 (F). Histograms of results of triplicate experiments are shown.
AF-2 domain. ER␣ was also truncated to amino acid 378
Bands were quantitated using the FluorChem digital imaging system
(ER378), leaving regions A–D and the N-terminal third
and AlphaEaseFC software (Alpha Innotech Corp, San Leandro, CA).
(G and H) Interaction of ER␤ (G) and VDR (H) with GST-LCoR and
of the LBD (Figure 4D), or to amino acid 282 in region
D (HE15) or 180, which encodes the A/B domain (data
not shown). While ER378 bound specifically to GST-
Nuclear Receptor Corepressor LCoR
Figure 5. LCoR Is a Nuclear Receptor Core-
(A, C, D, F, and H) LCoR represses ER␣-, GR-,
PR- and VDR-dependent transactivation.
COS-7 cells were cotransfected with expres-
sion vectors for ER␣ HEG0 (A and C), GR (D),
PR (F), or VDR (H), ERE3-TATA-pXP2 (A and
C), GRE5/pXP2 (D and F), or VDRE3tk/pXP2
(H) luciferase reporter vectors, pCMV-␤-gal
as internal control, and LCoR/pSG5 or
LSKAA/pSG5 expression vectors as indicated.
Cells were treated with 10
M of hormones
(solid bars) or vehicle (open bars). Normalized
luciferase activities (RLU) are the means ⫾
SEM from at least three experiments. (A) (in-
set) Control Western blot of ER␣ from ex-
tracts of COS-7 cells transfected with ER␣
HEG0 and 0, 500, or 1000 ng of LCoR/pSG5
in the absence or presence of estradiol. (C)
LCoR represses TIF-2 coactivation of ER␣.
Cells were transfected as in (A) with LCoR,
TIF-2, or TIF2.1 as indicated. (J) A GAL4-
LCoR fusion protein represses transactiva-
tion. COS-7 cells were transfected with 750
ng of 17-mer-5tk/pXp2, with indicated amounts
of GAL4-LCoR/pSG5, 1000 ng of pSG5, or
GAL4/pSG5. Normalized luciferase activities
(RLU) are the means ⫾ SEM from at least
three experiments. (B, E, G, I, and K). Differing
effects of HDAC inhibitor TSA on repression
by LCoR. Transfections were performed as
in the left-hand panels except that TSA (3 M)
LCoR but not TIF-2.1 in a hormone-independent manner residues contributing to ligand-independent interaction
with LCoR are located between ER␣ amino acids 283(Figure 4D), no such interaction was observed with HE15
or the A/B domain (data not shown), suggesting that and 377.
Interaction of LCoR with helix 3 was further probed
using GST fusions of ER␣ point mutants T347A, H356R,
N359S, and K362E. Helix 3 forms a critical part of the
static region of the coactivator binding pocket (Shiau et
al., 1998), and the integrity of lysine 362 at the C terminus
of helix 3 (Brzozowski et al., 1997) is essential for ligand-
dependent binding of p160 coactivators (Henttu et al.,
1997). While the K362A mutation disrupted both TIF-2.1
and LCoR binding, mutations T347A, H356R, and N359S
had a minimal effect on interaction of TIF-2.1, but par-
tially or completely abolished binding of LCoR (Figures
4E and 4F). The above data indicate that LCoR and TIF-
2.1 recognize overlapping binding sites, although LCoR
interacts with residues on helix 3 that are distinct from
those recognized by TIF-2.1.
Binding of LCoR to other nuclear receptors was also
analyzed by GST pull-down assays, which showed that
LCoR also bound LBDs of ER␤, VDR, RARs ␣, ␤, and ␥,
and RXR␣ in a ligand-dependent manner (Figures 4G
and 4H, and data not shown). Taken together, the above
results indicate that LCoR binds to the LBDs of several
nuclear receptors in a hormone-dependent or partially
hormone-dependent manner, and the interaction of
LCoR with the static portion (helix 3) of the coactivator
binding pocket of ER␣ differs from than that of TIF-2.1.
LCoR Is a Repressor of Ligand-Dependent
Transcription Induced by Class I
and Class II Nuclear Receptors
The effects of LCoR on transactivation by nuclear recep-
tors were tested by transient transfection in COS-7 cells
(Figure 5), which revealed that LCoR is a repressor of
ligand-dependent transcription of class I and II recep-
tors. Coexpression of LCoR produced a dose-depen-
dent repression of hormone-dependent transactivation
by ER␣ which was abolished by mutation of the NR box,
Figure 6. LCoR Interacts Directly with Specific HDACs
as the LSKAA mutant had no effect on ER␣ function
(A) HDACs 1, 3, 4, and 6 were in vitro translated and incubated with
(Figure 5A). Repression of estrogen-dependent gene ex-
GST alone or with GST-LCoR or GST-LSKAA fusion proteins. The
pression was not due to downregulation of ER␣ protein
input (lane 1) represents 10% of the amount of labeled protein used
in cells cotransfected with LCoR (Figure 5A, inset). Simi-
in the assays.
lar results were obtained in MCF-7 and HEK293 cells
(B) Association of tagged LCoR or LCoR-LSKAA with HDAC3. Ly-
(data not shown). Consistent with LCoR and TIF-2 recog-
sates from COS-7 cells transiently transfected with HA-HDAC3 and
nizing overlapping binding sites on ER␣, LCoR re-
Flag-LCoR or Flag-LSKAA were precipitated with anti-Flag antibody.
Cell extract and immunocomplexes were analyzed by Western blot-
pressed estrogen-dependent expression coactivated
ting with anti-HDAC3 or anti-Flag.
by TIF2 or TIF2.1 (Figure 5C). Repressive effects of 1 g
(C) Endogenous LCoR coimmunoprecipitates with endogenous
of transfected LCoR on ligand-activated transcription on
HDAC3. Immunoprecipitations from MCF-7 cell extracts were per-
the order of 2.2- to 5-fold were observed in experiments
formed with either rabbit control IgG or anti-HDAC3 antibody, and
with the glucocorticoid, progesterone, and vitamin D
immunoprecipitates were probed for HDAC3 or LCoR as indicated.
receptors (Figures 5D, 5F, and 5H). In each case, muta-
(D) Association of LCoR and LCoR-LSKAA with HDAC6. Lysates
from COS-7 cells transiently cotransfected with HA-Flag-HDAC6
tion of the NR box disrupted transcriptional repression.
and HA-LCoR or HA-LSKAA were precipitated with anti-Flag anti-
Moreover, GAL4-LCoR fusion repressed the activity of
body, and the immunocomplexes were analyzed by Western blotting
the 5 ⫻ 17-mer-tk promoter in a dose-dependent man-
with anti-HA or anti-Flag.
ner by 4-fold (Figure 5J), whereas free LCoR had no
(E) Endogenous LCoR coimmunoprecipitates with endogenous
effect on the 5 ⫻ 17-mer-tk promoter (data not shown).
HDAC6. Immunoprecipitations from MCF-7 cell extracts were per-
The mechanism of action of LCoR was investigated by
formed with either rabbit control IgG or anti-HDAC6 antibody, and
immunoprecipitates were probed for HDAC6 or LCoR as indicated.
analyzing the effect of the HDAC inhibitor trichostatin A
(TSA) on repression of ligand-dependent transcription.
Remarkably, while TSA completely abolished LCoR-
dependent repression of ER␣ and GR function (Figures LCoR Interacts Selectively with Histone Deacetylases
Pull-down assays performed with GST-LCoR and GST-7B and 7E), it had little or no effect on repression of PR
or VDR, or on repression by GAL-LCoR (Figures 5G, 5I, LSKAA to screen for potential interactions with class I
HDACs 1 and 3, and class II HDACs 4 and 6 revealedand 5K). This suggests that LCoR may function by
HDAC-dependent and -independent mechanisms. that both LCoR proteins interacted with HDACs 3 and
Nuclear Receptor Corepressor LCoR
Figure 7. LCoR Interacts with C-Terminal
(A) Schematic representation of LCoR show-
ing CtBP binding sites 1 and 2, and the posi-
tion of the Mfe1 site used to create C-ter-
minally truncated LCoR.
(B) GST pull-down assays were performed
with in vitro translated CtBP1, and GST con-
trol (pGEX) or fusions with LCoR, LCoR-
LSKAA, or LCoR-Mfe1 deletion mutant.
(C) GST pull-down assays were performed
with in vitro translated CtBP1, and GST con-
trol (pGEX) or fusions with LCoR, LCoR-
LSKAA, or LCoR mutated in CtBP binding
sites 1 (m1), 2 (m2), or 1 and 2 (m1⫹2). All
GST fusion proteins were expressed at similar
levels (data not shown).
(D) LCoR coimmunoprecipitates with CtBPs.
Extracts of MCF-7 cells were immunoprecipi-
tated with rabbit control IgG or with a rabbit
polyclonal anti-CtBP antibody, and immuno-
precipitates were probed for CtBP1, CtBP2,
(E and F) Colocalization of LCoR and CtBP1
(E) or CtBP2 (F) by confocal microscopy (see
Experimental Procedures for details).
(G) Mutation of CtBP binding motifs attenu-
ates repression by LCoR. COS-7 cells were
cotransfected with expression vectors for
ER␣ or GR or PR as indicated, along with
ERE3-TATA-pXP2 or GRE5/pXP2 as appro-
priate, and either wild-type LCoR or LCoR
mutated in CtBP binding motifs 1 or 2 as indi-
6, but not with HDACs 1 and 4 (Figure 6A). Reciprocal be consistent with CtBP and its associated factors con-
tributing to the TSA-insensitive repression of the PRcoimmunoprecipitation experiments revealed an inter-
action between epitope-tagged LCoR or LCoR-LSKAA observed above.
and HDAC3 (Figure 6B and data not shown). Moreover,
interaction between endogenous LCoR and HDAC3 was
confirmed by coimmunoprecipitation with an anti-
HDAC3 antibody from extracts of MCF-7 cells (Figure
We have identified LCoR, a corepressor that is widely
6C). Identical results were obtained in extracts of
expressed in human adult and fetal tissues and cell lines.
HEK293 cells (data not shown). Similarly, HA-LCoR and
LCoR function differs from those of NCoR and SMRT
HA-LCoR-LSKAA were coimmunoprecipitated with HA-
as it is recruited to receptors through an NR box in the
Flag-HDAC6 by an anti-Flag antibody (Figure 6D), and
presence of agonist. Highly homologous murine LCoR
endogenous LCoR coimmunoprecipitated with HDAC6
is expressed in two-cell embryos, suggesting that it
from extracts of MCF-7 cells (Figure 6E). Taken together,
functions from the earliest stages of embryonic develop-
these results indicate that LCoR can function to couple
ment. LCoR is most highly expressed in the placenta
specific HDACs to ligand-activated nuclear receptors.
and at near term is predominantly present in syncytiotro-
phoblasts. Receptors for estrogen, progesterone, and
glucocorticoids are expressed in the syncytiotropho-LCoR Interacts with C-Terminal Binding Protein
(CtBP) Corepressors blast layer, which represents a barrier between the ma-
ternal and the fetal circulation and is a critical site ofAnalysis of LCoR sequence (Figure 7A) revealed
PLDLTVR (aa 64) and VLDLSTK (aa 82) motifs that are steroid hormone signaling, biosynthesis, and catabo-
lism (Pepe and Albrecht, 1995; Whittle et al., 2001). Thehomologous to the PLDLS/TXR/K sequence defined as
a binding site for the corepressor CtBP1 (Vo et al., 2001). function of LCoR as an attenuator of nuclear receptor
signaling suggests that it may be an important modula-CtBP1, which was originally found as a protein that inter-
acts with the C terminus of E1A, functions by HDAC- tor of steroid hormone signaling in syncytiotrophoblasts.
LCoR contains a putative helix-loop-helix domain.dependent and -independent mechanisms (Chinna-
durai, 2002) and is highly homologous to CtBP2 (Sewalt Multiple repeats of an HLH domain are required for high-
affinity site-specific DNA binding of Drosophila pip-et al., 1999). GST pull-down assays revealed an interac-
tion between CtBP1 and wild-type LCoR, the LSKAA squeak proteins (Lehmann et al., 1998). Similarly, muta-
tion of one of the two HLH motifs in the MBLK-1 genemutant, and an LCoR mutant lacking the C-terminal half
of the protein (LCoR-Mfe1). CtBP1 binding was abol- strongly reduced site-specific DNA binding (Takeuchi et
al., 2001). The pipsqueak domain is homologous to mo-ished only when both binding sites in LCoR were mu-
tated (m1⫹2; Figure 7C). While NADH can modulate tifs found once in a number of prokaryotic and eukary-
otic proteins that interact with DNA, such as recombi-CtBP function (Zhang et al., 2002), no effect of NADH
was seen on its interaction with LCoR in vitro (data not nases (Lehmann et al., 1998; Sigmund and Lehmann,
2002), suggesting that LCoR itself may interact with DNA.shown).
CtBP1 and 2 are most efficiently immunoprecipitated Analysis of the interaction of LCoR with nuclear recep-
tors by BRET, coimmunoprecipitation, and GST pull-with an antibody that recognizes both proteins. Western
analysis suggested that the immunoprecipitates of down assays indicates that LCoR binds to receptor
LBDs in a ligand-dependent or partially ligand-depen-MCF-7 cells contained mostly CtBP1 (Figure 7D). Signifi-
cantly, LCoR was coimmunoprecipitated with CtBP pro- dent manner. Moreover, the dependence of LCoR bind-
ing to ER␣ on the integrity of its LXXLL motif and theteins under these conditions (Figure 7D). A similar coim-
munoprecipitation of LCoR was observed from extracts integrity of ER␣ helix 12 indicates that LCoR associates
with the same hydrophobic pocket in the LBD as p160of HEK293 cells (data not shown). In addition, immuno-
cytochemical analysis of LCoR and CtBP1 expression in coactivators. However, while mutation of K362 (helix
3) disrupted binding of both LCoR and TIF-2.1, LCoRMCF-7 cells revealed a strongly overlapping expression
pattern of the two proteins in discrete nuclear bodies binding was more sensitive to mutation of other helix 3
amino acids than TIF-2.1. Of particular note, LCoR bind-(Figure 7E). Similarly, the expression patterns of LCoR
and CtBP2 overlapped in MCF-7 cell nuclei (Figure 7F). ing was sensitive to the integrity of residue 347 of ER␣,
which lies outside binding groove residues 354–362 rec-Note that no fluorescence signal was seen in control
experiments where specific antibody was removed or ognized by the NR box II peptide of TIF-2 (GRIP1; Shiau
et al., 1998), suggesting that LCoR recognizes an ex-replaced with control IgG (data not shown). Mutation of
CtBP binding sites partially reduced the capacity of tended region of helix 3. LCoR residues outside the
LXXLL motif may thus contact the ER␣ LBD.LCoR to repress ligand-dependent transcription by ER␣
and the GR (Figure 7G), and consistent with the effect LCoR inhibited ligand-dependent transactivation by
nuclear receptors in a dose-dependent manner up toon the wild-type protein, TSA completely abolished the
residual repression of ER␣ by LCoR mutated in both 5-fold and functioned as a repressor when coupled to
the GAL4 DNA binding domain. While LCoR and p160binding sites (data not shown). Significantly, mutation
of site 2 or both sites largely abolished repression of PR- coactivators both bind in an agonist-dependent manner
to coactivator binding pockets, several results indicatedependent transactivation. Taken together, the above
data shows that binding of CtBPs contributes to tran- that the repression observed by LCoR was not simply
a result of blockage of p160 recruitment. Rather, LCoRscriptional repression by LCoR. Moreover, the greater
dependence on the CtBP binding sites of LCoR for re- recruits multiple factors that act to repress transcription.
While the HDAC inhibitor TSA abolished repressionpression of progesterone-induced transactivation would
Nuclear Receptor Corepressor LCoR
by LCoR of estrogen- and glucocorticoid-dependent LCoR acts gene specifically or is a general attenuator
transcription, the compound had little or no effect on
of ligand-dependent transactivation. The action of core-
repression of progesterone- or vitamin D-dependent
pressors such as LCoR that recognize agonist-bound
transcription or repression by GAL-LCoR, indicating
receptors is perhaps counterintuitive. However, their
HDAC-dependent and -independent modes of action.
existence suggests that there exist signals that act to
LCoR interacted with HDACs 3 and 6 but not HDAC1
attenuate the consequences of hormone-induced re-
or HDAC4, in vitro, and interactions with HDACs 3 and 6
ceptor function. Such effects would provide a counter-
were confirmed in coimmunoprecipitations. Preliminary
balance to signaling that augments hormone-induced
experiments indicate that HDACs 3 and 6 interact with
transactivation; for example, the stimulatory effects of
distinct regions of LCoR in the C-terminal half of the
MAP kinase signaling on ER␣ function (Kato et al., 1995).
protein (our unpublished data). HDACs 3 and 6 are class
One of the keys to understanding the function of LCoR
I and II enzymes, respectively. Unlike other class II en-
will be to determine the mechanisms modulating its ago-
zymes, HDAC6 contains two catalytic domains (Bertos
nist-dependent interaction with nuclear receptors. If
et al., 2001; Khochbin et al., 2001) and has not previously
LCoR acts to attenuate the function of agonist-bound
been associated with nuclear receptor corepressor
receptors, then it is likely that posttranslational modifi-
complexes. HDAC6 is both cytoplasmic and nuclear,
cation or LCoR and/or receptors will affect the relative
and recent studies have revealed its capacity to deacet-
affinities of LCoR and p160s for coactivator binding
ylate tubulin (Hubbert et al., 2002), suggesting that it
pockets. LCoR contains several putative phosphoryla-
may have broad substrate specificity.
tion motifs, including a number of MAP kinase sites in
Several biochemical studies to date have character-
the region of the NR box, as well as potential sites for
ized different corepressor complexes associated with
protein kinases A and C, raising the possibility that its
nuclear receptors, which include different HDACs (Glass
interaction with ligand-bound nuclear receptors may be
and Rosenfeld, 2000; Rosenfeld and Glass, 2001). Using
modulated by phosphorylation. In addition, LCoR con-
SMRT affinity chromatography, HDAC3 was identified
tains a consensus leptomycin B-sensitive nuclear export
as a component of a multiprotein complex that also
L; aa149–164), suggesting that its ac-
contained transducin ␤-like protein, TBL1, a homolog
cess to receptors may be regulated by nuclear export
of the groucho corepressor (Guenther et al., 2000). NCoR
under some conditions. Such a mechanism would be
was also found to be part of a large complex purified
analogous to a recent study showing that NCoR core-
by HDAC3 affinity chromatography (Wen et al., 2000).
pression of NF-B signaling can be attenuated by nu-
Whether LCoR is also a component of these complexes
clear export (Baek et al., 2002).
or different complex(es) remains to be seen. Studies to
In summary, we have identified a nuclear receptor
date suggest that NCoR and SMRT may interact with
corepressor LCoR, which is widely expressed through-
varying stability with distinct corepressor complexes
out mammalian development and represses ligand-
that include multiple HDACs, indicating that composi-
dependent nuclear receptor transactivation by recruit-
tions of individual corepressor complexes are not fixed.
ment of multiple factors. Our studies suggest that LCoR
Significantly, we also found that LCoR interacts with
is an important attenuator of nuclear receptor signaling
the corepressor CtBP1 through tandem consensus
during fetal development and in the adult.
CtBP-interaction motifs. Like LCoR, the sensitivity of
repression by CtBPs to TSA is dependent on the pro-
moter tested, indicative of HDAC-dependent and -inde-
Note that descriptions of antibodies, plasmid constructions, North-
pendent modes of action (Chinnadurai, 2002). CtBP pro-
ern blotting, and transfections are provided in the supplemental
teins interact with several different transcriptional
data at http://www.molecule.org/cgi/content/full/11/1/139/DC1.
repressors, including the nuclear receptor corepressor
RIP140 (Vo et al., 2001). The TSA-sensitive and -insensi-
Isolation of LCoR cDNA Sequences
tive actions of LCoR are analogous to another CtBP-
A yeast two-hybrid screen (2 ⫻ 10
transformants; Clontech human
interacting repressor Ikaros, which is composed of
fetal kidney cDNA Matchmaker library PT1020-1; Palo Alto, CA) with
distinct domains mediating repression by HDAC-depen-
an ER␣-LBD bait in the presence of 10
M estradiol yielded 10
dent and -independent mechanisms (Koipally and Geor-
colonies, of which six were dependent on estradiol for
LacZ expression. Three clones contained 1.2 kb inserts identical to
gopoulos, 2002a, 2002b). CtBP binding to Ikaros con-
coactivator AIB-1 (Anzick et al., 1997), and one contained an insert
tributes to its HDAC-independent mode of action
of 1.3 kb of LCoR sequence. 1.6 ⫻ 10
human gt11 prostate cDNA
(Koipally and Georgopoulos, 2002a). CtBPs also associ-
clones (Clontech, HL1131b) were screened for more LCoR se-
ate with specific polycomb group (PcG) repressor com-
quence, yielding five clones containing LCoR sequences 1–1417,
plexes (Sewalt et al., 1999), and HDAC-independent
462–1376, 704–1406, 1122–2915, and 1214–3016. Multiple alignment
repression of transcription by CtBP has been linked to
of the different cDNA clones was performed (CAP program; INFO-
its association with PcG complexes (Dahiya et al., 2001).
BIOGEN site http://www.infobiogen.fr). Homologies to ESTs and
proteins were found using BLAST2 and PSI-BLAST, respectively,
Our initial experiments indicate that LCoR also associ-
employing standard parameters and matrices.
ates with components of PcG complexes (our unpub-
Immunocytochemistry and In Situ Hybridization
Our studies have suggested that LCoR can act as a
MCF-7 cells were cultivated on collagen IV-treated microscope
corepressor for several receptors. However, it will be
slides in 6-well plates, fixed with 2% paraformaldehyde for 15 min
essential to verify the effects of LCoR on regulation of
at room temperature, washed (3⫻) with PBS, and permeabilized
endogenous nuclear receptor target genes using chro-
with 0.2% Triton X100, 5% BSA, 10% horse serum in PBS. Cells
matin immunoprecipitation assays. In addition, overex-
were then incubated with ␣-LCoR (1:500), and ␣CtBP1 or ␣CtBP2
(1:50) in buffer B (0.2% Triton X100, 5% BSA in PBS) for 1 hr at
pression/knockdown experiments will determine whether
room temperature. Cells were washed (3⫻) with PBS and incubated J.H.W. are chercheurs boursier of the Fonds de Recherche en Sante
bec.with goat anti-rabbit-Cy2 and donkey anti-goat Cy3 (1:300) in buffer
B for 1 hr at room temperature. Slides were mounted with Immuno-
Fluore Mounting Medium (ICN, Aurora, OH) and visualized using a
Received: March 20, 2002
Zeiss LSM 510 confocal microscope at 63⫻ magnification. In situ
Revised: October 22, 2002
hybridization was carried out (Han et al., 1996) using 443 bp sense
and antisense LCoR probes, and a hybridization temperature of
60⬚C and maximum wash conditions of 0.1⫻ SSC at 65⬚C.
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from the Canadian Institutes of Health Research (CIHR) to J.H.W.
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