Ligand-dependent nuclear receptor corepressor LCoR functions by histone deacetylase-dependent and -independent mechanisms.
ABSTRACT 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.
- SourceAvailable from: biu.ac.il[show abstract] [hide abstract]
ABSTRACT: Over the past few years, the long-standing idea that covalent modification of chromatin can play a role in determining states of gene activity has been confirmed. Eukaryotic genes can be silenced by deacetylation of acetyl-lysine moieties in the N-terminal tails of histones. Recent work links histone deacetylases with an increasing number of repressors, suggesting that deacetylation might be a rather pervasive feature of transcriptional repression systems.Trends in Biochemical Sciences 04/2000; 25(3):121-6. · 13.08 Impact Factor
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ABSTRACT: The ligand-binding domain of nuclear receptors contains a transcriptional activation function (AF-2) that mediates hormone-dependent binding of coactivator proteins. Scanning surface mutagenesis on the human thyroid hormone receptor was performed to define the site that binds the coactivators, glucocorticoid receptor-interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC-1). The residues involved encircle a small surface that contains a hydrophobic cleft. Ligand activation of transcription involves formation of this surface by folding the carboxyl-terminal alpha helix against a scaffold of three other helices. These features may represent general ones for nuclear receptors.Science 07/1998; 280(5370):1747-9. · 31.03 Impact Factor
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ABSTRACT: Pipsqueak (Psq) belongs to a family of proteins defined by a phylogenetically old protein-protein interaction motif. Like the GAGA factor and other members of this family, Psq is an important developmental regulator in Drosophila, having pleiotropic functions during oogenesis, embryonic pattern formation, and adult development. The GAGA factor controls the transcriptional activation of homeotic genes and other genes by binding to control elements containing the GAGAG consensus motif. Binding is associated with formation of an open chromatin structure that makes the control regions accessible to transcriptional activators. We show here that Psq contains a novel DNA-binding domain, which binds, like the GAGA factor zinc finger DNA-binding domain, to target sites containing the GAGAG consensus motif. Binding is suppressed, as in the GAGA factor and other proteins of the family, by the associated protein-protein interaction motif. The DNA-binding domain, which we call the Psq domain, is identical with a previously identified region consisting of four tandem repeats of a conserved 50-amino acid sequence, the Psq motif. The Psq domain seems to be structurally related to known DNA-binding domains, both in its repetitive character and in the putative three-alpha-helix structure of the Psq motif, but it lacks the conserved sequence signatures of the classical eukaryotic DNA-binding motifs. Psq may thus represent the prototype of a new family of DNA-binding proteins.Journal of Biological Chemistry 11/1998; 273(43):28504-9. · 4.65 Impact Factor
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-
minal AF-2 helix (helix 12) to create a binding pocket
recognized by coactivators.
function (Robyr et al., 2000; Glass and Rosenfeld, 2000;
Dilworth and Chambon, 2001; Rosenfeld and Glass,
2001). Their diversity suggests that transcriptional acti-
vation byreceptors occurs through recruitmentof multi-
ple factors acting sequentially or combinatorially. Co-
activators of the p160 family, SRC1/NCoA1, TIF-2/
GRIP-1, and pCIP/AIB1/RAC3/ACTR/TRAM-1 (Onate et
al., 1995; Chakravarti et al., 1996; Hong et al., 1996;
Voegelet al.,1996;Anzicket al.,1997;Chen etal.,1997),
which interact with ligand-bound receptors through
LXXLL motifs or NR boxes (Voegel et al., 1996; Heery
et al., 1997).Cocrystallographic studies of ligand-bound
receptors revealed ?-helical NR boxes oriented within a
clamp composedof conservedresidues inhelices 3and
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 CREB
binding protein (CBP) and its homolog p300 (Kamei et
al., 1996; Chen et al., 1997; Torchia et al., 1997). Several
coactivators including CBP/p300 and associated factor
p/CAF possess histone acetyltransferase activity, re-
quired for chromatin remodeling (Ogryzko et al., 1996;
Yang et al., 1996; Chen et al., 1997; Kurokawa et al.,
1998) and subsequent access of the transcriptional ma-
chinery to promoters.
Corepressors NCoR and SMRT mediate ligand-inde-
pendent repression by thyroid and retinoic acid recep-
tors (Horlein et al., 1995; Chen and Evans, 1995; Perissi
et al., 1999) and recruit multiprotein complexes impli-
cated in transcriptional repression and histone deacety-
lation (Alland et al., 1997; Hassig et al., 1997; Heinzel et
al., 1997; Kadosh and Struhl, 1997; Laherty et al., 1997;
Nagy et al., 1997; Pazin and Kadonaga, 1997). Histone
deacetylases (HDACs) fall into three classes based on
homology, domain structure, subcellular localization,
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
combinations of ancillary proteins and class I or class
II HDACs (Rosenfeld and Glass, 2001), suggesting that
their function depends on cell type, combinations of
transcription factors bound to specific promoters, and
phase of the cell cycle.
Here, we have identified a ligand-dependent core-
pressor (LCoR) that interacts with ER? and other class
I and class II nuclear receptors through a single NR box.
LCoR, which is expressed from the earliest stages of
mammalian development,functions inan HDAC-depen-
multiple cofactors. LCoR represents a distinct class of
Isabelle Fernandes,1Yolande Bastien,1
Timothy Wai,1Karen Nygard,5Roberto Lin,1
Olivier Cormier,4Han S. Lee,1Frankie Eng,1
Nicholas R. Bertos,3Nadine Pelletier,3
Sylvie Mader,4Victor K.M. Han,5
Xiang-Jiao Yang,3and John H. White1,2,*
1Department of Physiology
2Department of Medicine
3Department of Oncology
Montreal, Quebec, H3G 1Y6
4Department of Biochemistry
University of Montreal
Montreal, Quebec, H3C 3J7
5Department of Paediatrics
Department of Obstetrics and Gynecology
Department of Biochemistry and Anatomy
MRC Group in Fetal and Neonatal Health
Lawson Research Institute
University of Western Ontario
London, Ontario, N6A 4V2
LCoR (ligand-dependent corepressor) is a transcrip-
tional corepressor widely expressed in fetal and adult
tissues that is recruited to agonist-bound nuclear re-
ceptors through a single LXXLL motif. LCoR binding
to estrogen receptor ? 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 recep-
tor-dependent fashion, indicating HDAC-dependent
and -independent modes of action. LCoR binds di-
rectly to specific HDACs in vitro and in vivo. Moreover,
LCoR functions by recruiting C-terminal binding pro-
tifs and colocalizes with CtBPs in the nucleus. LCoR
nist-activated nuclear receptor signaling by multiple
Nuclear receptors are ligand-regulated transcription
factors whose activities are controlled by a range of
lipophilic extracellular signals. They directly regulate
transcription of genes whose products control many
aspects of physiology and metabolism (Chawla et al.,
domains, A–F. Many N-terminal A/B regions contain
transactivation domains (activating function-1; AF-1),
ligand binding domain (LBD) (Tora et al., 1989; Robyr et
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
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 4747bpcDNA (AB058698)iden-
tified from a human brain library (Nagase et
al., 2001) containing an extra 5?UTR exon is
indicated at the bottom.
the Human Genome Browser (http://genome.
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-
ac.uk) and Network Protein Sequence Analy-
nuclear receptor corepressor that acts to attenuate sig-
naling by agonist-bound receptors.
339 that is homologous to a simple nuclear localization
signal (NLS) of the SV40 large T antigen-type. The NLS
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%
homologous to motifs encoded by the Eip93F, T01C1.3,
and MBLK-1 genes of Drosophila, C. elegans, and Hon-
(Figure 1C). The domain also bears 35% homology to
the pipsqueak motif (PSQ) repeated four times in the
tor pipsqueak (Lehmann et al., 1998).
Identification of LCoR
LCoR was isolated from a yeast two-hybrid library as a
cDNA containing a 1299 bp open reading frame (433
amino acids;47,006 kDa; Figures1A and 1D)encoding a
protein that interacted with the ER? LBD in an estradiol-
dependent manner.Additional cDNAswereobtainedfrom
a human prostate cDNA library, and several expressed
sequence tags (ESTs; Figure 1A). Human sequences
were also highly homologous (?95%) to several mouse
ESTs, including multiple clones from a two-cell embryo
library (data not shown), indicating that LCoR is ex-
exons on chromosome 10q24.1, including four short
5?UTR exons that contain several in-frame stop codons
(Figure 1B and data not shown). A human brain EST
(Nagase et al., 2001) contains a single exon insert that
frame and contains an upstream stop codon (Figures
1A and 1B). The initiator ATG of LCoR lies within a con-
sensus Kozak sequence RNNatgY (Kozak, 1996).
LCoR bears only limited resemblance to known co-
regulators. There is a single LXXLL motif (NR box) at
amino acid 53 and a PRKKRGR motif at amino acid
LCoR Is Widely Expressed in Fetal and Adult Tissues
observed in placenta, the cerebellum, and corpus callo-
sum of the brain, adult kidney and a number of fetal
tissues (see Supplemental Figure S2 at http://www.
cell lines (Figure 2A), with highest levels of expression
observed in intestinal Caco-2 cells and embryonic
dant in MDA-MB361 breast carcinoma cells, expression
was weaker in MDA-MB231 and MCF-7 breast cancer
lines (Figure 2A). Along with the EST data cited above,
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
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, andcolon carcinomalines,
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 b35S-labeled LCoR
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
Given the robust expression of LCoR transcripts in
placenta and the complex placental steroid physiology,
LCoR expression was investigated further by in situ hy-
bridization analysis of a section of human placenta (Fig-
ure 2B). The results reveal that LCoR is predominantly
expressed in the syncytiotrophoblast layer of terminally
differentiated cells, which acts as a barrier between ma-
ternal circulation and the fetus whose function is critical
for controlling maternal hormonal signals that modulate
fetal metabolism and development (Pepe and Albrecht,
mone-treated MCF-7 cells (Wijayaratne and McDonnell,
by bioluminescence resonance energy transfer (BRET)
in living COS-7 cells transiently cotransfected with plas-
mids expressing ER?-EYFP and LCoR-rluc fusion pro-
teins. BRET and its variant fluorescence resonance en-
ergy transfer (FRET) have been used in the past to study
receptor-coregulator interactions (Llopis et al., 2000).
Treatment with estradiol or diethylstilbestrol (DES) en-
with agonist-dependent interaction of LCoR and ER?,
fen (OHT) or raloxifene had no significant effect. More-
over, mutation of the NR box of LCoR to LSKAA largely
disrupted hormone-dependent interaction and reduced
hormone-independent interaction of the two proteins
by approximately 2-fold (Figure 3C), indicating that the
LCoR LXXLL motif is essential for ligand-dependent in-
teraction with ER?.
Agonist-Dependent Interaction of LCoR
and ER? In Vivo
peptide detected a protein of approximately 50 kDa in
MCF-7, HEK293, and COS-7 cell extracts (Figure 3A),
in excellent agreement with cDNA cloning data. The
antibody also specifically detected several LCoR fusion
proteins and deletion mutants (data not shown). Immu-
nocytochemical studies with the antibody in all three
lines revealed a nuclear protein (data not shown and
nous LCoR coimmunoprecipitated with endogenous
ER? in an estradiol-dependent manner from MCF-7 cell
extracts (Figure 3B). No immunoprecipitation of ER?
or LCoR was observed when anti-ER? antibody was
replaced by control IgG (Figure 3B). Note that reduced
ER? expression after estradiol treatment is consistent
with enhanced turnover of the receptor observed in hor-
Interaction of LCoR with Nuclear Receptor
Ligand Binding Domains In Vitro
In vitro translated LCoR selectively bound to the ER?
LBD fused to GST (GST-ER?-LBD) in a partially estro-
gen-dependent manner (Figure 4A). Consistent with
BRET analyses, antiestrogens OHT, raloxifene, or ICI
164,384 did not induce interaction of LCoR with ER?
(Figure 4A), and hormone-dependent binding of ER?
was abolished by mutation of the LCoR NR box (LSKAA;
Figure 4B). Similar results were obtained with GST-ER?
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
mouse IgG or mouse monoclonal anti-ER? antibody from extracts of
MCF-7 cells treated for 4 hr with vehicle (?) or estradiol (E2).
(C) Bioluminescence resonance energy transfer (BRET) assays on
COS-7 cells transiently cotransfected with plasmids expressing
EYFP-ER? and rluc-LCoR or rluc-LCoR-LSKAA fusion proteins and
lated as described in the Experimental Procedures. The data shown
represent the mean ? SEM of three experiments.
Figure 4. Characterization of LCoR Interaction In Vitro with ER?,
ER?, and VDR by GST Pull-Down Assay
Estradiol (E2), hydroxytamoxifen (OHT), raloxifene (Ral), and
ICI164,384 (ICI), vitamin D3 (D3) were added to 10?6M as indicated.
Inputs (lanes 1) represent 10% of the amount of labeled protein
used in assays.
(A) Ligand-dependent interaction of in vitro translated LCoR with
(B and D) Interaction of in vitro translated ER? (HEG0; [B]) or ER378
(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
(E and F) Interaction of GST fusions of wild-type ER? LBD or LBD
mutants T347A, H356R, N359S, and K362A with LCoR (E) or TIF-
2.1 (F). Histograms of results of triplicate experiments are shown.
(G and H) Interaction of ER? (G) and VDR (H) with GST-LCoR and
shown). Double point mutation of ER? helix 12 (L539A,
L540A; mAF-2) abolished ligand-dependent binding of
LCoR (Figure 4C), demonstrating the importance of the
AF-2 domain. ER? was also truncated to amino acid 378
(ER378), leaving regions A–D and the N-terminal third
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, andH) LCoRrepresses 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?7M of hormones
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
of GAL4-LCoR/pSG5, 1000 ng of pSG5, or
GAL4/pSG5. Normalized luciferase activities
(RLU) are the means ? SEM from at least
effects of HDAC inhibitor TSA on repression
by LCoR. Transfections were performed as
or the A/B domain (data not shown), suggesting that
residues contributing to ligand-independent interaction
with LCoR are located between ER? amino acids 283
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
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
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
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,
as the LSKAA mutant had no effect on ER? function
pression was not due to downregulation of ER? protein
in cells cotransfected with LCoR (Figure 5A, inset). Simi-
lar results were obtained in MCF-7 and HEK293 cells
nizing overlapping binding sites on ER?, LCoR re-
pressed estrogen-dependent expression coactivated
by TIF2 or TIF2.1 (Figure 5C). Repressive effects of 1 ?g
the order of 2.2- to 5-fold were observed in experiments
with the glucocorticoid, progesterone, and vitamin D
receptors (Figures 5D, 5F, and 5H). In each case, muta-
tion of the NR box disrupted transcriptional repression.
Moreover, GAL4-LCoR fusion repressed the activity of
the 5 ? 17-mer-tk promoter in a dose-dependent man-
ner by 4-fold (Figure 5J), whereas free LCoR had no
effect on the 5 ? 17-mer-tk promoter (data not shown).
The mechanism of action of LCoR was investigated by
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
7B and 7E), it had little or no effect on repression of PR
or VDR, or on repression by GAL-LCoR (Figures 5G, 5I,
and 5K). This suggests that LCoR may function by
HDAC-dependent and -independent mechanisms.
Figure 6. LCoR Interacts Directly with Specific HDACs
(A) HDACs 1, 3, 4, and 6 were in vitro translated and incubated with
GST alone or with GST-LCoR or GST-LSKAA fusion proteins. The
input (lane 1) represents 10% of the amount of labeled protein used
in the assays.
(B) Association of tagged LCoR or LCoR-LSKAA with HDAC3. Ly-
sates from COS-7 cells transiently transfected with HA-HDAC3 and
Cell extract and immunocomplexes were analyzed by Western blot-
ting with anti-HDAC3 or anti-Flag.
(C) Endogenous LCoR coimmunoprecipitates with endogenous
HDAC3. Immunoprecipitations from MCF-7 cell extracts were per-
formed with either rabbit control IgG or anti-HDAC3 antibody, and
immunoprecipitates were probed for HDAC3 or LCoR as indicated.
(D) Association of LCoR and LCoR-LSKAA with HDAC6. Lysates
from COS-7 cells transiently cotransfected with HA-Flag-HDAC6
and HA-LCoR or HA-LSKAA were precipitated with anti-Flag anti-
with anti-HA or anti-Flag.
(E) Endogenous LCoR coimmunoprecipitates with endogenous
HDAC6. Immunoprecipitations from MCF-7 cell extracts were per-
formed with either rabbit control IgG or anti-HDAC6 antibody, and
immunoprecipitates were probed for HDAC6 or LCoR as indicated.
LCoR Interacts Selectively with Histone Deacetylases
Pull-down assays performed with GST-LCoR and GST-
LSKAA to screen for potential interactions with class I
HDACs 1 and 3, and class II HDACs 4 and 6 revealed
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
levels (data not shown).
(D) LCoR coimmunoprecipitates with CtBPs.
Extracts ofMCF-7 cellswere 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
coimmunoprecipitation experiments revealed an inter-
action between epitope-tagged LCoR or LCoR-LSKAA
and HDAC3 (Figure 6B and data not shown). Moreover,
interactionbetween endogenousLCoRand HDAC3was
confirmed by coimmunoprecipitation with an anti-
HDAC3 antibody from extracts of MCF-7 cells (Figure
6C). Identical results were obtained in extracts of
HEK293 cells (data not shown). Similarly, HA-LCoR and
HA-LCoR-LSKAA were coimmunoprecipitated with HA-
Flag-HDAC6 by an anti-Flag antibody (Figure 6D), and
endogenous LCoR coimmunoprecipitated with HDAC6
these results indicate that LCoR can function to couple
specific HDACs to ligand-activated nuclear receptors.
be consistent with CtBP and its associated factors con-
tributing to the TSA-insensitive repression of the PR
We have identified LCoR, a corepressor that is widely
LCoR function differs from those of NCoR and SMRT
as it is recruited to receptors through an NR box in the
presence of agonist. Highly homologous murine LCoR
is expressed in two-cell embryos, suggesting that it
ment. LCoR is most highly expressed in the placenta
phoblasts. Receptors for estrogen, progesterone, and
glucocorticoids are expressed in the syncytiotropho-
blast layer, which represents a barrier between the ma-
ternal and the fetal circulation and is a critical site of
steroid hormone signaling, biosynthesis, and catabo-
lism (Pepe and Albrecht, 1995; Whittle et al., 2001). The
function of LCoR as an attenuator of nuclear receptor
signaling suggests that it may be an important modula-
LCoR contains a putative helix-loop-helix domain.
affinity site-specific DNA binding of Drosophila pip-
squeak proteins (Lehmann et al., 1998). Similarly, muta-
tion of one of the two HLH motifs in the MBLK-1 gene
strongly reduced site-specific DNA binding (Takeuchi et
al., 2001). The pipsqueak domain is homologous to mo-
tifs found once in a number of prokaryotic and eukary-
otic proteins that interact with DNA, such as recombi-
nases (Lehmann et al., 1998; Sigmund and Lehmann,
2002), suggesting that LCoR itself may interact with DNA.
tors by BRET, coimmunoprecipitation, and GST pull-
down assays indicates that LCoR binds to receptor
LBDs in a ligand-dependent or partially ligand-depen-
dent manner. Moreover, the dependence of LCoR bind-
ing to ER? on the integrity of its LXXLL motif and the
integrity of ER? helix 12 indicates that LCoR associates
with the same hydrophobic pocket in the LBD as p160
coactivators. However, while mutation of K362 (helix
3) disrupted binding of both LCoR and TIF-2.1, LCoR
binding was more sensitive to mutation of other helix 3
amino acids than TIF-2.1. Of particular note, LCoR bind-
ing was sensitive to the integrity of residue 347 of ER?,
which liesoutside bindinggroove residues354–362 rec-
ognized by the NR box II peptide of TIF-2 (GRIP1; Shiau
et al., 1998), suggesting that LCoR recognizes an ex-
tended region of helix 3. LCoR residues outside the
LXXLL motif may thus contact the ER? LBD.
LCoR inhibited ligand-dependent transactivation by
nuclear receptors in a dose-dependent manner up to
5-fold and functioned as a repressor when coupled to
the GAL4 DNA binding domain. While LCoR and p160
coactivators both bind in an agonist-dependent manner
to coactivator binding pockets, several results indicate
that the repression observed by LCoR was not simply
a result of blockage of p160 recruitment. Rather, LCoR
While the HDAC inhibitor TSA abolished repression
LCoR Interacts with C-Terminal Binding Protein
Analysis of LCoR sequence (Figure 7A) revealed
PLDLTVR (aa 64) and VLDLSTK (aa 82) motifs that are
homologous to the PLDLS/TXR/K sequence defined as
acts with the C terminus of E1A, functions by HDAC-
dependent and -independent mechanisms (Chinna-
durai, 2002) and is highly homologous to CtBP2 (Sewalt
et al., 1999). GST pull-down assays revealed an interac-
tion between CtBP1 and wild-type LCoR, the LSKAA
mutant, and an LCoR mutant lacking the C-terminal half
of the protein (LCoR-Mfe1). CtBP1 binding was abol-
ished only when both binding sites in LCoR were mu-
tated (m1?2; Figure 7C). While NADH can modulate
CtBP function (Zhang et al., 2002), no effect of NADH
was seen on its interaction with LCoR in vitro (data not
CtBP1 and 2 are most efficiently immunoprecipitated
with an antibody that recognizes both proteins. Western
analysis suggested that the immunoprecipitates of
teins under these conditions (Figure 7D). A similar coim-
munoprecipitation of LCoR was observed from extracts
of HEK293 cells (data not shown). In addition, immuno-
cytochemical analysisof LCoRand CtBP1expression in
MCF-7 cells revealed a strongly overlapping expression
pattern of the two proteins in discrete nuclear bodies
(Figure 7E). Similarly, the expression patterns of LCoR
and CtBP2 overlapped in MCF-7 cell nuclei (Figure 7F).
Note that no fluorescence signal was seen in control
experiments where specific antibody was removed or
replaced with control IgG (data not shown). Mutation of
CtBP binding sites partially reduced the capacity of
LCoR to repress ligand-dependent transcription by ER?
and the GR (Figure 7G), and consistent with the effect
on the wild-type protein, TSA completely abolished the
residual repression of ER? by LCoR mutated in both
binding sites (data not shown). Significantly, mutation
dependent transactivation. Taken together, the above
data shows that binding of CtBPs contributes to tran-
scriptional repression by LCoR. Moreover, the greater
dependence on the CtBP binding sites of LCoR for re-
pression of progesterone-induced transactivation would
Nuclear Receptor Corepressor LCoR
by LCoR of estrogen- and glucocorticoid-dependent
transcription, the compound had little or no effect on
repression of progesterone- or vitamin D-dependent
transcription or repression by GAL-LCoR, indicating
HDAC-dependent and -independent modes of action.
LCoR interacted with HDACs 3 and 6 but not HDAC1
or HDAC4, in vitro, and interactions with HDACs 3 and 6
were confirmed in coimmunoprecipitations. Preliminary
experiments indicate that HDACs 3 and 6 interact with
distinct regions of LCoR in the C-terminal half of the
protein (our unpublished data). HDACs 3 and 6 are class
I and II enzymes, respectively. Unlike other class II en-
zymes, HDAC6 contains two catalytic domains (Bertos
been associated with nuclear receptor corepressor
complexes. HDAC6 is both cytoplasmic and nuclear,
and recent studies have revealed its capacity to deacet-
ylate tubulin (Hubbert et al., 2002), suggesting that it
may have broad substrate specificity.
Several biochemical studies to date have character-
ized different corepressor complexes associated with
and Rosenfeld, 2000; Rosenfeld and Glass, 2001). Using
SMRT affinity chromatography, HDAC3 was identified
as a component of a multiprotein complex that also
contained transducin ?-like protein, TBL1, a homolog
was also found to be part of a large complex purified
by HDAC3 affinity chromatography (Wen et al., 2000).
Whether LCoR is also a component of these complexes
or different complex(es) remains to be seen. Studies to
date suggest that NCoR and SMRT may interact with
varying stability with distinct corepressor complexes
that include multiple HDACs, indicating that composi-
tions of individual corepressor complexes are not fixed.
Significantly, we also found that LCoR interacts with
the corepressor CtBP1 through tandem consensus
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-
teins interact with several different transcriptional
repressors, including the nuclear receptor corepressor
RIP140 (Vo et al., 2001). The TSA-sensitive and -insensi-
tive actions of LCoR are analogous to another CtBP-
interacting repressor Ikaros, which is composed of
dentand -independentmechanisms(Koipally andGeor-
gopoulos, 2002a, 2002b). CtBP binding to Ikaros con-
tributes to its HDAC-independent mode of action
(Koipally and Georgopoulos, 2002a). CtBPs also associ-
ate with specific polycomb group (PcG) repressor com-
plexes (Sewalt et al., 1999), and HDAC-independent
repression of transcription by CtBP has been linked to
itsassociation withPcGcomplexes(Dahiya etal.,2001).
Our initial experiments indicate that LCoR also associ-
ates with components of PcG complexes (our unpub-
Our studies have suggested that LCoR can act as a
corepressor for several receptors. However, it will be
essential to verify the effects of LCoR on regulation of
endogenous nuclear receptor target genes using chro-
matin immunoprecipitation assays. In addition, overex-
pression/knockdown experiments will determine whether
LCoR acts gene specifically or is a general attenuator
of ligand-dependent transactivation. The action of core-
pressors such as LCoR that recognize agonist-bound
receptors is perhaps counterintuitive. However, their
existence suggests that there exist signals that act to
attenuate the consequences of hormone-induced re-
ceptor function. Such effects would provide a counter-
balance to signaling that augments hormone-induced
transactivation; for example, the stimulatory effects of
nist-dependent interaction with nuclear receptors. If
LCoR acts to attenuate the function of agonist-bound
receptors, then it is likely that posttranslational modifi-
cation or LCoR and/or receptors will affect the relative
affinities of LCoR and p160s for coactivator binding
pockets. LCoR contains several putative phosphoryla-
tion motifs, including a number of MAP kinase sites in
the region of the NR box, as well as potential sites for
protein kinases A and C, raising the possibility that its
interaction with ligand-bound nuclear receptors may be
modulated by phosphorylation. In addition, LCoR con-
cess to receptors may be regulated by nuclear export
under some conditions. Such a mechanism would be
analogous to a recent study showing that NCoR core-
pression of NF-?B signaling can be attenuated by nu-
clear export (Baek et al., 2002).
In summary, we have identified a nuclear receptor
corepressor LCoR, which is widely expressed through-
out mammalian development and represses ligand-
dependent nuclear receptor transactivation by recruit-
ment of multiple factors. Our studies suggest that LCoR
is an important attenuator of nuclear receptor signaling
during fetal development and in the adult.
Note that descriptions of antibodies, plasmid constructions, North-
ern blotting, and transfections are provided in the supplemental
data at http://www.molecule.org/cgi/content/full/11/1/139/DC1.
Isolation of LCoR cDNA Sequences
A yeast two-hybrid screen (2 ? 106transformants; Clontech human
fetal kidney cDNA Matchmaker library PT1020-1; Palo Alto, CA) with
an ER?-LBD bait in the presence of 10?6M estradiol yielded 10
His?/LacZ?colonies, of which six were dependent on estradiol for
LacZ expression. Three clones contained 1.2 kb inserts identical to
coactivator AIB-1 (Anzick et al., 1997), and one contained an insert
of 1.3 kb of LCoR sequence. 1.6 ? 106human ?gt11 prostate cDNA
clones (Clontech, HL1131b) were screened for more LCoR se-
quence, yielding five clones containing LCoR sequences 1–1417,
of the different cDNA clones was performed (CAP program; INFO-
BIOGEN site http://www.infobiogen.fr). Homologies to ESTs and
proteins were found using BLAST2 and PSI-BLAST, respectively,
employing standard parameters and matrices.
Immunocytochemistry and In Situ Hybridization
MCF-7 cells were cultivated on collagen IV-treated microscope
slides in 6-well plates, fixed with 2% paraformaldehyde for 15 min
at room temperature, washed (3?) with PBS, and permeabilized
with 0.2% Triton X100, 5% BSA, 10% horse serum in PBS. Cells
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
room temperature. Cells were washed (3?) with PBS and incubated
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
Zeiss LSM 510 confocal microscope at 63? magnification. In situ
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.
J.H.W. are chercheurs boursier of the Fonds de Recherche en Sante ´
du Que ´bec.
Received: March 20, 2002
Revised: October 22, 2002
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GST Pull-Down Assays and Immunoprecipitations
GST pull-down assays were performed as described (Eng et al.,
1998), with the exception that assays performed with in vitro trans-
lated ER378 included two more washes made with the GST buffer
containing 150 mM NaCl. For immunoprecipitations of tagged pro-
teins, COS-7 cells in 100 mm dishes were transfected with 6 ?g of
HA-LCoR and/or 6 ?g of HA-Flag-HDAC6 or with 6 ?g of Flag-LCoR
and/or 6 ?g of HA-HDAC3 and pSG5 carrier. Forty-eight hours after
transfection, cells were lysed 30 min at 4?C in 1 ml of JLB (20 mM
Tris-HCl [pH 8], 150 mM KCl, 10% glycerol, 0.1% IGEPAL CA-630,
and complete protease inhibitor cocktail; Boehringer-Mannheim,
Laval, Quebec, Canada). Cell debris were pelleted by centrifugation
(14,000 rpm, 5 min), and proteins were immunoprecipitated from
600 ?l of supernatant by incubation for 1 hr at 4?C with 4 ?g of
incubation with protein A?G agarose or protein-A agarose beads
for anti-Flag and anti-HDAC3, respectively. Beads were washed
(3?) with JLB. Bound immunocomplexes were boiled in Laemmli
buffer, separated by 10% SDS-PAGE, and blotted on PVDF mem-
brane with ?-Flag M2-peroxidase, ?-HDAC3, ?-HA-peroxidase
(1:500), and detected by enhanced chemiluminescence (NEN Life
Science Products, Boston, MA). For immunoprecipitation of endog-
enous HDAC3 or HDAC6, MCF-7 cells in 150 mm dishes were lysed
in 2 ml of JLB. Supernatants were cleared, incubated with 4 ?g of
?HDAC6 or ?HDAC3 or control rabbit IgG in the presence of protein
A agarose, and Western blotted as above. For ER? or CtBP, MCF-7
cells were lysed in 2 ml of 150 mM NaCl, 10 mM Tris-HCl (pH 7.4),
0.2 mM Na orthovanadate, 1 mM EDTA, 1 mM EGTA, 1% Triton-
100X, 0.5% IGEPAL CA-630, protease inhibitor cocktail, and immu-
noprecipitated as above with 4 ?g of ?CtBP or ?ER? antibodies, or
corresponding control IgG in the presence of protein A or protein
A?G agarose, respectively. Dilutions of specific antibodies used for
Western blotting were: LCoR, HDAC3, and HDAC6 (1:1000); CtBP1,
CtBP2, and ER? (1:100).
COS-7 cells in 6-well plates were transfected with 250 ng of LCoR-
rluc alone or with 2.5 ?g of ER?-EYFP, and treated 24 hr later with
10?7M estradiol or OHT for 18 hr. Cells were washed (2?) with PBS
and harvested with 500 ?l of PBS, 5 mM EDTA. Twenty thousand
cells (90 ?l) were incubated with 5 ?M final of coelenterazine H in
96-well microplates (3610, Costar, Blainville, Quebec, Canada) as
signals were quantified with a 1420 VICTOR2-multilabel counter
(Wallac-Perkin Elmer, Boston, MA), allowing sequential integration
diately after coelenterazine H addition, and ten repeated measures
sion at 470) ? Cf]/(emission 470), where Cf corresponded to (emis-
sion at 470/emission at 595) for the rluc-LCoR expressed alone in
the same experiments.
We thank Genevieve Melanc ¸on and Peter Ulycznyj for help with
BRET assays and Jacynthe Laliberte ´ for technical assistance with
confocal microscopy. Thiswork was supported bygrants MT-11704
from the Canadian Institutes of Health Research (CIHR) to J.H.W.
and MT-13147 to S.M. I.F. was supported by postdoctoral fellow-
V.K.M.H. is the holder of a Canada Research Chair in Perinatal
Research. X.-J.Y. is the holder of a CIHR scholarship. S.M. and
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