Thyroid Hormone Receptor
Dimerization
Function Maps to a
Conserved Subregion of the Ligand
Binding Domain
Jae Woon Lee, Tod Gulick,
and David D. Moore
Department of Molecular Biology
Massachusetts
General Hospital
Boston, Massachusetts
02114
Thyroid hormone receptors (TRs) bind as dimers to
specific DNA response elements.
We have used a
genetic approach to identify amino acid sequences
required for dimerization
of the TRP isoform. Bacteria expressing
a chimeric repressor composed
of
the DNA binding domain of the bacteriophage
X cl
repressor
fused to the TRP ligand binding domain
are immune to X infection
as a consequence
of
homodimerization
activity provided by the receptor
sequences.
The phenotypes
of deletions
and point
mutations
of the TRP sequences
map dimerization
activity to a subregion
of the ligand binding domain
that is highly conserved among all members of the
nuclear hormone
receptor
superfamily.
These results confirm and extend previous findings indicating that this subregion
plays an important role in the
dimerization
of TR@ and other superfamily
members.
(Molecular
Endocrinology
6: 1867-1873,
1992)
Previous studies have mapped dimerization
activity
to various domains of the conserved structure shared
by superfamily members. In the case of the glucocorticoid receptor, direct structural analysis has demonstrated a homodimerization
interface within the DNA
binding or C domain (15). Sequences just distal to the
DNA binding domain (16) and in the C-terminal part of
the ligand binding (E) domain (17, 18) have been implicated in homodimerization
of the estrogen receptor.
The analogous subregion of the E domain has been
proposed to be involved in heterodimerization
of both
TR and the retinoic acid receptors (RARs) (19-21). This
part of the E domain includes heptad repeats of hydrophobic residues that have been proposed to form a
leucine zipper dimerization
interface (22). However,
interpretation
of many of the studies with TR is complicated by the fact that dimerization activity is associated
with receptor domains where nuclear localization, transcriptional activation, and ligand binding activities reside, and methodologies
used depend on these properties for scoring dimerization.
We have used a recently developed genetic approach
to the analysis of protein-protein
interactions (23) to
map amino acid sequences required for homodimerization of the p-isoform of rat TR (rTR). This system,
based on genetic screening of the function of bacteriophage X cl repressor fusion proteins in Escherichia co/i,
permits segregation of dimerization from transcriptional
regulation and other activities. The cl repressor consists
of an amino-terminal
DNA binding domain (DBD) and a
functionally and structurally
distinct dimerization
domain. The repressor acts to shut off viral gene expression by binding to specific operator sites in essential
regulatory regions of the viral genome, rendering bacteria expressing intact cl immune to viral infection (24).
Since dimerization
of the repressor
is essential for
efficient DNA binding, expression of the cl DBD alone
is not sufficient to confer immunity. However, repressor
function can be rescued by fusion with a heterologous
dimerization domain (23). Here we describe the activity
of chimeric repressors in which dimerization function is
provided by the ligand binding domain of TRB.
INTRODUCTION
The nuclear hormone receptor superfamily is a group
of proteins linked by conserved structure and function
that includes the receptors for a variety of small hydrophobic compounds such as steroids, thyroid hormone
(T3), and retinoids. All of the members of the superfamily examined to date function as transcriptional
regulators by binding to DNA as dimers (reviewed in Refs. l3). While the steroid receptors bind as homodimers
to
simple palindromic response elements, head-to-head or
tail-to-tail inverted repeats and direct repeats of a hexameric consensus can function as T3 response elements. T3 receptors (TRs) can bind to such sites as
homodimers (4-7) and the binding affinity of the homodimers to a wide variety of wild type and mutant
elements correlates strongly with their response to T3
(4). TRs can also bind with even higher affinity to the
same array of sites as heterodimers with the retinoid X
receptors (RXRs; 8-l 4).
0888-8809/92/1867-1873$03.00/0
Molecular
Endocrmology
CopyrIght 0 1992 by The Endocrine
Society
1867
MOL
1868
ENDO.
1992
Vol6No.
11
RESULTS
A Chimeric Repressor Including the Hinge and
Ligand Binding Domains of Rat TR@ Confers
Immunity to X Infection
A
DBD
TRP
In the vector pYCl84cl, expression of the bacteriophage Xcl repressorDBD (aminoacids l-l 12) is under
the control of an isopropylthiogalactoside(IPTG)-inducible promoter. A fragment of the rTR/I (25) encompassing the C-terminalend of the DBD and the entire hinge
and ligand binding domains(D, E, and F, amino acids
169-461) was inserted into this vector to generate
pYCcl/TRP (Fig. IA). As shown in Fig. lB, E. co/i
transformed with pYCcl/TRP are immuneto high titer
Xinfection when expressionof the chimericgene product isinduced(+IPTG), but not inthe absenceof inducer
(-IPTG). The presenceor absenceof T3 has no effect
on the immunityphenotype of the chimera,as expected
from previous findingsthat the hormoneis not required
for T3 receptors to interact with specific DNA binding
sites in viva (26-28) or in vitro (4, 29, 30). Bacteria
transformedwith pYCcl are susceptibleto X infection
even under inducing conditions, and cells containing
either plasmidare susceptibleto infection by a hybrid
strain of X with an immunity region derived from 480, a
related bacteriophagethat is not subject to repression
by X cl. Thus, the chimericrepressor acts like the wild
type cl protein in conferring specific immunity to X
infection, demonstratingthat the hingeand ligandbinding domainsof TRPcontain a homodimerizationactivity.
The chimeric repressor expressed from pYCcl/TR@
bindsT3 specificallyand is presentat low concentration
in the immunecells. Crude extracts of cells grown in
the presenceof IPTG were incubated with [‘251]T3in
the presenceor absenceof an excess of unlabeledT3
and subjectedto molecularsieve chromatography.The
amount of T3 specificallybound at saturation indicates
that approximately 50 chimera monomersare present
per cell, a concentration similarto that of the cl repressor in a normallysogen(24). This low levelof expression
is a reflection of the low copy number of the pACYC
vector and the weaknessof its P-lactamasepromoter,
both of which are componentsof a strategy designed
to maximizethe sensitivity of the assay. Specific ligand
bindingwas taken to indicate proper protein folding of
the E domain of the chimera, as expected from the
previously describednormalligandbindingaffinity of E.
co/i-expressednative TRP(31). The apparent mol wt of
the specific T3 complex indicated the formation of
dimersor higherorder oligomersof the chimericprotein.
This is consistent with a recent report that a purified
fragment of the rTRcv1consistingonly of the E domain
can form dimersin solution(32).
TRj3 Homodimerization
Function Maps to a
Conserved Portion of the Ligand Binding Domain
The immunity phenotype of deletion mutants was used
to identify regionsof the ligandbindingdomainessential
HEPTAD
El
3
1
A/B
D
C
E
F
X
DBD
DIMER
CI
1
km m
cI/TRP
6
pYCclTRP
pYCl84cl
-IPTG
+IPTG
0
pfu:
lrl” 1: lo”
480
h
000
000
Fig. 1. The E Domain of TF@ Restores
X Immunity
Function
to The X Repressor
DBD
A, The primary
structures
of wild type TR@, X cl repressor
and a chimeric
repressor
protein are depicted.
TRP consists
of the N-terminal
A/B domain, the DBD, the hinge or D domain,
the E domain, and the C-terminal
F domain. The positions
of
the conserved
El subdomain
and the heptad repeats of hydrophobic
residues (19, 22) are indicated. The repressor
consists of an N-terminal
DBD and a C-terminal
dimerization
domain (DIMER).
B, Freshly
poured lawns of E. co/i (strain
XL1 B) transformed
with plasmids expressing
either the DBD
of the cl repressor
(pYC184cl)
or the chimeric
repressor
(pYCclTRP)
were infected by spotting
with 100-111 aliquots of
dilutions of lysates of the X mutant KH54 (X) or Charon
(480).
The titer and pattern of the aliquots is indicated.
The absence
or presence
of the inducer IPTG is indicated.
1
Receptor Dimerization
1869
for dimerization
activity. Mutants with short deletions
extending into the hinge (D) domain (Fig. 2, Dl and D2)
maintain immunity function, consistent with studies
demonstrating
that this region is not necessary for
function of steroid receptors (33,34). However, repressor function was also observed in several additional
chimeras retaining only the proximal portion of the D
domain. This finding is in agreement
with previous
results mapping an independent
but subsidiary dimerization function to this region of the estrogen receptor
(16) and TRa (6). This region was not included in the
further series of E domain deletion and point mutants.
A number of chimeras with internal or C-terminal
deletions in the region of the E domain proposed to
form a leucine zipper-like
dimerization
interface are
functional repressors (mutants D4-9 and 011). The
phenotypes of these deletion mutants rule out a primary
role for this region in homodimerization.
This is consistent with some recent results with TRa (6), and with the
fact that the primary structure of this region is not
generally consistent with a coiled coil/leucine zipper.
However, results reported for the estrogen receptor
(17, 18) support some role in homodimerization
for a
conserved subregion at the C-terminal portion of the E
domain. This subregion has also been implicated in TR
(19-21) and RXR (9, 10, 13) heterodimerization.
In
these studies the activity of this subregion was only
examined in the presence of other receptor dimerization
domains, and our results indicate that it is neither
necessary nor sufficient for homodimerization
of the
chimeric repressor.
Together, the N- and C-terminal deletions map a
region essential for TRP homodimerization
to amino
acids 283-313.
This interval consists largely of the
subregion called El (35), see Fig. 4A), the most strongly
conserved portion of the E domain. Dimerization function is eliminated in a C-terminal truncation that removes
part of El (D12) and by two small deletion mutations
that lie entirely within this subregion (D13 and 14).
To exclude the possibility that the phenotype of these
mutants results from an indirect alteration in the structure of the E domain, rather than a specific effect on
dimerization, we introduced a series of previously studied amino acid substitutions into the El subdomain of
the cl/TRP chimera. These mutations have been shown
to inactivate the transcriptional
regulatory function of
the receptor in cotransfections
and to prevent interaction with the T3 receptor accessory protein (TRAP) (36,
37). The alterations of TRP amino acids 288, 290, and
300 were of particular interest, since these mutant TRs
have been shown to bind T3 with normal affinity and
could, therefore, be confidently assumed to have an
authentic tertiary structure. As shown in Fig. 2, two of
these three substitutions (mutants L29OS and D300A)
inactivate function of the fusion protein. The Ala to Asp
substitution at TRP residue 287 that inactivates ligand
binding in mutant receptors also inactivates immunity
function when introduced into the chimera.
To directly verify these in vivo findings and to exclude
attribution of the immunity phenotype to potential differences in the concentration
of mutant chimeras in
bacteria, we examined the effect of El point mutations
in a cell-free system. Full-length wild type and mutant
fusion proteins were produced by in vitro transcription
and translation, and their dimerization
function was
examined in gel mobility shift assays (Fig. 3). As ex-
#
F
..(
w
~-----’
v,
+
)
e’
k-----.’
+.....,
w ......-(~
F
,
b-----F ......-( -*...m
w
+--*
F-~---~
~-~----’
~-~~---~
-
HEPTAD
El
mx
8
,
I
,
I
I
I
,,,,,/-,,>”
---
....----.---zx,.
>-----.-----..mz”
,................
-
(=1
zzzz2m
.
........
“,,,z~z,A
v-n,,-
J
,
l
.
l
r------
-
WT (169-461)
Dl
D2
D3
D4
D5
D6
D7
D8
D9
DlO
Dll
012
013
D14
Pl
P2
P3
P4
MUTATED
AMINO
ACIDS
d169-182
d169-199
d169-282
d333-343
d314-356
d321-360
d379-423
d379-440
d359-442
d281-428
d378-461
d292-461
d286-293
d287-305
A287D
K2881
L29OS
D300A
h
IMMUNITY
++
-I-+
++
+
++
+
+
++
++
+
++
++
-
Fig. 2. TR$ Homodimerization Activity Maps to the Conserved
El Subdomain
The indicated deletion mutations
were introduced
into the cl/TR@ chimera. Deleted amino acids
in the rTRp sequence.
Amino acids substituted
in each mutant are as noted: the wild type amino
position and the substituted
amino acid, e.g. in A287D an alanine at position 287 is replaced by an
immunity
to high titer X infection
as shown in Fig. 1; +, immunity
to infection
by titers 1 OO-fold
plaques on a sensitive host; -, full sensitivity
to infection.
are listed according
to position
acid is listed, followed
by the
aspartic acid (36, 37). ++, Full
higher than necessary
to yield
MOL
1870
END0.1992
cI/TR
CT DBD
L29OS
D3OOA
operator
NS
Vo16No.11
+
+
+
+
+
+
+
+
+
+
+ + + +
+
+
+ + + +
\\
.\
Fig. 3. Mobility
Shift Assays Confirm
Genetic
Mapping
of
Dimerization
Activity to El
The wild type chimeric
cl/TRP (cl/TR),
X repressor
DBD
alone (cl DBD), and two substitution
mutant chimeric repressors (L29OS and D300A) were produced
by in vitro transcription and translation,
and equivalent
amounts
of the proteins
were used for gel shift analysis. Binding reactions
included the
indicated
proteins
with no competitor
(lanes l-4)
a 40-fold
excess of a nonspecific
competitor
oligonucleotide
(NS), or a
40-fold excess of the unlabeled operator
oligonucleotide.
petted from previous results demonstrating
that dimerization is essential for high affinity DNA binding by the
repressor, the cl N-terminal DBD alone showed no
binding to a symmetric X operator site. However, substantial binding was observed with the wild type cl/TR
chimera, demonstrating
that the receptor sequences
can confer dimerization function. In contrast, binding of
equivalent amounts of either of the nonfunctional substitution mutant proteins (L29OS and D300A) was dramatically reduced. These data directly confirm that the
wild type TRP chimera forms dimers in vitro substantially more effectively than the El point mutants. We
conclude that immunity phenotype is an accurate assay
of homodimerization
and that the conserved El subregion has a direct role in dimerization of TRP.
DISCUSSION
The results described here demonstrate
that the E
domain of the rTF@ isoform contains a dimerization
activity that can restore repressor function to the Nterminal DBD of the bacteriophage
X cl protein. The
highly conserved El subregion is the only portion of
the TR E domain that is essential for this dimerization
function. Since all of the members of the nuclear receptor superfamily examined to date bind as dimers to their
response elements, we suggest that the strong con-
servation of the amino acid sequence of the El subdomain throughout the superfamily may be a reflection
of a conserved role for this element in dimerization.
This subregion has been associated with other functions, particularly the interactions of the glucocotticoid
and estrogen receptors with HSPSO (38-40). Since the
TRs (41), and perhaps other members of the superfamily (42) do not interact with HSPSO, this function is not
likely to be the basis for the conservation of this subregion.
Although the results described here are based on
analysis of TR homodimerization,
they may also be
relevant to heterodimerization.
General support for this
possibility is found in the strong similarity in DNA binding
specificity of TR homodimers
and TR/RXR heterodimers (8-13) which suggests that the protein-protein
contacts of the two complexes may be very similar.
More specific support is provided by the effects of El
mutations on interaction of TRP with TRAP, a protein
activity that stimulates binding of TR to T3 response
elements (43,44), apparently by formation of TR/TRAP
heterodimers (5, 45). The two TRP substitution mutations within El that prevent homodimerization
of the
chimeric repressor also strongly decrease interaction of
mutant receptors with TRAP in vitro and decrease
function of the receptor in cotransfections
(36, 37).
Moreover, the substitution at amino acid 288 that does
not prevent homodimerization
of the chimeric repressor
has a significantly weaker effect on the in vitro interaction with TRAP (37). Recent results demonstrate that
the TRAP effects can be explained by the activities of
the RXRs, which form strong heterodimers
with the
TRs and with other receptors. Thus, the precise overlap
between the effects of the El mutations on activity of
the repressor chimeras and on TRAP interactions suggests that El is similarly involved in homodimerization
of TRP and in its heterodimerization
with RXRs.
Further evidence for a conserved role for the El
segment in heterodimerization
is provided by the fact
that deletion of that subregion of RXRP prevents heterodimerization
with TR (13). Strong additional support
is provided by a recent preliminary report that introduction of mutations similar to those described here into
the vitamin D receptor (VDR) and RARa proteins
strongly decreases their function in cotransfections
and
their ability to cross-link to RXRP or to a 63-kilodalton
protein with TRAP activity (46; Rosen, E., and R. Koenig, personal communication).
Thus, although recent
biochemical evidence supports a primary role for TR/
RXR heterodimers
rather than homodimers
(30) we
conclude that the analysis of homodimerization
by the
approach described here can provide important information on the protein-protein
interactions of TRP.
El sequence similarity is high among the members
of the family (T3, retinoic acid, vitamin D, and 9-&sretinoic acid receptors and the orphan superfamily
member COUP-TF, chicken ovalbumin upstream promoter transcription
factor) reported to form heterodimers (8-l 3, 19, 20, 22, 47). This region is also strongly
conserved among the steroid receptors, all of which
Receptor
1871
Dimerization
apparently bind DNA only as homodimers.
In several
positions (shaded in Fig. 4A), the particular amino acid
conserved in one of the two groups is different from
that conserved in the other group. These positions may
contribute to the specificity of the dimeric interactions
of each group.
Although additional sequences in the E domain are
not required for TR homodimerization
in the results
described here, the conserved E3 subregion located
near the C terminus of the E domain (35) appears to be
essential for heterodimerization
of TRs, RARs, and
RXRs (10, 13, 20, 21). This short subregion, which
includes heptad 9 of Forman and Samuels (22) is also
required for homodimerization
of the estrogen receptor
(17, 18). It is interesting that there is some direct amino
acid sequence similarity between the E3 subdomain
and a portion of the El subdomain
in the group of
proteins that form heterodimers.
The VDR shows the
best match, with 6 of 11 identical residues plus a single
conservative
substitution,
while TR@ is more typical,
with 3 of 11 identities and two conservative substitutions (Fig. 4B). Further analysis will be necessary to
determine whether the El and E3 subdomains have
coordinate functions in dimeric interactions. The genetic
system described here may be useful in defining the
specificity in these interactions.
MATERIALS
RARa
VDR
RXRa
COUP
** *
*
VDFAKKLPMFCELPCEDQIILLKG
VEFAKOLPGFTTLTIADOITLLKA
IGFAKIiIPGFRDLTSED~IVLLKS
VEWAKRIPHFSELPLDDQVILLRA
VEWARNIPFFPDLQITDQVSLLRL
GR
MR
PR
AR
VKWAKAIPGFRNLHLDDQMTLLQY
VKWAKVLPGFKNLPLEDOITLIOY
VKWSKSLPGFRNLHIDD&TLI;Y
VKWAKALPGFRNLHVDDQMAVIQY
TRP
I
III
II
I
I
III1
II
B.
pYCl84cl
is a derivative
of pACYC177
(48) in which expression of the N-terminal
DBD (amino acids l-l 12) of X cl is
controlled
by a derivative
of the B-lactamase
promoter
containing a lactose operator
(kindly provided
by C. Bunker
and
R. Kingston,
Department
of Molecular
Biology, Massachusetts
General Hospital).
pACYCl77
contains
the origin of replication
from pl5A and is a low copy replicon compatible
with plasmids
like pBR322
and pUC19 that contain the colE1 origin. pYCcl/
TR@ was constructed
by insertion of a fragment
from the rat
TRB coding region encompassing
amino acids 169 to the Cterminus
(461) into this vector.
Deletions
and previously
described amino acid substitutions
(36, 37) were introduced
into
pYCcl/TRp
using convenient
restriction
sites and standard
methods (49). In some cases, Bal31 exonuclease
was used to
create a random
set of deletions
from a single site. Both
substitution
and deletion
mutations
were verified
by direct
DNA sequencing.
For DNA binding studies,
repressor
coding
regions were subcloned
into a derivative
of pBluescriptSK
that
contains a mammalian
consensus
sequence
for efficient translation initiation.
X Immunity
TRP
FAKKLPMFCELP
WPKLLMKVTDLR
El
E3
VDR
FAKMIPGRFDLT
YAKMIQKLADLR
El
E3
Fig. 4. El and E3 Sequences
A, Conservation
of the El subdomain
among superfamily
members.
The top group includes
El sequences
from rTR@
and RAFta, VDR, and RXRa, all of which have been reported
to form heterodimers
with TR, as well as COUP-TF,
an orphan
member of the superfamily
reported
to form heterodimers
with
RXR. TRP sequence
is from amino acids 284-306
(25). Positions altered by substitution
mutations
are indicated
by asterisks. The bottom
group includes
four steroid receptors,
all
thought
to form only homodimers.
GR, Glucocorticoid
receptor; MR, mineralocorticoid
receptor;
PR progesterone
receptor; AR androgen
receptor.
Except for rTRB, human cDNA
sequences
are indicated (see Ref. 53 and refs. therein). Positions conserved
in both groups are indicated by bars. Positions
that show conservation
of different
amino acids in the two
groups
are stippled.
B, Similarity
of El and E3 sequences
from TRB and VDR. The entire E3 sequence
and only a portion
of the El sequence
is included. Identical or conserved
amino
acids are shaded.
AND METHODS
Assay
Lawns of E. co/i strains containing
various
plasmids
were
infected by spotting
with O.l-ml aliquots of serial dilutions of
bacteriophage
X lysates containing
from 1 02-1 0’ plaque forming units. X Strains included
X KH54, which has X host range
and immunity,
and Charon3,
which has X host range but an
immunity
region from the related bacteriophage
$80 and is not
sensitive to X repressor
(50). Infected
lawns were incubated
at 37 C for 8-l 8 h.
DNA Binding
Assay
Proteins were produced
in vitro using bacteriophage
T7 RNA
polymerase
and rabbit reticulocyte
lysates as described
(51).
Equivalent
amounts
of full-length
versions
of each, as determined by sodium dodecyl
sulfate polyacrylamide
gel electrophoresis
and autoradiography
of %-labeled
products,
were
used in DNA binding reactions.
Binding reactions included 0.2
fmol of each protein and 1.5 pmol of a double-stranded
32Plabeled oligonucleotide
probe containinq
a consensus
h-operator site (5’-AATTCCACATGCAACCATTATCACCGCCG’GTGATAATAGTCGG-3’)
in a buffer consistina
of 25 mM Tris IDH
7.5) 50 mM KCI, 10% glycerol,
6 mM Ca&,
1.5 mM MgCI,,
0.3 mM EDTA, 0.5 mM dithiothreitoi,
50 Kg/ml BSA, and 50
@g/ml polydl.dC)
(52). In some reactions,
a 40-fold excess of
unlabeled
X-operator
oligonucleotide
or an unrelated
doublestranded
oligonucleotide
were added as competitor.
Free and
protein-bound
DNA were resolved by electrophoresis
on a 5%
MOL
i 872
ENDO.
Vol6No.
1992
polyacrylamide
(49).
gel in 0.5~
Tris-buffered
EDTA
as described
14.
Acknowledgments
We thank Chris Bunker,
Bob Kingston,
Jim Hu, and Ron
Koenig for helpful discussions,
Chris Bunker and Bob Kingston
for pYCl84cl,
and Ron Koenig for the mutations
affecting El.
15.
16.
Received
July 9, 1992. Revision received August 18, 1992.
Accepted
August 18, 1992.
Address requests
for reprints to: Dr. David Moore, Department of Molecular
Bioloav.
Massachusetts
General Hospital,
Boston, Massachusetts
?I21 14.
This work was supported
by PHS Grant DK-43382
from
the NIDDK and by a grant from Hoechst AG.
17.
ia.
19.
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regulation
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4. Williams GR, Harney JW, Forman BM, Samuels HH, Brent
GA 1991 Oligomeric
binding of T3 receptor
is required for
maximal T3 response.
J Biol Chem 266:19636-19644
5. Lazar MA, Berrodin TJ, Harding HP 1991 Differential
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binding by monomeric,
homodimeric
and potential heteromeric forms of the thyroid
hormone
receptor.
Mol Cell
Biol 11:5005-5015
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for thyroid hormone
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with a nuclear protein. Mol Endocrinol
5:1909-1920
7. Holloway
JM, Glass CK, Adler S, Nelson CA, Rosenfeld
MG 1990 The C-terminal
interaction
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hormone
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dictate positive or negative
transcriptional
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elements.
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G, Pfahl M
1992 Retinoid X receptor
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