Heterodimerization of thyroid hormone (TH) receptor with H-2RIIBP (RXRB) enhances DNA binding and TH-dependent transcriptional activation

Abstract

Steroid/TH receptors mediate transcriptional induction of promoters containing hormone response elements (HREs) through an unclear mechanism that involves receptor binding to both hormone and a HRE. Here we demonstrate that both HRE binding and the transcriptional inducing activities of one member of this family, TH receptor, were markedly enhanced by heterodimerization with H-2RIIBP, a non-TH-binding member of the steroid hormone receptor superfamily. H-2RIIBP, the mouse homologue of human retinoic acid-related receptor, was shown to form stable heterodimers with the TH receptor either in solution or when bound to a TH response element. The results presented indicate that it might be necessary for the TH receptor or other members of this superfamily to have specific partners for heterodimer formation to elicit maximal hormone-specific gene regulation from particular HREs.

Full text

Proc.
Nati.
Acad.
Sci.
USA
Vol.
89,
pp.
5572-5576,
June
1992
Biochemistry
Heterodimerization
of
thyroid
hormone
(TH)
receptor
with
H-2RIIBP
(RXRf3)
enhances
DNA
binding
and
TH-dependent
transcriptional
activation
PAUL
L.
HALLENBECK*,
MICHAEL
S.
MARKSt,
ROLAND
E.
LIPPOLDT*,
KEIKO
OZATOt,
AND
VERA
M.
NIKODEMt
*Genetics
and
Biochemistry
Branch,
National
Institute
of
Diabetes
and
Digestive
and
Kidney
Diseases;
and
tLaboratory
of
Developmental
and
Molecular
Immunity,
National
Institute
of
Child
Health
and
Human
Development,
Bethesda,
MD
20892
Communicated
by
Joseph
E.
Rall,
March
6,
1992
(received
for
review
November
25,
1991)
ABSTRACT
Steroid/TH
receptors
mediate
transcrip-
tional
induction
of
promoters
containing
hormone
response
elements
(HREs)
through
an
unclear
mechanism
that
involves
receptor
binding
to
both
hormone
and
a
HRE.
Here
we
demonstrate
that
both
HRE
binding
and
the
transcriptional
inducing
activities
of
one
member
of
this
family,
TH
receptor,
were
markedly
enhanced
by
heterodimerization
with
H-
2RIBP,
a
non-TH-binding
member
of
the
steroid
hormone
receptor
superfamily.
H-2RIBP,
the
mouse
homologue
of
human
retinoic
acid-related
receptor,
was
shown
to
form
stable
heterodimers
with
the
TH
receptor
either
in
solution
or
when
bound
to
a
TH
response
element.
The
results
presented
indicate
that
it
might
be
necessary
for
the
TH
receptor
or
other
members
of
this
superfamily
to
have
specific
partners
for
heterodimer
formation
to
elicit
maximal
hormone-specific
gene
regulation
from
particular
BREs.
Steroid/TH-dependent
transcriptional
activation
is
elicited
by
the
binding
of
a
steroid/TH
receptor-hormone
complex
to
specific
DNA
regulatory
sequences
found
near
or
within
genes
whose
transcriptional
control
is
responsive
to
the
hormone.
Although
many
reports
have
demonstrated
that
nuclear
factors
form
heterodim
r-s
with
known
transcription
factors
to
modify
specific
DNA
binding
and
transcription
(1-8),
much
less
is
known
about
the
requirement
of
nuclear
factors for
steroid/TH-dependent
transcription
(9-11).
Re-
cent
reports
have
demonstrated
that
several
uncharacterized
nuclear
proteins
present
in
a
variety
of
tissues
and
cell
lines
enhance
binding
of
thyroid
hormone
receptor
(THR)
(10,
12-16),
retinoic
acid
receptor
(17),
and
vitamin
D
receptor
(18)
to
particular
hormone
response
elements
(HREs).
Al-
though
the
THR
has
been
shown
to
bind
to
several
HREs
as
a
homodimer
(14,
19,
20),
analogous
to
steroid
hormone
receptors
binding
to
HREs
(21-25),
it
is
still
unclear
whether
the
THR
can
act
alone
or
with
another
protein(s)
to
regulate
expression
from
TH-controllable
genes.
We
investigated
the
possibility
that
another
protein,
namely,
the
H-2
region
II
binding
protein
(H-2RIIBP)
(26),
could
alter
the
DNA
binding
or
transcriptional
inducing
activity
of
rat
THRa
(rTHRa).
H-2RIIBP
was
believed
to
interact
with
rTHRa
through
a
TH
response
element
(TRE)
since
it
was
cloned
by
virtue
of
its
ability
to
bind
a
DNA
sequence
within
the
promoter
of
the
major
histocompatibility
complex
class
I
gene
that
contained
the
consensus
THR
binding
half-site
motif
AGGTCA
(26,
27).
H-2RIIBP,
a
ubiq-
uitously
expressed
nuclear
protein
that
is
the
mouse
homo-
logue
of
the
human
retinoic
acid-related
receptor
(28-30),
was
also
subsequently
shown
to
regulate
the
expression
of
this
gene
(31).
Further,
the
DNA
binding
and
dimerization
domains
of
H-2RIIBP
are
similar
to
the
THR
and
the
retinoic
acid
receptor
(26,
32,
33),
thereby
classifying
it
in
this
subgroup
of
the
superfamily
and
making
it
an
ideal
candidate
for
interacting
with
the
THR.
We
report
here
that
H-2RIIBP
enhanced
rTHRa
binding
to
the
rat
malic
enzyme
TRE
(ME-TRE)
(34,
35)
and
rTHRa-
TH-dependent
transcriptional
activation.
The
mechanism
appears
to
be
at
least
partially
the
result
of heterodimer
formation
since
H-2RIIBP-rTHRa
heterodimers
were
de-
tected
in
solution
and
bound
to
the
ME-TRE.
MATERIALS
AND
METHODS
Preparation
of
Nuclear
Extracts
and
DNA
Binding.
Nuclear
extracts
containing
high
levels
of
either
H-2RIIBP
(H2)
or
rTHRa
(R)
were
prepared
from
H-2RIIBP
(27)
or
rTHRa
(T.
Mitsuhashi
and
V.
M.
Nikoden,
unpublished
data)
recombi-
nant
baculovirus-infected
Sf9
cells.
The
level
of
rTHRa
and
H-2RIIBP
in
Sf9
nuclear
extracts
was
determined
by
Coomassie
blue
staining
of
specific
bands
resolved
by
SDS/
PAGE
compared
to
known
quantities
of
bovine
serum
albu-
min
(BSA).
The
estimated
concentration
of
rTHRa
was
3300
fmol/,l
in
nuclear
extract
containing
8
,ug
of
total
protein
per
Al
and
the
concentration
of
H-2RIIBP
was
15,700
fmol/hl
in
nuclear
extract
containing
2.9
,g
of
total
protein
per
Al.
ME-TRE
was
prepared
from
pTK1AM3
(34)
as
a
72-base-pair
HindIII-EcoRI
restriction
fragment.
DNA
binding
assays
were
performed
in
lx
BB
[20
mM
Hepes,
pH
7.9/2
mM
MgCl2/10%0
(vol/vol)
glycerol/1
mM
dithiothreitol/0.1%
Nonidet
P-40/80
mM
KCl/0.1
mM
EDTA/2
mM
sodium
pyrophosphate/0.4
mM
sodium
ortho-
vanadate/10
mM
sodium
fluoride/aprotinin
2
,g/ml/10
,AM
leupeptin/0.2
mM
phenylmethylsulfonyl
fluoride)
and
5
pAg
of
poly[d(A-T)]
(Pharmacia)
in
a
total
volume
of
20
,l.
After
a
4-h
incubation
on
ice,
mixtures
were
subjected
to
electro-
phoresis
either
on
a
4-20%
linear
polyacrylamide
gradient
gel
[30%
(wt/vol)
acrylamide/0.8%
N,N'-methylenebisacryla-
mide]
for
at
least
24
h
at
150
V
and
4°C
or
5%
gels
for
3-6
h
at
150
V.
Electrophoresis
buffer
was
0.5
x
TBE
(45
mM
Tris
borate/45
mM
boric
acid/2
mM
EDTA).
Molecular
mass
standards
were
from
Pharmacia
(BSA,
68
kDa;
lactate
de-
hydrogenase,
140
kDa;
catalase,
232
kDa;
ferritin,
440
kDa;
thyroglobulin,
669
kDa)
and
were
detected
as
described
(36).
For
gradient
gels,
DNA
binding
reaction
mixtures
contained
2
ug
of
nuclear
protein
corresponding
to
825
fmol
of
rTHRa
and/or
0.03
,g
of
nuclear
protein
containing
165
fmol
of
H-2RIIBP.
A
DNA
binding
assay
mixture
with
16
,g
of
Abbreviations:
TH,
thyroid
hormone;
HRE,
hormone
response
ele-
ment;
THR,
TH
receptor;
r,
rat;
TRE,
TH
response
element;
ME-
TRE,
malic
enzyme
TRE;
BSA,
bovine
serum
albumin;
DSS,
dissuc-
cinimidyl
suberate;
TK,
thymidine
kinase;
CAT,
chloramphenicol
acetyltransferase.
tTo
whom
reprint
requests
should
be
addressed.
5572
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.

Proc.
Natl.
Acad.
Sci.
USA
89
(1992)
5573
nuclear
protein
from
wild-type
baculovirus-infected
Sf9
cells
was
used
as
a
control.
Complexes
were
labeled
by
the
inclusion
of
-10
fmol
of
32P-labeled
ME-TRE
(5000
cpm)
or
0.038
t&Ci
of
[125I]TH
(3,
5,
3'-triiodothyronine;
NEN;
spe-
cific
activity,
2200
Ci/mmol;
1
Ci
=
37
GBq).
Identification
of
ME-TRE-Bound
Complexes
with
Antibod-
ies
to
rTHRa
or
H-2RIIBP.
Anti-H-2RIIBP
monoclonal
an-
tibody
MOK
13-17,
specific
for
H-2RIIBP,
was
prepared
as
described
(27).
Anti-rTHRa
antiserum
was
prepared
by
in-
jecting
95%
pure
denatured
rTHRa
isolated
from
recombi-
nant
baculovirus-infected
Sf9
cells
into
mice
and
inducing
ascites
fluid
production
as
described
(37,
38).
Ascites
fluid
was
confirmed
to
contain
antibody
specific
to
rTHRa
(see
Fig.
4).
The
effect
of
antisera
on
protein-DNA
complex
formation
was
performed
by
the
addition
of
either
MOK
13-17
(3
1.l)
or
anti-rTHRa
antiserum
(3
pl)
to
DNA
binding
reaction
mixtures
as
described
above,
with
the
exception
that
only
0.5
pug
of
nuclear
protein
corresponding
to
=206
fmol
of
rTHRa
and/or
0.00018
,ug
of
nuclear
protein
containing
-1
fmol
of
H-2RIIBP
were
utilized.
Antisera
was
added
45
min
after
commencement
of
DNA
binding
and
continued
for
2
h
before
protein-DNA
complexes
were
separated
by
PAGE
in
5%
gels.
Chemical
Cross-Linking
of
rTHRa
Homodimer
and
rTHRa/H-2RIIBP
Heterodimer.
In
vitro-translated
35S5
labeled
rTHRa
was
prepared
as
described
(35).
Labeled
rTHRa,
20,000
cpm,
was
combined
with
Sf9
nuclear
extract
containing
rTHRa,
H-2RIIBP,
or
nuclear
protein
from
wild-
type
baculovirus-infected
cells
in
a
total
of
50
,l
of
lx
BB.
After
incubation
for
4
h
at
0-40C,
the
cross-linking
agent
dissuccinimidyl
suberate
(DSS)
(Pierce,
dissolved
in
dime-
thyl
sulfoxide)
was
added
to
a
final
concentration
of
0.25
mM.
After
an
additional
30-min
incubation
at
4°C,
reactions
were
terminated
by
the
addition
of
1
M
NH4Cl
and
SDS-
solubilization
buffer
containing
5%
(vol/vol)
2-mercaptoeth-
anol,
heated
at
100°C
for
5
min,
and
subjected
to
SDS/PAGE.
Cross-linking
of
unlabeled
rTHRa
to
H-2RIIBP
was
per-
formed
essentially
as
described
above
except
products
were
transferred
to
a
nitrocellulose
filter
after
SDS/PAGE,
blocked
with
1%
BSA
in
phosphate-buffered
saline
(PBS),
and
incubated
overnight
with
rTHRa
antiserum
in
PBS,
and
antibody-bound
products
were
detected
with
125I-labeled
protein
A
as
described
(37).
Transfections.
Transfections
and
construction
of
the
ME-
TRE-thymidine
kinase
(TK)-chloramphenicol
acetyltrans-
ferase
(CAT)
reporter
plasmid
(pTK1AM3)
has
been
de-
scribed
(34).
Plasmid
pTK1AM3
was
electroporated
into
4.4
x
106
NIH
3T3
cells
with
pRSVH-2RIIBP
(27,
31)
and/or
pRSVRTRa
expression
plasmids
(34).
After
transfections
cells
were
equally
divided
into
four
60-mm
dishes,
two
containing
0.1
,uM
TH
in
medium
and
two
containing
vehicle,
and
cultured
for
72
h
before
assaying
CAT
activity
as
described
(34).
Samples
were
corrected
for
transfection
ef-
ficiency
by
utilizing
a
f-galactosidase
reporter
construct
included
in
every
transfection.
All
transfections
were
per-
formed
in
duplicate
at
least
three
times.
RESULTS
To
study
whether
H-2RIIBP
could
form
a
heterodimer
with
THR
and/or
enhance
binding
of
the
THR
to
TREs
and
to
determine
whether
THR
or
H-2RIIBP
could
bind
a
TRE
as
a
homodimer,
we
used
the
ME-TRE
from
the
rat
malic
enzyme
promoter
positions
(-280
to
-263).
The
ME-TRE
has
been
shown
(34,
35)
to
contain
two
half-sites,
both
of
which
were
essential
for
THRa
binding
and
function.
Mobility-gel-shift
assays
using
pore
gradient
gels
(36)
allowed
us
to
estimate
the
molecular
mass
of
protein-DNA
complexes
containing
32p-
labeled
ME-TRE
or
[1251]TH.
A
nuclear
extract
(2
pg
of
protein)
from
Sf9
cells
containing
=825
fmol
of
rTHRa
(46
kDa)
formed
an
=140-kDa
complex
with
the
ME-TRE
(48
kDa)
(Fig.
1,
lane
3).
However
the
ME-TRE
did
not
form
a
complex
with
any
protein
in
wild-
type
baculovirus-infected
nuclear
extract
(16
pzg)
alone
(Fig.
1,
lane
12).
These
results
suggest
that
rTHRa
was
bound
to
the
ME-TRE
as
a
homodimer.
Additionally,
rTHRa
in
a
nuclear
extract
from
Sf9
cells
formed
a
complex
with
the
palindromic
rat
growth
hormone
TRE
that
had
nearly
the
same
mobility
as
the
rTHRa-ME-TRE
complex
(data
not
shown).
THR
has
been
shown
(15,
19)
to
bind
to
the
palin-
dromic
rat
growth
hormone
TRE
as
a
homodimer.
Interestingly,
nuclear
extract
from
Sf9
cells
containing
=165
fmol
of
H-2RIIBP
(0.03
,g
of
nuclear
protein)
or
60,000
fmol
of
H-2RIIBP
(12
,ug
of
nuclear
protein)
did
not
bind
the
ME-TRE
under
these
conditions
(Fig.
1,
lane
9,
and
data
not
shown,
respectively).
However,
addition
of
0.03
,ug
of
nu-
clear
extract
containing
165
fmol
of
H-2RIIBP
(52
kDa)
to
825
fmol
of
rTHRa
(2.0
,g
of
nuclear
protein)
in
the
DNA
binding
reaction
mixture
resulted
in
a
slower
migrating
complex
than
was
observed
with
the
ME-TRE
bound
to
rTHRa
alone
(Fig.
1,
lanes
6
and
3,
respectively).
This
slower
migrating
complex
had
a
molecular
mass
of
-145
kDa,
as
predicted
for
a
heterodimer
of
rTHRa-H-2RIIBP
bound
to
the
ME-TRE.
Addition
of
0.03
,ug
of
wild-type
baculovirus-infected
Sf9
cell
nuclear
protein
to
2.0
,g
of
rTHRa-containing
nuclear
pro-
tein
did
not
affect
rTHRa
binding
to
the
ME-TRE
(data
not
shown).
Addition
of
H-2RIIBP
to
the
DNA
binding
reaction
mix-
ture
containing
rTHRa
also
increased
the
apparent
binding
affinity
of
rTHRa
for
the
ME-TRE
since
there
was
a
6-fold
increase
in
the
amount
of
ME-TRE
bound
in
the
presence
of
rTHRa
plus
H-2RIIBP
compared
to
rTHRa
alone
(Fig.
1,
lanes
6
and
3,
respectively).
Further,
this
6-fold
increase
in
ME-TRE
binding
was
solely
due
to
the
formation
of
the
slower
migrating
complex,
as
no
complex
corresponding
to
the
rTHRa
homodimer
bound
to
the
ME-TRE
was
observed
1
2
3
4
5
6
78
9
10
11
12
..
t
9.
*
kDar
-669
-
440
*TH--
-3
-232
-140
-69
32p
ME-TRE-
-
FIG.
1.
Binding
of
rTHRa
to
the
ME-TRE
in
the
presence
or
absence
of
H-2RIIBP
and/or
TH.
Autoradiograph
of
a
4-20o
PAGE
gradient
gel.
Lanes:
1-3,
nuclear
extracts
containing
rTHRa
alone
(-825
fmol/2
,ug
of
nuclear
protein);
4-6,
both
rTHRa
(825
fmol/2
,ug
of
nuclear
protein)
and
H-2RIIBP
(=165
fmol/0.03
,ug
of
nuclear
protein);
7-9,
H-2RIIBP
alone
(=165
fmol/0.03
,g
of
nuclear
pro-
tein);
10-12,
wild-type
viral
extract
(16
,ug
of
nuclear
protein).
Extracts
were
incubated
in
the
presence
of
10
ng
of
unlabeled
ME-TRE
and
0.038
.Ci
of
[125I]TH
Qanes
1,
4,
7,
and
10;
1
Ci
=
37
GBq)
or
5000
cpm
(=0.5
ng)
of
32P-labeled
ME-TRE
(lanes
2,
3,
5,
6,
8,
9,
11,
and
12).
Incubations
were
in
the
presence
of
5
,uM
TH
(lanes
2,
5,
8,
and
11)
or
absence
of
TH
(lanes
3,
6,
9,
and
12).
Solid
or
open
arrows
indicate
rTHRa
homodimer
or
rTHRa-H-2RIIBP
heterodimer
bound
to
ME-TRE,
respectively.
*TH
designates
free
[1251]TH,
which
migrates
anomously
on
these
gels
presumably
from
interaction
with
the
gel
matrix.
Dotted
arrow
indicates
high
molec-
ular
mass
aggregated
rTHRa-containing
complex
bound
to
[125I]TH.
Amount
of
complexes
was
quantitated
by
laser
densitometry.
Biochemistry:
Hallenbeck
et
al.

5574
Biochemistry:
Hallenbeck
et
al.
(Fig.
1,
lanes
6
and
3,
respectively).
Although
only
the
slower
migrating
complex
was
observed
when
DNA
binding
reaction
mixtures
contained
4-fold
less
H-2RIIBP
than
rTHRa,
as
seen
in
Fig.
1,
both
homodimer
and
heterodimer
ME-TRE
complexes
could
be
detected
in
approximately
equal
amounts
when
using
206-fold
less
H-2RIIBP
than
rTHRa
(data
not
shown
and
Fig.
2).
Therefore,
although
H-2RIIBP
alone
did
not
bind
to
the
ME-TRE
in
the
gel-shift
assay,
it
enhanced
rTHRa
binding
to
the
ME-TRE,
presumably
by
heterodimer
formation.
Addition
of
TH
to
the
binding
reaction
increased
the
formation
of
both
the
hetero-
and
homodimer-32P-labeled
ME-TRE
complexes
=2-fold
and
slightly
accelerated
the
mobility
of
these
complexes
(Fig.
1,
lane
5
compared
with
lane
6
and
lane
2
compared
with
lane
3,
respectively).
It
is
likely
that
TH
binding
to
rTHRa
induced
a
conformational
change
that
resulted
in
the
altered
mobility
of
both
com-
plexes.
Moreover,
when
[125I]TH
was
included
in
the
binding
reaction
mixture
containing
unlabeled
ME-TRE,
labeled
complexes
formed
that
comigrated
with
the
analogous
com-
plexes
formed
in
the
presence
of
unlabeled
TH
and
32p-
labeled
ME-TRE
(Fig.
1,
lanes
1,
2,
and
4,
5,
respectively).
There
was
6-fold
more
[125I]TH
bound
to
the
putative
het-
erodimer-ME-TRE
complex
than
to
the
homodimer-ME-
TRE
complex
(Fig.
1,
lanes
4
and
1,
respectively).
This
was
precisely
the
same
ratio
determined
for
the
amount
of
32p-
labeled
ME-TRE
bound
in
the
presence
of
unlabeled
TH
(Fig.
1,
lanes
5
and
2,
respectively).
Therefore,
the
rTHRa
ho-
modimer-ME-TRE
complex
and
the
putative
heterodimer-
ME-TRE
complex
bound
virtually
the
same
amount
of
TH
per
mol
of
ME-TRE
bound.
Similar
results
were
obtained
using
various
concentrations
of
rTHRa,
H-2RIIBP,
and
ME-TRE
(data
not
shown).
To
further
demonstrate
that
the
putative
heterodimer-ME-
TRE
complex
contained
both
rTHRa
and
H-2RIIBP,
we
examined
whether
antisera
specific
for
either
rTHRa
or
H-2RIIBP
included
in
the
DNA
binding
reaction
mixture
altered
the
formation
of
this
complex.
By
utilizing
5%
poly-
acrylamide
gels
to
separate
ME-TRE-bound
complexes
and
206-fold
less
H-2RIIBP
than
rTHRa
in
the
DNA
binding
reaction
mixture,
we
achieved
separation
of
approximately
equivalent
amounts
of
the
homodimer-ME-TRE
complex
and
the
putative
heterodimer-ME-TRE
complex.
Preim-
mune
serum
had
no
effect
on
the
formation of
the
ho-
modimer-ME-TRE
complex
observed
with
rTHRa
alone
(Fig.
2,
lane
6)
or
with
both
rTHRa
and
H-2RIIBP
(Fig.
2,
lane
5,
lower
band)
or
the
formation
of
the
putative
het-
erodimer-ME-TRE
complex
observed
only
with
both
rTHRa
and
H-2RIIBP
(Fig.
2,
lane
5,
upper
band).
Addition
of
rTHRa-specific
antisera
nearly
abolished
formation
of
the
putative
heterodimeric
complex
and
the
rTHRa
ho-
modimeric
complex
(lanes
2
and
3,
respectively).
However,
FIG.
2.
Identification
of
rTHRa
and
H-2RIIBP
bound
to
ME-
TRE
as
a
heterodimer
with
antisera
specific
for
H-2RIIBP
or
rTHRa.
Approximately
206
fmol
of
rTHRa
(0.5
jug
of
nuclear
protein)
and/or
1
fmol
of
H-2RIIBP
(0.00018
,ug
of
nuclear
protein)
were
utilized
and
electrophoresed
on
a
5%
polyacrylamide
gel.
One
of
the
following
sera
at
3
p1
was
added
to
a
ME-TRE
binding
reaction
mixture
containing
H-2RIIBP
alone
(lanes
1,
4,
and
7),
both
H-2RIIBP
and
rTHRa
(lanes
2,
5,
and
8),
or
rTHRa
alone
(lanes
3,
6,
and
9):
rTHRa
antiserum
(lanes
1-3),
preimmune
serum
(lanes
4-6),
or
H-2RIIBP-
specific
antiserum
(lanes
7-9).
Solid
and
open
arrows
refer
to
rTHRa
homodimer
and
rTHRa-H-2RIIBP
heterodimer
bound
to
the
ME-
TRE,
respectively.
H-2RIIBP-specific
antisera
added
to
the
binding
reaction
mixture
inhibited
the
formation
of
the
heterodimer-ME-TRE
complex
but
not
the
rTHRa
homodimer-ME-TRE
complex
(lanes
8
and
9,
respectively).
These
data,
combined
with
those
presented
in
Fig.
1,
indicate
that
the
slower
migrating
ME-TRE-bound
complex
formed
in
the
presence
of
both
H-2RIIBP
and
rTHRa
is
a
heterodimer
of
H-2RIIBP
and
rTHRa
bound
to
the
ME-TRE.
We
investigated
whether
rTHRa
homodimers
or
rTHRa-
H-2RIIBP
heterodimers
could
form
in
solution
in
the
absence
of
DNA
by
using
the
cross-linking
reagent
DSS.
The
rTHRa-
containing
complexes
were
detected
with
35S-labeled
rTHRa
or
with
rTHRa-specific
antisera.
In
vitro-translated
35S-
labeled
rTHRa
was
cross-linked
with
excess
unlabeled
Sf9-
cell-derived
H-2RIIBP
or
rTHRa.
SDS/PAGE
of
the
puta-
tive
cross-linked
complexes
and
autoradiography
revealed
the
presence
of
a
35S-labeled-rTHRa-containing
cross-linked
complex
(95-98
kDa),
seen
only
in
the
presence
of
both
the
cross-linking
agent
DSS
and
H-2RIIBP
(Fig.
3A,
lane
1).
Furthermore,
by
using
Western
blot
analysis
and
the
rTHRa-
specific
antibody
for
detection,
a
similar
complex
was
de-
tected
when
nuclear
extracts containing
rTHRa
and
H-
2RIIBP
from
recombinant
baculovirus-infected
Sf9
cells
were
mixed
prior
to
cross-linking
(Fig.
3B,
lane
1).
No
cross-linked
35S-labeled
rTHRa
complex
was
observed
in
the
absence
of
H-2RIIBP
(Fig.
3A,
lanes
3
and
4)
or
after
addition
of
nuclear
extract
from
wild-type
baculovirus-infected
Sf9
cells
(Fig.
3A,
lanes
5
and
6).
Similarly,
no
DSS-dependent
cross-linked
rTHRa-containing
complex
was
detected
by
Western
blot
analysis
with
the
rTHRa
antibody
in
rTHRa-
containing
nuclear
extract
alone
(Fig.
3B,
lanes
3
and
4)
or
in
that
extract
mixed
with
extract
from
wild-type
baculovirus-
infected
cells
(Fig.
3B,
lanes
5
and
6).
Despite
the
presence
of
the
easily
detectable
heterodimer
in
either
analysis,
no
DSS-specific
cross-linked
rTHRa
homodimer
was
detected.
Since
H-2RIIBP
formed
heterodimers
with
rTHRa
in
so-
lution
and
when
bound
to
the
ME-TRE,
we
anticipated
that
2C.
f
*
1
d
._
a ._
,_
*
Wj
...
.
..
~ ~ ~ ~ ~ ~ ~ ~
~~~~4
FIG.
3.
Chemical
cross-linking
of
rTHRa
to
H-2RIIBP.
(A)
Cross-linking
of
[35S]methionine-labeled
rTHRa
to
H-2RIIBP.
Au-
toradiograph
of
SDS/PAGE-separated
products
resulting
from
in-
cubation
of
[35S]methionine-labeled
rTHRa
synthesized
in
an
in
vitro
translation
system
(Promega)
with
300
fmol
of
unlabeled
H-2RIIBP
(lanes
1
and
2),
300
fmol
of
rTHRa
(lanes
3
and
4),
or
8
,gg
of
nuclear
extract
from
wild-type
baculovirus-infected
Sf9
cells
Qanes
5
and
6)
for
4
h
at
4°C
in
1
x
BB
before
the
addition
of
the
cross-linking
reagent
DSS
to
a
final
concentration
of
0.25
mM
(lanes
1,
3,
and
5).
After
an
additional
30-min
incubation,
samples
were
quenched
by
the
addition
of
1
ul
of
1
M
NH4Cl,
analyzed
by
SDS/PAGE
in
a
7%
gel,
and
autoradiographed.
(B)
Identification
of
cross-linked
products
with
rTHRa-specific
antiserum.
rTHRa
(33
pmol,
lanes
1-4)
and
H-
2RIIBP
(5
,mol,
lanes
1,
2,
5,
and
6)
were
cross-linked
with
DSS
as
described
above
(lanes
1,
3,
and
5),
subjected
to
SDS/PAGE
in
a
10%6
gel,
transferred
to
a
nitrocellulose
filter,
and
probed
for
rTHRa-
containing
species
with
rTHRa
antisera
and
17-I-labeled
protein
A.
Molecular
mass
standards
are
as
follows:
myosin,
200
kDa;
phos-
phorylase
b,
97
kDa;
BSA,
69
kDa;
ovalbumin,
46
kDa.
*,
Non-
DSS-dependent
complex
of
unknown
composition.
Proc.
Natl.
Acad
Sci.
USA
89
(1992)

Proc.
Natl.
Acad.
Sci.
USA
89
(1992)
5575
expression
of
H-2RIIBP
in
cells
expressing
rTHRa
should
affect
the
rTHRa-TH-ME-TRE-dependent
transcriptional
activation
of
a
reporter
gene
(34).
Transfection
of
12.20
ug
of
only
the
H-2RIIBP
expression
plasmid
(RSVH-2RIIBP)
with
the
ME-TRE-TK-CAT
reporter
plasmid
had
no
appreciable
effect
on
CAT
expression
in
the
presence
or
absence
of
TH
(Fig.
4A).
As
expected,
transfection
of
12.20
Ag
of
the
rTHRa
expression
(RSVRTHRa)
plasmid
in
the
presence
of
TH
resulted
in
a
24-fold
increase
in
CAT
expression
when
compared
to
the
amount
of
CAT
expressed
in
the
absence
of
TH
as
reported
(34).
Nevertheless,
when
RSVRTHRa
ex-
pression
plasmid
(12.20
pug)
was
cotransfected
with
gradually
increasing
amount
of
RSVH-2RIIBP,
higher
TH-stimulated
CAT
expression
was
observed
(Fig.
4A).
Equivalent
amounts
of
RSVH-2RIIBP
and
RSVRTHRa
expression
plasmid
co-
transfected
in
the
presence
of
TH
resulted
in
an
=80-fold
increase
in
TH-stimulated
CAT
expression
compared
to
the
level
of
CAT
expression
in
the
absence
of
TH.
Thus,
addition
of
RSVH-2RIIBP
expression
plasmid
resulted
in
a
3-fold
increase
in
TH-stimulated
CAT
expression
above
that
ob-
served
with
RSVRTHRa
alone
(Fig.
4A).
Similar
enhance-
ment
was
observed
when
utilizing
either
the
palindromic
TRE
derived
from
the
rat
growth
hormone
gene
or
by
replacing
the
entire
ME-TRE-TK
portion
of
the
reporter
plasmid
with
the
full-length
ME
promoter
containing
the
TRE
but
lacking
the
TK
promoter
(data
not
shown).
Further,
we
sought
to
determine
whether
the
concentration
of
rTHRa
affected
the
observed
RSVH-2RIIBP
dose-
dependent
stimulation
of
rTHRa-TH-responsive
CAT
ex-
pression.
In
this
experiment
an
increasing
amount
of
the
RSVRTHRa
expression
plasmid
was
added
to
12.20
pug
of
RSVH2RIIBP.
Surprisingly,
H-2RIIBP-enhanced
rTHRa-
mediated
transcription
was
dependent
on
a
minimal
concen-
tration
of
cotransfected
RSVRTHRa
(Fig.
4B).
Cotransfec-
tion
of
12.20
,ug
of
RSVH-2RIIBP
with
0.03
ug
or
0.13
ug
of
RSVRTHRa,
corresponding
to
a
5-
or
17-fold
increase
in
TH-dependent
transcriptional
activation
in
the
presence
of
RSVRTHRa
alone,
had
very
little
effect
on
CAT
expression
(Fig.
4B).
However,
inclusion
of
higher
amounts
of
RS-
VRTHRa
(0.60-12.20
,ug)
resulted
in
a
H-2RIIBP-dependent
increase
in
TH-mediated
transcription
of
3-
to
4-fold
(Fig.
4B).
We
hypothesize
that
when
a
limiting
amount
of
RS-
VRTHRa
was
transfected
into
NIH
3T3
cells,
it
complexed
with
endogenous
H-2RIIBP-like
protein,
and
thus,
an
excess
of
H-2RIIBP
had
little
effect
on
CAT
expression.
However,
this
could
be
reversed
by
adding
more
RSVRTHRa
to
that
amount
of
RSVH-2RIIBP.
We
therefore
suggest
the
exis-
tence
of
a
limited
amount
of
endogenous
H-2RIIBP-like
factor
in
NIH
3T3
cells,
which
would
be
necessary
for
maximal
TH-
and
THR-mediated
induction
of
gene
expres-
sion.
A
B
0.03
0.13
0.60
12.20
12.20
12.20
H5-H
H17H
H395-
DISCUSSION
Our
results
demonstrate
that
H-2RIIBP
formed
a
het-
erodimer
with
THR
and
that
this
heterodimer
formation
resulted
in
an
apparent
increase
of
affinity
of
THR
for
a
TRE.
These
results
are
similar
to
those
reported
by
others
where
protein(s)
from
crude
or
partially
purified
nuclear
extracts
derived
from
GH3
cells
(13,
39,
40),
JEG-3
cells
(13,
14),
235-1
cells
(13),
or
liver
(15,
16)
were
shown
to
enhance
the
binding
of
THR
to
a
TRE
despite
the
ability
of
these
extracts
to
bind
a
TRE
weakly
(13)
or
not
at
all
(15).
The
observation
that
some
of
these
protein(s),
generally
referred
to
as
THR
auxiliary
proteins,
were
also
shown
to
alter
the
mobility
of
a
THR-bound
TRE
complex
in
polyacrylamide
gels
suggests
that
they
may
also
be
binding
the
TRE
(15,
16).
Similarly,
H-2RIIBP
altered
the
mobility
of
the
rTHRa-ME-TRE
com-
plex
by
forming
a
heterodimer
with
rTHRa
on
the
TRE.
Whether
any
of
the
enhancing
activities
in
these
cells
could
be
ascribed
to
H-2RIIBP
present
in
these
extracts
must
wait
for
further
purification
and
characterization
of
those
enhanc-
ing
activities.
H-2RIIBP
is
present
in
appreciable
levels
in
a
variety
of
tissues
and
cell
lines
(26),
but
it
is
unlikely
that
all
the
enhancing
activity
is
due
to
H-2RIIBP
since
the
estimated
molecular
mass
of
the
suspected
protein(s)
is
in
the
range
of
42-63
kDa
rather
than
44
kDa,
the
molecular
mass
of
H-
2RIIBP
(26).
However,
the
data
presented
here
suggest
that
it
was
the
formation
of
heterodimers
of
rTHRa
and
H-2RIIBP
in
solu-
tion
that
modulated
the
TRE
binding
activity
of
rTHRa,
since
H-2RIIBP
bound
the
ME-TRE
only
in
the
presence
of
rTHRa
in
our
DNA
binding
assay.
Further,
rTHRa
formed
stable
heterodimers
with
H-2RIIBP
even
in
the
absence
of
the
ME-TRE.
Nevertheless,
it
is
still
unclear
how
H-2RIIBP
increased
the
apparent
binding
affinity
of
rTHRa
for
the
TRE.
H-2RIIBP
could
just
enable
more
rTHRa
to
bind
a
TRE
by
simply
increasing
the
amount
of
species
capable
of
binding
this
TRE
through
heterodimer
formation
(an
increase
in
binding
capacity)
and/or
the
heterodimer
actually
could
have
a
higher
affinity
for
the
TRE.
Either
scenario
raises
interest-
ing
questions
regarding
the
specificity
of
heterodimer
bind-
ing,
including
the
role
of
TH
in
these
processes.
The
coexpression
of
the
H-2RIIBP
with
the
rTHRa
ex-
pression
plasmid
in
NIH
3T3
cells
also
led
to
a
3-
to
4-fold
increase
in
the
amount
of
TH-rTHRa-ME-TRE-TK-
dependent
expression
of
the
reporter
gene
over
that
observed
with
rTHRa
and
TH
alone.
Thus
by
considering
these
results
and
the
binding
studies,
we
speculate
that
the
H-2RIIBP-
enhanced
transcription
was
the
result
of
H-2RIIBP
recruiting
rTHRa
to
bind
more
efficiently.
However,
it
is
also
possible
that
H-2RIIBP
may
have
the
ability
to
interact
with
the
transcriptional
machinery
and,
thereby,
directly
contribute
to
increased
transcriptional
activation.
FIG.
4.
Effect
of
rTHRa
and
H-2RIIBP
on
rTHRa-TH-ME-TRE-dependent
expression
of
the
CAT
reporter
gene.
(A)
Enhancement
of
TH-
dependent
transcriptional
activity
by
H-2RIIBP.
Reporter
plasmid
pTK1AM3
(ME-TRE-TK-CAT)
was
electroporated
into
NIH
3T3
cells
with
H-
2RIIBP
(pRSVH-2RIIBP),
rTHRa
(RSVRTHRa),
both,
or
control
plasmid
alone
(RSVANEO).
Ac-
tivity
at
100%
refers
to
a
24-fold
increase
in
CAT
expression
obtained
with
12.20
,g
of
transfected
RSVRTHRa
in
the
presence
and
absence
of
TH.
(B)
Dependence
of
H-2RIIBP-mediated
enhancement
T
h¶
of
rTHRa-TH-responsive
transcription
on
the
con-
centration
of
RSVRTHRa.
Activity
at
100%
refers
3.30
12.20
to
the
increase
in
CAT
expression
obtained
with
-
12.20
-
12.20
transfected
RSVRTHRa
alone
in
the
presence
and
-43H
H24-1
absence
of
TH.
Biochemistry:
Hallenbeck
et
al.

5576
Biochemistry:
Hallenbeck
et
al.
The
possibility
that
rTHRa
interacts
with
a
H-2RIIBP-like
protein
or
H-2RIIBP
itself
to
elicit
maximal
TH-dependent
regulation
of
gene
expression
in
vivo
is
suggested
by
our
experiments
in
NIH
3T3
cells.
We
demonstrated
that
a
certain
concentration
of
rTHRa
expression
plasmid,
corre-
sponding
to
that
which
we
hypothesize
is
required
to
titrate
endogenous
H-2RIIBP-like
protein(s),
was
necessary
to
ob-
serve
a
H-2RIIBP-dependent
increase
in
transcription.
The
existence
of
a
natural
heterodimer
partner
is
supported
further
by
studies
that
demonstrated
that
the
ability
of
THR
to
heterodimerize
and
the
transcriptional
activity
of
THR
were
not
mutually
exclusive.
Several
mutations
constructed
in
the
C
terminus
of
THR
abolished
the
ability
of
THR
to
form
a
heterodimer
with
an
enhancing
protein(s)
present
in
235-1
cells
and
to
activate
transcription
(41).
The
requirement
for
THR
to interact
with
a
natural
heterodimer
partner
might
also
explain
why
THR
can
only
mediate
TH-induced
gene
ex-
pression
from
some
HREs,
regardless
of
the
observed
high
binding
affinity
that
the
receptor
has
for
some
non-TH-
responsive
HREs
in
vitro
(42-46).
H-2RIIBP
and
rTHRa
join
a
growing
list
of
transcriptional
regulatory
proteins
(4,
8,
47),
including
Jun/Fos
(1,
2,
5,
7)
and
Myc/Max
(3),
that
form
heterodimers
with
altered
bind-
ing
and
transcriptional
activation
through
respective
cis-
acting
DNA
elements.
Thus,
our
results
of
heterodimer-
induced
modulation
of
DNA
binding
and
transcriptional
activation
underscore
the
diversity
of
transcriptional
regula-
tion
in
the
steroid/TH
receptor
superfamily
and
offer
insight
into
the
hormone-specific
regulation
of
gene
expression.
Note
Added
in
Proof.
Since
this
manuscript
was
submitted
for
review,
four
papers
have
been
published
on
the
same
subject
(48-51).
In
general,
our
results
are
concordant
with
these.
We
thank
Dr.
J.
Robbins
for
his
critical
reading
of
this
manuscript,
Dr.
T.
Mitsuhashi
for
preparation
of
baculovirus
TH
recombinant
construct,
all
members
of
the
laboratory
of
V.M.N.
and
Dr.
D.
Sackett
and
Dr.
S.
Simmons
for
discussions.
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