(Received for publication, May 9, 1994; and in revised form, December 5, 1994)
From the
Direct repeats of the hexamer AGGTCA can serve as response elements for vitamin D, thyroid hormone, or retinoic acid. The specificity of the response appears to reside in the spacing between the hexamers, with response elements for vitamin D restricted to direct repeats separated by a 3-base pair (bp) spacer, thyroid hormone a 4-bp spacer, and retinoic acid a 5-bp spacer (3-4-5 rule). Recently we have shown that the optimum thyroid hormone receptor binding site consists of an 8-bp sequence (TAAGGTCA), not a hexamer. Therefore we tested whether the 3-4-5 rule is valid for octamer sequence direct repeats. In transfection experiments octamer direct repeats with 3-, 4-, or 5-bp spacers conferred equivalently strong thyroid hormone responses, although a repeat with a 9-bp spacer was substantially weaker. For the 4- and 5-bp spacer constructs, the 5` half-site octamer had as strong an influence on thyroid hormone induction as did the 3` half-site octamer, although for the 3-bp spacer construct the 5` octamer was marginally less potent than the 3` octamer. Transfection and gel shift experiments did not suggest a simple correlation between the binding of thyroid hormone receptor-retinoid X receptor heterodimers and thyroid hormone induction from these response elements. We conclude that half-site sequence can override the effect of spacing in determining the hormone responsiveness of a direct repeat response element. In addition, the thyroid hormone response may not be due simply to the binding of thyroid hormone receptor-retinoid X receptor heterodimers to the DNA.
Thyroid hormone (3,5,3`-triiodothyronine; T) (
)receptors (TRs) are members of a large family of zinc
finger transcription factors which also includes the receptors for all
known steroid hormones, retinoic acid (RA) and
1,25-dihydroxycholecalciferol (vitamin D
; vitD). In the
case of thyroid hormone-responsive genes, T
regulation is
dependent on the binding of TRs to specific cis-acting sequences, or
T
response elements
(TREs)(1, 2, 3, 4, 5) . The
well characterized positive TRE in the promoter of the rat growth
hormone (GH) gene has been shown to consist of three copies of a
loosely conserved hexamer motif, AGGTCA(6, 7) . This
hexamer has also been characterized as a binding half-site for several
other related members of this family of transcription factors,
including retinoic acid receptors (RARs), retinoid X receptors (RXRs)
and the vitamin D receptor
(VDR)(8, 9, 10, 11) . It is
recognized that these receptors can bind as dimers to direct repeats
(DR) of this hexamer(8, 9, 10, 11) .
Differential spacing between hexamer half-sites has been proposed to
account for the specificity of the T
-, RA-, and
vitD-dependent responses of otherwise equivalent DR response elements.
Thus, a DR of the hexamer AGGTCA has been shown to function
specifically as a vitD response element (VDRE) if the hexamers are
separated by 3 base pairs (bp), as a TRE if the spacer is 4 bp, and as
an RA response element (RARE) if the spacer is 5 bp (3-4-5
rule)(8, 9) .
Recently, we and others have shown
that nucleotides surrounding the hexamer sequence can influence TR
binding and function(12, 13, 14) . A protocol
of sequential electrophoretic mobility shift assays (EMSAs) and
polymerase chain reaction (PCR) amplifications allowed us to identify
the optimal TR1 monomer binding sequence from an original pool of
random DNA sequences(12) . This optimal binding sequence,
5`-TAAGGTCA, is 2 bp longer than the consensus hexamer sequence. We
have demonstrated that the 2 additional 5` bp are important for the
binding and function of TRs (12) and that a single octamer
sequence can function as a TRE but not as an RARE, VDRE, or RXR
response element (RXRE)(15) .
The 3-4-5 rule was based on
functional studies of the effect of variable spacing between hexamer
sequences. In retrospect, the hexamer AGGTCA represents an incomplete
TR binding sequence. Therefore we wanted to evaluate the effect of
spacing on the ligand responses of reporter genes containing optimal
octamer TR1 binding sequences arranged as DR response elements. In
the transfection studies described below, we demonstrate that octamer
DRs with 3-, 4-, and 5-bp spacers (
)all function as equally
strong TREs. Thus, the two upstream nucleotides of the receptor binding
site play a prominent role in determining the strength and specificity
of T
DR response elements.
In addition, constructs were made in which one of the two half-sites contained the octamer sequence (TAAGGTCA) while the other contained the hexamer (GCAGGTCA). For example, ptk86DR3 had the octamer in the upstream position, whereas ptk68DR3 had the octamer in the downstream position.
The protein-DNA binding reactions were
performed in 35 µl of 20 mM HEPES, pH 7.8, 50 mM KCl, 1 mM dithiothreitol, 0.1% Nonidet P-40, and 20%
glycerol. The incubations included varying amounts of receptor(s), 1.4
µg of poly(dIdC), and 80,000 cpm of labeled DNA. Reactions
were incubated at room temperature for 40 min and then subjected to
electrophoresis in 6% polyacrylamide gels (29:1
acrylamide:bisacrylamide) in 0.25
TBE buffer (22 mM Tris base, 22 mM boric acid, 0.5 mM EDTA) at
room temperature. The gels were fixed in 30% methanol/10% acetic acid,
dried, and exposed to film with an intensifying screen for 1-6 h
at -70 °C as indicated.
Figure 1:
Octamer direct
repeat response elements with 3-5-bp spacers have equivalent
T responses. The octamer 5`-TAAGGTCA was inserted as a
single copy into the reporter plasmid pUTKAT3 in a forward orientation (MA) or as direct repeats with 3-, 4-, 5-, or 9-bp
spacers (8DR3-8DR9) and transfected into JEG-3
cells along with the internal control plasmid pRSVGH and an expression
vector for TR
1. Cells were cultured for 2 days ±
T
, and then the cell lysates were assayed for CAT activity
and the media for human GH. CAT activity was normalized using GH and
presented in arbitrary CAT/human growth hormone units. Results are the
mean ± S.E. for at least four independent
transfections.
Figure 2:
Hexamer direct repeat response elements
conform to the 3-4-5 rule. The sequence 5`-GCAGGTCA was arranged as a
direct repeat with a 3-, 4-, 5-, or 9-bp spacer in the reporter plasmid
pUTKAT3 (6DR3-6DR9). Transfections were
performed as described in Fig. 1. Ligand responsiveness (-fold
CAT induction) is defined as CAT/human GH for cells cultured with
T divided by CAT/human GH for cells cultured in the absence
of T
. Results are the mean ± S.E. for at least four
independent transfections. Basal CAT activities were similar for all 4
reporter plasmids.
Figure 3: Octamer direct repeat response elements obey the 3-4-5 rule as VDREs and RAREs. Transfections were performed as described in Fig. 1. Octamer direct repeat response elements with 3-5-bp spacers (8DR3-8DR5) were cotransfected with either the VDR or RAR expression plasmid, and CAT induction by the appropriate ligand was determined. Results are the mean ± S.E. for at least four independent transfections. CAT activities in the absence of ligand were similar for all three reporter plasmids.
Figure 4:
TRRXR heterodimers bind equivalently
to 6DR4 and the 8DR3-5 elements. EMSAs were performed in the
presence of T
with
P-labeled DNA probes
derived from reporter plasmids containing the elements 6DR4, 8DR3,
8DR4, or 8DR5. The probe labeled C is derived from the empty
reporter plasmid vector (pUTKAT3). Incubation with RXR
alone
failed to yield detectable complexes with any of the probes (left panel). Incubation with TR
1 alone yielded
monomer-DNA complexes only with the 8DR probes (middlepanel), consistent with their enhanced affinity relative
to the hexamer TRE sequence for TR monomers. Incubation with TR
1
plus RXR
yielded heterodimer-DNA complexes with 6DR4 and all three
8DR probes (right panel).
Since TR homodimers might contribute to the
T responsiveness of DR TREs, we also evaluated TR homodimer
formation on the 8DRs by performing EMSAs with a 10-fold increased dose
of TR
1. TR homodimers did not form on the single octamer element,
but did form on 8DR3-8DR5 and 8DR9 with approximately equal
intensity (data not shown). Since 8DR9 is a weak TRE, the strength of
8DR3-8DR5 as TREs cannot be directly correlated with the strength
of TR homodimer binding as measured by EMSA. Although the EMSA data do
not allow one to conclude whether TR monomers, TR homodimers, or
TR
RXR heterodimers actually are involved in gene activation from
the 8DR elements, they do indicate that all of these complexes could
potentially contribute to the T
response.
Despite much progress over the last several years, the molecular basis of hormone-specific regulation of gene expression is still unclear. An essential component of a hormone response is the binding of the appropriate receptors to the regulatory regions of responsive genes. In order to understand this process relative to the ErbA superfamily of hormone receptors, it is necessary to determine the optimum binding sequence of each particular receptor, as well as the degree of cross-binding possible with other closely related members of this gene family.
Previous investigators did not know a priori the exact size or sequence of the optimal TR binding site, so a
consensus binding sequence was derived from limited mutagenesis of a
complex TRE in the rat growth hormone promoter and sequence comparisons
of the positive and negative TREs of a limited number of
T-responsive genes. The result was an initial
characterization of the TR binding site as containing 6 nucleotides
(5`-AGGTCA) and the observation that positive TREs consisted of complex
arrangements of loosely conserved sites. In the case of DR elements,
the effect of spacing was believed to be essential in conferring
ligand-specific responses, with vitD responses being restricted to
elements with a 3-bp spacer, T
responses to elements with a
4-bp spacer, and RA responses to elements with a 5-bp spacer (3-4-5
rule). However, these studies were based on suboptimal TR binding
sites.
In the present report we demonstrate that DRs of the optimal
(octamer) TR binding sequence are less affected by alterations in
spacing than had been previously proposed according to the 3-4-5 rule.
Thus, 8DR3, 8DR4, and 8DR5 all are capable of eliciting an equally
strong T response. However, interposing an additional
half-turn of DNA helix between the receptor binding sites (8DR9)
weakens the magnitude of T
induction. This suggests that
the induction from 8DR3, 8DR4, and 8DR5 involves a protein-protein
interaction in addition to DNA binding. This interaction can occur with
half-site spacings of 3, 4, and 5 bp, but not 9 bp. It is perhaps
surprising that 8DR9 is no more active than a single octamer sequence
as a TRE. The explanation for this observation is not known. Although
it could reflect an adverse effect of phasing on T
induction, this TRE is more than 100 bp from the transcriptional
start site and we are unaware of published data to demonstrate such
effects for comparably positioned TREs. Alternatively, perhaps the
number of TR monomers bound to DNA is not the rate-limiting factor for
determining the level of T
induction. In any case, it is
clear that the 3-4-5 rule cannot be used to predict the specificity of
hormone induction from these octamer DR response elements. Although the
3-4-5 rule represents a major advance in our understanding of a
variable (spacing) that restricts the specificity of hormone induction
from hexamer DR elements, this rule is less influential when optimal TR
binding sequences are present. We conclude that the affinity of the DNA
half-site sequence for TR has at least as great an influence on
conferring ligand response as does the spacing of DRs (at least for
spacings of 3-5 bp).
TRs can bind to direct repeat TREs as
monomers, homodimers, or heterodimers with a variety of other ErbA
superfamily members such as
RXRs(23, 24, 25, 26) ,
RARs(33) , VDR(34) , and chicken ovalbumin upstream
promoter-transcription factor(35) . It is not known which of
these complexes are important transcriptional regulators in
vivo. However, indirect evidence has led to the notion that
TRRXR heterodimers may be the most important functional complex
within the cell, at least for traditional hexameric DR4
TREs(23, 24, 25, 26, 27, 28) .
TR
RXR heterodimers have been shown to bind DNA with the 5`
half-site occupied by RXR and the 3` half-site by
TR(29, 30) . Thus, if the transcriptional response
from 8DR3-5 were due entirely to the binding of TR
RXR
heterodimers, one would expect that the octamer would be required only
for the 3` half-site. This is especially the case since the optimal
half-site sequence for RXR
binding is GGGGTCA, not
TAAGGTCA(36) . However, a direct test using reporters
comprising one octamer and one hexamer half-site failed to substantiate
this prediction. Indeed, 86DR4 was as potent as 68DR4, and also as
potent as 8DR4. Although neither 86DR5 nor 68DR5 was quite as potent as
8DR5, the 5` octamer was again as potent as the 3` octamer. These data
argue against TR
RXR heterodimers as being the sole activators of
transcription from the 8DR elements. This conclusion is further
supported by EMSA data (Fig. 4), which demonstrate that 6DR4
binds TR
RXR heterodimers as well as the 8DRs, even though it is
weaker as a TRE. It is likely that part of the T
induction
from the 8DR elements is accounted for by TR
monomers(12, 15) , and it is possible that TR
homodimers or TR heterodimers with other co-regulators also may account
for some of the induction.
When the octamer elements were tested for
RA and vitD induction, the 5-bp spacer (8DR5) was found to be the most
potent RARE, and the 3-bp spacer (8DR3) had the greatest vitD response.
Thus, in contrast to the above data for TRs, the specificity of
induction by RAR and VDR on these sequences does conform to the 3-4-5
rule. However, it appears that TAAGGTCA does not represent the optimal
binding site for RAR (37) or VDR(38, 39) . It
is possible that variably spaced DR response elements composed of the
optimal binding sequences for RAR or VDR will show RA and vitD
responses that do not conform to the 3-4-5 rule, analogous to what is
shown here for T and 8DR3-5.
As noted above, it
appears that T-responsive cells may contain a diverse
population of TR-containing complexes ranging from TR monomers to
several different TR dimers and potentially larger complexes. The
ability of a particular DNA sequence to confer a T
response
in a particular cell or tissue may depend on the affinity of different
specific TR-containing complexes for that sequence. Since a T
response requires the binding of a TR-containing complex, TREs
appear to require at least one TR binding site. Genes whose regulatory
regions contain one or more optimal TR binding sites may have their
T
response transduced by TR monomers, homodimers,
heterodimers, and/or larger order complexes. In those cases where the
individual half-sites are suboptimal for TR binding, a TR homo- or
heterodimer may still possess sufficient cooperative affinity such that
it may confer T
responsiveness. In order to understand the
relative contribution of each TR-containing complex to conferring
T
responsiveness in various tissues, it will be necessary
to understand how TRs bind to different arrangements of optimal and
suboptimal sites. In addition it will be necessary to understand how
each of the potential dimerization partners of TRs bind to their own
optimal and suboptimal sites.
Since many members of this receptor
family are closely related, a particular DNA sequence may function as a
high affinity site for one receptor complex and a modest affinity site
for several other receptor complexes. With the close proximity of two
sites, some sequences can function as response elements for multiple
receptors (e.g. a palindromic sequence can function as a TRE
and an RARE; (40) ) and other arrangements will be more
restricted in their potential receptor interactions (e.g. TAAGGTCA can function as a TRE but not an RXRE, RARE, or VDRE; (15) ). We believe it is reasonable to propose that a
T-responsive cell contains a limited variety of TR
complexes (monomers, specific heterodimers, etc.) and that each TR
complex will confer the greatest ligand response to a set of regulatory
elements that bind a particular complex with the highest affinity and
in a conformation supportive of trans-activation. For example,
TR monomers will confer the greatest T
response from a
single octamer sequence 5`-TAAGGTCA placed in a forward orientation
relative to the transcriptional start site(12) . In this view,
TR homodimers will confer maximal T
responses to sites
composed of two copies of 5`-TAAGGTCA. Based on studies showing that
RXR binds 5` to TR in TR
RXR
heterodimers(29, 30) , we hypothesize that TR
RXR
heterodimers will confer the greatest T
response from an
element composed of an optimal TR binding site in close proximity and
downstream of an optimal RXR binding site. Thus, changes in the
composition of the pool of TR complexes could alter the genetic
response of a cell to T
. It is possible that the presence
or absence of a particular TR complex may account for the
tissue-specific effects of T
. Before one can understand
physiologic and pathologic hormone or vitamin regulation at the
transcriptional level, it will be necessary to characterize the optimal
binding sites for a large group of related zinc finger receptors (such
as RARs, RXRs, and chicken ovalbumin upstream promoter-transcription
factor), determine how different combinations of sequences confer
ligand-restricted changes in gene expression through specific receptor
combinations, and identify these sequences as the actual regulatory
elements of specific sets of genes modulated by the relevant hormone
receptors.