(Received for publication, October 2, 1996, and in revised form, December 31, 1996)
From the Section of Microbiology, Division of Biological Sciences, University of California, Davis, California 95616
Retinoid X receptors (RXRs) are transcription factors that traditionally have been thought to bind DNA as protein dimers. Recently, however, it has been recognized that RXRs can also bind to DNA as protein tetramers. Receptor tetramers form cooperatively on response elements containing suitably reiterated half-sites, and play an important role in determining the specificity of DNA recognition by different nuclear receptors. We report here that RXR tetramers exhibit significant functional plasticity, and form on response elements possessing diverse half-site orientations and spacings. This ability of RXRs to form tetramers and related oligomers appears to contribute to the synergistic transcriptional activation observed when multiple, spatially separated response elements are introduced into a single promoter. Oligomerization may therefore be a common paradigm for DNA recognition and combinatorial regulation by several different classes of transcription factors.
Nuclear hormone receptors are hormone-regulated transcription
factors, and include the steroid receptors, vitamin D3
receptors, thyroid hormone receptors, and two distinct categories of
retinoid receptors: retinoic acid receptors
(RARs)1 and retinoid X receptors (RXRs)
(reviewed in Refs. 1-7). RXRs in particular play important roles in
vertebrate development and differentiation, both autonomously in
response to 9-cis-retinoic acid and combinatorially in
conjunction with other nuclear hormone receptors (8-14). Three
different genetic loci (denoted ,
, and
) encode RXRs
(8-14).
RXRs, in common with other nuclear hormone receptors, function by
binding to specific DNA sequences (denoted hormone response elements,
HREs) and regulating the transcription of adjacent target genes in
response to hormone (1-7). The nuclear hormone receptors share a
common structural organization comprising a DNA-binding domain
(including a central zinc-finger domain), a hormone-binding domain, and
a variety of motifs involved in interactions with the transcriptional
machinery (Fig. 1 and Refs. 1-7). Most nuclear hormone
receptors can bind to DNA as protein dimers, with each receptor
molecule recognizing a "half-site," a conserved 6-8-base DNA
sequence (15-18). HREs have therefore been traditionally viewed as
composed of two half-sites. Both the sequence of the individual half-sites and their spacing and orientation contribute to the specificity of DNA recognition (15-25). The protein-protein interfaces involved in receptor dimer formation have been mapped to both zinc-finger and hormone-binding domains (15-18, 24-26).
It is now clear, however, that receptor dimers are not the only paradigm for DNA recognition, and that certain members of the nuclear receptor family can also bind to DNA as protein complexes larger than dimer in size (27). These high order oligomers include receptor trimers, tetramers, and pentamers, and bind with high cooperativity to response elements containing suitably reiterated half-sites (27). RXR tetramers have also been observed in solution (28-30). Oligomer formation by RXRs permits recognition of DNA sequences that are not recognized by receptor dimers, and contributes to isoform-specific promoter utilization (27).
We wished to investigate the nature of these newly elucidated receptor oligomers. Here, we report that RXR tetramer formation is relatively plastic in character, and that tetramers form on response elements possessing a wide variety of half-site orientations and spacings. In fact, receptor oligomers can form cooperatively on spatially separated response elements and may contribute to the synergistic gene activation observed when multiple response elements are introduced into a single promoter. Intriguingly, the half-site spacing and orientation required for tetramer formation are non-equivalent at different positions in the response element, suggesting a corresponding anisotropism in the RXR tetramer. High order oligomer formation may be a common means of generating DNA specificity and combinatorial regulation for a variety of transcription factors.
The wild-type, N
terminus, and the chimeric mouse RXR proteins were obtained as nuclear
extracts from Sf9 cells infected with the appropriate recombinant
baculovirus (27). The construction of the
N terminus RXR
,
N
terminus RXR
, and the
·
RXR chimera was described previously
(27). The TR·RXR chimera represents a fusion of codons 1-159 of the
avian thyroid hormone receptor
-1 sequence to codons 228-464 of
mouse RXR
. The
N
C RXR
, representing codons 139-228, was
isolated as glutathione S-transferase fusion protein from
Escherichia coli bearing a suitable pGEX plasmid vector
(31). No significant functional differences were detected when the same
receptor was isolated from the two different expression systems (27).
Oligonucleotides were obtained as complementary, single-stranded DNAs
(Operon Incorporated) and were annealed to create double-stranded DNAs
with 4-base overhangs. For use as probes in electrophoretic mobility
shift experiments, the overhangs were filled in with radiolabeled
nucleotides and Klenow fragment of DNA polymerase I (31, 32). For use
as response elements, the double-stranded DNAs were introduced into the
SalI site of pD33-CAT (31). An adjacent, cryptic half-site
present in the pD33-CAT vector itself (AGGTCG) was removed from these
constructs by PstI and HindIII cleavage,
converting the overhanging ends to blunt with T4 DNA polymerase, and
religation.
Receptor preparations (estimated as 20-30 ng of receptor/µl) were incubated at 25 °C for 20-25 min with radiolabeled oligonucleotide probe (40,000-60,000 cpm, 20-60 ng of DNA) in 15 µl of binding buffer (10 mM Tris-Cl, pH 7.5, 3% glycerol, 13.3 µg/µl bovine serum albumin, 66.7 mM KCl, 2 mM MgCl2, and 133 µg/ml poly(deoxyinosine-deoxycytosine)). The resulting DNA-protein complexes were then resolved by non-denaturing electrophoresis through a 5% polyacrylamide, 0.13% bisacrylamide gel (either at 25 °C and 200 V, or 4 °C and 320 V) and were visualized by autoradiography (31, 32).
Transient TransfectionsDrosophila SL-2 cells
were maintained at room temperature in Schneider's medium supplemented
with 14% fetal bovine serum. Transient transfections were performed
using a calcium phosphate co-precipitation protocol and typically
employing 1 µg of pD33-CAT reporter, 0.15 µg of pHSP82-lacZ (used
as an internal standard), and 0.1 µg of either
pA5C-RXR or pA5C-RXR
expression
plasmid/35-mm plate (31). After incubation in the absence or presence
of 100 nM 9-cis-retinoic acid (Ligand Research),
the transfected cells were harvested, and the chloroamphenicol
acetyltransferase activity was determined relative to that of the
-galactosidase control (31).
Both RXR and
can efficiently bind to
reiterated elements containing four half-sites oriented as direct
repeats (Fig. 2A). These protein-DNA
complexes migrate at a position characteristic of 4-fold occupancy
(4R), and form with extremely high cooperativity and no
evidence of prior dimer formation (27). We have therefore defined these
RXR
and RXR
complexes as protein tetramers (27). In contrast,
RXR
primarily forms dimers (2R) and neither binds these
reiterated elements as tetramers in vitro nor efficiently mediates gene activation through them in vivo (Fig.
2A and Ref. 27). It should be noted that at high protein
concentrations, RXR
can occupy all four half-sites on a reiterated
element by the non-cooperative binding of two independent RXR
dimers
(Fig. 2A); this is, however, in clear contrast to the highly
cooperative binding of tetramers observed with RXR
and
(Fig.
2A).
The various complexes observed in Fig. 2A are mediated by
the corresponding RXR isoforms, as demonstrated by the ability of RXR-directed antisera to supershift the complex, and by the absence of
complex formation by equivalent protein preparations isolated from
uninfected Sf9 cells, or from cells infected by non-recombinant baculovirus (Fig. 2, A and
B).2 Furthermore, analogous
complexes were formed by purified glutathione S-transferase-RXR fusion proteins isolated from E. coli, suggesting that RXR is likely the only protein necessary for
complex formation (27).
Our assignment of stoichiometry to the various complexes in Fig.
2A was based initially on titration experiments with
elements containing differing numbers of half-sites; a stepwise
decrease in the mobility of the complex occurred corresponding to the
number of half-sites in the element (27). A more absolute assignment of
stoichiometry was obtained by a combinatorial experiment. We compared
the migration pattern of DNA complexes generated by an N-terminal
deletion of RXR (which produces a rapidly migrating DNA-protein
complex) with that of the full-length RXR
(which produces a slower
migrating complex) (Fig. 2, C and D). Mixing these two different receptor derivatives on a two-half site element produced a single intermediate complex, the combinatorial result predicted if this element is indeed bound by receptor dimers
(i.e. RR, Rr, rr). In contrast, mixing these same
two receptor derivatives with our four half-site element produced three
intermediate complexes (Fig. 2D) precisely the number
predicted for combinatorial mixtures of a receptor tetramer
(i.e. RRRR, RRRr, RRrr,
Rrrr, rrrr). We therefore conclude that the RXR
and
RXR
complexes observed on DNA elements containing four half-sites do
indeed represent 4-fold occupancy by receptor tetramers.
What RXR domains are involved in
tetramer formation? We have previously shown that the N terminus of RXR
confers the differing oligomerization properties of the ,
, and
isoforms (27). Exchanging the N termini of the RXR
and
isoforms exchanges the oligomerization phenotype (Fig.
3A). However, the N terminus plays a negative
role in oligomerization, not a positive one; deletion of the N terminus
does not affect oligomer formation by RXR
, and actually enhances
oligomerization by RXR
(
N-
and
N-
; Fig. 3A).
Thus, the actual oligomerization interface(s) must lie outside the
N-terminal domain. Indeed, deletion of both the N terminus and the
C-terminal hormone-binding domain disrupted cooperative tetramer
formation without abolishing dimerization (
N
C
; Fig.
3A). We conclude that the zinc-finger domain is sufficient for dimer formation, but that additional, C-terminal sequences are
necessary for tetramer formation.
We next tested if the oligomerization properties associated with the C
terminus of RXR were transferable to another nuclear receptor. Thyroid
hormone receptor--1 (TR
-1) binds DNA as a dimer, but does not
appear to form tetramers on 4-fold reiterated TR response elements
(Refs. 1-7, and data not shown). We therefore replaced the C-terminal
hormone-binding domain of TR
-1 with that of RXR
(Fig. 1).
Although this chimera readily bound to a reiterated TR response element
as a homodimer, it failed to form the cooperative tetramers
characteristic of the RXR
parent (Fig. 3B). It was conceivable that the TR N terminus might disrupt tetramer formation by
the TR·RXR
chimeras, analogous to the negative effects of the N
terminus of RXR
. However, deletion of the N terminus failed to
confer tetramer formation on either the TR·RXR chimera, or on the
native TR (Fig. 3B and data not shown). We conclude that the
C terminus of RXR is necessary for oligomerization, but is not
sufficient under the conditions employed here.
Nuclear hormone receptor dimers can
recognize a variety of half-site orientations and spacings (15-17,
23-25). RXR homodimers, for example, bind efficiently to direct,
convergent, and divergent repeats of half-sites spaced by 1, 0, or 2 bases, respectively. To dissect the topological constraints operative
on the RXR tetramer, we first explored the effects of varying the
spacing of the half-sites within the 4-fold reiterated element.
Introduction of a 2- or 10-base spacer at the center of the element
(i.e. between half-sites 2 and 3) had little or no
detectable effect on tetramer formation by RXR (Fig.
4A), indicating that the oligomer could
readily accommodate changes in spacing at this location. Formation of tetramers by RXR
was slightly destabilized, though not abolished, by
introduction of 5 bases at the same location (Fig. 4A)
presumably reflecting the non-integral rotation about the DNA
imposed by this spacer. None of these changes in spacing conferred
tetramer binding on the RXR
isoform (Fig. 4A).
In contrast to its relative lack of effect at the center of the
element, the 10-base insertion abolished tetramer formation by RXR
when introduced between half-sites 1 and 2, or between half-sites 3 and
4 (Fig. 4B). Instead of tetramers, RXR
bound to these
elements relatively weakly, and as a broad complex migrating in a
position characteristic of 3-fold occupancy (denoted 3R; Fig. 4B). We attribute this broadening or smearing
phenomenon to an instability of this trimeric complex during the
electrophoretic separation, a feature that was observed with several
other non-optimal elements. Apparently the 10-base insertion at these
flanking sites interferes with recruitment of a fourth receptor
molecule to the DNA, resulting in formation of a receptor trimer. We
conclude that the receptor interface between half-sites 2 and 3 is
relatively insensitive to spacing, whereas the receptor interfaces
between half-sites 1 and 2, or 3 and 4, are
spacing-dependent.
We next varied the relative orientations of the half-sites in the
reiterated element. Direct (>>>>), convergent (>><<), and divergent (<<>> or ><><) orientations between the central
half-sites 2 and 3 were all compatible with tetramer formation (Figs.
2A and 5). Similarly, both direct (>>>>)
and convergent (><><) repeat orientations were acceptable between
half-sites 1 and 2 or between 3 and 4 (Figs. 2A and 5).
However, inversion of just a single half-site at position 3 (>><>)
strongly inhibited tetramer formation and produced an unstable trimer,
presumably reflecting a non-optimal orientation of this half-site
relative to half-site 4 (Fig. 5). Similar elements lacking half-site 4 (>><) or containing a dysfunctional half-site at position 3 (>>8>) also failed to form tetrameric complexes (Fig. 5). Thus,
most, but not all orientations of individual half-sites could function
to recruit an RXR tetramer.
It should be noted that identical receptor preparations were employed in these experiments, and that the overall length and base composition of the different probes were kept as similar as possible; we therefore attribute the changes in mobility of the various protein-DNA complexes to alterations in tetramer formation, rather than arising from changes in protein modification or DNA conformation.
RXRThe N terminus of the RXR isoform
prevents tetramer formation on the consensus reiterated element
composed exclusively of direct repeats (Fig. 2A). If this
interference with tetramer formation is due to a steric hindrance
mediated by the RXR
N terminus, it might be partially relieved on
DNA elements with a different half-site topology. Indeed, although
tetramers of RXR
also failed to form on most of the elements tested
(e.g. Fig. 4A), reiterated elements with a
divergent orientation between half-sites 2 and 3 bound four molecules
of RXR
with detectable cooperativity, i.e. with some
degree of tetramer formation (compare the binding of RXR
to the
<<>> or ><>< orientations versus the >><<
orientation; Fig. 6). Nonetheless, the extent of
tetramer formation by RXR
on these divergent elements was
significantly less than observed for RXR
(compare Fig. 6 to Fig.
5).
RXR Tetramer Formation in Vitro Correlated with Reporter Gene Activation in Vivo
We tested the ability of the various response
elements to mediate transcriptional activation in transient
transfections (Fig. 7). Generally, the reiterated
elements that permitted tetramer formation by RXR in
vitro also conferred efficient RXR
-mediated gene activation in
transient transfection assay, whereas elements with half-site spacings
or orientations that disrupted tetramer formation failed to activate
reporter gene expression. This correlation was observed over a range of
receptor DNA concentrations (data not shown). Similarly, the
tetramer-deficient RXR
isoform exhibited a much lower activity on
all of the reiterated elements tested (Fig. 7), despite RXR
and
possessing near equal transcriptional activities on elements composed
of only two half-sites (Ref. 27 and data not shown) and despite the use
of identical expression vectors and transfection conditions. One
dramatic exception to this general correlation was noted, however; our
element possessing an inversion of the third half-site (>><>)
conferred extremely strong transcriptional activation by RXR
,
despite failing to allow efficient tetramer formation in
vitro (Fig. 7B).
Hormone has been reported to enhance dimer formation by RXRs on
two-half-site elements (13). We therefore asked if the presence of
hormone could influence RXR tetramer formation, perhaps accounting for
the apparent discrepancy between our DNA binding studies (performed in
the absence of hormone) and the transfection studies (measured in its
presence). Indeed, inclusion of 9-cis-retinoic acid in the
DNA binding assay significantly enhanced RXR tetramer formation on
several of the reiterated elements tested. This effect was particularly
strong for the prototype direct repeat element (>>>>; Fig.
8A) and for the element containing an
inverted third half-site (>><>; Fig. 8B), accounting at
least partly for the strong activity of the latter element in transient
transfections. Notably, hormone did not enhance RXR
tetramer
formation on the elements that were inactive in the transient
transfection assays, nor did hormone confer tetramer binding by RXR
on any of the elements tested (data not shown). Under these conditions,
inclusion of 9-cis-retinoic acid had only modest effects on
dimer formation by RXR
, as determined on elements containing only
two half-sites (Fig. 8A and data not shown).
Multiple, Spatially Distinct HREs May Function in an Analogous Manner as Reiterated Elements by Recruiting Receptor Oligomers
It
was intriguing that insertion of a 10-base spacer in the center of a
reiterated element was fully compatible with tetramer formation and
with reporter gene activation. This modified element is, in essence,
two dimeric response elements (>>) separated by one turn of the DNA.
It is known that multiple copies of a dimeric response element, when
introduced into a promoter, can stimulate reporter gene expression to
levels much greater than that seen with one copy (e.g.
33-36). To test if this synergy on separated dimeric response elements
is related to the ability of receptors to form high order oligomers, we
compared activation by RXR versus RXR
on reporter
genes containing multiple dimeric response elements (each separated by
18 bases). Indeed, increasing the number of dimeric response elements
resulted in a synergistic increase in reporter gene activation by
RXR
, but had only a much weaker effect on RXR
(Fig.
9, compare B to A). Thus the
ability of a receptor to form oligomers on a single reiterated
half-site element closely parallels the ability of the same receptor to
mount a synergistic response on multiple, "separate" dimeric
elements. This suggests that formation of high order receptor-DNA
complexes may underlie both phenomena.
The specificity of nuclear hormone receptors for their cognate response elements has traditionally been believed to operate at two levels: (a) recognition of the nucleotide sequence of the individual half-sites by each receptor monomer, mediated by contacts between the receptor zinc-finger domain and bases in the DNA, and (b) recognition of the spacing and orientation of the half-sites in an element, determined by the nature of the protein-protein interface in the receptor dimer (17-25). More recently, however, we have suggested that there is a third level of DNA recognition, conferred by the ability of certain nuclear receptors to bind cooperatively as high order oligomers to response elements containing highly reiterated half-sites (27). The ultimate affinity of a receptor for a given response element appears to be the combined consequence of all three components, and the destabilizing effects of a non-optimal half-site sequence can be counteracted if the half-sites are highly reiterated (27).
Many nuclear hormone receptors display a wide diversity in their ability to recognize dimeric response elements containing a variety of half-site orientations and spacings (1-7, 18-25). In common with these dimers, the RXR tetramer also exhibits a broad plasticity in its ability to accommodate a variety of half-site orientations and spacings in the reiterated elements examined here. Not all orientations were acceptable between all half-sites (see below), but a surprising mixture of direct, convergent, and divergent orientations could be efficiently bound by receptor tetramers in vitro and mediate reporter gene activation in vivo.
Tetramers of RXR have also been observed in solution (28-30). Although probably reflecting a similar phenomena, it is unclear if these free tetramers are the immediate precursors of the DNA-bound complexes we observe here. The tetramers in solution are destabilized by hormone (28-30), in contrast to the hormone-mediated enhancement of tetramer formation we observe on DNA. In addition, for the RXR tetramers in solution to be precursors to DNA-associated tetramers, either they must pre-exist as a mixed population of differing monomer orientations or they must rearrange on DNA binding so as to allow recognition of a wide range of different half-site orientations and spacings.
At Least Two Distinct Protein-Protein Interfaces Are Involved in Receptor OligomerizationDimers of nuclear hormone receptors are
stabilized by protein-protein interfaces that map to both zinc-finger
and hormone-binding domains (Fig. 10A, and
Refs. 18-26). The protein-protein interface in the zinc-finger domain
is thought to dictate recognition of half-site spacing and orientation;
small changes in the spacing or orientation of half-sites can
destabilize dimer binding by preventing formation of the proper
receptor-receptor contacts in this zinc-finger domain (15, 17, 19, 20,
24, 25). In contrast, the dimerization interface in the hormone-binding domain exhibits significant functional plasticity, and is able to
stabilize receptor dimer formation on response elements displaying a
variety of half-site orientations and spacings (see Refs. 24-26, and
references therein). This contrast between the tight topological constraints imposed by the zinc-finger dimerization domain, compared with the more permissive nature of the hormone-binding domain interface, implies either the presence of a flexible "swivel" between the zinc-finger and hormone-binding domain, or the existence of
multiple, alternative dimerization interfaces that are
differentially invoked with the different half-site orientations
(24-26).
In light of this work on dimers, it is notable that the topological requirements for tetramer formation are non-equivalent at the different half-sites in the reiterated elements. Most striking, the spacer between half-sites 2 and 3 can vary significantly in length without effect on RXR tetramer formation, whereas similar changes in the spacing between half-sites 1 and 2, or between half-sites 3 and 4, strongly impair tetramer formation. These requirements suggest that the interactions between RXRs at half-sites 1 and 2 (or between 3 and 4) may be mediated through the zinc-finger domain interfaces previously described for two-half-site elements. In contrast, the ability of half-sites 2 and 3 to accommodate a variety of spacers is consistent with the properties observed for the hormone-binding domain interface.
Thus the RXR tetramer may be thought of as a "dimer of dimers," with one dimer bound to half-sites 1 and 2, and a second, interacting dimer cooperatively bound to half-sites 3 and 4. Two possible conceptualizations, both consistent with the data presented here, are shown in Fig. 10. In the first model (Fig. 10B), the interface holding the two dimers together is the same hormone-binding domain interface as that previously described for receptor dimers, but now oriented away from the zinc-finger domain interface. In the second model (Fig. 10C), the two receptor dimers are held together by a novel interface, also located within the hormone-binding domain, but now distinct from the domain previously characterized in stabilization of receptor dimers. In either model, additional determinants mapping outside of the hormone-binding domain would function in certain contexts to restrict tetramer formation (as observed for the N-terminal deletions and the TR·RXR chimera).
The Ability of Receptors to Bind Cooperatively to Reiterated Half-sites May Contribute to the Synergistic Transcriptional Activation Observed with Multiple "Dimeric" Response ElementsNaturally occurring, hormone-responsive promoters often possess multiple copies of the relevant response element (e.g. Refs. 33-37). These multiple response elements appear to act synergistically to enhance the magnitude of the hormone response (33-39). We demonstrate here a correlation between the ability of different RXRs to bind to a single, tetrameric response element, and the ability of these same receptors to synergistically activate transcription from multiple, dimeric response elements. We suggest that multiple, dimeric response elements are functionally equivalent to a single reiterated element containing a large central insertion. Thus, the synergistic effects observed with multiple HREs may reflect, in part, a cooperative binding of high order receptor oligomers to these repeated elements (36).
Recognition of Reiterated Elements May Extend to Other Members of the Nuclear Hormone Receptor Family, and Might Provide a Potential Nexus for Combinatorial Transcriptional RegulationIt is notable that many naturally occurring response elements comprise three or more half-sites (e.g. Refs. 38-40). It is tempting to suggest that these multiple half-sites are recognized by either homo- or hetero-oligomeric forms of receptors. There are several attractive consequences of such a hypothesis. (a) The same naturally occurring elements often contain non-optimal half-site sequences that, in dimeric elements, are destabilizing for receptor binding. In the reiterated elements, the destabilizing effects of the non-optimal half-sites would be compensated by the multiple DNA-protein contacts provided by the cooperative binding of a receptor oligomer. (b) Reiterated sites also provide a potent nexus for combinatorial regulation, perhaps recruiting mixed oligomers of different receptors. The constituents of the receptor oligomer might vary from cell to cell, or from response element to response element, permitting the transcriptional response to be precisely tailored in a cell-type and promoter-specific manner.
It is intriguing that the STAT family of transcription factors also bind cooperatively to reiterated DNA sites, a phenomenon that plays an important role in DNA recognition by, and functional interactions between different STAT family members (41). We suggest that oligomer formation, as observed here for RXR, may be a common means of generating DNA specificity and combinatorial regulation for a variety of transcription factors.
We gratefully thank Catherine Judelson for superb technical assistance, R. M. Evans and K. Ozato for providing RXR clones, K. R. Yamamoto for providing the pD33-CAT plasmid, and L. Schneider for supplying the SL-2 cells.