(Received for publication, December 19, 1995; and in revised form, March 1, 1996 )
From the
The mouse mammary tumor virus promoter has been shown to be inducible by glucocorticoids and progesterone. Although steroid hormone receptors bind with high affinity to palindromic response elements, the hormone-responsive region of the mouse mammary tumor virus promoter contains a pair of directly repeated half-sites that are important for hormone inducibility. Recent experiments have also indicated that direct repeats can function as estrogen response elements. Here, we have investigated DNA binding by steroid receptors to direct repeats and provide evidence using gel retardation assays, methylation interference, and gene transfer experiments that direct repeats of TGTTCT or RGGTCA motifs function as response elements for glucocorticoid (GR) or estrogen receptors (ER), respectively, by binding receptor homodimers. Specific GR- or ER-DNA complexes were observed on direct repeats with different spacings between half-sites, indicating that binding of steroid receptors to direct repeats is more flexible than binding to palindromic elements. This flexibility was further emphasized by the observation that the GR could also bind to everted repeats of TGTTCT motifs separated by 9 base pairs. The isolated DNA binding domains of the GR and ER bound cooperatively to palindromes, but no evidence was observed for cooperative binding to direct repeats. Under similar conditions the DNA binding domains of retinoid receptors retinoid X receptor and retinoic acid receptor bound to direct repeats cooperatively as heterodimers. Similarly, ER derivative HE15, which lacks a functional ligand binding domain, bound palindromic response elements but failed to bind direct repeats. These results indicate that the dimerization domain in the ligand binding domain is essential for binding of steroid receptors to direct repeats and that the dimerization domain in the D-box of the DNA binding domain is not functional under these conditions. Moreover, the results suggest that steroid receptor DNA binding domains may lack dimerization domains outside the D-box, which would function in binding to direct repeats, in contrast to receptors for retinoids and thyroid hormone. A comparison of the mechanisms of binding of steroid receptors and retinoid and thyroid hormone receptors to direct repeats is presented.
The nuclear receptors are a family of transcriptional enhancer
factors that bind to specific DNA sequences in target promoters known
as response
elements(1, 2, 3, 4, 5) .
Specific members of the nuclear receptor family represent the primary
intracellular targets for small lipid-soluble molecules, such as
steroid and thyroid hormones, retinoids, and vitamin D, and
act as ligand-inducible transcriptional regulators. These
ligand-inducible receptors control a wide spectrum of developmental and
physiological processes through modulating the transcription of target
genes.
Sequence analyses of nuclear receptors have shown that they
are composed of a series of conserved
domains(1, 2, 3, 4, 5, 6) .
The DNA binding domain (region C) is the most highly conserved and
contains a 66-68-amino acid core composed of two zinc fingers
that form a single structural
domain(7, 8, 9, 10) . Three amino
acids adjacent to the N-terminal zinc finger, known as the P-box, are
critical for DNA sequence
recognition(11, 12, 13) . A subfamily of
receptors, those for glucocorticoids (GR), progesterone, androgens, and
mineralocorticoids, contain P-box amino acids Gly, Ser, and Val and
recognize response elements composed of half-sites with the consensus
AGAACA. In contrast, the discriminatory amino acids Glu, Gly, and Ala
are found in the P-box of the estrogen receptor (ER), ()which binds to response elements with consensus RGGTCA
half-sites. DNA binding domains (DBDs) of receptors for thyroid
hormone, vitamin D
, and retinoids contain a similar P-box
(Glu, Gly, Gly) and recognize response elements containing the
consensus RGG/TTCA.
The ligand binding domain (LBD) is the second
most highly conserved among nuclear receptors. In addition to binding
ligand, LBDs contain regions controlling transcriptional regulation,
and receptor
dimerization(14, 15, 16, 17, 18, 19, 20) ,
which stabilizes receptor DNA binding. The action of LBD dimerization
domains varies among receptors. In the absence of ligand, steroid
receptors can form homodimers in solution through the dimerization
domain in the LBD(17, 19, 21) . In contrast
receptors for thyroid hormone, vitamin D, and retinoids
form heterodimers with the retinoid X receptors
(RXRs)(21, 22, 23, 24, 25) .
Several studies have suggested that interaction with RXRs is stimulated
by binding of ligand to the heterodimeric partner, whereas
homodimerization of RXRs is stimulated by binding of their cognate
ligand 9-cis-retinoic
acid(5, 26, 27) .
Hormone response
elements for steroid receptors have been characterized as palindromic
sequences, containing two half-sites separated by 3 base pairs. The
estrogen response element (ERE) in the vitellogenin A2 gene of Xenopus laevis contains the perfectly palindromic element
AGGTCANNNTGACCT(28) . A number of estrogen-responsive genes
contain imperfectly palindromic EREs either in isolation or in multiple
arrays (29, 30, 31, 32, 33, 34, 35) .
Similarly, the imperfectly palindromic glucocorticoid response element
(GRE) tgtACANNNTGTTCT has been found in the rat tyrosine
aminotransferase gene(36) . Altering the spacing between
half-sites disrupts the binding of steroid receptors to palindromic
response elements(37, 38) . In contrast to steroid
receptors, RXR homodimers, or heterodimers of RXR with receptors for
thyroid hormone, vitamin D, retinoids, or a number of
orphan receptors preferentially recognize response elements composed of
direct repeats of cognate
half-sites(39, 40, 41) . Different
combinations of heterodimers discriminate between response elements
based on the spacing between half-sites. Structure-function studies
have shown that heterodimers bind with RXR in the 5`
position(42, 43, 44, 45) . The
isolated DBDs of RXR and its heterodimeric partners can bind
cooperatively to directs repeats, and discrimination between direct
repeats with different spacings is controlled by the specific
conformation of each DBD heterodimer (42, 43, 44, 45, 46, 47) .
Cooperative binding of steroid receptor DBDs to palindromic response elements is controlled by a dimerization domain in the second zinc finger of the DBD, known as the D-box(12, 48, 49) . Substitution of the D-boxes of the GR or ER with the corresponding sequences of the thyroid hormone receptor (TR), or retinoic acid receptor (RAR) disrupts cooperative binding of DBDs to palindromic response elements, indicating that these regions of RAR and TR cannot homodimerize(48, 49) . Heterodimerization between RXRs and RARs on response elements composed of half-sites separated by 5 bp (DR5) occurs through interaction of the D-box of RXRs with a region of the N-terminal zinc finger of RAR(46) . In contrast, RXR/RAR heterodimers formed on response elements containing half-sites separated by 2 bp (DR2) require the extreme C terminus of the RXR DBD and a distinct N-terminal domain of the RAR DBD(47) . Interestingly, the corresponding N-terminal domain of RXR, and the extreme C terminus of the RXR DBD, but not the D-box, have been implicated in binding of RXR homodimers to DR1 response elements(47) . This indicates that multiple regions of RAR and RXR DBDs are capable of acting as dimerization interfaces.
Recent
studies have suggested that direct repeats of RGGTCA half-sites can
function as EREs(50, 51, 52, 53) .
Moreover, the glucocorticoid-responsive region of the MMTV promoter
contains a promoter proximal direct repeat of TGTTCT motifs required
for optimal ligand inducibility(54, 55) . We therefore
investigated the mechanism of binding of the ER and the GR to direct
repeats. Evidence is provided that the GR is capable of binding to the
TGTTCT repeats of the MMTV promoter as a homodimer. Similarly, the ER
can homodimerize on direct repeats. The roles of receptor dimerization
domains in the binding of GR or ER homodimers to direct repeats and
palindromes have been investigated using a series of truncated
receptors. A comparison of the mechanisms of DNA binding by steroid
receptors and receptors for thyroid hormone, vitamin D, and
retinoids is also presented.
Figure 3:
Binding of the estrogen receptor to direct
repeats of RGGTCA motifs. A, oligonucleotides and expression
vectors used in this study. P and D refer to
palindrome and direct repeat, respectively, and the number following
refers to the inter-half-site spacing. The ER cDNAs encoded by the
expression vectors HEG0 and CE0 are shown below. B, binding of
the human estrogen receptor to ERE-P3 and ERE-D6 response elements. Gel
retardation assays with ERE-P3, -D6 and D6M1 oligonucleotides were
performed with control extracts or extracts of HeLa cells transiently
transfected with a human estrogen receptor (HEG0) expression
vector as indicated. C, an experiment identical to that of B, except that HeLa cells were transfected with a chicken
estrogen receptor (CE0) expression vector. D,
analysis of retarded complexes formed on ERE-P3 or ERE-D5
oligonucleotides using the human ER and anti-ER monoclonal antibody F3 (56) . Aliquots of extracts of COS-7 cells transiently
transfected with human ER expression vector HEG0 (lanes 2 and
6-8), or parental expression vector (lanes 3-5)
were incubated in the absence (lanes 1, 3, and 6) or
presence of antibody as indicated (lanes 2, 4, 5, 7, and 8) prior to loading on 5% polyacrylamide gels. E,
specific binding of the ER to radiolabeled ERE-P3 (100,000 cpm, 20
fmol) was competed with increasing amounts of competitor
oligonucleotide as indicated (open squares, ERE-P3; black
squares, ERE-D6; black triangles, ERE-D6M1; black
circles, GRE-P3). Competitor oligonucleotide (fmol) was plotted on
a logarithmic scale.
Figure 1:
Binding of the GR
to direct and everted repeats of TGTTCT motifs. A,
oligonucleotides and expression vector used in this study. P,
D, and E refer to palindrome, direct repeat, and everted
repeat, respectively, and the number following refers to the
inter-half-site spacing. The GR cDNA encoded by the expression vector
HG1 is shown below. B, binding of HG1 to GRE-P3 and GRE-D9
oligonucleotides. Lanes 1-5 show gel retardation assays
performed with extracts of COS-7 cells transiently transfected with an
HG1 expression vector, using the ERE and GRE oligonucleotides as
indicated. Lanes 6-10 show the same experiment performed
with extracts of untransfected COS-7 cells. The complex formed on
GRE-P3, and GRE-D9, but not on ERE-P3 or GRE oligonucleotides GRE-D9M1
and GRE-D9M2, which contain mutations in the upstream or downstream
half-sites, is indicated by the arrowhead. C,
analysis of retarded complexes formed by partially purified human GR
using anti-GR polyclonal antibody. Oligonucleotide probes were
incubated with anti-GR antibody alone (lanes 1, 4, 7, 10, and 13), with 0.5 µl of partially purified human GR alone (lanes 2, 5, 8, 11, and 14), or with a mixture of GR and
anti-GR antibody (lanes 3, 6, 9, 12, and 15).
GR-specific retarded and supershifted complexes are indicated by single and double arrowheads, respectively, and the
nonspecific complex formed in the presence of antibody alone is
indicated by the asterisk. D, competition of unlabeled GRE
oligonucleotides for binding of the GR to GRE-P3. Aliquots of extracts
of transiently transfected COS-7 cells expressing the GR were incubated
with a mixture of 20 fmol of
P-labeled GRE-P3
oligonucleotide and increasing concentrations of unlabeled competitor
probe as indicated (open squares, GRE-P3; filled
squares, GRE-D9; filled triangles, GRE-D9M1; open
triangles, GRE-D9M2; open circles, ERE-P3). Competitor
oligonucleotide (femtomoles) was plotted on a logarithmic
scale.
For gel retardation assays, cells were harvested by combining those scraped from two or three 3-cm plates in 500 µl of ice-cold phosphate-buffered saline. Cells were centrifuged at 2500 rpm for 10 min at 4 °C, the supernatant was carefully removed, and the pellet was resuspended in 30 µl of high salt extraction buffer (25 mM Tris (pH 7.9), 0.3 mM DTT, 0.1 EDTA, 400 mM NaCl, 10% (v/v) glycerol). Cells were lysed by 3 cycles of freezing at -70 °C and thawing at room temperature, then centrifuged at 10,000 rpm for 10 min at 4 °C. Supernatants were stored at -70 °C. For CAT assays, cells from individual plates were washed with 1.0 ml of phosphate-buffered saline, incubated for 15 min at room temperature in 250 µl of lysis buffer (Promega), harvested by scraping, and transferred to an Eppendorf tube and then centrifuged at 10,000 rpm for 5 min. Extracts were stored at -70 °C.
We performed gel retardation experiments to test whether homodimers of the GR were capable of binding to this sequence. A number of oligonucleotides were used in this study (Fig. 1A), including the wild-type sequence from the MMTV promoter (GRE-D9), as well as mutant sequences in which either the upstream or downstream half-sites were disrupted (GRE-D9M1 and GRE-D9M2, respectively). A perfectly palindromic GRE (GRE-P3) was used as a positive control. The results show that a complex was formed on the GRE-P3 oligonucleotide upon incubation with extracts of COS-7 cells transiently transfected with a vector expressing the human GR, but not in extracts of cells transfected with a control vector (Fig. 1B, lanes 2 and 7; arrowhead). A similar complex was formed on the GRE-D9 oligonucleotide only in the presence of the GR (Fig. 1B, lanes 3 and 8). No such complex was formed on oligonucleotides GRE-D9M1 or GRE-D9M2 where either the upstream or downstream half-sites have been disrupted (Fig. 1B, lanes 4, 5, 9, and 10). Taken together, these results indicate that a complex is formed on an oligonucleotide containing TGTTCT half-sites separated by 9 bp in the presence of the human GR and that formation of this complex requires the integrity of both half-sites. Moreover, this complex comigrates with the one formed on palindromic response elements, suggesting that it corresponds to receptor homodimers.
Given the significant levels of nonspecific binding in COS-7 cell extracts, the presence of the GR in retarded complexes formed on GRE oligonucleotides was further tested using receptor purified from insect cells overexpressing the GR, along with polyclonal antibody raised against the human GR (Fig. 1C). A nonspecific complex was observed when oligonucleotides were incubated with antibody in the absence of GR-containing extract, but this complex did not correspond to the GR (Fig. 1C, lanes 1, 4, 7, 10, and 13; asterisk). The GR-specific complex formed on GRE-P3 is further retarded by incubation with a polyclonal anti-human GR antibody (Fig. 1C, lanes 2 and 3; arrowheads), confirming the presence of the GR. Similarly, a specific complex was formed on the GRE-D9 oligonucleotide, which was further shifted in the presence of antibody (Fig. 1C, lanes 5 and 6). Consistent with the results of Fig. 1B where no GR-specific binding was observed to oligonucleotides containing a single half-site, no specific complexes are formed on the GRE-D9M1 or GRE-D9M2 oligonucleotides, either in the absence or presence of antibody (Fig. 1C, lanes 11, 12, 14, and 15). Taken together, the results of Fig. 1demonstrate that the GR can bind to direct repeats of TGTTCT motifs.
These results suggest that the GR DNA binding domain rotates in converting from a palindromic DNA binding mode to a direct repeat binding mode (see also Fig. 8). The flexibility of DNA binding by the GR is substantiated by the observation that specific retarded complexes formed on oligonucleotide GRE-E9 (see Fig. 1), which contains an everted repeat of TGTTCT motifs separated by 9 bp (Fig. 1C, lanes 8 and 9).
Figure 8: Comparison of mechanisms of binding to direct repeats and palindromes by steroid and retinoid receptors. For clarity only the DNA and ligand binding domains of receptors are shown. Dimerization domains are indicated. The relative geometry of the individual domains in binding to palindromes and direct repeats is indicated by the orientation of the labeling of each domain. DNA half-sites are indicated by arrows. Dimerization domains in ligand and DNA binding domains are indicated by cross-hatches. A, schematic representation of conformations adopted by steroid receptor homodimers in binding to palindromes, direct repeats, and everted repeats. B, binding of RAR/RXR heterodimers to direct repeats (note dimerization of DNA binding domains). C, binding of homodimers of steroid receptors to direct repeats of widely spaced half-sites with accompanying looping out of intervening DNA.
Competition experiments were also performed with GRE oligonucleotides to analyze the relative efficiencies of palindromic GREs, direct repeats, and single half-sites in competing for GR binding. A typical result (Fig. 1D) showed that the GRE-D9 oligonucleotide competes 3.5-fold less efficiently for binding to the GR than GRE-P3 palindrome and 3.5-6-fold more efficiently than the GRE-D9M1 and GRE-D9M2 mutants, which contain only single half-sites. Taken together the above results indicate that the GR can bind cooperatively to direct repeats, suggesting that direct repeats may serve as response elements in vivo.
Figure 2:
Dexamethasone inducibility of
promoter-reporter constructs containing GRE-D9 response elements. A, schematic representation of recombinants used in transient
transfections. B, typical CAT assay performed using extracts
of CV-1 cells transiently transfected with the recombinants shown in A and treated with dexamethasone (25 nM) as
indicated. Extract volumes were adjusted for transfection efficiency by
normalization to -galactosidase activity (see ``Experimental
Procedures''). C, relative CAT acetylation determined
from a total of three experiments. -Fold inductions varied by
±15%.
The presence of the the human ER in specific retarded complexes observed on direct repeats was confirmed using the monoclonal F3 antibody that recognizes an epitope in the C-terminal F region of the receptor (Fig. 3D) and the ERE-D5 oligonucleotide. In the presence of antibody, complexes formed only in extracts of cells transfected with an HEG0 expression vector (Fig. 3D, lanes 1 and 6) were further retarded (Fig. 3D, lanes 2, 7, and 8), indicating that, like the palindromic ERE, the ERE-D5 oligonucleotide binds the ER. These results are further supported by competition experiments which showed that ERE-D6 competes 8-12-fold more efficiently for ER binding than the ERE-D6M1 mutant (Fig. 3E and data not known). Taken together, the results of Fig. 3indicate that the ER can bind cooperatively to directly repeated half-sites.
The
participation of both RGGTCA half-sites in ER binding to the ERE-D6
oligonucleotide was confirmed by methylation interference experiments
which indicated that methylation of G residues in either half-site
disrupted the formation of retarded complexes (Fig. 4).
Oligonucleotides methylated at G residues present in either half-site
were significantly under-represented in ER-specific retarded complexes (Fig. 4, asterisks). Note that methylation of the 5` G
residue of the GGGTCA half-site disrupted complex formation,
implicating this residue in ER binding (Fig. 4, left
panel). The consensus half-site for palindromic EREs is often
given as GGTCA. However, a survey of weaker imperfectly palindromic
elements often shows a purine at the -1
position(29, 30, 31, 32, 33, 34, 35) .
In this respect EREs are similar to response elements for receptors of
retinoids, thyroid hormone, and vitamin D.
Figure 4: Methylation interference analysis of ER binding to direct repeats. Methylation interference experiments (see ``Experimental Procedures'' for details) were performed with the ERE-D6 oligonucleotide and extracts of HeLa cells expressing HEG0. Cleavage products were resolved on a 15% urea/acrylamide gel. T, F, and B refer to total, free, and bound oligonucleotide, respectively. G residues in the two half-sites of ERE-D6 are indicated. B, G residues whose methylation reduces binding by >50%, as determined by densitometric scanning of the autoradiogram, are indicated by asterisks.
Figure 5: Flexibility of binding of steroid receptors to direct repeats. A, oligonucleotides used in this study. P and D refer to palindrome and direct repeat, respectively, and the number following refers to the inter-half-site spacing. For oligonucleotides ERE-D2 to ERE-D5 only, the top strand is shown. B, gel retardation assays performed with HEG0 expressed in COS-7 cells. Extracts were incubated with oligonucleotides ERE-D2 to ERE-D6, as indicated. C, gel retardation assays performed with HG1 expressed in COS-7 cells or with control COS-7 extracts with oligonucleotides GRE-D6, D9, and P3 as indicated. Specific complexes are indicated by arrowheads.
Similarly, a GR-specific complex was formed, as expected, on the GRE-D9 sequence derived from the MMTV promoter (Fig. 5C, lanes 3 and 4), but also on the related oligonucleotide GRE-D6 with 6-bp spacing between half-sites (Fig. 5C, lanes 5 and 6). We have also seen binding of the GR to the GRE of the hepatitis B promoter(63) , which is a direct repeat of TGTCCT motifs separated by 6 bp (data not shown). While these are not exhaustive series of oligonucleotides, it is clear from the above results that a stringent inter-half-site spacing requirement (of 3 bp) for steroid receptor binding to palindromic EREs or GREs is not applicable to response elements formed from direct repeats.
Figure 6: Role of N- and C-terminal domains of steroid receptors in binding to direct repeats. A, the full-length ER cDNA encoded by the expression vectore HEG0, along with N- and C-terminally truncated receptor derivatives encoded by HE15 and HEG19 expression vectors are shown. Oligonucleotides and expression vectors used in this study are shown below. P and D refer to palindrome and direct repeat, respectively, and the number following refers to the inter-half-site spacing. B, gel retardation assay performed with control COS-7 extracts or extracts of COS-7 transfected with an HEG19 expression vector using ERE-P3, ERE-D6, or ERE-D6M1 oligonucleotides as indicated. C, gel retardation assay performed with control COS-7 extracts or extracts of COS-7 transfected with an HE15 expression vector using ERE-P3, ERE-D6, or ERE-D6M1 oligonucleotides as indicated.
The requirement for the ligand binding domain in binding to direct repeats was further supported by studies with the ER and GR DNA binding domains expressed at high levels in E. coli. The ER DBD derivative HE81 (Fig. 7A) binds highly cooperatively as a dimer to palindromic response elements (Fig. 7B, lane 1, and (49) ). Previous studies have shown that this cooperativity requires a functional dimerization domain located in the D-box, which is situated in the C-terminal zinc finger of the DBD (49) . No binding was observed to the ERE-D6 oligonucleotide in bacterial extracts not expressing HE81 (Fig. 7B, lane 2). A complex, corresponding to an HE81 monomer was formed on ERE-D6M1, which contains a single half-site (Fig. 7B, lane 4). A similar monomeric complex was formed on ERE-D6, which contains a direct repeat. No evidence for dimer formation was seen (Fig. 7B, lane 3, and data not shown). We have also tested binding of the ER DBD to the ERE-D5 element over a range of concentrations. Formation of a dimeric complex was observed on the ERE-P3 element (Fig. 7C, lane 1), even at the lowest concentration tested (data not shown). However, no evidence for dimer formation was seen on ERE-D5, even at concentrations which saturate ERE-P3 (Fig. 7C, lanes 1-5).
Figure 7:
DNA binding to direct repeats by the
isolated DNA binding domains of the estrogen, glucocorticoid, and
retinoid receptors. A, receptor DNA binding domain sequences
encoded by bacterial expression vectors are indicated. The
oligonucleotide RARE-D5 is indicated below. ERE- and GRE-containing
oligonucleotides are described in previous figures. B, gel
retardation assay performed with control fractions or 1 M heparin-agarose fractions of E. coli expressing the
estrogen receptor DNA binding domain derivative HE81 using ERE-P3,
ERE-D6, or ERE-D6M1 oligonucleotides as indicated. Monomeric (M) and dimeric (D) complexes are indicated. C, gel retardation assay performed with control extracts or
extracts of E. coli expressing the estrogen receptor DNA
binding domain derivative HE81 using ERE-P3 or ERE-D5 oligonucleotides
as indicated. Monomeric (M) and dimeric (D) complexes
are indicated. Assays shown in lanes 1-5 were performed
with 2 µl of 4-, 4-, 8-, 16-, and 32-fold dilutions of extracts,
respectively. D, gel retardation assay performed with extracts
of E. coli expressing the glucocorticoid receptor DNA binding
domain derivative GR-DBD using ERE-P3, GRE-P3, GRE-D9, GRE-D9M1, or
GRE-D6 oligonucleotides as indicated. Monomeric (M) and
dimeric (D) complexes are indicated. E, gel
retardation assay performed with extracts of E. coli expressing DNA binding domain derivatives of retinoid receptors
RAR or RXR
(44) using the RARE-D5 oligonucleotide.
Assays were performed with 0.25 µl (lanes 1 and 2) or 0.5 µl (lanes 4 and 5) of
RAR
- or RXR
-containing extracts alone or with 0.125 µl (lane 3) or 0.25 µl (lane 6) of each extract
combined. Heterodimers of RAR
and RXR
DNA binding domains are
indicated by the arrowhead, and monomers and homodimers of
RAR
DNA binding domains are indicated by the asterisks.
A
similar result was obtained when binding of the GR DBD (Fig. 7A) to direct repeats was analyzed. While
formation of a dimeric complex was readily observed on the palindromic
oligonucleotide GRE-P3, but not on ERE-P3 (Fig. 7D, lanes 1 and 2), no dimer formation was observed on the half-site
of GRE-D9M1 or the direct repeat of GRE-D9 (Fig. 7D, lanes 3 and 4). In addition, no dimeric complexes were formed on
the GRE-D6 oligonucleotide (Fig. 7D, lane 5). The above
experiments have been repeated several times, over a range of DBD
concentrations, using crude bacterial extracts expressing the ER or GR
DBDs, or with fractions of DBD purified to 50-80%
homogeneity, and no evidence for cooperative DNA binding was observed
(data not shown).
In contrast to the results with steroid receptor
DBDs, and consistent with previous findings(44) , cooperative
DNA binding was readily seen with combinations of RAR and RXR
DBDs expressed in E. coli (Fig. 7E). Formation
of heterodimeric complexes was observed (Fig. 7E, lanes 3 and 6) under conditions where only low levels of
monomeric complex are seen when RAR
or RXR
are incubated
alone. Taken together, the results of Fig. 6and Fig. 7indicate that the dimerization domain in the D-box of
steroid receptor DBDs does not function in binding to direct repeats
and suggest that steroid receptor DBDs may lack the dimerization
interfaces located outside the D-box similar to those found in retinoid
receptors. In addition, the results indicate that the dimerization
function located in the ligand binding domain is essential for binding
to direct repeats.
The binding of either the ER or the GR
to direct repeats is weaker than to palindromes. However, it should be
pointed out that most GREs and EREs defined to date are not perfectly
palindromic and are therefore bound more weakly by their corresponding
receptors. The imperfect palindrome, AaGtTCANNNTGACCC, from the
estrogen-responsive Xenopus vitellogenin B1 gene, competes
8-fold less efficiently for ER binding than a perfectly palindromic
ERE(64) . Our results indicated that the direct repeat of
ERE-D6 competes 6-10-fold less efficiently for ER binding than
the palindromic sequence ERE-P3 and 8-12-fold more efficiently
than an oligonucleotide containing only a single half-site (Fig. 3). This suggests that direct repeats of consensus
half-sites could bind the ER roughly as efficiently as physiological
imperfect palindromes. It is noteworthy that molecular genetic
experiments in yeast have identified a number of DNA sequences that
bind ER very weakly as potential EREs(52) . In a similar study,
a direct repeat of RGGTCA motifs separated by 3 bp mediated an estrogen
response(51) .
Direct repeats, like imperfectly palindromic elements, can be found in multiple arrays upstream of target promoters(50, 54, 55) . Multiple imperfect palindromes of glucocorticoid- or estrogen-responsive promoters have been shown to combine to give a synergistic response to hormone(33, 34, 36) . In the MMTV promoter, proximal response sequences, containing the sequences studied here, combine with more distal sites for an optimal response to glucocorticoid(55) . Similarly, there is strong synergism between the four AGGTCA half-sites found far upstream of the chicken ovalbumin gene(50) . In agreement with these results, our findings indicate that insertion of three, but not one, copies of the GRE-D9 element upstream of a heterologous promoter renders the promoter inducible by dexamethasone (Fig. 2).
Studies with RAR, TR, and RXR DBDs have suggested that cooperative binding to direct repeats only occurs on elements with specific inter-half-site spacings(42, 43, 44, 45, 46, 47) . Cooperativity between RAR and RXR DBDs is observed on elements with 2- or 5-bp spacings, with a different set of dimerization interfaces required in each case(46, 47) . Binding of RAR/RXR heterodimers can occur to half-sites separated by 10 bp; however, specific interactions between the DBDs are apparently not required, and similar to the results presented here, DNA binding occurs exclusively through dimerization of the ligand binding domains(47) . It remains possible that there exist (a) specific inter-half-site spacing(s) of direct repeats where cooperative binding by DBDs of steroid receptors can occur. However, accumulating evidence suggests that dimerization by DBDs is not necessary for direct repeats to function as response elements for steroid receptors and that, unlike binding to palindromic elements, binding of steroid receptors to direct repeats is very flexible. Each of the four RGGTCA half-sites located upstream of the chicken ovalbumin gene is separated by over 100 bp from its neighbor, and cooperative binding to two of the half-sites separated by 36 bp has been observed(50) . Molecular genetic studies in yeast have suggested that direct repeats with 3-bp spacings can function as EREs(51, 52) . The two GRE half-sites of the MMTV promoter, and hepatitis B virus promoter, are separated by 9 and 6 bp respectively. Finally, our DNA binding studies have suggested that steroid receptors will bind cooperatively to direct repeats with different spacings ( Fig. 1and Fig. 3-5).
The lack of apparent alternative dimerization
domains in the DBDs of steroid receptors may account for the
accumulating evidence of wide flexibility in their binding to direct
repeats. Just as the ER and GR are constrained by D-box dimerization in
recognition of palindromic elements, binding to direct repeats by
heterodimers of RXR with retinoic acid, and thyroid hormone receptors
is restricted by protein-protein interactions (Fig. 8B), which hold the DBDs in specific
conformations. In contrast, binding of steroid receptor homodimers in
the absence of DBD dimerization would be only constrained by a
combination of the flexibility of the linker region between the DBD and
the LBD and the flexibilty of the DNA template. Thus binding to widely
spaced half-sites, for example those upstream of the chicken ovalbumin
gene(50) , would be accompanied by a looping out of the
intervening DNA (Fig. 8C). Interestingly in this
regard, recent studies with the response element of the
F-crystallin promoter (68) showed that RXR/TR and RXR/RAR
heterodimers can bind with high affinity to everted repeats separated
by 6 or 8 bp. The everted half-sites of the
F-crystallin response
element are separated by an 8 bp (A + T)-rich linker, similar to
the (A + T)-rich linker of the MMTV D9 element (see Fig. 1), and it was speculated that local DNA melting may
contribute to flexibility of DNA binding(68) .
In summary, the studies presented here extend our definition of the possible DNA sequences that can constitute steroid response elements, as defined by sequences recognized by receptor homodimers. They suggest that the DBDs of steroid receptors, similar to those of retinoid and thyroid hormone receptors, can adopt different conformations in recognizing response elements composed of either palindromes or direct repeats. The results also emphasize the importance of dimerization domains in controlling response element recognition by steroid receptors.