(Received for publication, June 6, 1995; and in revised form, September 8, 1995)
From the
Two precisely arranged proenkephalin cAMP response elements
(CREs) behave as a single protein binding site. The experiments
described support a model in which a secondary structural change
creates a new binding site, which is made up of sequences from both of
the CREs. The CRE-binding protein (CREB) binds CRE-1, but binding there
is entirely dependent on the presence of CRE-2. Electron spectroscopic
images show that a CREB dimer occupies twice as much DNA in the
proenkephalin gene as in the prodynorphin gene. The enhancer region is
sensitive to P nuclease in a CREB concentration-dependent
manner, and sensitivity is strand-specific, indicating
protein-stabilized structural change. DNase I analysis shows that in
the native proenkephalin gene, CREB binds both CRE-1 and CRE-2. In
vivo, both CREs are occupied in the transcriptionally active
proenkephalin gene, while neither is in the silent gene. Whereas CREB
can bind CRE-2, mutation or elimination of either proenkephalin CRE
alters response to second messengers and transcription factors. Thus,
binding to CRE-2 alone is not sufficient. Specific and efficient
transcription of the proenkephalin gene requires the presence of both
CREs, precisely arranged to allow them to form a single protein binding
site.
A limited number of sequence-specific DNA-binding proteins is
sufficient for the precise and complex transcriptional regulation in
the brain. Within each gene, the number, type, and arrangement of
binding sites allow the assembly of a gene-specific transcriptional
machine(1) . Studies of the genes that encode precursors to
endogenous opiate receptor ligands have clarified aspects of
transcriptional control and its contribution to the overall regulation
of these peptides. Opioid peptides are neuromodulators and circulating
hormones that play a role in nociception, adaptation, learning, and
development (2) . The gene that encodes preproenkephalin is
transcriptionally responsive to a variety of stimuli, including growth
factors, neurotransmitters, cell depolarization, and hormones;
remarkably, the several discrete pathways that carry the messages of
the cell surface events converge at a 27-base pair region comprising
two cAMP-responsive enhancers(3, 4) . The two CREs ()are not, however, equivalent. CRE-2 appears to be a
typical enhancer. Its sequence is similar to that of phorbol ester and
cAMP response elements, and it binds factors that mediate the
transcriptional response to second
messengers(4, 5, 6, 7) . Tandem
copies of CRE-2 can respond to stimuli that mimic receptor occupation,
but by itself CRE-2 is not able to support efficient transcription or
receptor response(5, 7, 8, 9) . In
contrast, CRE-1 is apparently not a binding site for transcription
factors known to respond to second messengers nor are multiple copies
of CRE-1 sufficient to confer a response to cAMP(7) . Yet point
mutations in or deletion of CRE-1 alters the transcriptional response
to cell surface ligands, to second messengers, and to CRE-binding
proteins(3, 4, 9) . Comparison of sequences
from several species, including human(10) , rat(11) ,
hamster(12) , and guinea pig (13) , reveals that the
27-base pair region containing the CREs is identical. The arrangement
of the CREs with respect to each other as well as the context of each
CRE must therefore be critical. Altering the spacing between the two
proenkephalin CREs alters transcriptional regulation(4) . That
context is significant is further emphasized by the observation that
the 8-bp CTGCGTCA sequence of CRE-2 is exactly that of a critical
transcription factor-binding element of another opioid peptide gene,
prodynorphin; yet the two elements mediate different responses to
identical stimuli and transcription factors(14, 15) .
What mechanism requires that the arrangement of CRE-1 and CRE-2 be precisely maintained? A direct role for CRE-1 in the action of CRE-binding proteins is suggested by the observation that response to CREB protein is altered when CRE-2 is intact but CRE-1 is deleted(9) . Recent studies show CREB to be a mediator of the proenkephalin transcriptional response in the brain. The use of a fusion transgene implicates CREB in enhancing proenkephalin expression in response to stress within the mouse hypothalamus(16) . Within rat striatum, CREB is the likely factor that acts via the proenkephalin enhancer to effect transcription both in control and in haloperidol-treated animals (6) and in response to amphetamines(17) .
Proenkephalin transcription is sensitive to point mutations in or loss of either CRE, even though CRE-1 does not by itself bind CRE-binding proteins such as CREB/ATF or AP-1. Although CRE-2 is known to bind such transcription factors, CRE-2 cannot function efficiently in the absence of CRE-1(3, 4, 9) . What makes the CREs effective together? We have previously studied the nearly palindromic region that contains the CREs. By intrastrand hydrogen bonding, an alternative CRE can be created from the nearly complementary sequences of CRE-1 and CRE-2(9, 18, 19) . The hairpin containing the site is stable under physiological conditions(18) , and its structure has been analyzed by NMR spectroscopy(9, 19) . The alternative binding site differs from that of CRE-2 by the presence of two GT mismatched base pairs(9, 18, 19) , and it binds CREB protein with higher affinity than does the native duplex site(9) . The presence of the T in one of the GT base pairs appears critical to the increase in binding affinity(9) . Studies on the structure of this region have relied entirely on synthetic oligonucleotides and have been limited to the 23-base pair region.
To test the biological relevance of the alternative site, we have studied protein-proenkephalin complexes in very large molecules of DNA. The formation of a site consisting of both CRE-1 and CRE-2 would require an alteration in the DNA structure. In the alternative binding site, both CRE-1 and CRE-2 would directly interact with CREB protein. Furthermore, both CREs should bind protein during the formation of active transcription complexes. Quantitative electron microscopy shows that the DNA in CREB-proenkephalin complexes does adopt a different conformation from that in CREB-prodynorphin complexes. In the native gene, the CREB footprint spans both proenkephalin CREs, whereas in a mutant lacking CRE-1 the footprint is limited to the region of CRE-2. In vivo analysis shows that both CREs are occupied in the actively transcribed gene but that neither is occupied in the silent gene. Thus, an alternative conformation of the DNA creates a single protein binding site required for the proenkephalin-specific transcriptional machine.
Figure 3: Protein binding and expression with mutant CREs. A, sequences of native and mutant proenkephalin duplexes and of GT hairpin. GT hairpin is formed from the top strand of native duplex(9, 18, 19) . The numbering is for the human proenkephalin gene(9) . The boxes in duplex sequences mark locations of CREs; in the GT hairpin, the boxes indicate the non-Watson-Crick GT base pairs(9, 18, 19) , and the horizontal arrows mark the CREs. The vertical arrows indicate altered base pairs. B, gel shift analysis of binding of purified 6-histidine CREB to oligonucleotide probes. Only the top portion of the gel, with the shifted complexes, is shown. SOM, somatostatin; NAT, native proenkephalin. The other labels are as in A. C, fractional acetylation of chloramphenicol for the native and mutant proenkephalin plasmids as determined by transient transfection analysis. Numbers are the average for three dishes. D, Northern analysis of total RNA from C6 and R2C cultured cells. The positions of 28 S and 18 S rRNA were determined by ethidium bromide staining. The arrow indicates the position of 1.4-kilobase proenkephalin mRNA. E, immunoblot analysis of C6 cell extract with anti-phosphorylated-CREB serum. Molecular weight positions were based on migration of pre-stained standards.
Figure 1: Electron spectroscopic images of unstained proenkephalin and prodynorphin DNA fragments and of CREB complexes with fragments. a, 1322-bp proenkephalin gene fragment without protein. b, CREB-proenkephalin complex. c, 2924-bp prodynorphin gene fragment without protein. d, CREB-prodynorphin complex. e, map of proenkephalin gene fragment indicating positions of CREs. f, map of prodynorphin gene fragment. The boxes indicate the region of CREB binding determined by physical mapping. The images shown are from a mock reaction (no protein added) (a and c) and of complexes (b and d) located at the correct positions on the fragments as determined by contour length measurements. The bar represents 132 nm.
Structural
information is derived from phosphorus mapping (Fig. 2). For
each complex, reference electron microscopic images (Fig. 2, left panels) and net phosphorus images (Fig. 2, center panels) are generated. The phosphorus image shows only
the DNA within an intact protein-DNA complex. Superposition of the
phosphorus (DNA) image on the mass image (Fig. 2, right
panels) creates a picture of DNA within the complex and allows
measurement of the mass of DNA within the complex. With respect to the
CREB-proenkephalin and CREB-prodynorphin complexes, this approach
allows direct comparison of the DNA within two different complexes in
which the protein component is the same. Binding of CREB to the
proenkephalin fragment alters the path of the DNA (Fig. 2, a, b, and c). The DNA appears throughout the
complex (Fig. 2, a, b, and c, right panel). Mass analysis indicates that the
CREB-proenkephalin complex contains about 60 bp of DNA and 87 kDa of
protein (Table 1). The protein mass is close to the predicted
80-kDa mass of a CREB dimer(26) . Binding of TBP monomers to
the same proenkephalin DNA fragment shows the alteration in DNA to be
characteristic of the CREB-proenkephalin complex rather than
characteristic of the DNA fragment itself (Fig. 2e).
TBP monomer induces bending in the DNA fragment at the TATAA box, but
the DNA folds neatly along the edge of the protein DNA complex, without
the distortion observed in the CREB-proenkephalin DNA complex (Fig. 2e). TBP monomer-proenkephalin DNA complexes
contain about 17 bp of DNA (Table 1) and about 25.5 kDa of
protein ( Table 1and (35) ). TBP binds both as monomer
and dimer (not shown), and the degree of bending is proportional to the
number of bound TBP molecules. ()Physical contact between
ATF/CREB factors and TFIID has been reported(36, 37) .
In preliminary studies of complexes containing both CREB and TBP, we
have not observed interaction between the two (data not shown). Thus,
interaction of CREB with TFIID would involve a protein other than TBP,
as has been suggested(37) .
Figure 2: Electron spectroscopic images of protein-DNA complexes. a-c, three independent CREB complexes on the proenkephalin gene fragment. d, CREB complex on the prodynorphin gene fragment. e, TBP complex on the proenkephalin gene fragment. For each complex, reference (mass) images are shown on the left, net phosphorus images in the center, and the superposition of the mass and net phosphorus images are shown on the right. The bar represents 16 nm (a-c), 19 nm (d), and 21 nm (e).
Analysis of CREB binding to the prodynorphin fragment (Fig. 2d and Table 1) indicates that the CREB-prodynorphin DNA complex contains an intermediate amount of DNA as compared with CREB-proenkephalin DNA and TBP monomer-proenkephalin DNA complexes (Table 1). Most of the DNA in the prodynorphin complex appears to be along the side of the complex (e.g.Fig. 2d, right panel). The protein mass in both prodynorphin and proenkephalin (83 and 87 kDa, respectively; Table 1) is consistent with the binding of a CREB dimer(26) . However, only about half as much DNA (33 versus 59 bp) is contained in the prodynorphin complexes with CREB dimer as compared with proenkephalin (Table 1). Thus, although a CREB dimer binds within each enhancer, the DNA within the complexes is in different conformation in prodynorphin versus proenkephalin.
Figure 4: DNase I analysis of CREB-CRE complexes formed on supercoiled CAT fusion plasmids. Footprints of top strand (see also Fig. 3A) are shown. A, analysis of native and mutant proenkephalin CRE-CREB complexes. The native sequence of the region of CREs is shown at left. Sequence markers were dideoxy chain termination reactions (not shown) as described under ``Materials and Methods.'' The arrows indicate bases mutated in one of the mutant plasmids, with specific mutations as indicated in Fig. 3A. The rectangles in the sequence enclose CRE-1 (top) and CRE-2. The positions of the CREs in the gel are shown by the two rectangles along the side. Filled circles alongside images of the gels indicate the positions of the mutated bases. For each set, the leftmost lane is 0 CREB control, with increasing concentration of protein as described under ``Materials and Methods.'' B, DNase I analysis of native prodynorphin CRE-3 in plasmid pCAT2.0dyn(14, 15) . CRE-3 contains 8-bp sequence (CTGCGTCA) like that of proenkephalin CRE-2. The positions of CRE within sequence of region and on gel are indicated by rectangles.
The mutant plasmid
with the seven bases substituted in the region of CRE-1 (Fig. 3A, sub) was used to
evaluate the contribution of CRE-1 to CREB binding. The substitutions
both change the sequence in the area of CRE-1 and reduce the
palindromic nature of the region so that CRE-1 and CRE-2 are not able
to form a CREB site of the type formed by the top strand of the native
duplex (the GT hairpin) (Fig. 3A). This substitution
altered the pattern of the footprint as compared with the wild type
(compare Fig. 4A, sub
and native). There is clearly still protection of CRE-2,
indicating that CREB still binds, but the protected region does not
extend upstream toward CRE-1. Whereas in the native gene the region of
CRE-1 is protected from DNase I digestion (along upper
rectangle, Fig. 4A, native), in the
plasmid without CRE-1 there is no protection of CRE-1 (along upper
rectangle, Fig. 4A, sub
).
In fact there is not protection even of the several bases 3` to CRE-1,
that is, between the CREs. As discussed above, the core sequence at
CRE-2 is identical to that of the high affinity CRE located at
-1541 in the rat prodynorphin gene(14, 15) .
DNase I analysis of that prodynorphin CRE in plasmid pCAT2.0dyn (14) yields a footprint that appears the same as that at CRE-2
in the sub
proenkephalin plasmid (Fig. 4, compare A, sub
, with B), indicating
that the 8-bp sequence they share is sufficient to produce the
footprint. In the native proenkephalin gene, however, the binding site
comprises CRE-1 and CRE-2. Thus, although a CREB dimer binds in each,
the DNA within the native is in different conformation from the DNA in
the prodynorphin and sub
complexes. In the native
proenkephalin gene CRE-1 participates directly in binding CREB protein.
Figure 5:
Nuclease P analysis of native
proenkephalin-CREB complexes. Top and bottom strands are as shown in Fig. 3A. For each gel, the leftmost lane is 0 CREB
control, with increasing CREB as described under ``Materials and
Methods.'' Sequence markers were dideoxy chain termination
reactions (not shown) as described under ``Materials and
Methods.'' Positions of the CREs are shown by the two
rectangles along the side.
Figure 6:
In vivo footprint analysis of
CREs in rat cells that express and in rat cells that do not express
proenkephalin. The upper gel image corresponds to the top
strand, and the lower image corresponds to the bottom strand (Fig. 3A). Endogenous proenkephalin gene is expressed
in C6 glioma cell line but not in R2C Leydig tumor cell line (Fig. 3D). The numbering of bases is according to the
rat sequence (10) , which is offset by three bases from the
human sequence (Fig. 3A) and which differs from the
human sequence outside the 27-bp region containing the CREs. The region
from -84 to -106 in the human gene (Fig. 3A) is identical to -81 to -103 in
the rat gene. The rectangles along the upper lane of
the gels indicate the positions of the CREs and correspond to the rectangles in the duplex sequence shown below the
images of the gels. Hairpins drawn to the right represent
stable structures formed from strands of the 23-bp region containing
the CREs(18, 19, 43) . The shaded areas in the hairpin structures indicate non-Watson-Crick base
pairs(9, 18, 19) . Naked DNA was purified and
then modified by DMS in vitro; in vivo DNA was
purified from cells treated with MeSO then DMS before
purification (see ``Materials and Methods''). + forsk DNA was from cells treated with forskolin then DMS. Residues
that are less reactive (open arrowheads) or more reactive (filled arrowheads) in cells expressing proenkephalin (C6
in vivo and C6 + forsk) are indicated along the
images of the gels and in the representations of the sequences of the
region of the CREs. Ratios to C6 naked for residues in the region of
the CREs are, for the top strand, at residue G
:
0.63 (C6 in vivo), 0.32 (C6 + forsk), 1.09 (R2C naked), and 0.90 (R2C in vivo); at
G
: 2.13, 1.86, 0.96, and 0.78; at
G
: 0.44, 0.32, 1.12, and 0.77; at
G
: 1.30, 1.44, 0.97, and 1.0. For the bottom strand
the ratios are at residue G
: 1.57, 1.44, 0.92, and
0.84; at A
: 1.78, 1.69, 1.09, and 0.67; and at
A
: 1.98, 1.73, 1.0, and
0.65.
In the bottom strand, no residue is protected but several show enhanced reactivity (Fig. 6, bottom gel). Only one G in the immediate vicinity of the CREs, that at -102, shows altered sensitivity. Gs at -106 and -73 are also more reactive in cells expressing proenkephalin (ratio of about 1.3:1 at -106 and about 1.8:1 at -73 for both C6 in vivo and C6 + forsk when compared with C6 naked). Within each CRE, however, there is an unusual A band only in the DNA from proenkephalin-expressing cells (C6 in vivo and C6 + forsk) (Fig. 6; -96 in CRE-1 and -85 in CRE-2). There is also a hypersensitive A at -112 that is specific to the cells expressing proenkephalin (Fig. 6). In C6 cells expressing the proenkephalin gene, these A bands are of approximately equal strength to the weakest of the Gs (see G at -84 and G at -111, Fig. 6, bottom gel). The environment of individual G residues, due to sequence and stacking effects, leads to varied sensitivity of Gs in the essentially G-specific DMS hot piperidine protocol (32) (e.g. G ladders from naked DNA, Fig. 6). The mechanism for the appearance of the A bands is not clear, but change in conformation and/or charge distribution can cause unusual methylation on A. Hot piperidine does not cleave N-1- or N-3-methylated As(32, 42) , but N-7-methyl A can be cleaved by the same mechanism as N-7-methyl G residues(42) . It is possible that change in charge distribution, perhaps due to AC base pairing(18) , causes some methylation at N-7 of As. In the synthetic proenkephalin enhancer, such AC base pairs are formed(18, 19) . The appearance of the three A bands on the bottom strand indicates that their environment is specifically altered in the vicinity of the CREs; within each CRE, one A residue is hypersensitive.
The pattern of modification of G and A residues within the two CREs is the same (Fig. 6, diagram of binding to duplex), showing that each CRE is similarly occupied during transcription but that neither is occupied in R2C cells not expressing the gene. There are no protected residues along the bottom strand, but the enhanced signals indicate structural alteration. Representation of the 23-bp region containing the CREs as hairpins (9, 18, 19, 43) suggests a possible mechanism for the pattern of DMS sensitivity: in the top strand hairpin, the residues that are similarly modified (hypersensitive at -88 and -99; protected at -86 and -97) appear as equivalent to each other in a symmetrical site (Fig. 6, top strand hairpin). Within the CREs, the abnormally strong A signals appear at sites of AC base pairs (Fig. 6, bottom strand hairpin). The A bands, as well as hypersensitivity of Gs, is consistent with structural change, perhaps including unusual base pairing.
The expression and binding data we have presented support the important observations of others (3, 4) that although CRE-2 can function independently as a binding site, it cannot by itself support efficient transcription. Studies on a series of mutant CREs emphasized that CRE-1 is critical in transcription(4) . Data presented in this manuscript show CRE-1 to be part of the binding site for a well characterized CRE-binding protein, and in vivo analysis indicates that specific occupation of both CREs correlates with transcriptional activity (Fig. 6).
Although DNase I footprinting shows that CRE-1 does bind CREB protein, binding there is dependent on binding at CRE-2. The point mutations that altered only CRE-2 eliminated binding in both CREs (Fig. 4A, -88C,-89A). Two mechanisms could account for the DNase I footprinting results. The CREs could be separate sites, each of which binds a CREB dimer. In this case, CRE-1 would be a separate but very poor binding site for CREB protein; bound protein at CRE-2 would cause a cooperative interaction, facilitating binding at CRE-1. Alternatively, together the CREs form a single binding site occupied by a single dimer. The electron spectroscopic imaging studies support a single dimer's using both CREs (Table 1); furthermore, the absence of binding at CRE-1, even at very high concentrations of CREB in the -88C,-89A mutant (Fig. 4A), argues against CRE-1 functioning as a separate CREB site. Therefore, the short region contains two different sites capable of binding factors. CRE-2 can bind CREB, but in the native gene CREB prefers a site composed of both CREs.
What then is
the nature of the binding site? One possibility is that bending
accommodates a single dimer's use of sequences in both CREs, that
is, each CRE would form a half-site. Because affinity for CRE-1 by
itself is so low (Fig. 4A, -88C,-89A), this model would predict that a
transcription factor scanning the DNA would recognize the CTGCGTCA at
CRE-2, a competent binding site (Fig. 4A, sub and Fig. 4B), bind there,
and then adopt as part of its site the neighboring CRE-1. The mechanism
by which the protein would straddle the region to select the low
affinity CRE-1 as part of its site is unclear. Protein-induced bending
might provide energy for adjustment from binding at CRE-2 alone to
using each CRE as a half-site. However, if this model is correct, CRE-1
is auxiliary, a premise that is not supported by studies with mutant
enhancers. Analysis of point mutations throughout the region shows that
mutations in either CRE can have similar effects on
transcription(4) . In vivo footprinting supports the
functional equivalence of the CREs, because the pattern of modification
in the transcribed gene is the same in both CREs (Fig. 6). Such
a pattern is unlikely for two sites of very different affinities placed
side by side. Furthermore, the very low affinity for CRE-1 by itself
argues against the protein adjusting its binding to use that site.
Thus, the data are not consistent with this model.
A second model
proposes that a structural change creates a site different from either
CRE. We have previously demonstrated that the enhancer region adopts an
alternative structure that can contribute to transcriptional
regulation(9, 18, 19, 42) . This
structure creates a unique site that uses CRE-1 and CRE-2 from one
strand and that includes two GT base pairs (Refs. 9, 18, and 19 and Fig. 3A). The alternative site has higher affinity for
CREB than does CRE-2 ( (9) and Fig. 3), and in vitro analysis has demonstrated that increased affinity is dependent on
a T in one of the GT base pairs(9) . The higher affinity for
this site suggests that a transcription factor scanning the DNA could
bind there and stabilize the structure. Because formation of the
alternative site depends on sequences in both CREs, point mutations in
either CRE would be as likely to affect transcription, as has been
shown(4) . The experiments described here support this model.
P nuclease sensitivity of the enhancer region is CREB
concentration-dependent, indicating protein-stabilized structural
change, and CREB-CRE complexes increase nuclease P
sensitivity of the enhancer region in a strand-specific manner,
suggesting differential occupation of the strands. In vivo footprinting shows not only transcription-dependent occupation of
both CREs but also shows that the pattern of base modification is the
same within both CREs (Fig. 6, sequence diagrams). The
similarity in protection and enhancement is striking; the same bases
are altered to the same extent. A shared site would depend equally on
the CREs, so the protection and enhancement are consistent with protein
binding to a site made up of CRE-1 and CRE-2 (Fig. 6, top strand
hairpin). In addition, there are specifically enhanced A bands on the
bottom strand. A structural basis for this unusual methylation could be
the conformational change in the CRE region; the hypersensitive A in
each CRE corresponds to a mismatched AC base pair, the environment of
which alters charge distribution (18) (Fig. 6, bottom
strand hairpin).
The data presented here provide the physical basis for the previously observed essential role of CRE-1 in regulating proenkephalin transcription; CRE-1 acts in concert with CRE-2 to create the transcription factor binding site. The DNA must adopt a secondary structure that accommodates a single binding site made up of sequences from CRE-1 and CRE-2, so that they are equivalent contributors to CREB binding. Although the conformational details of the structure require further study, we have shown that both CREs have similar and specific roles as factor binding sites during transcription. The existence of alternative sites within the short region could contribute in several ways to precise transcriptional regulation. Binding of a single factor to both CREs might assure proper orientation for assembly of the transcriptional machinery. The presence of three sites (CRE-1, CRE-2, and the site made up of both) allows for flexibility in binding to factors of differing affinities and sequence specificities.