From the Howard Hughes Medical Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0650
Received for publication, February 16, 2001
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ABSTRACT |
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Heterodimeric transcription regulatory proteins
can bind to palindromic recognition elements in two opposite
orientations. We have developed a gel-based fluorescence resonance
energy transfer assay for quantifying heterodimer orientation
preferences. Fos-Jun heterodimers bind in opposite orientations to AP-1
sites with different flanking sequences. The effects of individual
amino acid and base pair substitutions on heterodimer binding
orientation were quantified. Base pairs at positions ±6 and ±10
relative to the center of the AP-1 site were the principal determinants
of Fos-Jun binding orientation. Amino acid residues of opposite charge adjacent to the basic regions of Fos and Jun had independent effects on
heterodimer orientation. Exchange of these amino acid residues between
the basic region-leucine zipper domains of Fos and Jun reversed the
binding orientation. Heterodimers formed by full-length Fos and Jun
exhibited the same changes in binding orientation in response to amino
acid and base pair substitutions. The preferred orientation of
heterodimer binding affected the stability of Fos-Jun-NFAT1 complexes
at composite regulatory elements. Changes in heterodimer orientation
preference altered the transcriptional activity and the promoter
selectivity of Fos-Jun-NFAT1 complexes. Consequently, the orientation
of Fos-Jun binding can influence transcriptional activity by altering
cooperative interactions with other transcription regulatory proteins.
Regulation of the transcriptional activities of the myriad of
genes in mammalian genomes requires combinatorial interactions among
multiple transcription regulatory proteins within promoter and enhancer
regions (reviewed in Ref. 1). Whether the regulation occurs at the
level of chromatin remodeling, coactivator recruitment, or assembly of
the transcription machinery, specification of a unique target in the
genome requires cooperation among multiple transcription factors
(2-5). The cooperative interactions among many transcription
regulatory proteins require juxtaposition of contact surfaces that are
either part of or tightly coupled to the DNA binding domain (6-10).
Interactions among such proteins generally require a specific spacing
and orientation of the DNA recognition sequences to allow cooperative
complex formation.
Many mammalian transcription regulatory proteins form heterodimers that
recognize palindromic DNA sequence elements. Such heterodimers can
potentially bind to their recognition sequences in either of two
opposite orientations. Heterodimers that bind in opposite orientations
can differ in their interactions with transcription factors that bind
to adjacent regulatory elements. The binding orientations of such
heterodimers may be controlled by recognition of asymmetric base pairs
flanking the palindromic core sequence element or through interactions
with other transcription factors (8, 11-14). Promoter elements are
classically defined based on their position- and
orientation-dependent transcriptional activities. The
orientation of heterodimer binding to asymmetric regulatory elements
can influence their transcriptional activities (13, 15-18). The
mechanisms whereby the orientation of heterodimer binding influences
transcriptional activity remain to be characterized.
Fos and Jun are members of the
bZIP1 family of transcription
factors and bind to palindromic AP-1 regulatory elements. The x-ray
crystal structure of the bZIP domains of Fos and Jun bound to the AP-1
site revealed that the heterodimer could bind to the recognition
element in both orientations (19). In solution, Fos-Jun heterodimers
bind to different AP-1 sites in opposite preferred orientations (12,
18, 20, 21). The orientation of heterodimer binding is affected by
sequences flanking the palindromic core AP-1 recognition element and
amino acid residues adjacent to the basic regions of Fos and Jun (12,
14). No direct contacts between the amino acid residues adjacent to the
basic regions of Fos-Jun and the base pairs flanking the AP-1 site are
observed in the x-ray crystal structure (19). Thus, the orientation of Fos-Jun binding appears to be controlled by indirect recognition of
differences in DNA structure between flanking sequences on opposite
sides of the AP-1 site.
Fos and Jun activate different genes in different cell types and in
response to different extracellular signals (22). These differences in
Fos-Jun regulatory specificity are mediated at least in part by
cooperative interactions with structurally unrelated transcription
factors. Fos-Jun heterodimers can physically and functionally interact
with members of the NFAT, Ets, Smad, and nuclear hormone receptor
transcription factor families (10, 23, 24). The interaction between
Fos-Jun and NFAT1 (NFATp, NFATc2) has been characterized in greatest
detail. Cooperative binding by Fos-Jun and NFAT1 to composite
regulatory elements in cytokine gene promoters requires a specific
orientation of Fos-Jun binding (8, 11). The x-ray crystal structure of
the Fos-Jun-NFAT1 complex at the ARRE2 element shows a specific contact interface between NFAT1 and one face of the leucine zipper (7). The
interaction with NFAT1 can reverse the orientation of heterodimer binding (8). However, the preferred orientation of Fos-Jun binding can
influence the stability and the transcriptional activity of
Fos-Jun-NFAT1 complexes (18).
We have investigated the structural basis and the functional
significance of the opposite orientations of Fos-Jun heterodimer binding at different AP-1 sites. To compare the effects of individual base pairs and amino acid residues on heterodimer orientation, we
developed an approach for determination of the free energy of Fos-Jun
heterodimer reorientation at different AP-1 sites. The functional
consequences of opposite orientations of Fos-Jun heterodimer binding
were examined by comparing the stabilities and transcriptional
activities of Fos-Jun-NFAT1 complexes formed by heterodimers with
opposite orientation preferences.
Preparation of Fluorescent Oligonucleotides and
Proteins--
Oligonucleotides containing the sequences listed in Fig.
6 with symmetrical CTC extensions on both ends were synthesized with fluorescein on the 5' ends. Duplexes formed by one labeled and one
unlabeled strand were purified by gel electrophoresis. Proteins encompassing amino acid residues 139-200 of Fos and 257-318 of Jun
were expressed and purified to homogeneity as described (8, 12, 14).
The amino acid substitutions indicated in the figures replaced residues
at positions 139-141 and 257-259, respectively. The proteins were
labeled by incubation with Texas Red maleimide (Molecular Probes) and
purified as described (8, 12, 14). The FosRI and JunRI calibration
standards contained R155I and R273I substitutions, respectively.
Full-length Fos and Jun (with or without R155I or R273I substitutions)
as well as the DNA binding domain of NFAT1 (residues 396-692) were
expressed and purified as described (8, 25, 26).
The gelFRET Assay for the Orientation of Fos-Jun Heterodimer
Binding--
The orientation of heterodimer binding was determined
based on the ratio of acceptor to donor fluorescence emissions from donor fluorophores (fluorescein) linked to either end of an
oligonucleotide to an acceptor fluorophore (Texas Red) linked to either
subunit of the heterodimer. Complexes were formed by incubation of 2-6 µM Fos-Jun heterodimers in which one subunit was labeled
with Texas Red with 500 nM oligonucleotides labeled with
fluorescein on either the left or the right end. NFAT1 was added to the
reactions indicated at 1 µM. The complexes were separated
by native 8% PAGE from the free components as well as from homodimer
and nonspecific complexes. The gels were scanned using a FluorImagerFSI
fluorescence scanner (Molecular Dynamics) with 530 ± 15 nm band
pass and 610 nm cut-on filters. To calculate the emission from each
fluorophore, the emission through each filter was compared with the
emissions of calibration standards containing only donor or acceptor fluorophores.
Quantitative Analysis of Heterodimer Binding
Orientation--
The donor (FL) and acceptor (TR) emissions of
complexes labeled on the left (L) or the right (R) end were used to
calculate the acceptor to donor ratio (TR/FL). The acceptor to donor
ratios of complexes labeled on opposite ends of the oligonucleotide
were used to obtain the end preference (EP) value for each complex (EP = (TRL/FLL)/(TRL/FLL + TRR/FLR). The end preference value of a
population containing two binding orientations represents the average
of the end preference values of fully oriented complexes bound in
opposite orientations weighted by the fraction of complexes bound in
each orientation. In order to calibrate the relationship between end
preference values and heterodimer binding orientation, we used either
Fos-Jun-NFAT1 or Fos-JunRI and FosRI-Jun calibration standards. The end
preferences of Fos-Jun-NFAT1 complexes labeled on Fos and on Jun were
assumed to reflect the end preferences of fully oriented heterodimers
(EPJun-Fos). The end preferences of the heterodimer bound
in the opposite orientation (EPFos-Jun) were derived by
assuming that the end preferences of complexes bound in opposite
orientations were equivalent for complexes labeled on different
subunits (EPJun-FosTR = EPFos-JunTR). The
calculated end preference values of fully oriented Fos-Jun heterodimers
(EPFos-Jun and EPJun-Fos) were used to
determine the fraction of heterodimers bound in each orientation
(fFos-Jun and fJun-Fos)
based on fFos-Jun = (EP
Fos-Jun heterodimers that bind to the AP-1 site in opposite
orientations contact the central base pair using arginines from different subunits (Fig. 1, insets). Replacement of the
arginine residue in different subunits by an isoleucine shifts the
binding orientation in opposite directions (12, 20). To use these heterodimers as calibration standards, we assume that contacts to the
central base pair influence the free energy of reorientation independent of interactions with flanking sequences
( Measurement of Fos-Jun-NFAT1 Complex
Dissociation--
Fos-Jun-NFAT1 complexes bound to composite
recognition elements were prepared by incubation of 50 nM
Fos-JunTR (with or without arginine substitutions as
indicated) and 100 nM NFAT1 with 20 nM
oligonucleotide labeled with fluorescein on the right end in 20 mM Tris-Cl (pH 7.6), 50 mM NaCl, 5% glycerol
(v/v), 5 mM dithiothreitol, 0.5 mg/ml bovine serum albumin
for 10 min at 25 °C. Sonicated herring DNA was added to 1 mg
ml In Vitro Transcription Analysis--
Templates for in
vitro transcription were constructed by insertion of single
composite AP-1-NFAT recognition elements (shown in Fig. 8B)
at position The x-ray crystal structures of Fos-Jun heterodimers bound to an
AP-1 site revealed that Fos-Jun can bind to the AP-1 site in two
orientations that are related by an ~180° rotation about the dimer
axis (Fig. 1) (19). Studies of the
orientation of Fos-Jun binding in solution demonstrated that the
heterodimer binds to different AP-1 sites in opposite orientations
(12). To investigate the nucleic acid and protein determinants of the orientation of Fos-Jun binding, we used gel-based fluorescence resonance energy transfer (gelFRET) to determine the orientations of
Fos-Jun heterodimers at different AP-1 sites (Fig.
2).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EPJun-Fos)/ (EPFos-Jun
EPJun-Fos) and fJun-Fos = 1
fFos-Jun. We assume that the fraction of
heterodimers bound in each orientation represents the equilibrium
distribution (Fos-Jun
Jun-Fos). Studies of the rate of
heterodimer reorientation indicate that this reaction reaches
equilibrium rapidly during incubation of the samples prior to gel
electrophoresis (18). The intermediates in the reorientation reaction
do not influence our analysis providing that they do not represent a
major fraction of the complexes observed under the experimental
conditions. The free energy of reorientation (
GORI) is calculated based on the fraction of
heterodimers in each orientation at equilibrium
(
GORI = RT
ln(fJun-Fos/ fFos-Jun)). At each AP-1 recognition site, the free energy of reorientation reflects the difference in binding energies
(
GBIND) between the Jun-Fos and Fos-Jun
orientation isomers (
GORI =
G
G
GORI =
GFLANK +
GCENTRAL). Thus, inversion of the central
base pair reverses its effect on the free energy of reorientation
(
GCENTRAL(C:G) =
GCENTRAL(G:C)). We also assume that
substitution of the arginine residues in the basic regions of Fos and
Jun does not alter the effect of flanking sequences on the orientation
preference (
G
G
G
G
G
G
G
G
G
G
G
G
1 with continuous stirring to compete for
dissociated proteins. The change in energy transfer was monitored as a
function of time by excitation of fluorescein at 480 nm and measurement
of fluorescein emission at 520 nm and Texas Red emission at 610 nm.
50 of the basal c-fos promoter (27). The
promoters were fused to 390- or 242-base pair G-less transcription units. Two templates (10 µg/ml each) that generate transcripts of 390 and 242 bases in length and differ by a single base pair at the center
of the AP-1 site were mixed with an AdML190 control template (2 µg/ml). This mixture of templates was incubated with 0.8 µM full-length Fos-Jun heterodimers (with or without
arginine substitutions as indicated) and 1 µM NFAT1 in 40 mM HEPES (pH 7.6), 30 mM KCl, 5 mM
MgCl2, 1 mM dithiothreitol, 4% glycerol, and
1% polyvinyl alcohol at 30 °C for 5 min. Nuclear extract (50 µg
of protein) prepared from Namalwa cells containing low endogenous Fos-Jun and NFAT1 activities was added, and the reactions were incubated at 30 °C for 5 min. 500 µM ATP and CTP, 100 µM 3'O-methyl-GTP, 10 µM UTP, 15 µCi of [
-32P]UTP, and 10 mM creatine
phosphate were added, and transcription was allowed to proceed for 30 min at 30 °C. The transcripts were treated for 30 min at 30 °C
with 100 units of RNase T1 and were analyzed by
polyacrylamide gel electrophoresis and quantitated by PhosphorImager analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Fos-Jun heterodimers can bind the AP-1
site in two opposite orientations. Structures of Fos-Jun
heterodimers bound to the same AP-1 site in opposite orientations (19).
The sequences below show the positions and numbering of the
nucleotide bases in one strand, and the insets above show
the contacts to the central base pair for each complex. Under
equilibrium conditions in solution, the relative concentrations of the
two complexes were determined by the difference in Gibbs free energy
between the two binding orientations
( GORI).
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Fig. 2.
Sequences flanking the AP-1 site and amino
acid residues adjacent to the basic regions of Fos and Jun determine
the orientation of heterodimer binding. A, gelFRET
analysis of Fos-Jun heterodimer binding orientations at the site used
for crystallization of the Fos-Jun-AP-1 complex (X site). Heterodimers
formed by Fos and Jun containing the amino acid residues indicated
(above the lanes) adjacent to their basic regions
were incubated with AP-1 site oligonucleotides labeled with fluorescein
on either the left (L) or the right
(R) end as indicated below the lanes.
Each pair of lanes contained the same heterodimer labeled with Texas
Red (TR) on the subunit indicated above the
lanes. The complexes (upper bands) were separated
by PAGE from free DNA (lower bands) and nonspecific
complexes. The gel was scanned using a laser (488 nm) that excites the
fluorescein donor, and the donor (green) and acceptor
(red) emissions at each position in the gel were measured.
The images corresponding to donor and acceptor emissions were
superimposed. Thus, orange bands correspond to complexes
with high energy transfer, and green bands correspond to
complexes with low energy transfer. The diagrams below the
gel indicate the preferred binding orientations of the
heterodimers in the corresponding lanes. The result is representative
of three independent experiments. B, end preferences of
heterodimers formed by different Fos and Jun subunits at binding sites
containing different combinations of flanking sequences. The end
preferences of heterodimers labeled on Fos (red bars) and
Jun (blue bars) containing the amino acid residues indicated
(below the bars) adjacent to their basic regions
are shown pairwise at the binding site indicated above
each graph. A high end preference indicates that the labeled
subunit favors binding to the left half-site, whereas a low end
preference indicates that the labeled subunit favors binding to the
right half-site (arrow on right). The end
preferences of control heterodimers containing the amino acid
substitutions R155I in Fos and R273I in Jun (indicated by
RI) are shown in the subpanels on the right. The
X site corresponds to the sequence used for crystallization of the
Fos-Jun-AP-1 complex (19). The Xrev site contains the same
flanking sequences on the opposite sides of the core AP-1 site. The M
site contains nearly symmetrical flanking sequences
(italics). The XM and MX sites contain flanking sequences
from the left side of the X site and the right side of the M site and
vice versa. Standard deviations are shown for end preferences at the X
and M sites from three or more independent experiments.
gelFRET Analysis of the Orientation of Fos-Jun Heterodimer Binding-- The gelFRET assay of the orientation of Fos-Jun heterodimer binding is based on measurement of the relative efficiencies of energy transfer from donor fluorophores placed on opposite ends of an oligonucleotide to an acceptor fluorophore linked to one subunit of the heterodimer in complexes separated by gel electrophoresis (Fig. 2A) (12). Complexes in which the donor and acceptor fluorophores are on the same side of the binding site are predicted to exhibit higher efficiencies of energy transfer (orange bands) than complexes in which the donor and acceptor fluorophores are on opposite sides of the binding site (green bands). The orientation preferences of different complexes are compared by calculation of the relative efficiencies of energy transfer from opposite ends of the oligonucleotide (Fig. 2B, end preference). A high end preference indicates that the labeled subunit favors binding to the left half-site, whereas a low end preference indicates that the labeled subunit favors binding to the right half-site. To confirm that differences in the relative efficiencies of energy transfer from opposite ends of the oligonucleotide reflect differences in heterodimer orientation, we compared the end preferences of complexes labeled on different subunits. Complexes with a high end preference when labeled on one subunit exhibited a low end preference when labeled on the other subunit. Reciprocal changes in the relative efficiencies of energy transfer from opposite ends of the oligonucleotide to different subunits of the heterodimer were interpreted to reflect changes in heterodimer orientation.
The basic regions of Fos and Jun contact the symmetrical half-sites of the core AP-1 recognition element (19). Amino acid residues adjacent to the basic regions can influence recognition of sequences flanking the core binding element (12, 20). Fos contains a cluster of positively charged amino acid residues immediately adjacent to the basic region, whereas Jun contains negatively charged residues on the amino-terminal side of the basic region. We examined the effects of the substitution of positively and negatively charged amino acid residues adjacent to the basic regions of Fos and Jun on the orientation of heterodimer binding at the site used for crystallization of the Fos-Jun-AP-1 complex (X site). Complexes formed by Fos-Jun heterodimers containing different amino acid substitutions exhibited different efficiencies of energy transfer from donor fluorophores linked to opposite ends of the oligonucleotide to acceptor fluorophores linked to either Fos or Jun (Fig. 2, X). In complexes formed by heterodimers containing amino acid residues of opposite charge (FosKRR-JunEEE and FosEEE-JunKRR), the subunits with positively charged residues exhibited preferential energy transfer from the right end, whereas the subunits containing negatively charged residues favored energy transfer from the left end of the oligonucleotide. These differences in energy transfer from opposite ends of the oligonucleotide are consistent with opposite orientations of binding by heterodimers in which amino acid residues adjacent to the basic regions were exchanged between Fos and Jun.
Influence of Sequences Flanking the AP-1 Site-- To determine the relative effects of the core AP-1 recognition element and flanking sequences on the orientation of heterodimer binding, we exchanged the flanking sequences between the left and the right sides of the binding site. The exchange of flanking sequences reversed the binding orientations of all heterodimers (Fig. 2B, Xrev). The slight asymmetry between the end preferences of complexes at the X and Xrev sites reflects the influence of the core AP-1 recognition element on heterodimer orientations. Thus, sequences flanking the X site had a much larger effect on heterodimer binding orientations than the asymmetric base pair at the center of the AP-1 site.
To identify the sequences that determine the orientation of Fos-Jun binding at the X site, we first compared the effects of the amino acid substitutions on the orientation of heterodimer binding at a site with different flanking sequences (Fig. 2B, M). Heterodimers formed by Fos and Jun bZIP domains containing amino acid residues of opposite charge (FosKRR-JunEEE and FosEEE-JunKRR) exhibited opposite end preferences at this site compared with the X site (compare M and X in Fig. 2B). The amino acid substitutions had smaller effects on heterodimer end preferences at the M site, which contained nearly symmetrical flanking sequences. Since the core AP-1 recognition sequences were identical between these two sites, sequences flanking the core AP-1 recognition elements must determine the opposite binding orientations.
The binding orientation reflects the balance between the relative binding affinities of the two subunits in the heterodimer for opposite sides of the binding site. To locate the sequences that determine the opposite binding orientations at the X and M sites, we examined the effects of exchange of flanking sequences on the binding orientation (Fig. 2B). Complexes formed on oligonucleotides that contained flanking sequences from the left side of the X site and the right side of the M site exhibited end preferences more similar to those observed at the X site (Fig. 2B, compare XM and X). Conversely, complexes formed on oligonucleotides that contained flanking sequences from the left side of the M site and the right side of the X site exhibited end preferences more similar to those observed at the M site (Fig. 2B, compare MX and M). The subunits containing positively charged amino acid residues (FosKRR and JunKRR) exhibited a stronger preference for binding to the left side of the X site than the XM site. Conversely, subunits containing negatively charged residues (FosEEE and JunEEE) exhibited a modest preference for binding to the left side of the M site but had little effect on the orientation preference at the MX site. Thus, both the left and the right sides of the X and M sites affected the orientation of heterodimer binding, but differences between the left sides of the X and M sites contributed more to the opposite binding orientations at these sites.
Influence of Single Base Pair Substitutions--
The X and M sites
differ at multiple positions flanking the core AP-1 recognition
element. Both sites contained symmetrical base pairs to positions ±5
from the center of the AP-1 site. The binding orientations at these
sites must therefore be determined by sequences more than 5 base pairs
from the center of the AP-1 site. To investigate the contributions of
individual flanking base pairs to the opposite binding orientations at
the X and M sites, we exchanged single base pairs between these sites
(Fig. 3). Replacement of the T:A base
pair at the 6 position from the center of the X site by a G:C base
pair shifted the heterodimer orientations toward increased binding by
subunits with positively charged amino acid residues (FosKRR and
JunKRR) and reduced binding by subunits with negatively charged
residues (FosEEE and JunEEE) to the left half-site (Fig. 3,
X-6G). Thus, the asymmetric base pairs at the ±6 positions
of the X site contributed to the opposite orientations of heterodimer
binding at the X and the M sites.
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The base substitution at the 6 position of the X site reduced but did
not eliminate the effects of amino acid substitutions adjacent to the
basic regions of Fos and Jun on the orientation of heterodimer binding.
Since all base pairs up to and including the ±6 positions at the X-6G
site were symmetry-related, sequences further from the center of the
AP-1 site must influence the binding orientation. Replacement of base
pairs at the
7 or the
8 positions of the X site by T:A and A:T base
pairs from the M site caused relatively little change in heterodimer
end preferences (Fig. 3, X-7T and X-8A). Thus,
DNA sequences more than 8 base pairs from the center of the AP-1
recognition site or a specific combination of flanking base pairs must
contribute to the difference in heterodimer orientations at the X and M sites.
The X and M sites contain the same base pairs at the ±10 positions in
symmetrically reversed locations. The A:T base pair at position 10
and the G:C base pair at position +10 are the only asymmetric base
pairs flanking the M site. Amino acid substitutions adjacent to the
basic regions of Fos and Jun had no significant effect on heterodimer
orientations at binding sites with symmetrical flanking sequences (Fig.
6, site S). The asymmetric base pairs at the ±10 positions
must therefore mediate the effects of amino acid substitutions in Fos
and Jun on heterodimer end preferences at the M site. To confirm the
role of the base pairs at the ±10 positions in the opposite binding
orientations at the X and M sites, we substituted the base pairs at the
±10 positions of the M site by the corresponding base pairs from the X
site (Fig. 3, M-10C+10T). Replacement of these base pairs in
the M site reversed the effects of amino acid substitutions adjacent to
the basic regions of Fos and Jun on heterodimer end preferences. These
base pairs had the same effect on heterodimer end preferences at a binding site containing a symmetrical CG dinucleotide at the center of
the binding site (data not shown). Consequently, the asymmetric base
pairs at positions ±10 contributed to the opposite orientations of
heterodimer binding at the X and M sites.
Quantitative Analysis of Heterodimer Orientation Preferences-- The differences between the end preferences of various Fos-Jun complexes allow determination of their relative orientation preferences. However, it is not possible to determine the absolute orientation preference of a specific complex without knowing the relationship between the end preference values and the fraction of complexes bound in each orientation. To determine the absolute orientation preferences of Fos-Jun heterodimers, we used calibration standards to estimate the end preferences of fully oriented complexes. The ratio between the two binding orientations was then calculated based on the proportionality between differences in end preference and orientation preference (see under "Experimental Procedures"). We describe two independent strategies for calibration of the gelFRET assay. We used both approaches to quantify the contributions of individual base pairs to the opposite orientations of heterodimer binding at the X and M sites.
The cooperative interaction between Fos-Jun and NFAT1 requires a specific orientation of Fos-Jun heterodimer binding (8, 9, 11). Thus, quaternary Fos-Jun-NFAT1 complexes provide calibration standards that are predicted to exhibit a strong preference for binding in one orientation. One limitation of this calibration strategy is that the heterodimers can be oriented in only one of the two opposite orientations. Furthermore, heterodimers with strong orientation preferences in the absence of NFAT1 can influence the end preferences of Fos-Jun-NFAT1 complexes (8). NFAT1 binding may also influence energy transfer through mechanisms unrelated to the orientation of heterodimer binding. For purposes of these experiments, we assumed that 1) the end preferences of Fos-Jun-NFAT1 complexes represented a good estimate of the end preferences of fully oriented Fos-Jun heterodimers and that 2) the end preferences of complexes bound in opposite orientations were equivalent for complexes labeled on different subunits.
To test the validity of the calibration of the gelFRET assay using
Fos-Jun-NFAT1 complexes, we compared the effects of symmetry-related single base pair substitutions on opposite sides of the AP-1 site on
the orientation of heterodimer binding (Fig.
4). These base substitutions were
predicted to have effects of equal magnitudes but opposite directions
on the orientation preference. The base pairs in the NFAT recognition
sequence had a moderate effect on heterodimer end preferences in the
absence of the base substitutions (Fig. 4A, N). Single base
pair substitutions at the 6 and +6 positions shifted the end
preferences in opposite directions (Fig. 4A, N-6T and
N+6A). These base substitutions had converse effects on the
end preferences of all heterodimers. The subunits containing negatively
charged amino acid residues adjacent to the basic region (FosEEE and
JunEEE) favored binding to the half-sites proximal to the base
substitutions, whereas the subunits containing positively charged
residues (FosKRR and JunKRR) favored binding to the distal half-sites.
The end preferences of quaternary Fos-Jun-NFAT1 complexes were
virtually unaffected by the base substitutions flanking the AP-1 site
(Fig. 4B, N-6T, N and N+6A). The fraction of
Fos-Jun heterodimers bound in each orientation was calculated based on the end preferences of the Fos-Jun heterodimer in the absence of NFAT1
and the same Fos-Jun heterodimer in the Fos-Jun-NFAT1 complex (see
under "Experimental Procedures").
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The Free Energy of Heterodimer Reorientation--
To facilitate
comparison of the orientation preferences of different complexes, we
define the free energy of reorientation (GORI) as the difference in Gibbs free
energies between complexes formed by opposite orientations of
heterodimer binding (Jun-Fos versus Fos-Jun). This
difference is equivalent to the difference in binding energies between
heterodimers bound in opposite orientations to the same recognition
site (
GORI =
G
G
To compare the effects of individual amino acid or base pair
substitutions on the orientation preference, we calculate the difference in the free energy of reorientation between complexes with
and without the substitution (GORI). This
difference represents the change in the free energy of reorientation
caused by the substitution in the specific context examined. The
changes in the free energy of reorientation caused by the
symmetry-related
6T and +6A substitutions at the N site were nearly
identical in magnitude but of opposite signs, indicating that these
base substitutions had equal effects in opposite directions on the
orientation preferences of all complexes (Fig. 4C).
The use of Fos-Jun-NFAT1 complexes for calibration of the gelFRET assay
is directly applicable only to binding sites that can be placed
adjacent to an NFAT recognition sequence. To develop a more general
calibration strategy, we exploited the effects of amino acid (RI)
substitutions in the basic regions of Fos and Jun that shift the
binding orientation in opposite directions (Figs. 2, 3, and
4D). The end preferences of these complexes were used to
calibrate the gelFRET assay (see under "Experimental Procedures"). To test the validity of calibration of the gelFRET assay using heterodimers in which the arginine residue in either subunit was replaced, we compared the changes in the free energy of reorientation (GORI) calculated based on FosRI-Jun and
Fos-JunRI calibration standards (Fig. 4E) with that obtained
by calibration using Fos-Jun-NFAT1 complexes (Fig. 4C). The
effects of the flanking base pair substitutions on the free energy of
reorientation were virtually identical based on both approaches. The
symmetry-related base substitutions on opposite sides of the AP-1 site
had equivalent effects in opposite directions on the orientation
preferences of all heterodimers. The effects of the
6T and +6A
substitutions were therefore independent of other asymmetric base pairs
in the N site. Fos labeled with Texas Red exhibited a slightly greater
preference for occupying the half-site distal from the base
substitution. This may reflect a small effect of the fluorophore on the
orientation preferences of the heterodimers. However, this trend does
not interfere with analysis of either the effects of amino acid
substitutions or the influence of flanking base pairs on the
orientation of heterodimer binding since the effect was small and
affected all heterodimers to a similar extent. The comparable results
from calibration of the gelFRET assay using Fos-Jun-NFAT1 complexes as
well as FosRI-Jun and Fos-JunRI heterodimers validate the quantitative
analysis of the orientation of Fos-Jun heterodimer binding.
Independent Effects of Amino Acid Substitutions in Fos and in Jun
on Orientation Preference--
Amino acid residues adjacent to the
basic regions of both Fos and Jun influence the orientation of
heterodimer binding. To determine if these amino acid residues affected
the binding orientation independently or interacted with each other, we
compared the effects of all amino acid substitutions in complexes
formed with different dimerization partners (Fig.
5). The effect of each amino acid substitution was calculated based on the difference in the free energy
of reorientation (GORI) between
heterodimers that differ only in the amino acid residues adjacent to
the basic region of one subunit. Each amino acid substitution caused a
characteristic change in the free energy of reorientation that was
virtually unaffected by the dimerization partner, indicating that
substitutions in the two subunits affected heterodimer orientation
independently (i.e. substitution of JunEEE by JunKAE caused
the same change in the free energy of reorientation in heterodimers
with either FosKRR or FosEEE, Fig. 5). The free energy change caused by
a particular amino acid substitution resulted in an increase in the
orientation preference of some complexes but a decrease in the
orientation preference of other complexes (i.e. substitution of JunEEE by JunKAE reduced the orientation preference of heterodimers with FosEEE but increased the orientation preference of heterodimers with FosKRR at the N-6T site, Fig. 4A).
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The amino acid substitutions had opposite effects on heterodimer
orientations at the N-6T and N+6A sites (Fig. 5, compare upper and lower panels). These effects were not
perfectly reciprocal because of the small effects of additional
asymmetric base pairs at the N site on heterodimer orientation. The
change in the free energy of reorientation caused by exchange of the
amino acid residues adjacent to the basic regions of Fos and Jun
(bars on the right in Fig. 5) provides a measure
of the influence of electrostatic interactions on heterodimer
orientation at each site (Fig.
6).
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Quantitative Effects of Base Substitutions on Orientation Preference-- Calibration of the gelFRET assay makes it possible to compare the quantitative effects of base substitutions at different binding sites on the orientation preferences of Fos-Jun heterodimers. We determined the change in the free energy of reorientation caused by the exchange of charged amino acid residues between Fos and Jun at each binding site (Fig. 6). The changes in the free energy of reorientation were determined separately for heterodimers labeled on Fos and on Jun to eliminate the possibility that the differences in orientation preferences were caused by the fluorophore label and to obtain independent verification of the results.
As shown by the qualitative analysis of end preferences, substitution
of the the T:A base pair at the 6 position of the X site by a G:C
base pair from the M site had a larger effect on heterodimer
orientation than the substitutions at the
7 or
8 positions (Fig. 6,
X-6G, X-7T, and X-8A). The complementary
substitution of a G:C base pair at the
6 position in the M site by a
T:A base pair had a reciprocal effect on heterodimer orientation (Fig. 6, M-6T). Likewise, complementary base substitutions at the
7 and
8 positions of the M site had small effects on heterodimer orientation (Fig. 6, M-7C and M-8C). Exchange of
the asymmetric base pairs at the ±10 positions of the M site reversed
the change in the free energy of reorientation caused by the charged
amino acid residues (Fig. 6, M-10C and M+10T).
Likewise, exchange of the sequences flanking the X site reversed the
effect of electrostatic interactions on the free energy of
reorientation (Fig. 6, Xrev). Substitution of the
G:C base pair at the
5 position of a symmetrical binding site by an
A:T base pair from the X site had little effect on heterodimer binding
orientations (Fig. 6, compare S and S-5A). A
binding site containing asymmetric base pairs at all positions with the
exception for the ±6 and ±10 positions exhibited little effect of the
exchange of amino acid residues on heterodimer orientation (Fig. 6,
MX). Thus, the asymmetric base pairs at the ±6 and ±10 positions were the principal determinants of the difference in heterodimer binding orientations at the X and M sites.
To investigate the influence of sequence context on the effects of
asymmetric base pairs on heterodimer orientation, we compared the
effects of base substitutions at the ±6 positions in different binding
sites. The base substitutions at the 6 and +6 positions in the N site
had effects of similar magnitudes on heterodimer binding orientations
(Fig. 6, compare N-6T and N+6A versus
N). Thus, the other asymmetric base pairs in the N site did
not alter the effects of these base substitutions. The same base
substitutions in the M-6T and X-6G sites had slightly smaller effects
on heterodimer orientations (compare with the M and X sites). To
investigate the effects of these base pairs in a different sequence
context and at a different position flanking the AP-1 site, we inserted these base pairs at the ±6 and ±7 positions of otherwise symmetrical binding sites. Insertion of the asymmetric (T:A and C:G) base pairs at
the ±6 positions favored the same orientation of heterodimer binding
as they did at the N-6T, M-6T, X, and XM sites. However, the effect of
the asymmetric base pairs at the ±6 positions on the change in the
free energy of reorientation at this binding site was smaller than
their effect at other binding sites (Fig. 6, I-6T I+6C).
Insertion of the same asymmetric base pairs at the ±7 positions also
favored heterodimer binding in the same orientation. Moreover, the
effect of the asymmetric base pairs at ±7 positions on the change in
the free energy of reorientation at this binding site was similar to
their effect at the ±6 positions (Fig. 6, I-7T I+7C). Thus,
the same asymmetric base pairs at the ±6 and ±7 positions can have
similar effects on heterodimer orientation preferences, and neighboring
base pairs can influence the magnitudes of these effects.
Orientation Preferences of Full-length Fos and Jun--
Previous
studies of the orientation of Fos-Jun binding have been limited to
analysis of truncated peptides encompassing the bZIP domains (8, 11,
12, 20). These truncated peptides differ from native Fos and Jun both
in length and in amino acid sequence. Regions outside the bZIP domains
of Fos and Jun are essential for transcription activation (28) and
influence DNA structure in the Fos-Jun-AP-1 complex (21, 26). To
investigate the orientation preferences of full-length Fos and Jun, we
examined the end preferences of heterodimers formed by full-length Fos and Jun with labeled Fos and Jun bZIP domains (Fig.
7). The end preferences of heterodimers
formed by full-length Fos and Jun with the corresponding bZIP domains
were similar to those of the same heterodimers formed by FosKRR and
JunEEE (Fig. 7, compare upper and lower panels).
Amino acid substitutions adjacent to the basic regions of the bZIP
domains had comparable effects on the end preferences of heterodimers
formed by both full-length and truncated Fos and Jun. Symmetry-related
base substitutions on opposite sides of the AP-1 site had converse
effects on the end preferences of heterodimers formed by the
full-length proteins (Fig. 6, compare 6T and +6A
sites). NFAT1 shifted the end preferences of complexes formed by
both full-length and truncated proteins to a similar extent. In
addition, replacement of the arginine residues that can contact the
central base pair had comparable effects on the end preferences of
heterodimers formed by full-length as well as truncated Fos and Jun.
Consequently, the factors that determine the orientation of heterodimer
binding are likely to be closely related for the truncated proteins
encompassing the bZIP domains and for native Fos and Jun.
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Functional Effects of the Orientation Preference of Fos-Jun
Binding--
The preferred orientation of Fos-Jun heterodimer binding
may influence interactions with transcription factors that bind to adjacent recognition sequences. We examined the influence of the preferred orientation of heterodimer binding on cooperative DNA binding
and transcription activation by Fos-Jun and NFAT1. The dissociation of
Fos-Jun heterodimers from composite regulatory elements was measured in
the presence and absence of NFAT1 by monitoring the
time-dependent change in fluorescence resonance energy
transfer following addition of unlabeled competitor DNA (Fig.
8A). We examined the
dissociation rates of heterodimers formed by wild type Fos-Jun as well
as heterodimers in which the arginine that can contact the central
guanine was replaced by an isoleucine in either subunit. The
dissociation rates were compared at binding sites containing central
C:G or G:C base pairs that favor opposite orientations of binding by
heterodimers in which one arginine was replaced (12, 20). In the
absence of NFAT1, there was no significant difference (less than
2-fold) between the dissociation rates from these two binding sites for
any of the heterodimers (data not shown). In the presence of NFAT1,
the dissociation rate of wild type Fos-Jun heterodimers was reduced by
~100-fold at both binding sites (Fig. 8A, upper panels).
The similar dissociation rates at both sites are consistent with the weak orientation preference of Fos-Jun heterodimer binding at these
sites in the absence of NFAT1 (8) and the small effect of the central
base pair on the orientations of heterodimers formed by wild type Fos
and Jun.
|
In contrast, Fos-Jun-NFAT1 complexes formed by heterodimers in which the arginine in Fos was replaced by an isoleucine (FosRI-Jun) exhibited a more than 10-fold slower dissociation rate at the binding site containing a central G:C base pair than at the site containing a C:G base pair (Fig. 8A, middle panels). The effect of the central base pair was even larger for heterodimers in which the arginine in Jun was replaced by an isoleucine (Fos-JunRI), but the relative stabilities of Fos-Jun-NFAT1 complexes at the two binding sites were reversed (Fig. 8A, lower panels). In both cases, the slower dissociation rate was observed for complexes in which the preferred orientation of heterodimer binding in the absence of NFAT1 was the same as the orientation in the Fos-Jun-NFAT1 complex. Thus, different combinations of the same amino acid and base pair substitutions have distinct effects on the stability of the Fos-Jun-NFAT1 complex depending on their effects on the orientation of heterodimer binding. The larger difference in the stabilities of complexes at the two binding sites for Fos-JunRI heterodimers compared with FosRI-Jun heterodimers is consistent with the preferential interaction between the central guanine and the arginine in Fos (12, 20). Consequently, the preferred orientation of Fos-Jun heterodimer binding can influence cooperative interactions with other transcription factors at composite regulatory elements.
The differences in Fos-Jun-NFAT1 complex stabilities caused by opposite orientations of Fos-Jun binding may influence the transcriptional activities of Fos-Jun-NFAT1 complexes. We examined transcription activation by full-length Fos-Jun and NFAT1 using in vitro transcription reactions containing two templates with composite NFAT-AP-1 regulatory elements that favored opposite orientations of heterodimer binding (Fig. 8B). The promoters on the two templates differed by a single base pair at the center of the AP-1 site that controls the preferred binding orientations of heterodimers in which one of the arginines that can contact this base pair has been replaced (12, 20). The promoters were linked to transcription units of different lengths to allow comparison of the transcriptional activities of the two promoters in the same reaction. In the absence of added NFAT1, heterodimers formed by wild type Fos and Jun as well as subunits containing arginine substitutions exhibited modest activation of both promoters. In the presence of NFAT1, wild type Fos-Jun heterodimers exhibited robust activation of both promoters. In contrast, heterodimers in which the arginine in Fos was replaced (FosRI-Jun) preferentially activated transcription from the promoter containing a central G:C base pair. Heterodimers in which the arginine in Jun was replaced (Fos-JunRI) exhibited an even greater bias but selectively activated transcription from the promoter containing a central C:G base pair. Thus, heterodimers with opposite orientation preferences exhibited reciprocal patterns of transcription activation at promoters that differed by single base pairs. The ratio between the relative efficiencies of transcription activation by heterodimers with opposite orientation preferences at the two promoters was 6. The difference in the lengths of the transcription units did not influence the efficiencies of transcription activation since the relative amounts of the two transcripts were reversed by exchange of the promoters between the two transcription units (data not shown). Thus, the orientation preference of Fos-Jun heterodimer binding can influence both transcriptional activity and promoter selectivity.
The influence of the preferred orientation of Fos-Jun binding on
transcriptional activity at these promoters was presumably due to their
cooperative interaction with NFAT1. However, heterodimers in which the
arginine residue in one subunit was replaced exhibited differential
activation of the two promoters even in the absence of added NFAT1
(Fig. 8B, upper panel, lanes 3 and 4).
To confirm that the influence of heterodimer orientation preference on
transcriptional activity reflected the interaction with NFAT1, we
examined transcription activation on templates where the NFAT
recognition sequence was moved to the opposite side of the AP-1 site by
substitution of 2 base pairs on each side of the AP-1 site (Fig.
8B, lower panel). To reduce the effects of endogenous
activators in the nuclear extract, transcription was performed in the
presence of competitor oligonucleotides. Under these conditions,
heterodimers in which the arginine residue was replaced exhibited
comparable activation of both promoters in the absence of added NFAT1.
In the presence of added NFAT1, the heterodimers in which different
arginine residues were replaced exhibited selective activation of
different promoters. The ratio between the relative transcriptional
activities of heterodimers with opposite orientation preferences at the
two promoters was 15. Significantly, the relative activities of
promoters containing central C:G and G:C base pairs were reversed by
transfer of the NFAT recognition sequence to the opposite side of the
AP-1 site. Consequently, the orientation preference of heterodimer
binding can influence the synergistic activation of transcription by
Fos-Jun and NFAT1 at composite regulatory elements.
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DISCUSSION |
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Synergistic control of gene expression frequently depends on a specific arrangement of regulatory elements within promoter and enhancer regions (2, 3, 5). The structural organization of transcription factor complexes is therefore likely to have a crucial role in the control of transcriptional activity. The sequence of the DNA binding site can influence the conformation of transcription regulatory proteins (29). The functions of transcription factor complexes can be affected by conformational changes caused by differences in the sequence of the regulatory element (30-32). Opposite orientations of heterodimer binding represent perhaps the most dramatic conformational change in terms of its effects on interactions with adjacent regulatory proteins. The gelFRET assay provides a quantitative tool for analysis of conformational variation in nucleoprotein complexes.
Quantitative analysis of the orientation preferences of Fos-Jun heterodimers at different AP-1 sites demonstrated that base pairs that are far from the core AP-1 recognition sequence can influence the orientation of Fos-Jun heterodimer binding. The sequences flanking AP-1 sites in different promoters and enhancers are distinct, suggesting that the preferred orientation of Fos-Jun binding varies between different regulatory regions. It is also possible that transcription factor binding to sequences adjacent to the AP-1 site can influence the orientation of Fos-Jun binding even in the absence of direct contacts between the proteins. This mechanism could contribute to the regulatory interactions that have been observed between Fos-Jun and a variety of structurally unrelated transcription factors that bind to adjacent recognition elements (10, 23, 24).
The effects of base substitutions at the ±6 positions as well as amino acid substitutions adjacent to the basic regions on heterodimer orientation are highly correlated with their effects on DNA bending in the Fos-Jun-AP-1 complex (12, 14, 20). The largest effects of asymmetric base pairs on heterodimer orientation were observed at the ±6 and ±10 positions where the major or minor groove faces the same side of the DNA helix as the amino acid residues adjacent to the basic region that control the binding orientation. This is consistent with the preferred mode of DNA bending by roll into the major and minor grooves (33). Base pairs flanking the core AP-1 recognition element affected both the binding orientation and DNA bending by Fos and Jun without the requirement for direct amino acid-nucleotide contacts (12). One mechanism whereby flanking DNA sequences influence the orientation of Fos-Jun binding involves electrostatic interactions between charged amino acid residues in Fos-Jun and the negatively charged phosphodiester backbone of DNA (14).
The free energy of Fos-Jun heterodimer reorientation at all binding sites examined was relatively small compared with the free energy of Fos-Jun binding to the AP-1 site. It was also smaller than the free energy of Fos-Jun interactions with NFAT1 since NFAT1 can reverse the orientation of Fos-Jun binding at all composite regulatory elements examined (8). Thus, the preferred orientation of heterodimer binding does not prevent formation of Fos-Jun-NFAT1 complexes at binding sites where Fos-Jun alone bind in the disfavored orientation. However, the orientation preference of Fos-Jun binding affected the relative stabilities of Fos-Jun-NFAT1 complexes at different composite regulatory elements. Moreover, the orientation preference affected the relative transcriptional activities of templates containing composite regulatory elements with opposite preferred orientations of heterodimer binding. Thus, the orientation preference of heterodimer binding can control both the transcriptional activity and promoter selectivity of Fos-Jun-NFAT1 complexes.
The arginine substitutions that shift the preferred orientation of Fos-Jun heterodimer binding also reduce the binding affinity of Fos-Jun heterodimers. However, the reduction in binding affinity did not cause the orientation-dependent transcriptional activity since the same change in binding affinity was observed regardless of the central base pair, and the heterodimer with a higher binding affinity (Fos-JunRI) exhibited the greater orientation dependence. Moreover, transcription activation by Fos-Jun-NFAT1 complexes formed by wild type Fos-Jun heterodimers was also affected by the preferred orientation of heterodimer binding (18). A Fos-Jun-NFAT1 complex at a particular regulatory element is formed in competition with all other accessible sites. Thus, the orientation preference of Fos-Jun heterodimer binding is likely to influence the occupancy and the transcriptional activity of composite regulatory elements in the cell.
The influence of the orientation of heterodimer binding on
transcriptional activity and promoter selectivity emphasizes the importance of conformational variation among transcription regulatory proteins for transcriptional activity. Such conformational variability can influence both cooperative interactions among transcription factors
bound to composite regulatory elements as well as interactions with
coactivators and corepressors (13, 31, 32). The conformational variations of transcription regulatory proteins are likely to represent
equilibria among several conformations. The balance of such equilibria
can be controlled by relatively subtle structural determinants. This
provides the potential for control of transcriptional activity through
shifts in the equilibrium between different conformational states.
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ACKNOWLEDGEMENTS |
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We are grateful to the members of the Kerppola laboratory for critical reading of the manuscript and stimulating discussions.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a Rackham Merit Fellowship.
§ To whom correspondence should be addressed. Tel.: 734-615-1703; Fax: 734-615-3397; E-mail: kerppola@umich.edu; URL, www.umich. edu/~hhmi/.
Published, JBC Papers in Press, March 19, 2001, DOI 10.1074/jbc.M101494200
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ABBREVIATIONS |
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The abbreviations used are: bZIP, basic region-leucine zipper; gelFRET, gel-based fluorescence resonance energy transfer; NFAT, nuclear factor of activated T cells; FL, fluorescein; TR, Texas Red; EP, end preference; PAGE, polyacrylamide gel electrophoresis.
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