(Received for publication, November 1, 1995; and in revised form, January 18, 1996)
From the
The nuclear hormone receptors regulate target gene expression in response to hormones of extracellular origin. The DNA binding specificity of these receptors therefore plays the critical role of defining the precise repertoire of target genes that respond to a given hormone. We report here an analysis of the DNA binding specificity of the thyroid hormone receptor (c-ErbA protein) and that of an oncogenic derivative, the v-ErbA protein. These otherwise closely similar proteins exhibit quite divergent DNA sequence specificities at multiple positions within the DNA binding site. The thyroid hormone receptor (c-ErbA protein) exhibits a particularly broad DNA specificity, whereas the v-ErbA protein is comparatively quite specific. Intriguingly, these differences in DNA recognition largely map to an N-terminal receptor domain not traditionally implicated in DNA binding, and are further influenced by heterodimer formation with retinoid X receptors. We propose that the N terminus of nuclear hormone receptors plays an critical role in DNA recognition by altering the conformation of the receptor domains that make the actual base-specific contacts.
The nuclear hormone receptors are a family of ligand-regulated
transcription factors that mediate cellular responses to a broad range
of small hydrophobic
hormones(1, 2, 3, 4, 5) .
Members of the family include the steroid receptors, the retinoid acid
receptors (RARs), ()retinoid X receptors (RXRs), and the
thyroid hormone receptors (T
Rs). Additional diversity is
generated within individual receptor classes by the expression of
multiple receptor isoforms; for example, T
Rs are encoded by
two separate loci,
and
(3, 4) . Despite
this diversity, the different nuclear hormone receptors share a common
mode of action, functioning by binding to specific DNA sequences and
regulating expression of nearby target
genes(1, 2, 3, 4, 6, 7) .
The DNA recognition properties of each receptor play the critical
role of defining the specific repertoire of target genes that respond
to a given hormone. Most receptors bind to DNA as protein dimers, with
each receptor molecule binding to a ``half-site,'' a
conserved 6-8-nucleotide DNA
sequence(7, 8, 9, 10, 11) .
Recognition of the sequence of each half-site has generally been
believed to be mediated exclusively by a zinc-finger motif within the
center of each receptor (Fig. 1) (12, 13, 14) . Amino acids in the P-box helix
within this zinc-finger motif make direct contact with bases in the
major grove of the DNA half-site, and altering amino acids in the P-box
can alter the half-site specificity of the
receptor(14, 15, 16, 17, 18, 19, 20, 21, 22) .
An additional -helix, the A-box located at the C-terminal extreme
of the zinc-finger motif, makes minor groove contacts with bases at the
5` end of the half-site(14, 23) .
Figure 1: Schematic of the c-Erb A and v-Erb A proteins. Schematic representations of c- and v-Erb A proteins are presented (A) with the DNA binding and hormone binding domains indicated. Alterations in v-Erb A relative to c-Erb A are also shown, including N- and C-terminal deletions, 13 internal amino acid substitutions (vertical bars), and N-terminal retroviral-derived Gag sequences. Amino acids involved in the novel DNA recognition properties of v-Erb A that were altered in this study (mutants C32Y and S61G) are highlighted. Although having no known effect on DNA half-site specificity, codon 78 (a lysine in c-Erb A and a threonine in v-Erb A) was also altered in our studies to produce a v-Erb A allele (S61G/T78K) with a fully c-Erb A-like zinc finger domain (18) . The relevant amino acid sequences are also presented (B). (c) denotes the c-Erb A sequence, and (v) denotes the v-Erb A sequence.
Aberrant forms of
nuclear hormone receptors are involved in several forms of cancer. The
v-erbA oncogene, for example, is a mutated copy of the host
cell gene (c-erbA) for
TR
-1(24, 25) . V-ErbA has sustained a
number of mutations relative to its T
R
progenitor (Fig. 1); as a result, v-ErbA is impaired in hormone binding and
in transcriptional activation and appears to function in the cancer
cell as a constitutive
repressor(26, 27, 28, 29, 30) .
In addition v-ErbA exhibits a distinct DNA specificity from that of
c-ErbA
; although both v-ErbA and c-ErbA efficiently recognize
AGGTCA half-sites, only the c-ErbA polypeptide also recognizes AGGACA
half-sites(18, 20, 31, 32) . This
alteration in DNA specificity is due, in part, to the presence of a
serine at codon 61 in the P-box of v-ErbA that is a glycine in c-ErbA (Fig. 1) and is consistent with structural studies that place
codon 61 proximal to the fourth base of the
half-site(12, 13, 14, 18, 19, 20, 31) .
Unexpectedly, however, additional receptor domains N-terminal to the
zinc-finger also strongly influence the half-site specificity of the
ErbA polypeptides(18, 20, 32) . The amino
acid at codon 32, a cysteine in v-ErbA and a tyrosine in c-ErbA (Fig. 1), is particularly critical, and only by changing the
identity of both codon 32 and codon 61 is it possible to exchange the
DNA recognition properties of v-ErbA and
c-ErbA(18, 32) .
The analogous N-terminal domains of RARs, RXRs, and retinoid orphan receptors also play a critical role in DNA half-site specificity (32, 33, 34, 35, 36) . Unfortunately, this region of the nuclear receptors has not been modeled in structural studies, leaving the precise molecular basis of its actions unclear. Unlike the zinc-finger domain, it is doubtful that the N terminus makes direct base-specific contacts with the DNA half-site(14) .
We describe here a detailed dissection of the effects of these N-terminal receptor determinants on half-site recognition. We report that the contributions of the N terminus to DNA recognition specificity are at least as potent as those of the zinc-finger domain itself and can broadly influence sequence recognition over multiple locations in the half-site; we propose a conformational model to explain these effects. We also demonstrate that interactions with RXR, a heterodimeric partner for many nuclear hormone receptors, has a differential effect on the DNA half-site recognition properties of v-ErbA and c-ErbA, indicating that processes influencing heterodimer formation by nuclear hormone receptors may also alter their target gene specificity.
Figure 2:
Determination of the half-site
recognition specificity of the v-ErbA and TR/c-ErbA
proteins by electrophoretic mobility shift assay. A series of
radiolabeled oligonucleotide DR+4 probes, consisting of single
base substitutions in the consensus half-site element TAAGGTCA, were
tested for the ability to bind to T
R/c-ErbA (A) or
v-ErbA (B) in an electrophoretic mobility shift assay. Each
base substitution is identified, below the panel, as to location and
the nature of the base substitution; thus in our nomenclature,
-2A represents a AAGGTCA half-site sequence and +2T refers
to a TAATTCA half-site sequence, whereas +2G (or +4T, etc.)
is simply the consensus TAAGGTCA half-site. The locations of free
probe, probe bound to receptor monomers (arrowhead), and probe
bound to receptor dimers (arrows) are indicated. An asterisk indicates a non-specific probe-derived band observed
even in the absence of protein (data not
shown).
Each of the 25 possible
permutated half-sites were synthesized as a direct repeat with a 4-base
spacer (DR-4), an arrangement found in many physiological response
elements for c-ErbA(8, 9) . We then tested the ability
of c-ErbA or v-ErbA to bind to these elements in vitro using
an electrophoretic mobility shift assay. An autoradiograph of a typical
assay is shown in Fig. 2, whereas the results from multiple
assays were quantified, averaged, and are displayed in Fig. 3.
Several observations are of note. (a) Although c-ErbA bound
best to the consensus TAAGGTCA half-site, an extensive set of
variations of this consensus were also strongly recognized by the
receptor. This was particularly evident for the base substitutions at
half-site positions -2, -1, +1, +4, +5, and
+6, most of which were accommodated by c-ErbA with relatively
modest effects on binding. (b) In contrast, most substitutions
in the consensus half-site at positions +2 and +3 were highly
destabilizing for c-ErbA binding (with the exception of the +3T
substitution, which retained moderate TR binding).
Figure 3:
Half-site recognition properties of
TR/c-ErbA and v-ErbA proteins: quantified data.
Electrophoretic mobility shift assays were performed as in Fig. 2using either the c-ErbA protein (panel A) or the
v-ErbA protein (panel B). The total radiolabel migrating as
bound complex was quantified and is expressed relative to binding of
the consensus TAAGGTCA half-site DR+4 element (= 100%). At
least two separate experiments were performed for each oligonucleotide
substitution; the average and standard deviation are plotted. To
facilitate comparison of the c-ErbA and v-ErbA binding specificity, the
results were also expressed as a difference plot (binding by v-ErbA
minus binding by c-ErbA (panel C).
The v-ErbA protein demonstrated a very different, and generally more restrictive, DNA specificity pattern from that of c-ErbA. V-ErbA bound the consensus half-site element relatively strongly. However, v-ErbA bound weakly, or not at all, to many of the substitutions at +4, +5, and +6 that were strongly bound by c-ErbA (Fig. 3B; presented as a difference plot in Fig. 3C). v-ErbA, in common with c-ErbA, also failed to bind to most of the base substitutions at positions +2 and +3. However, substitutions at -1 proved an intriguing exception and were recognized as well, or better, by v-ErbA as by c-ErbA. In fact, our results indicate that the optimal v-ErbA half-site sequence was TGAGGTCA, in contrast to the optimal c-ErbA site, TAAGGTCA, but consistent with results obtained by a randomized binding site selection procedure(42) . Half-site sequences that exhibited differential recognition by v- and c-ErbA in these direct DNA binding studies were subsequently re-analyzed in DNA competition experiments with essentially the same results (Table 1). We conclude that differences in DNA recognition between v-ErbA and c-ErbA extend throughout the DNA half-site and are manifested as a more restrictive v-ErbA recognition of bases +3, +4, +5, and +6, and a broader recognition of bases at -1.
Figure 4: Half-site recognition properties of mutant v-Erb A proteins. Electrophoretic mobility shift assays were performed as in Fig. 2and Fig. 3, but using the C32Y v-Erb A N-terminal domain mutant (A), the S61G/T78K v-Erb A zinc-finger mutant (B), or the triple C32Y/S61G/T78K v-Erb A mutant (C). The total radiolabel migrating as bound complex was quantified and is expressed relative to binding of the consensus TAAGGTCA half-site DR-4 element (100%). The same results, expressed as a difference plot (binding by wild-type v-Erb A minus binding by each mutant v-Erb A), are also shown (D-F). At least two separate experiments were performed for each oligonucleotide substitution; the average and standard deviation are plotted.
Compared to the zinc-finger swap, just changing codon 32 in the v-ErbA N terminus to that of c-ErbA (a mutant denoted C32Y; Fig. 1) had a greater overall effect on DNA recognition, significantly enhancing recognition of the +3T, +4C, +4G, +5G, +5T, +6C, +6G, and +6T substitutions, and decreasing binding to -1G (Fig. 4A). In these aspects the specificity of the C32Y v-ErbA mutant nearly paralleled that of c-ErbA (compare Fig. 3A and 4A, and compare the difference plots in Fig. 4D and 3C). Nonetheless, the C32Y mutation alone failed to confer a strong, c-ErbA-like recognition of the +4A or +5A substitutions. Indeed, a triple v-ErbA mutation, denoted C32Y/S61G/T78K, significantly enhanced recognition of the +4A and +5A substitutions and yielded an overall DNA recognition pattern indistinguishable from that of c-ErbA (compare Fig. 4C and 3A, and compare the difference plots in Fig. 4F and 3C). Parallel results were obtained in DNA competition experiments (Table 1). We conclude that virtually all the differences in DNA recognition noted for v- and c-ErbA are accounted for by amino acid differences in the N terminus and zinc-finger domains of these proteins, and that the N-terminal domain plays a dominant role in this phenomenon.
Figure 5:
Effects of heterodimer formation with RXR
on DNA half-site specificity of TR/c-ErbA and v-ErbA.
Electrophoretic mobility shift assays were performed as in Fig. 2and 3, but using RXR
c-ErbA heterodimers (panel
A) or RXR
v-ErbA heterodimers (panel B) in place of
the homodimers previously described. The total radiolabel migrating as
bound complex was quantified and is expressed relative to binding of
the consensus TAAGGTCA half-site DR+4 element (= 100%). At
least two separate experiments were performed for each oligonucleotide
substitution; the average and standard deviation are plotted. To
facilitate comparison, the probe bound by the RXR
ErbA heterodimer
in each panel (open bars) is compared to the probe bound by
the analogous ErbA homodimer (closed bars). A difference plot
(binding by RXR
v-ErbA minus binding by c-ErbA
RXR) is also
shown (panel C).
Surprisingly, formation of heterodimers with RXR had non-identical effects on v-ErbA relative to c-ErbA. In common with c-ErbA, heterodimerization of v-ErbA with RXR enhanced binding to half-site substitutions at the -2, +4, +5, and +6 positions (Fig. 5B). In contrast, however, heterodimerization with RXR had much less of an effect on recognition of the +1, +2, and +3 half-site positions by v-ErbA than by c-ErbA (Fig. 5B). As a consequence, certain differences between v- and c-ErbA in half-site recognition (at positions -1, +5, and +6) were muted by heterodimerization with RXR, whereas novel differences in half-site recognition (at positions +1, +2, and +3) were created (compare the difference plots in Fig. 5C and Fig. 3C).
How can this promiscuity of DNA sequence recognition be reconciled with the specific base contacts seen in structural studies? Studies with non-cognate elements suggest that the precise receptor-DNA contacts can vary with modest changes in orientation or conformation of the receptor accommodating non-consensus sequences(12, 45, 46) . It is also likely that the specific base pair-receptor contacts, typically hydrogen bonds or van der Waals' interactions, contribute only modestly to the change in free energy of receptor upon DNA binding, compared with the stronger, sequence-independent ionic contacts between the receptor and the DNA backbone(12, 45, 46) . In fact, base-specific interactions may function chiefly to prevent receptor binding to inappropriate DNA sequences (by ``clashing'' sterically or otherwise with the amino acids in the receptor P- and A-boxes) rather than by contributing positively to binding by stabilizing contacts to the correct base pairs.
How might the N-terminal region
be capable of such effects? The N-terminal domain has not been included
in the structural studies reported to date, and therefore its precise
actions must remain speculative. It appears unlikely, however, that the
v-ErbA N terminus can make direct contact with the bases in the AGGTCA
core hexanucleotide, precluding a direct effect on base pair
recognition. We also disfavor a model in which the N terminus mediates
its effects indirectly by altering the conformation of the target DNA;
although both v-ErbA and c-ErbA induce DNA bending, the extent of the
bend is independent of the nature of the N terminus. ()Instead, we suggest that the receptor N terminus acts
indirectly by influencing the tertiary conformation of the zinc-finger
P- and A-box helices. We propose that although the P- and A-box amino
acids make the actual discriminatory contacts with bases in the DNA,
the N terminus can alter the precise position or orientation of the
zinc-finger recognition helices and thus can define in a global fashion
the exact base contacts made, and the stringency of the protein-DNA
interaction. Perhaps, due to these interactions with the N terminus,
the P-box helix of c-ErbA is slightly tilted away from the 3` end of
the half-site relative to the P-box helix of v-ErbA, resulting in the
enhanced ability of c-ErbA to accommodate a broader range of base
substitutions over these positions. In fact, the zinc-finger domain is
known to exhibit some flexibility in its location relative to the DNA
major groove(45) . Furthermore, a conformational coupling
between the receptor N terminus and the zinc-finger domain is
consistent with previous mutagenesis studies, and with the apparent
proximity of these two domains, as suggested by the limited
crystallographic data available(14, 32) .