©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
DNA Recognition by Normal and Oncogenic Thyroid Hormone Receptors
UNEXPECTED DIVERSITY IN HALF-SITE SPECIFICITY CONTROLLED BY NON-ZINC-FINGER DETERMINANTS (*)

(Received for publication, November 1, 1995; and in revised form, January 18, 1996)

Catherine Judelson Martin L. Privalsky (§)

From the Section of Microbiology, Division of Biological Sciences, University of California, Davis, California 95616

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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), (^1)retinoid X receptors (RXRs), and the thyroid hormone receptors (T(3)Rs). Additional diversity is generated within individual receptor classes by the expression of multiple receptor isoforms; for example, T(3)Rs are encoded by two separate loci, alpha and beta(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 alpha-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 T(3)Ralpha-1(24, 25) . V-ErbA has sustained a number of mutations relative to its T(3)Ralpha 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-ErbAalpha; 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.


EXPERIMENTAL PROCEDURES

Source of Proteins and Oligonucleotide Probes

The wild-type v-ErbA, avian c-ErbAalpha-1, and avian RXR receptors were obtained as nuclear extracts from recombinant baculovirus-infected Sf9 cells(18) . The C32Y, S61G/T78K, and C32Y/S61G/T78K mutant v-ErbA polypeptides were isolated as glutathione S-transferase fusion proteins from Escherichia coli transfected with the appropriate pGEX recombinant plasmid vectors(32, 37) ; no significant differences were observed in the DNA binding specificities of ErbA proteins synthesized in the bacterial system compared to the same polypeptide encoded in the baculovirus system (data not shown). The different oligonucleotide probes employed in the binding experiments were synthesized on a Milligen Cyclone Plus using phosphoramidite chemistry. Each probe was created as two complementary oligonucleotides with four base overhangs (e.g. for the consensus, 5`-TCGAA TAAGG TCAAA TAAGG TCAGA G-3` and 5`-TCGAC TCTGA CCTTA TTTGA CCTTA T-3`), the oligonucleotides were annealed, and the double-stranded DNAs were radiolabeled by fill-in using [-P]dGTP and Klenow fragment of E. coli DNA polymerase.

DNA Binding Assays

Standard electrophoretic mobility shift assays were employed as described previously(18, 38) . Briefly, the radiolabeled oligonucleotide probe (typically 1-3.5 times 10^5 counts/min, approximately 20-30 ng of DNA) was incubated with the protein of interest in 15 µl of binding buffer (10 mM Tris-Cl, pH 7.5, 50 mM KCl, 5% glycerol, 13.3 mg/ml bovine serum albumin, and 133 µg/ml poly(deoxyinosinebullet deoxycytosine)) for 15 min at room temperature. Any protein-DNA complexes that formed were then resolved by electrophoresis through a 4.5% polyacrylamide, 0.12% bisacrylamide gel at 200 V for 75 min. The resulting electrophoretograms were dried, visualized by autoradiography, and quantified by use of a Betascan analyzer. In the absence of RXRs, c-ErbA and v-ErbA generally produced both monomeric and dimeric complexes, probably due to the low cooperativity with which the c-Erb Aalpha isoform forms homodimers(7) . Perhaps as a consequence of this low cooperativity, the ratio of monomers to dimers formed by a given ErbA protein did not significantly vary from one response element to another ( Fig. 2and quantification data not shown). Competition assays were performed by binding the receptor of interest to 27 ng of the radiolabeled consensus sequence in the presence of differing amounts (0, 125, 500, or 1000 ng) of the unlabeled competing DNA.


Figure 2: Determination of the half-site recognition specificity of the v-ErbA and T(3)R/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(3)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).




RESULTS

The Different DNA Binding Specificities of v-ErbA and c-ErbA Extend over Multiple Positions within the DNA Half-site

We wished to further explore the mechanisms by which the N terminus of the ErbA protein influences DNA recognition specificity. We first tested if the influence of the N-terminal and P-box amino acids on DNA recognition was localized to the fourth base position analyzed previously, or if their effects extended over additional positions in the half-site. We began with an optimized consensus half-site (TAAGGTCA), systematically altered each position to the alternative three bases, and tested the ability of each substituted DNA sequence to bind to ErbA. Our use of an 8-base half-site in the current study follows from the recent demonstration that alterations in bases 5` to the traditionally defined hexanucleotide half-site can also influence DNA recognition(21, 39, 40, 41, 42) . These 5` bases are probably recognized through interactions with the receptor A-box helix(14, 23) . To allow comparison with previous work, we number the original hexanucleotide half-site sequence as +1 thorough +6, and denote the two 5` bases as the -2 and -1 positions.

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 T(3)R binding).


Figure 3: Half-site recognition properties of T(3)R/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.



The N-terminal Domain, Not the Zinc-finger region, Plays the Dominant Role in Defining the Different Half-site Specificities of v- and c-ErbA

We next examined the relative contributions of the N-terminal and zinc-finger domains to the differing DNA half-site specificities of c- and v-ErbA. The zinc-finger domain of the nuclear hormone receptors mediates most of the known protein/base pair contacts (12, 13, 14) . Nonetheless, complete replacement of the v-ErbA zinc-finger domain with that of c-ErbA (a mutant denoted S61G/T78K, Fig. 1) produced only relatively modest effects on the half-site specificity of the v-ErbA protein (compare Fig. 3B and 4B). Most evident was an increase in v-ErbA binding to the +4A substitution, although some enhancement of binding to the +3T, +4C, +5A, +5G, +5T, and +6G substitutions was also observed. A difference plot (probe bound by wild-type v-ErbA minus that bound by the mutant; Fig. 4E) is also presented to allow comparison to Fig. 3C.


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.

RXR Heterodimerization Further Broadens the Half-site Recognition Properties of Both c-ErbA and v-ErbA, but in a Non-equivalent Fashion

Many nuclear receptors can form heterodimers with other members of the nuclear hormone receptor family (reviewed in Refs. 43 and 44). In fact, RXRbulletc-ErbA heterodimers exhibit higher overall DNA binding affinity and enhanced target gene activation than do c-ErbA homodimers, and may be the dominant form of c-ErbA in living cells(43, 44) . We therefore tested the effect of RXRbulletc-ErbA heterodimerization on DNA half-site specificity. Interestingly, heterodimerization with RXR further extended the already broad DNA recognition properties of c-ErbA to allow recognition of virtually all the single base substitutions tested. Base substitutions at -2, -1, +4, +5 and +6 that were moderately well recognized by c-ErbA homodimers were now strongly recognized by RXRbulletc-ErbA heterodimers (Fig. 5A). Furthermore, base substitutions at +1, +2, and +3 that exhibited no detectable binding by c-ErbA homodimers were now strongly recognized by the RXRbulletc-ErbA heterodimer (Fig. 5A). The ability of the RXRbulletc-ErbA heterodimer to bind to each permutated half-site is expressed relative to its ability to bind the consensus sequence; thus our results reflect actual alterations in sequence specificity. All binding experiments were performed in DNA probe excess, under conditions such that only the binding of receptor heterodimers was measured; no binding of RXR homodimers was detected under these conditions.


Figure 5: Effects of heterodimer formation with RXR on DNA half-site specificity of T(3)R/c-ErbA and v-ErbA. Electrophoretic mobility shift assays were performed as in Fig. 2and 3, but using RXRbulletc-ErbA heterodimers (panel A) or RXRbulletv-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 RXRbulletErbA heterodimer in each panel (open bars) is compared to the probe bound by the analogous ErbA homodimer (closed bars). A difference plot (binding by RXRbulletv-ErbA minus binding by c-ErbAbulletRXR) 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).


DISCUSSION

The c-ErbA Protein Exhibits a Broad Ability to Accommodate Half-site Variations That Diverge from the Optimal Consensus Sequence

Structural modeling indicates that the base pairs in the DNA half-site are contacted through a web of multiple interactions with amino acids in both the P- and A-box of the receptor(12, 13, 14) . These multiple protein-base-specific DNA contacts, both direct and through immobilized water molecules, might be expected to impose a strict DNA sequence specificity on these receptors. In practice, however, nuclear hormone receptors exhibit a relatively promiscuous DNA sequence specificity and conversely can accept a surprising variety of P- and A-box amino acid substitutions without abolishing DNA binding(18, 19, 20, 21, 22) . In fact, as demonstrated here, c-ErbA homodimers exhibit a near absolute specificity for the consensus half-site sequence only at positions +2 and +3 (TAAGGTCA), whereas a broad range of substitutions are permissible at nearly all other positions. Although obtained with artificial elements, these results are consistent with the considerable sequence variation found in naturally occurring response elements(8, 9) .

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.

v-ErbA Exhibits a Restrictive Half-site Specificity That Maps to a Single Base Change Outside of the Traditionally Defined DNA Binding Domain

Although structurally closely related to c-ErbA, the v-ErbA protein exhibits a much more narrow DNA sequence specificity over much of the half-site sequence. Intriguingly, although there is a difference in the P-box between v- and c-ErbA, at codon 61, alteration of this amino acid produces only modest effects on DNA recognition. In fact, in a crystallographic analysis(14) , this codon does not make any base-specific contacts with the consensus half-site, although it potentially can make unfavorable contacts with incorrect half-sites (19, 20, 22) . On the other hand, alteration of codon 32 in the N terminus of the receptor (and outside of the previously defined DNA binding domain) dramatically broadens the sequence recognition properties of v-ErbA to more closely approximate those of c-ErbA; mutation of both N-terminal and zinc-finger domains confers a fully c-ErbA specificity.

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. (^2)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) .

RXR Heterodimer Formation Alters the DNA Half-site Specificity of v- and c-ErbA, but in a Non-equivalent Manner

Many nuclear hormone receptors can bind to DNA both as homodimers, and as heterodimers with other members of the receptor family (reviewed in (43) and (44) ). RXRbulletc-ErbA heterodimers exhibit an increased overall affinity for DNA and elevated transcriptional activation properties compared to those of homodimers. We demonstrate here that heterodimerization with RXRs can also alter the half-site specificity of c-ErbA and v-ErbA. For c-ErbA, this is manifested as a further broadening of half-site specificity, including a novel recognition of half-site substitutions (at +2 and +3) that are not detectably bound by c-ErbA homodimers. Perhaps reflecting a reciprocal phenomenon, the inhibitory effects of receptor P-box amino acid substitutions on DNA binding can also be counteracted by RXR heterodimerization. (^3)It is possible that the inherently high overall affinity for DNA of the RXRbulletc-ErbA heterodimer simply overwhelms the destabilizing effects of these response element or amino acid substitutions. However, it is striking that recognition of certain half-site substitutions is relatively unaffected by RXR heterodimerization, whereas recognition of others is greatly enhanced. This was particularly evident for v-ErbA, which as an RXR heterodimer exhibits significantly enhanced binding to base substitutions at +4 to +6, but not to substitutions at +2 and +3. Notably, the N-terminal domain of ErbA forms part of the heterodimer interface with RXR(14) ; perhaps RXR influences half-site specificity in the heterodimer through this interaction with the ErbA N terminus, altering, in turn, the conformation of the ErbA zinc-finger domain.

v-ErbA, c-ErbA, and Target Genes in the Cancer Cell

It is intriguing that the DNA recognition properties of the v-ErbA oncoprotein homodimer are both broader (at -1) and more narrow (at +4, +5, and +6) than those of the c-ErbA/T(3)R progenitor. v-ErbA acts in the cancer cell by interfering with erythroid differentiation, apparently by binding to and repressing target genes normally activated by c-ErbA, RARs, and possibly RXRs(26, 27, 28, 29, 30, 38, 47) . The extended recognition specificity of v-ErbA at the -1 position may contribute to its ability to compete for, and repress target genes for heterologous receptors such as RARs. Conversely, the more narrow specificity of v-ErbA at +4 to +6 may prevent repression of c-ErbA-responsive genes critical for cell proliferation(18) . It is intriguing in this regard to note the different effects of RXR heterodimerization on the DNA specificity of c-ErbA and v-ErbA. By enhancing v-ErbA recognition of base substitutions at +4 to +6, RXR heterodimerization would permit v-ErbA to bind to, and repress certain sets of c-ErbA target genes that are not repressed by v-ErbA homodimers. Conversely, by enhancing c-ErbA, but not v-ErbA, recognition of half-site substitutions at +2 and +3, RXR heterodimerization would create novel targets for c-ErbA activation that cannot be repressed by v-ErbA homo- or heterodimers. At the high levels of v-ErbA expression observed in the cancer cell, it is likely that both homodimeric and heterodimeric v-ErbA species co-exist, and that the precise balance between these species has an important influence in determining the effects of v-ErbA on the neoplastic phenotype.


FOOTNOTES

*
This work was supported by United States Public Health Service/National Institutes of Health Grant CA53394. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 916-752-3013; Fax: 916-752-9014; mlprivalsky{at}ucdavis.edu.

(^1)
The abbreviations used are: RAR, retinoid acid receptor; RXR, retinoid X receptor; T(3)R, thyroid hormone receptor.

(^2)
J. Hamaguchi and M. L. Privalsky, unpublished data.

(^3)
C. Nelson, C., S. C. Hendy, J. S. Faris, and P. J. Romaniuk, manuscript in preparation.


ACKNOWLEDGEMENTS

We are especially grateful to H. W. Chen for providing reagents, assistance, and helpful discussion, to J. Hamaguchi for providing Sf9 cell extracts, and to P. Romaniuk for sharing information and many excellent suggestions prior to publication.


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