BACKGROUND
the nuclear receptors are a class of transcriptional regulatory proteins that function in physiology and development in animals  <cit> .
they are conserved from sponges to mammals  <cit> , but this class of proteins has seen significant amplification in the number of genes and elaboration of the amino acid sequences in nematode species  <cit> : while humans have  <dig> predicted nuclear receptor genes and drosophila melanogaster has  <dig>  caenorhabditis elegans and c. briggsae each have over  <dig>  most nuclear receptors have two major conserved functional domains: the dna-binding domain  and the ligand-binding domain .
these two domains are normally found in that order from n-terminus to c-terminus, although there are unorthodox nuclear receptors that have no apparent lbd, such as knirps and odr- <dig>  <cit> , and some that have no dbd, such as dax <dig> and shp  <cit> .
the nuclear receptor dbd is responsible for binding to a specific dna response element  in the promoters of target genes, relatively weak dimerization contacts, and nuclear localization  <cit> .
the dbd consists of two c- <dig> zinc finger structures at its n-terminal portion and a c-terminal extension , which is sometimes grouped with the "hinge region" between the dbd and lbd .
two key regions in the dbd have been shown to play major roles in nre binding specificity by making direct and water-mediated hydrogen bond contacts with specific dna bases.
the p box consists of six conserved amino acids in an alpha-helical region that begins in the c-terminal portion of the first zinc finger  <cit> .
the a box is a poorly-conserved c-terminal portion of the cte, and is thought to play a role in modulating dna specificity, although this function is not as well understood  <cit> .
a few other conserved amino acids c-terminal to the p box also contact dna bases, and several other amino acids spread throughout the dbd make contact with the dna phosphodiester backbone.
two other regions of the dbd play key roles in dimerization, although some nuclear receptors are unable to dimerize without the strong dimerization function of the lbd.
in the dbd, the d box, located in the n-terminal portion of the second zinc finger, and the alpha-helical t box of the cte have been demonstrated to play direct roles in dimerization between dbd partners  <cit> .
in most cases, nuclear receptors bind to cognate nre's via the dbd, however there are examples of nuclear receptors that can provide some regulatory function without binding to dna through their dbd  <cit> .
the lbd was named based on its function in binding lipophilic hormones in vertebrates and insects  <cit> .
however, some nuclear receptors, such as nurr <dig>  are clearly ligand-independent  <cit>  and many more are believed to be .
the lbd functions in binding coactivator and corepressor proteins, which in turn interact with other transcription factors or modulate chromatin structure in order to affect changes in transcriptional rates of target genes.
the lbd also plays a major role in dimerization and nuclear localization  <cit> .
the nr2e subclass of nuclear receptors is evolutionarily conserved in both vertebrates and invertebrates.
the founding member, tailless , was first identified by a genetic approach in drosophila, where it functions in anterior-posterior pattern formation and central nervous system development  <cit> .
the vertebrate ortholog, tlx , functions in limbic system and adult neural stem cell development  <cit> .
the c. elegans ortholog, nhr- <dig>  appears to function in various aspects of hypodermal and neural development and organogenesis  <cit> .
the drosophila gene dissatisfaction  encodes a possible tll paralog  that functions in the development of sex-specific neural and muscular features  <cit> .
the final nr2e gene member is represented by pnr  in vertebrates, and fax- <dig>  in c. elegans.
in humans and mice, pnr functions in specifying photoreceptor identity in the retina  <cit> , while in c. elegans, fax- <dig> functions in specification of interneuron identity  <cit> .
one phylogenetic analysis of pnr and fax- <dig> family members from different species suggested a possible early divergence between the two genes, with subsequent loss of fax- <dig> from vertebrate lineages and loss of pnr from nematode lineages  <cit> .
the identification of dna binding specificities for different nuclear receptors has been fruitful in predicting downstream target genes and in understanding the molecular logic of how dbd amino acid sidechains interact with nre bases and the phosphodiester backbone.
studies on drosophila tll and vertebrate tlx binding specificities suggested that the nr2e receptors bind to half-sites with a distinctive sequence, aagtca, instead of the more common nre half-site aggtca  <cit> .
dsf also preferentially binds aagtca half-sites, and like tll and tlx can bind either as a monomer or dimer  <cit> .
initial dna-binding studies showed that pnr also bound to aagtca half-sites, but only as a dimer bound to directly-repeated half-sites separated by a single base-pair   <cit> .
thus an adenine residue at the second position of the nre half-site has been suggested to be a defining feature of nr2e half-site specificity.
examination of the p boxes of nr2e subclass members suggested a possible amino acid that could mediate the difference in specificity between this subclass and other nuclear receptors.
the nuclear receptors rxr, ngf1-b, rar, reverb, ecr and usp all preferentially bind aggtca half-sites and have p boxes with the amino acid sequence cegckg  <cit> .
in contrast, the nr2e class members all have the p box sequence cgcg.
the fifth amino acid in the p box, lysine- <dig> , has been shown to make direct hydrogen bond contacts to the base at the second position of the nre in co-crystal structures for nuclear receptors that bind the aggtca half-site  <cit> .
therefore, it has been suggested that lysine- <dig> may confer specificity for binding a guanine residue at nre position two.
in contrast, a serine or alanine at dbd position  <dig>  as found in the nr2e nuclear receptors, may confer specificity for binding an adenine base at nre position two  <cit> .
a structural study that examined rxr-rar binding to dr <dig> dimeric aggtca sites identified the amino acids that make direct and water-mediated contacts to hexamer bases  <cit> .
guanine- <dig> of the half-site was contacted by tyr- <dig>  glu- <dig>  and lys- <dig> of rxr and rar.
these amino acids also made contact to other half-site bases, and other amino acids in the dbd and cte made direct and water-mediated contacts to other half-sites bases and to the phosphodiester backbone.
tyr- <dig> is common to all nr2e dbd's  and many other nuclear receptors that bind aggtca half-sites, thus it is unlikely to mediate differences in nre binding specificity.
given these data, the amino acid present at dbd position  <dig>  in addition to the previously mentioned position  <dig>  could mediate binding specificities at position two of the nre half-site.
an understanding of dna binding specificity of the nr2e class of nuclear receptors requires a comparison of the dna-binding properties of nr2e <dig>  and nr2e <dig>  subclass members.
we have examined the dna binding properties of the fax- <dig> nuclear receptor of c. elegans, a pnr ortholog, and compared them to the nhr- <dig> nuclear receptor of c. elegans, a tll ortholog.
we find that unlike nr2e <dig> subclass members, fax- <dig> will bind half-sites with the sequence angtca and does not bind to monomeric sites.
we tested whether the conserved asn- <dig> in nr2e <dig> subclass members was a key determiner of specificity for binding to nre half-site position two.
changing asp- <dig> of nhr- <dig> to asn- <dig> was sufficient to change the binding preference of nhr- <dig> from aagtca half-sites to aggtca half-sites.
in contrast, changing asn- <dig> of fax- <dig> to asp- <dig> was not sufficient to change the dna-binding preference, suggesting that the role of dbd position  <dig> in binding specificity is context-dependent.
RESULTS
fax- <dig> and orthologs have a distinct p box
as a first step toward identifying amino acids that might confer subclass-specific dna binding activities, we aligned amino acid sequences of the dbd's, including the cte, of fax- <dig> and orthologs from vertebrate and invertebrate species and compared them to equivalent sequences from other nr2e class members .
this analysis included five members of the nr2e <dig> subclass  and six members of the nr2e <dig> subclass .
due to its similar dna-binding activity, dsf  was grouped with the nr2e <dig> subclass for this analysis.
subclasses and individual members within a subclass differed in the length of the d box.
while all nr2e <dig> subclass members had a d box containing eight amino acids, the nr2e <dig> subclass members had either nine , eleven  or twelve .
these differences might contribute to the different dimerization properties of tll as compared to pnr.
with fax- <dig> as the reference sequence, all the nr2e members evaluated were identical at  <dig> of  <dig>  amino acid residues, as expected for the strong evolutionary conservation observed for nuclear receptor dbd's.
the nr2e dbd's had variability within a subclass  at  <dig> of  <dig>  residues.
in some cases, the variability was limited to amino acids with similar chemical properties.
for example, position  <dig> can be leu, tyr or phe, all of which are hydrophobic residues.
the remaining nine amino acid residue positions of the dbd's were identical within a subclass, but different between subclasses.
for example, position  <dig> is a val in all members of the nr2e <dig> subclass and ile in all members of the nr2e <dig> subclass.
this last group identifies the amino acids that might play roles in differing dna-binding specificity of the nr2e <dig> subclass as compared to the nr2e <dig> subclass.
of particular note, position  <dig>  which is a p box residue predicted to contact the dna, has an asn in all members of the nr2e <dig> subclass and an asp in all members of the nr2e <dig> subclass.
fax- <dig> binds dr <dig> sites with relaxed specificity
we used the electrophoretic mobility shift assay  to test binding of bacterially-expressed and purified fax- <dig> protein .
binding of fax- <dig> protein to nre-containing dna  resulted in two discrete shifted bands, which were inhibited and super-shifted by the addition of anti-fax- <dig> antiserum .
the relative intensities of these two bands varied somewhat from experiment to experiment.
the lower band was neither preferentially reduced by competitor dna containing a monomeric binding site, nor was it preferentially observed in experiments using a monomeric binding sites , indicating that the two bands probably represent two different topological states of protein-dna complexes  rather than monomeric and dimeric protein-dna complexes.
because both bands showed similar behavior in competition and supershift experiments, we have summed both bands as being indicative of fax-1-dna binding throughout this paper.
fax- <dig> protein produced no discernible shifted products at these locations when allowed to bind negative control dna carrying a dimeric aattca sequence .
the first column indicates the name used to refer to each sequence in the text.
the second column shows the actual sequence tested.
the third column indicates the percentage of the labeled dna that was shifted to high mobility in emsa experiments using fax- <dig> protein.
the fourth column indicates standard deviation for each set of emsa experiments summarized.
we evaluated the significance of the data using one-way anova and fisher 95% confidence intervals for all pairwise comparisons.
dr1a and dr1g were not significantly different from each other.
dr1a was significantly different from all other sequences tested .
the difference between dr1g and dr1c or dr1t fell just outside the range of statistical significance.
binding data for mon <dig>  mon <dig>  dr3a, and drnc sites were not significantly different from each.
pairwise differences between hrws or hrsw and mon <dig> or mon <dig> were significant .
because the fax- <dig> vertebrate ortholog, pnr, has been shown to bind aagtca dimeric sites separated by one base-pair   <cit> , we first tested whether fax- <dig> would display the same binding activity.
fax- <dig> protein bound to dna containing aagtca dimeric sequences , and this binding could be competed with similar effectiveness by dna containing aagtca dr1a dimers or aggtca dr1g dimers, but not negative control aattca dimers .
thus fax- <dig> can bind aagtca dimers specifically, similar to pnr and tll.
we tested whether fax- <dig> could discriminate among different nucleotides at the second position, as tll does  <cit> .
unlike tll, fax- <dig> bound to aggtca dr1g dimers with avidity similar to that of aagtca dr1a dimers .
furthermore, fax- <dig> also bound dimers with pyrimidines in the second position: specific binding, although significantly weaker, was also observed with acgtca and atgtca dimers .
unlabeled dr1a dna competed more effectively for binding to dr1c sites than did unlabeled dr1c dna .
therefore, in contrast to tll and tlx, fax- <dig> does not discriminate among different nucleotides at the second hexamer position, although it binds purines more strongly than pyrimidines.
fax- <dig> does not bind monomeric hexamers but can bind a weak site adjacent to a strong site
we tested next whether fax- <dig> could bind both a single copy of the hexamer and a dimeric copy of the hexamer, like tll, tlx, and dsf, or could bind only a dimeric copy of the hexamer, like pnr.
when challenged to bind dna containing only a monomeric copy of the aagtca hexamer , fax- <dig> showed negligible binding that was not significantly different from negative controls .
consistent with this result, unlabeled dna containing monomeric aagtca sequences competed very poorly for binding to labeled aagtca dr1a or aggtca dr1g dimeric sequences .
in contrast to the results for monomeric sites, when we placed a predicted weak binding site  in register for dr <dig> binding with a strong site , creating heteromeric repeats in either of the two possible orders , we found weak, but significant, binding by fax- <dig> .
this binding was approximately 1/ <dig> as strong as binding to atgtca dimers and 1/ <dig> as strong as binding to aagtca dimers , and significantly different from drnc controls.
these data are consistent with a model where fax- <dig> binds as an obligate dimer, but binding to a strong site can facilitate binding to an adjacent weaker site through cooperativity.
fax- <dig> can bind dr <dig> sites but not dr <dig> sites
nuclear receptor dimerization confers limitations on the amount of separation between the two hexamer half-sites that will be tolerated by a given protein  <cit> .
our data show that fax- <dig> can bind dr <dig> sites, which separate the two hexamers with a single base-pair, similar to tll and pnr.
we tested whether increasing the separation between the two hexamers would influence the strength of fax- <dig> binding.
when aagtca hexamers that are separated by two base-pairs  were tested, fax- <dig> could bind these sequences, albeit not as strongly as dr <dig> sites .
the strength of binding, as measured by emsa, was similar to the strength of binding to acgtca dimers and about half that observed with aagtca dimers .
these data show that fax- <dig> prefers dr <dig> sites, but can also bind dr <dig> sites.
when we further increased the separation of the two aagtca hexamers to three base-pairs , we were no longer able to detect binding by fax- <dig> .
these data demonstrate that fax- <dig> dimerization requires relatively close proximity of the two hexamers, and also serve to reinforce the conclusion that fax- <dig> is unable to bind as a monomer.
dbd amino acid  <dig> partially mediates dna-binding specificity in fax- <dig> and nhr-67
in order to investigate differences between nr2e <dig> and nr2e <dig> subclass dna-binding properties and to allow for mutagenesis of the subclass-specific dbd amino acid  <dig>  we constructed fax- <dig> dbd::gal <dig> activation domain and nhr- <dig> dbd::gal <dig> activation domain fusion genes and tested their ability to activate transcription from promoters that carry different potential binding sites using the yeast one-hybrid system  <cit> .
as expected from our emsa analysis, the fax- <dig> dbd conferred significant β-galactosidase activity in one-hybrid studies with both aagtca dr1a and aggtca dr1g dimers in this assay , but did not with an aagtca monomer .
also consistent with previous results for the nr2e <dig> subclass  <cit> , the nhr- <dig> dbd conferred significant β-galactosidase activity with aagtca dr1a dimers , but not with aggtca dr1g dimers .
these data demonstrate that for fax- <dig> and nhr- <dig>  the dbd alone is sufficient to confer at least some half-site recognition and dimerization properties, and that the modality of binding inferred from our one-hybrid experiments yields data that are consistent with our direct assessment of fax- <dig> binding activity by emsa and the published direct assessment of tll and tlx binding  <cit> .
therefore, testing dna binding of mutated versions of fax- <dig> and nhr- <dig> by one-hybrid should yield data that are indicative of altered binding preference in vitro.
curiously, binding of fax- <dig> to aggtca dr1g dimers gave much stronger quantitative results than aagtca dr1a dimers in this assay, in contrast to the expected quantitative results from our emsa studies, which showed similar binding to both dimeric sites.
this difference may be due to fax- <dig> protein sequences that were not present in the fax- <dig> dbd construct or due to artifacts related to dna sequence context in the reporter constructs or yeast cellular environment.
given this result, and because of the indirect nature of the one-hybrid assay, we have not drawn conclusions about relative binding strength based on quantitative comparisons using this assay.
asn- <dig> of the fax- <dig> dbd is the only amino acid in the p box that is identical in all known nr2e <dig> subclass members and is different from the asp- <dig> that is consistently found in all known nr2e <dig> subclass members, including nhr- <dig> .
because nhr- <dig> and tll discriminate between nre half-sites that differ at the second position and fax- <dig> does not, we hypothesized that the difference between the different dna-binding specificities might depend on which amino acid is present at position  <dig>  this hypothesis is supported by structural data that show water-mediated hydrogen-bond contacts between this amino acid and the second position of the nre for nuclear receptors that bind dr <dig> dimers  <cit> .
to test this hypothesis, we mutated the fax- <dig> dbd in the fax- <dig> dbd::gal <dig> activation domain one-hybrid construct from the wild-type codon for asn to a codon for asp.
mutating the fax- <dig> dbd from asn- <dig> to asp- <dig> significantly increased the β-galactosidase activity observed in one-hybrid studies with aagtca dr1a sites approximately  <dig> -fold .
however, the mutation did not significantly decrease or abolish the activity observed with aggtca dr1g sites .
therefore, converting asn- <dig> to asp- <dig> in the context of a fax- <dig> dbd was not sufficient to change the inferred binding specificity to that of nhr- <dig>  due to the caveats in interpreting quantitative results from one-hybrid studies, it is unclear whether the asn to asp change results in increased binding of fax- <dig> to aagtca dr1a sites, although our results are consistent with that possibility.
more convincing results were obtained when we performed the reciprocal experiment in which the nhr- <dig> dbd was mutated from asp- <dig> to asn- <dig>  this mutation eliminated detectable β-galactosidase activity in one-hybrid studies with aagtca dr1a sites , and produced significant β-galactosidase activity in one-hybrid studies with aggtca dr1g sites .
from these data, we infer that while wild-type nhr- <dig> with asp- <dig> can bind aagtca sites but not aggtca sites, mutant nhr- <dig> with asn- <dig> can bind aggtca sites but not aagtca sites.
thus this single amino acid change had the effect of completely reversing the modality of nhr- <dig> binding activity.
in this context, an asn- <dig> changed the preference of binding at nre position two from adenine to guanine.
these results indicate that the amino acid present at position  <dig> in the dbd of nhr- <dig>  in particular, and perhaps nr2e nuclear receptors in general, is a mediator of dna binding specificity.
discussion
our findings demonstrate that fax- <dig> binds angtca half-sites that are separated by one base-pair and can bind aagtca half-sites that are separated by two base-pairs.
this contrasts it with tll and nhr- <dig>  which display a strong preference for aagtca half-sites.
fax- <dig> binds more strongly to sites that have a purine at the second position on this strand, but does not distinguish between aagtca and aggtca.
amino acid  <dig> of the dbd is known to contact position  <dig> of the nre, and it has been proposed that having a ser or ala at this position rather than a lys may allow for recognition of aagtca instead of aggtca half-sites  <cit> .
all known nr2e class receptors, including fax- <dig> and nhr- <dig>  have a ser or ala at position  <dig>  suggesting that they may be able to bind the aagtca sequence via this amino acid residue.
however, we have shown that a difference in dna-binding specificity exists between members of the nr2e <dig> and nr2e <dig> subclasses.
in the context of an nhr- <dig> dbd, the ability to bind guanine at the second position of the nre hexamer is conferred, in part, by the asn- <dig> found in all nr2e <dig> members, while the ability to bind adenine at the second position of the nre hexamer is conferred by the asp- <dig> found in nr2e <dig> members.
despite the important role of dbd position  <dig>  it is also clear that the amino acid at this position is not sufficient to confer a particular mode of binding specificity.
other amino acids in the dbd, perhaps others that are consistently different between classes, must also contribute to nre binding specificity.
an example of another amino acid residue that could participate in subclass specificity is position  <dig> of the dbd, which is consistently gln in nr2e <dig> and arg in nr2e <dig> and is predicted to make phosphate backbone contacts with the dna .
by analogy to co-crystal structures for rar-rxr heterodimers bound to dr <dig> sites  <cit> , asn- <dig> would be predicted to make water-mediated hydrogen bonds to the pyrimidine bases on the strand complementary to the adenine or guanine at the second position of the nre.
the implication of this comparison is that asp binds most favorably to thymine on the complementary strand, while asn binds favorably to cytosine and may be able to interact favorably with any of the four bases, depending on context.
asn can make a greater variety of h-bond contacts than asp due to the presence of the amide donor and acceptor in the r group of asn in comparison to the acceptor-only carboxyl of asp.
this greater range of potential h-bonding may be a contributor to the relaxed specificity observed for nr2e <dig> subclass dna binding.
inferences drawn from our data, the co-crystal structure of nuclear receptors that bind dr <dig> sites, and general data on nuclear receptor-nre interactions allow us to develop a model for the remainder of fax-1-nre interactions.
in co-crystal structures, the last four bases of the hexamer nre half site, gtca, are contacted by hydrogen bonds from dbd amino acid  <dig> and  <dig>  usually lysarg or argarg  <cit> .
in all fax- <dig> and tll subclass members, these positions are lysarg.
therefore, it seems reasonable to suggest that the lysarg dipeptide sequence of fax- <dig> contacts the last four base-pairs of the nre.
however, these amino acids are apparently not sufficient to confer this binding preference since the steroid receptors also have the lysarg sequence, yet bind a very different half-site  <cit> .
these and other observations raise the important caveat that the geometry of nuclear receptor- dna binding can vary among classes  <cit> ; therefore the implications of our study may be limited to the nr2e class.
we have not explored specificity at the first position of the nre half-site, which might also be somewhat degenerate.
based on the inference drawn from the rar-rxr co-crystal on dr <dig> sites, we would expect that the adenine at the first position of the fax- <dig> nre is contacted by ser- <dig> and arg- <dig> of the dbd.
the former amino acid residue is generally a ser among nr2e <dig> subclass members and can be either a ser or ala among nr2e <dig> subclass members.
amino acid position  <dig>  found in the t box, is a conserved arginine in all nr2e nuclear receptors.
while the general conservation of protein-dna contacts among nuclear receptors for which it has been directly tested is fairly good, we cannot be certain that other amino acids don't also participate directly in fax-1-nre binding.
furthermore, in all cases, other amino acids can contribute to dna-binding specificity through conformation-specific contacts with the phosphodiester backbone  <cit> .
the definition of potential dna-binding sites allows us to predict candidate in vivo targets in the genome of c. elegans.
there are  <dig> argtca dr <dig> sites  in the c. elegans genome.
if we expand the list to include those sites that have a pyrimidine at the second position, angtca dr <dig> sites, the number grows to  <dig> potential binding sites.
these sites are not near any of the genes that are known to be dependent on fax- <dig> for their regulation .
fax- <dig> may regulate these genes indirectly via another transcriptional regulator, by heterodimerization with another nuclear receptor that binds a different half-site, or by binding to other transcription factors at the promoter by a non-nre-dependent mechanism.
in addition, there are approximately  <dig> argtca dr <dig> sites in the c. elegans genome, to which fax- <dig> dimers may be able to bind in vivo.
expanding the list to include those sites that allow fax- <dig> dimeric binding via heteromeric sites that contain a weak site and a strong site  increases the number of potential fax- <dig> binding sites in the genome to over  <dig>  the sequences upstream of ncs- <dig> and opt- <dig>  both of which are regulated by fax- <dig>  contain sites that match this more degenerate binding site .
with so many potential binding sites in the genome, other factors such as cognate coactivators or corepressors may help define the functional relevance of any particular candidate nre.
these observations suggest that members of the nr2e <dig> subclass are able to form dimers on a much wider variety of dimeric nre's , as compared to the more restrictive nr2e <dig> subclass .
however, this does not mean that nr2e <dig> subclass members bind more sites in vivo; nr2e <dig> subclass monomers may also bind monomeric aagtca sites, of which there are over  <dig> .
studies on the human fax- <dig> ortholog, pnr, have demonstrated a similar dna-binding activity  <cit> .
pnr was found to bind to dr <dig> aagtca nre's, but not to monomeric aagtca sequences.
an unbiased screen for sequences that bind pnr revealed that, like fax- <dig>  it can bind sequences that vary at positions two and three of the half-site hexamer  <cit> .
this study identified a preferred pnr binding site with the sequence agrtcaaarrtca, a sequence that is consistent with our analysis of fax- <dig> dna binding.
while almost all binding sites revealed by this strategy include the tca sequence at the 3' end of the half-sites, greater variability was observed for all three bases at the 5'end of the half-sites, including nre position three in addition to nre position two.
because glu- <dig> of rar and rxr directly contact nre half-site position three in the rar-rxr co-crystal, it may be that asn- <dig> of fax- <dig> and pnr also mediates the relaxed specificity at position three of the nre.
therefore, the strong evolutionary conservation of the nr2e <dig> subclass dbd reflects, at least in part, the constraint of an evolutionarily-conserved nre binding site.
whether the dna-binding activity that appears to be conserved between nematode and vertebrate members of the nr2e <dig> subclass translates into conserved patterns of gene regulation has yet to be investigated.
pnr acts as a repressor of the transcription of cone-specific genes and may activate the transcription of rod-specific genes during vertebrate photoreceptor development  <cit> .
the vertebrate orthologs of some of the targets of fax- <dig> regulation are also transcribed in vertebrate photoreceptor cells  <cit> , which allows for the possibility of conserved gene regulatory patterns.
CONCLUSIONS
our results define subclass-specific dna-binding specificities for the nr2e <dig> and nr2e <dig> subclasses, and suggest that binding preferences for the second nucleotide position of the dna half-site are partially mediated by amino acid  <dig> of the nr2e dbd.
the repertoire of potential dimeric binding sites is much larger for members of the nr2e <dig> subclass.
fax- <dig>  like nr2e <dig> subclass member pnr, binds nre's as a homodimer with a strong preference for dr <dig> sites.
therefore, dimerization behavior may be a second conserved difference between the nr2e <dig> subclass, members of which can bind as a monomer or homodimer, and the obligate dimer nr2e <dig> subclass.

 the nuclear receptors are a class of transcriptional regulatory proteins that function in physiology and development in animals. they are conserved from sponges to mammals, but this class of proteins has seen significant amplification in the number of genes and elaboration of the amino acid sequences in nematode species: while humans have predicted nuclear receptor genes and drosophila melanogaster has caenorhabditis elegans and c. briggsae each have over most nuclear receptors have two major conserved functional domains: the dna-binding domain and the ligand-binding domain. these two domains are normally found in that order from n-terminus to c-terminus, although there are unorthodox nuclear receptors that have no apparent lbd, such as knirps and odr-, and some that have no dbd, such as dax and shp. the nuclear receptor dbd is responsible for binding to a specific dna response element in the promoters of target genes, relatively weak dimerization contacts, and nuclear localization. the dbd consists of two c- zinc finger structures at its n-terminal portion and a c-terminal extension, which is sometimes grouped with the "hinge region" between the dbd and lbd. two key regions in the dbd have been shown to play major roles in nre binding specificity by making direct and water-mediated hydrogen bond contacts with specific dna bases. the p box consists of six conserved amino acids in an alpha-helical region that begins in the c-terminal portion of the first zinc finger. the a box is a poorly-conserved c-terminal portion of the cte, and is thought to play a role in modulating dna specificity, although this function is not as well understood. a few other conserved amino acids c-terminal to the p box also contact dna bases, and several other amino acids spread throughout the dbd make contact with the dna phosphodiester backbone. two other regions of the dbd play key roles in dimerization, although some nuclear receptors are unable to dimerize without the strong dimerization function of the lbd. in the dbd, the d box, located in the n-terminal portion of the second zinc finger, and the alpha-helical t box of the cte have been demonstrated to play direct roles in dimerization between dbd partners. in most cases, nuclear receptors bind to cognate nre's via the dbd, however there are examples of nuclear receptors that can provide some regulatory function without binding to dna through their dbd. the lbd was named based on its function in binding lipophilic hormones in vertebrates and insects. however, some nuclear receptors, such as nurr are clearly ligand-independent and many more are believed to be. the lbd functions in binding coactivator and corepressor proteins, which in turn interact with other transcription factors or modulate chromatin structure in order to affect changes in transcriptional rates of target genes. the lbd also plays a major role in dimerization and nuclear localization. the nr2e subclass of nuclear receptors is evolutionarily conserved in both vertebrates and invertebrates. the founding member, tailless, was first identified by a genetic approach in drosophila, where it functions in anterior-posterior pattern formation and central nervous system development. the vertebrate ortholog, tlx, functions in limbic system and adult neural stem cell development. the c. elegans ortholog, nhr- appears to function in various aspects of hypodermal and neural development and organogenesis. the drosophila gene dissatisfaction encodes a possible tll paralog that functions in the development of sex-specific neural and muscular features. the final nr2e gene member is represented by pnr in vertebrates, and fax- in c. elegans. in humans and mice, pnr functions in specifying photoreceptor identity in the retina, while in c. elegans, fax- functions in specification of interneuron identity. one phylogenetic analysis of pnr and fax- family members from different species suggested a possible early divergence between the two genes, with subsequent loss of fax- from vertebrate lineages and loss of pnr from nematode lineages. the identification of dna binding specificities for different nuclear receptors has been fruitful in predicting downstream target genes and in understanding the molecular logic of how dbd amino acid sidechains interact with nre bases and the phosphodiester backbone. studies on drosophila tll and vertebrate tlx binding specificities suggested that the nr2e receptors bind to half-sites with a distinctive sequence, aagtca, instead of the more common nre half-site aggtca. dsf also preferentially binds aagtca half-sites, and like tll and tlx can bind either as a monomer or dimer. initial dna-binding studies showed that pnr also bound to aagtca half-sites, but only as a dimer bound to directly-repeated half-sites separated by a single base-pair. thus an adenine residue at the second position of the nre half-site has been suggested to be a defining feature of nr2e half-site specificity. examination of the p boxes of nr2e subclass members suggested a possible amino acid that could mediate the difference in specificity between this subclass and other nuclear receptors. the nuclear receptors rxr, ngf1-b, rar, reverb, ecr and usp all preferentially bind aggtca half-sites and have p boxes with the amino acid sequence cegckg. in contrast, the nr2e class members all have the p box sequence cgcg. the fifth amino acid in the p box, lysine-, has been shown to make direct hydrogen bond contacts to the base at the second position of the nre in co-crystal structures for nuclear receptors that bind the aggtca half-site. therefore, it has been suggested that lysine- may confer specificity for binding a guanine residue at nre position two. in contrast, a serine or alanine at dbd position as found in the nr2e nuclear receptors, may confer specificity for binding an adenine base at nre position two. a structural study that examined rxr-rar binding to dr dimeric aggtca sites identified the amino acids that make direct and water-mediated contacts to hexamer bases. guanine- of the half-site was contacted by tyr- glu- and lys- of rxr and rar. these amino acids also made contact to other half-site bases, and other amino acids in the dbd and cte made direct and water-mediated contacts to other half-sites bases and to the phosphodiester backbone. tyr- is common to all nr2e dbd's and many other nuclear receptors that bind aggtca half-sites, thus it is unlikely to mediate differences in nre binding specificity. given these data, the amino acid present at dbd position in addition to the previously mentioned position could mediate binding specificities at position two of the nre half-site. an understanding of dna binding specificity of the nr2e class of nuclear receptors requires a comparison of the dna-binding properties of nr2e and nr2e subclass members. we have examined the dna binding properties of the fax- nuclear receptor of c. elegans, a pnr ortholog, and compared them to the nhr- nuclear receptor of c. elegans, a tll ortholog. we find that unlike nr2e subclass members, fax- will bind half-sites with the sequence angtca and does not bind to monomeric sites. we tested whether the conserved asn- in nr2e subclass members was a key determiner of specificity for binding to nre half-site position two. changing asp- of nhr- to asn- was sufficient to change the binding preference of nhr- from aagtca half-sites to aggtca half-sites. in contrast, changing asn- of fax- to asp- was not sufficient to change the dna-binding preference, suggesting that the role of dbd position in binding specificity is context-dependent.  fax- and orthologs have a distinct p box as a first step toward identifying amino acids that might confer subclass-specific dna binding activities, we aligned amino acid sequences of the dbd's, including the cte, of fax- and orthologs from vertebrate and invertebrate species and compared them to equivalent sequences from other nr2e class members. this analysis included five members of the nr2e subclass and six members of the nr2e subclass. due to its similar dna-binding activity, dsf was grouped with the nr2e subclass for this analysis. subclasses and individual members within a subclass differed in the length of the d box. while all nr2e subclass members had a d box containing eight amino acids, the nr2e subclass members had either nine, eleven or twelve. these differences might contribute to the different dimerization properties of tll as compared to pnr. with fax- as the reference sequence, all the nr2e members evaluated were identical at of amino acid residues, as expected for the strong evolutionary conservation observed for nuclear receptor dbd's. the nr2e dbd's had variability within a subclass at of residues. in some cases, the variability was limited to amino acids with similar chemical properties. for example, position can be leu, tyr or phe, all of which are hydrophobic residues. the remaining nine amino acid residue positions of the dbd's were identical within a subclass, but different between subclasses. for example, position is a val in all members of the nr2e subclass and ile in all members of the nr2e subclass. this last group identifies the amino acids that might play roles in differing dna-binding specificity of the nr2e subclass as compared to the nr2e subclass. of particular note, position which is a p box residue predicted to contact the dna, has an asn in all members of the nr2e subclass and an asp in all members of the nr2e subclass. fax- binds dr sites with relaxed specificity we used the electrophoretic mobility shift assay to test binding of bacterially-expressed and purified fax- protein. binding of fax- protein to nre-containing dna resulted in two discrete shifted bands, which were inhibited and super-shifted by the addition of anti-fax- antiserum. the relative intensities of these two bands varied somewhat from experiment to experiment. the lower band was neither preferentially reduced by competitor dna containing a monomeric binding site, nor was it preferentially observed in experiments using a monomeric binding sites, indicating that the two bands probably represent two different topological states of protein-dna complexes rather than monomeric and dimeric protein-dna complexes. because both bands showed similar behavior in competition and supershift experiments, we have summed both bands as being indicative of fax-1-dna binding throughout this paper. fax- protein produced no discernible shifted products at these locations when allowed to bind negative control dna carrying a dimeric aattca sequence. the first column indicates the name used to refer to each sequence in the text. the second column shows the actual sequence tested. the third column indicates the percentage of the labeled dna that was shifted to high mobility in emsa experiments using fax- protein. the fourth column indicates standard deviation for each set of emsa experiments summarized. we evaluated the significance of the data using one-way anova and fisher 95% confidence intervals for all pairwise comparisons. dr1a and dr1g were not significantly different from each other. dr1a was significantly different from all other sequences tested. the difference between dr1g and dr1c or dr1t fell just outside the range of statistical significance. binding data for mon mon dr3a, and drnc sites were not significantly different from each. pairwise differences between hrws or hrsw and mon or mon were significant. because the fax- vertebrate ortholog, pnr, has been shown to bind aagtca dimeric sites separated by one base-pair, we first tested whether fax- would display the same binding activity. fax- protein bound to dna containing aagtca dimeric sequences, and this binding could be competed with similar effectiveness by dna containing aagtca dr1a dimers or aggtca dr1g dimers, but not negative control aattca dimers. thus fax- can bind aagtca dimers specifically, similar to pnr and tll. we tested whether fax- could discriminate among different nucleotides at the second position, as tll does. unlike tll, fax- bound to aggtca dr1g dimers with avidity similar to that of aagtca dr1a dimers. furthermore, fax- also bound dimers with pyrimidines in the second position: specific binding, although significantly weaker, was also observed with acgtca and atgtca dimers. unlabeled dr1a dna competed more effectively for binding to dr1c sites than did unlabeled dr1c dna. therefore, in contrast to tll and tlx, fax- does not discriminate among different nucleotides at the second hexamer position, although it binds purines more strongly than pyrimidines. fax- does not bind monomeric hexamers but can bind a weak site adjacent to a strong site we tested next whether fax- could bind both a single copy of the hexamer and a dimeric copy of the hexamer, like tll, tlx, and dsf, or could bind only a dimeric copy of the hexamer, like pnr. when challenged to bind dna containing only a monomeric copy of the aagtca hexamer, fax- showed negligible binding that was not significantly different from negative controls. consistent with this result, unlabeled dna containing monomeric aagtca sequences competed very poorly for binding to labeled aagtca dr1a or aggtca dr1g dimeric sequences. in contrast to the results for monomeric sites, when we placed a predicted weak binding site in register for dr binding with a strong site, creating heteromeric repeats in either of the two possible orders, we found weak, but significant, binding by fax-. this binding was approximately 1/ as strong as binding to atgtca dimers and 1/ as strong as binding to aagtca dimers, and significantly different from drnc controls. these data are consistent with a model where fax- binds as an obligate dimer, but binding to a strong site can facilitate binding to an adjacent weaker site through cooperativity. fax- can bind dr sites but not dr sites nuclear receptor dimerization confers limitations on the amount of separation between the two hexamer half-sites that will be tolerated by a given protein. our data show that fax- can bind dr sites, which separate the two hexamers with a single base-pair, similar to tll and pnr. we tested whether increasing the separation between the two hexamers would influence the strength of fax- binding. when aagtca hexamers that are separated by two base-pairs were tested, fax- could bind these sequences, albeit not as strongly as dr sites. the strength of binding, as measured by emsa, was similar to the strength of binding to acgtca dimers and about half that observed with aagtca dimers. these data show that fax- prefers dr sites, but can also bind dr sites. when we further increased the separation of the two aagtca hexamers to three base-pairs, we were no longer able to detect binding by fax-. these data demonstrate that fax- dimerization requires relatively close proximity of the two hexamers, and also serve to reinforce the conclusion that fax- is unable to bind as a monomer. dbd amino acid partially mediates dna-binding specificity in fax- and nhr-67 in order to investigate differences between nr2e and nr2e subclass dna-binding properties and to allow for mutagenesis of the subclass-specific dbd amino acid we constructed fax- dbd::gal activation domain and nhr- dbd::gal activation domain fusion genes and tested their ability to activate transcription from promoters that carry different potential binding sites using the yeast one-hybrid system. as expected from our emsa analysis, the fax- dbd conferred significant β-galactosidase activity in one-hybrid studies with both aagtca dr1a and aggtca dr1g dimers in this assay, but did not with an aagtca monomer. also consistent with previous results for the nr2e subclass, the nhr- dbd conferred significant β-galactosidase activity with aagtca dr1a dimers, but not with aggtca dr1g dimers. these data demonstrate that for fax- and nhr- the dbd alone is sufficient to confer at least some half-site recognition and dimerization properties, and that the modality of binding inferred from our one-hybrid experiments yields data that are consistent with our direct assessment of fax- binding activity by emsa and the published direct assessment of tll and tlx binding. therefore, testing dna binding of mutated versions of fax- and nhr- by one-hybrid should yield data that are indicative of altered binding preference in vitro. curiously, binding of fax- to aggtca dr1g dimers gave much stronger quantitative results than aagtca dr1a dimers in this assay, in contrast to the expected quantitative results from our emsa studies, which showed similar binding to both dimeric sites. this difference may be due to fax- protein sequences that were not present in the fax- dbd construct or due to artifacts related to dna sequence context in the reporter constructs or yeast cellular environment. given this result, and because of the indirect nature of the one-hybrid assay, we have not drawn conclusions about relative binding strength based on quantitative comparisons using this assay. asn- of the fax- dbd is the only amino acid in the p box that is identical in all known nr2e subclass members and is different from the asp- that is consistently found in all known nr2e subclass members, including nhr-. because nhr- and tll discriminate between nre half-sites that differ at the second position and fax- does not, we hypothesized that the difference between the different dna-binding specificities might depend on which amino acid is present at position this hypothesis is supported by structural data that show water-mediated hydrogen-bond contacts between this amino acid and the second position of the nre for nuclear receptors that bind dr dimers. to test this hypothesis, we mutated the fax- dbd in the fax- dbd::gal activation domain one-hybrid construct from the wild-type codon for asn to a codon for asp. mutating the fax- dbd from asn- to asp- significantly increased the β-galactosidase activity observed in one-hybrid studies with aagtca dr1a sites approximately -fold. however, the mutation did not significantly decrease or abolish the activity observed with aggtca dr1g sites. therefore, converting asn- to asp- in the context of a fax- dbd was not sufficient to change the inferred binding specificity to that of nhr- due to the caveats in interpreting quantitative results from one-hybrid studies, it is unclear whether the asn to asp change results in increased binding of fax- to aagtca dr1a sites, although our results are consistent with that possibility. more convincing results were obtained when we performed the reciprocal experiment in which the nhr- dbd was mutated from asp- to asn- this mutation eliminated detectable β-galactosidase activity in one-hybrid studies with aagtca dr1a sites, and produced significant β-galactosidase activity in one-hybrid studies with aggtca dr1g sites. from these data, we infer that while wild-type nhr- with asp- can bind aagtca sites but not aggtca sites, mutant nhr- with asn- can bind aggtca sites but not aagtca sites. thus this single amino acid change had the effect of completely reversing the modality of nhr- binding activity. in this context, an asn- changed the preference of binding at nre position two from adenine to guanine. these results indicate that the amino acid present at position in the dbd of nhr- in particular, and perhaps nr2e nuclear receptors in general, is a mediator of dna binding specificity. discussion our findings demonstrate that fax- binds angtca half-sites that are separated by one base-pair and can bind aagtca half-sites that are separated by two base-pairs. this contrasts it with tll and nhr- which display a strong preference for aagtca half-sites. fax- binds more strongly to sites that have a purine at the second position on this strand, but does not distinguish between aagtca and aggtca. amino acid of the dbd is known to contact position of the nre, and it has been proposed that having a ser or ala at this position rather than a lys may allow for recognition of aagtca instead of aggtca half-sites. all known nr2e class receptors, including fax- and nhr- have a ser or ala at position suggesting that they may be able to bind the aagtca sequence via this amino acid residue. however, we have shown that a difference in dna-binding specificity exists between members of the nr2e and nr2e subclasses. in the context of an nhr- dbd, the ability to bind guanine at the second position of the nre hexamer is conferred, in part, by the asn- found in all nr2e members, while the ability to bind adenine at the second position of the nre hexamer is conferred by the asp- found in nr2e members. despite the important role of dbd position it is also clear that the amino acid at this position is not sufficient to confer a particular mode of binding specificity. other amino acids in the dbd, perhaps others that are consistently different between classes, must also contribute to nre binding specificity. an example of another amino acid residue that could participate in subclass specificity is position of the dbd, which is consistently gln in nr2e and arg in nr2e and is predicted to make phosphate backbone contacts with the dna. by analogy to co-crystal structures for rar-rxr heterodimers bound to dr sites, asn- would be predicted to make water-mediated hydrogen bonds to the pyrimidine bases on the strand complementary to the adenine or guanine at the second position of the nre. the implication of this comparison is that asp binds most favorably to thymine on the complementary strand, while asn binds favorably to cytosine and may be able to interact favorably with any of the four bases, depending on context. asn can make a greater variety of h-bond contacts than asp due to the presence of the amide donor and acceptor in the r group of asn in comparison to the acceptor-only carboxyl of asp. this greater range of potential h-bonding may be a contributor to the relaxed specificity observed for nr2e subclass dna binding. inferences drawn from our data, the co-crystal structure of nuclear receptors that bind dr sites, and general data on nuclear receptor-nre interactions allow us to develop a model for the remainder of fax-1-nre interactions. in co-crystal structures, the last four bases of the hexamer nre half site, gtca, are contacted by hydrogen bonds from dbd amino acid and usually lysarg or argarg. in all fax- and tll subclass members, these positions are lysarg. therefore, it seems reasonable to suggest that the lysarg dipeptide sequence of fax- contacts the last four base-pairs of the nre. however, these amino acids are apparently not sufficient to confer this binding preference since the steroid receptors also have the lysarg sequence, yet bind a very different half-site. these and other observations raise the important caveat that the geometry of nuclear receptor- dna binding can vary among classes; therefore the implications of our study may be limited to the nr2e class. we have not explored specificity at the first position of the nre half-site, which might also be somewhat degenerate. based on the inference drawn from the rar-rxr co-crystal on dr sites, we would expect that the adenine at the first position of the fax- nre is contacted by ser- and arg- of the dbd. the former amino acid residue is generally a ser among nr2e subclass members and can be either a ser or ala among nr2e subclass members. amino acid position found in the t box, is a conserved arginine in all nr2e nuclear receptors. while the general conservation of protein-dna contacts among nuclear receptors for which it has been directly tested is fairly good, we cannot be certain that other amino acids don't also participate directly in fax-1-nre binding. furthermore, in all cases, other amino acids can contribute to dna-binding specificity through conformation-specific contacts with the phosphodiester backbone. the definition of potential dna-binding sites allows us to predict candidate in vivo targets in the genome of c. elegans. there are argtca dr sites in the c. elegans genome. if we expand the list to include those sites that have a pyrimidine at the second position, angtca dr sites, the number grows to potential binding sites. these sites are not near any of the genes that are known to be dependent on fax- for their regulation. fax- may regulate these genes indirectly via another transcriptional regulator, by heterodimerization with another nuclear receptor that binds a different half-site, or by binding to other transcription factors at the promoter by a non-nre-dependent mechanism. in addition, there are approximately argtca dr sites in the c. elegans genome, to which fax- dimers may be able to bind in vivo. expanding the list to include those sites that allow fax- dimeric binding via heteromeric sites that contain a weak site and a strong site increases the number of potential fax- binding sites in the genome to over the sequences upstream of ncs- and opt- both of which are regulated by fax- contain sites that match this more degenerate binding site. with so many potential binding sites in the genome, other factors such as cognate coactivators or corepressors may help define the functional relevance of any particular candidate nre. these observations suggest that members of the nr2e subclass are able to form dimers on a much wider variety of dimeric nre's, as compared to the more restrictive nr2e subclass. however, this does not mean that nr2e subclass members bind more sites in vivo; nr2e subclass monomers may also bind monomeric aagtca sites, of which there are over. studies on the human fax- ortholog, pnr, have demonstrated a similar dna-binding activity. pnr was found to bind to dr aagtca nre's, but not to monomeric aagtca sequences. an unbiased screen for sequences that bind pnr revealed that, like fax- it can bind sequences that vary at positions two and three of the half-site hexamer. this study identified a preferred pnr binding site with the sequence agrtcaaarrtca, a sequence that is consistent with our analysis of fax- dna binding. while almost all binding sites revealed by this strategy include the tca sequence at the 3' end of the half-sites, greater variability was observed for all three bases at the 5'end of the half-sites, including nre position three in addition to nre position two. because glu- of rar and rxr directly contact nre half-site position three in the rar-rxr co-crystal, it may be that asn- of fax- and pnr also mediates the relaxed specificity at position three of the nre. therefore, the strong evolutionary conservation of the nr2e subclass dbd reflects, at least in part, the constraint of an evolutionarily-conserved nre binding site. whether the dna-binding activity that appears to be conserved between nematode and vertebrate members of the nr2e subclass translates into conserved patterns of gene regulation has yet to be investigated. pnr acts as a repressor of the transcription of cone-specific genes and may activate the transcription of rod-specific genes during vertebrate photoreceptor development. the vertebrate orthologs of some of the targets of fax- regulation are also transcribed in vertebrate photoreceptor cells, which allows for the possibility of conserved gene regulatory patterns.  our results define subclass-specific dna-binding specificities for the nr2e and nr2e subclasses, and suggest that binding preferences for the second nucleotide position of the dna half-site are partially mediated by amino acid of the nr2e dbd. the repertoire of potential dimeric binding sites is much larger for members of the nr2e subclass. fax- like nr2e subclass member pnr, binds nre's as a homodimer with a strong preference for dr sites. therefore, dimerization behavior may be a second conserved difference between the nr2e subclass, members of which can bind as a monomer or homodimer, and the obligate dimer nr2e subclass. 
this amino acid position is part of the "p box" that plays a critical role in defining binding site specificity and has been shown to make hydrogen-bond contacts to the second position of the hexamer in co-crystal structures for other nuclear receptors. we show that nr2e subclass member fax- unlike nhr- and other nr2e subclass members, binds to hexamer half-sites with relaxed specificity: it will bind hexamers with the sequence angtca, although it prefers a purine to a pyrimidine at the second position. these findings identify subclass-specific dna-binding specificities and dimerization properties for the nr2e and nr2e subclasses. based on a phylogenetic analysis of dna-binding domains, we define two conserved groups of orthologous nr2e genes: the nr2e subclass, which includes c. elegans nhr- drosophila tailless and dissatisfaction, and vertebrate tlx, and the nr2e subclass, which includes c. elegans fax- and vertebrate pnr. the presence of a single strong site adjacent to a very weak site allows dimeric fax- binding, further increasing the number of dimeric binding sites to which fax- may bind in vivo. for the nr2e protein nhr- asp- permits binding to aagtca half-sites, while asn- permits binding to aggtca half-sites. pnr and tll nuclear receptors have been shown to bind the hexamer half-site aagtca, instead of the hexamer aggtca recognized by most other nuclear receptors, suggesting unique dna-binding properties for nr2e class members. the nuclear receptors of the nr2e class play important roles in pattern formation and nervous system development. the relaxed specificity allows fax- to bind a much larger repertoire of half-sites than nhr- while nr2e class proteins bind both monomeric and dimeric sites, the nr2e class proteins bind only dimeric sites.
 the nuclear receptors of the nr2e class play important roles in pattern formation and nervous system development. based on a phylogenetic analysis of dna-binding domains, we define two conserved groups of orthologous nr2e genes: the nr2e subclass, which includes c. elegans nhr- drosophila tailless and dissatisfaction, and vertebrate tlx, and the nr2e subclass, which includes c. elegans fax- and vertebrate pnr. pnr and tll nuclear receptors have been shown to bind the hexamer half-site aagtca, instead of the hexamer aggtca recognized by most other nuclear receptors, suggesting unique dna-binding properties for nr2e class members.  we show that nr2e subclass member fax- unlike nhr- and other nr2e subclass members, binds to hexamer half-sites with relaxed specificity: it will bind hexamers with the sequence angtca, although it prefers a purine to a pyrimidine at the second position. we use site-directed mutagenesis to demonstrate that the difference between fax- and nhr- binding preference is partially mediated by a conserved subclass-specific asparagine or aspartate residue at position of the dna-binding domain. this amino acid position is part of the "p box" that plays a critical role in defining binding site specificity and has been shown to make hydrogen-bond contacts to the second position of the hexamer in co-crystal structures for other nuclear receptors. the relaxed specificity allows fax- to bind a much larger repertoire of half-sites than nhr- while nr2e class proteins bind both monomeric and dimeric sites, the nr2e class proteins bind only dimeric sites. the presence of a single strong site adjacent to a very weak site allows dimeric fax- binding, further increasing the number of dimeric binding sites to which fax- may bind in vivo.  these findings identify subclass-specific dna-binding specificities and dimerization properties for the nr2e and nr2e subclasses. for the nr2e protein nhr- asp- permits binding to aagtca half-sites, while asn- permits binding to aggtca half-sites. the apparent conservation of dna-binding properties between vertebrate and nematode nr2e receptors allows for the possibility of evolutionarily-conserved regulatory patterns. 