Our findings demonstrate that FAX-1 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-67, which display a strong preference for AAGTCA half-sites. FAX-1 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 22 of the DBD is known to contact position 2 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-1 and NHR-67, have a Ser or Ala at position 22, 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 NR2E1 and NR2E3 subclasses. In the context of an NHR-67 DBD, the ability to bind guanine at the second position of the NRE hexamer is conferred, in part, by the Asn-19 found in all NR2E3 members, while the ability to bind adenine at the second position of the NRE hexamer is conferred by the Asp-19 found in NR2E1 members.
Despite the important role of DBD position 19, 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 58 of the DBD, which is consistently Gln in NR2E3 and Arg in NR2E1 and is predicted to make phosphate backbone contacts with the DNA (Fig. 1).
By analogy to co-crystal structures for RAR-RXR heterodimers bound to DR1 sites , Asn-19 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 NR2E3 subclass DNA binding.
Inferences drawn from our data, the co-crystal structure of nuclear receptors that bind DR1 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 26 and 27, usually LysArg or ArgArg . In all FAX-1 and Tll subclass members, these positions are LysArg. Therefore, it seems reasonable to suggest that the LysArg dipeptide sequence of FAX-1 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 [1, 17, 32, 36]; 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 DR1 sites, we would expect that the adenine at the first position of the FAX-1 NRE is contacted by Ser-22 and Arg-79 of the DBD. The former amino acid residue is generally a Ser among NR2E3 subclass members and can be either a Ser or Ala among NR2E1 subclass members. Amino acid position 79, 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 [3, 36].
The definition of potential DNA-binding sites allows us to predict candidate in vivo targets in the genome of C. elegans. There are 60 ARGTCA DR1 sites (assuming that the separating base-pair can vary) in the C. elegans genome. If we expand the list to include those sites that have a pyrimidine at the second position, ANGTCA DR1 sites, the number grows to 109 potential binding sites. These sites are not near any of the genes that are known to be dependent on fax-1 for their regulation (flp-1, ncs-1, nmr-1, nmr-2, and opt-3 ). fax-1 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 100 ARGTCA DR2 sites in the C. elegans genome, to which FAX-1 dimers may be able to bind in vivo.
Expanding the list to include those sites that allow FAX-1 dimeric binding via heteromeric sites that contain a weak site and a strong site (ANGTCANANNTCA or ANNTCANANGTCA) increases the number of potential FAX-1 binding sites in the genome to over 1200. The sequences upstream of ncs-1 and opt-3, both of which are regulated by fax-1, contain sites that match this more degenerate binding site (data not shown). 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 NR2E3 subclass are able to form dimers on a much wider variety of dimeric NRE's (over 1200), as compared to the more restrictive NR2E1 subclass (there are 36 AAGTCA DR1 sites). However, this does not mean that NR2E3 subclass members bind more sites in vivo; NR2E1 subclass monomers may also bind monomeric AAGTCA sites, of which there are over 20,000.
Studies on the human fax-1 ortholog, PNR, have demonstrated a similar DNA-binding activity [25, 28]. PNR was found to bind to DR1 AAGTCA NRE's, but not to monomeric AAGTCA sequences. An unbiased screen for sequences that bind PNR revealed that, like FAX-1, 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-1 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-19 of RAR and RXR directly contact NRE half-site position three in the RAR-RXR co-crystal, it may be that Asn-19 of FAX-1 and PNR also mediates the relaxed specificity at position three of the NRE. Therefore, the strong evolutionary conservation of the NR2E3 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 NR2E3 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 [28, 37]. The vertebrate orthologs of some of the targets of fax-1 regulation are also transcribed in vertebrate photoreceptor cells , which allows for the possibility of conserved gene regulatory patterns.