title: Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of Caenorhabditis elegans is partially mediated via a subclass-specific P-box residue.
abstract: BACKGROUND
  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 NR2E1 subclass, which includes C. elegans nhr-67, Drosophila tailless and dissatisfaction, and vertebrate Tlx (NR2E2, NR2E4, NR2E1), and the NR2E3 subclass, which includes C. elegans fax-1 and vertebrate PNR (NR2E5, NR2E3). 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.
  
  
  RESULTS
  We show that NR2E3 subclass member FAX-1, unlike NHR-67 and other NR2E1 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-1 and NHR-67 binding preference is partially mediated by a conserved subclass-specific asparagine or aspartate residue at position 19 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-1 to bind a much larger repertoire of half-sites than NHR-67. While NR2E1 class proteins bind both monomeric and dimeric sites, the NR2E3 class proteins bind only dimeric sites. The presence of a single strong site adjacent to a very weak site allows dimeric FAX-1 binding, further increasing the number of dimeric binding sites to which FAX-1 may bind in vivo.
  
  
  CONCLUSIONS
  These findings identify subclass-specific DNA-binding specificities and dimerization properties for the NR2E1 and NR2E3 subclasses. For the NR2E1 protein NHR-67, Asp-19 permits binding to AAGTCA half-sites, while Asn-19 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.
authors.affiliation: Biology Department, Muhlenberg College, Allentown, PA 18104, USA. stephendem@pcom.edu
authors.forename: Stephen D
authors.initials: SD
authors.lastname: DeMeo
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Lombel
authors.forename: Melissa
authors.initials: M
authors.lastname: Cronin
authors.forename: Eric L
authors.initials: EL
authors.lastname: Smith
authors.forename: Danielle R
authors.initials: DR
authors.lastname: Snowflack
authors.forename: Kristy
authors.initials: K
authors.lastname: Reinert
authors.forename: Sheila
authors.initials: S
authors.lastname: Clever
authors.forename: Bruce
authors.initials: B
authors.lastname: Wightman
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D018509:DNA, Helminth / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary / Q000502:physiology; D011956:Receptors, Cell Surface / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements / Q000502:physiology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D029742:Caenorhabditis elegans Proteins; D018509:DNA, Helminth; C405022:FAX-1 protein, C elegans; C520004:Nhr-67 protein, C elegans; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D001224:Aspartic Acid; D001216:Asparagine