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

Stephen D DeMeo^*¹^,2 , Rebecca M Lombel^*¹^,3 , Melissa Cronin¹ , Eric L Smith¹^,4 , Danielle R Snowflack¹^,5 , Kristy Reinert¹^,6 , Sheila Clever^*¹ and Bruce Wightman¹

¹Biology Department, Muhlenberg College, Allentown, PA 18104, USA

²Current address : Philadelphia College of Osteopathic Medicine, Philadelphia, PA 19131, USA

³Current address : Department of Pediatrics, University of Michigan Health System, Ann Arbor, MI 48109, USA

⁴Current address : Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY 10029, USA

⁵Current address : Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA

⁶Current address : School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA

author email corresponding author email* Contributed equally

BMC Molecular Biology 2008, 9:2doi:10.1186/1471-2199-9-2


Published:	7 January 2008

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.

Conclusion

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.