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DNA-binding specificity and in vivo targets of Caenorhabditis elegans nuclear factor I.

ABSTRACT: The conserved nuclear factor I (NFI) family of transcription factors is unique to animals and essential for mammalian development. The Caenorhabditis elegans genome encodes a single NFI family member, whereas vertebrate genomes encode 4 distinct NFI protein subtypes (A, B, C, and X). NFI-1-deficient worms exhibit abnormalities, including reduced lifespan, defects in movement and pharyngeal pumping, and delayed egg-laying. To explore the functional basis of these phenotypes, we sought to comprehensively identify NFI-1-bound loci in C. elegans. We first established NFI-1 DNA-binding specificity using an in vitro DNA-selection strategy. Analysis yielded a consensus motif of TTGGCA(N)(3)TGCCAA, which occurs 586 times in the genome, a 100-fold higher frequency than expected. We next asked which sites were occupied by NFI-1 in vivo by performing chromatin immunoprecipitation of NFI-1 followed by microarray hybridization. Only 55 genomic locations were identified, an unexpectedly small target set. In vivo NFI-1 binding sites tend to be upstream of genes involved in core cellular processes, such as chromatin remodeling, mRNA splicing, and translation. Remarkably, 59 out of 70 (84%) of the C. briggsae orthologs of the identified targets contain conserved NFI binding sites in their promoters. These experiments provide a foundation for understanding how NFI-1 is recruited to unexpectedly few in vivo sites to perform its developmental functions, despite a vast over-representation of its binding motif.

SUBMITTER: Whittle CM

PROVIDER: S-EPMC2715542 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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DNA-binding specificity and in vivo targets of Caenorhabditis elegans nuclear factor I.

Whittle Christina M CM Lazakovitch Elena E Gronostajski Richard M RM Lieb Jason D JD

Proceedings of the National Academy of Sciences of the United States of America 20090707 29

The conserved nuclear factor I (NFI) family of transcription factors is unique to animals and essential for mammalian development. The Caenorhabditis elegans genome encodes a single NFI family member, whereas vertebrate genomes encode 4 distinct NFI protein subtypes (A, B, C, and X). NFI-1-deficient worms exhibit abnormalities, including reduced lifespan, defects in movement and pharyngeal pumping, and delayed egg-laying. To explore the functional basis of these phenotypes, we sought to comprehe ...[more]

PMID: 19584245

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Project description: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.

Dataset Information

DNA-binding specificity and in vivo targets of Caenorhabditis elegans nuclear factor I.

Publications

DNA-binding specificity and in vivo targets of Caenorhabditis elegans nuclear factor I.

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