Genome-wide in Silico Identification of New Conserved and Functional Retinoic Acid Receptor Response Elements (Direct Repeats Separated by 5 bp)

Yannick N Anno,Sébastien Lalevée,Vincent Laudet,Amandine Chatagnon,Olivier Poch,Cécile Rochette-Egly,Gerard Benoit,Odile Lecompte,Eric Samarut

doi:10.1074/jbc.m111.263681

Abstract

The nuclear retinoic acid receptors interact with specific retinoic acid (RA) response elements (RAREs) located in the promoters of target genes to orchestrate transcriptional networks involved in cell growth and differentiation. Here we describe a genome-wide in silico analysis of consensus DR5 RAREs based on the recurrent RGKTSA motifs. More than 15,000 DR5 RAREs were identified and analyzed for their localization and conservation in vertebrates. We selected 138 elements located ±10 kb from transcription start sites and gene ends and conserved across more than 6 species. We also validated the functionality of these RAREs by analyzing their ability to bind retinoic acid receptors (ChIP sequencing experiments) as well as the RA regulation of the corresponding genes (RNA sequencing and quantitative real time PCR experiments). Such a strategy provided a global set of high confidence RAREs expanding the known experimentally validated RAREs repertoire associated to a series of new genes involved in cell signaling, development, and tumor suppression. Finally, the present work provides a valuable knowledge base for the analysis of a wider range of RA-target genes in different species.

Highlights

Retinoic acid (RA) receptors regulate gene expression through binding-specific response elements (RAREs)
We describe a genome-wide in silico analysis of consensus DR5 RAREs based on the recurrent RGKTSA motifs
Bioinformatic Genome-wide Research of DR5 RAREs Corresponding to the RGKTSA Motif—Only a few RAREs have been identified to date and associated to RA-target genes

Summary

Background

Retinoic acid (RA) receptors regulate gene expression through binding-specific response elements (RAREs). The development of high throughput technologies such as DNA microarrays revealed that within a given cell type or tissue, the RA response is composed of a huge and complex network of responsive genes (8 –10) Such techniques could not discriminate between direct primary and secondary target genes (which are modulated by the product of a primary target gene rather than by RXR/RAR heterodimers), and only a few of the RA target genes contained identified RAREs. More recently, chromatin immunoprecipitation coupled with array hybridization (ChIP-chip) allowed the identification of new RAR binding loci [11, 12]. Computational techniques were developed for the genome-wide identification of DR5 RAREs and for the characterization of their genomic and phylogenetic context In this way we amassed a collection of DR5 RAREs that is conserved across vertebrate species and that was validated for its occupancy and functionally analyzed for the RA-responsiveness of the associated genes. Such a strategy allowed us to characterize a new set of high confidence conserved DR5 RAREs associated to a series of new potential RA-target genes, providing a wider knowledge base for the analysis of the RA response in different species

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION