Nuclear receptor location analyses in mammalian genomes: from gene regulation to regulatory networks

Abstract

Rapid progress in mapping nuclear receptor binding sites, referred to as "location analysis," has recently been achieved through the use of chromatin immunoprecipitation approaches. Location analysis can be performed on a single locus or cover a complete genome, and the resulting datasets can be probed to identify direct target genes and/or investigate the molecular mechanisms by which nuclear receptors control gene expression. In addition, when coupled with other genetic and functional genomics investigative methods, location analysis has proven to be a powerful tool with which to identify novel biological functions of nuclear receptors and build transcriptional regulatory networks. Thus, the knowledge gained from several recent chromatin immunoprecipitation-based studies has challenged basic concepts of nuclear receptor action, offered new insights into gene-regulatory mechanisms, and led to the identification of nuclear receptor-controlled biological functions.

Fig. 1.

Transcriptome Complexity and Assignment of Binding Sites to Target Genes Binding sites for nuclear receptors identified by ChIP-based experiments are usually assigned to the nearest annotated gene. Binding sites located in proximal promoter regions can be assigned with high confidence to the associated gene, although specific regulation of one gene over another in transcriptional units sharing a bidirectional promoter is possible. Unambiguous assignment of a target gene to a binding site located far away from its TSS requires the use of the 3C technology. A locus control region can regulate the expression of an individual gene within a cluster in a spatiotemporal manner. Binding sites located far away from a known gene could actually be close to an unannotated promoter or in the vicinity of an unknown gene. A significant number of promoters have been found in the 3′-untranslated region of another gene. Finally, a binding site located on one chromosome can be used to bring a transcription factor close to a gene located on another chromosome. A gene in black represents the actual target of the nuclear receptor bound to a proximal or distal site; a gene in gray indicates the annotated gene nearest to a binding site but may not be the actual target of the receptor; a gene in white denotes the presence of an unannotated gene. UTR, Untranslated region.

Fig. 2.

Regulatory Networks, Phenotypic Analyses, and Biological Functions of Nuclear Receptors Nuclear receptors control the expression of a limited set of primary target genes. Some of these genes encode transcription factors, coregulator proteins, micro-RNAs, and posttranslational modifiers of proteins that will both influence the original primary response and lead to a broad secondary response. ChIP-based, expression profiling, and computational biology approaches are considered a useful set of tools with which to build regulatory networks (top). In parallel, phenotypic analyses using small interfering RNA knockdown, gene knock-out and overexpression, as well as physiological and pharmacological challenges in cell-based and animal models are used to study the overall functions of a particular nuclear receptor (bottom). When integrated, the two complementary approaches can lead to the identification of nuclear receptor-dependent biological functions for which we have a clear understanding of the molecular mechanisms underlying the observed phenotypes (middle). A nuclear receptor is represented by two blue ovals; genes are represented by green and red boxes for up- and down-regulated genes, respectively; gene products are represented by diverse shapes and colors. Arrows indicate activation; blunt arrows represent repression. siRNA, small interfering RNA.