199-205 Insight - Pennachio NS.indd

Multiple lines of evidence indicate that important functional properties are embedded in the non-coding portion of the human genome, but identifying and defining these features remains a major challenge. An initial estimate of the magnitude of functional non-coding DNA was derived from comparative analysis of the first available mammalian genomes (human and mouse), which indicated that fewer than half of the evolutionary constrained sequences in the human genome encode proteins, a prospect that gained further support when additional vertebrate genomes became available for comparative genomic analyses. The overall impact of these presumably functional non-coding sequences on human biology was initially unclear. A considerable urgency to define their locations and functions came from a growing number of known associations of non-coding sequence variants with common human diseases. Specifically, genome-wide association studies (GWAS) have revealed a large number of disease susceptibility regions that do not overlap protein-coding genes but rather map to non-coding intervals. For example, a 58-kilobase linkage disequilibrium block located at human chromosome 9p21 was shown to be reproducibly associated with an increased risk for coronary artery disease, yet the risk interval lies more than 60 kilobases away from the nearest known protein-coding gene. To estimate the global contribution of variation in non-coding sequences to phenotypic and disease traits, we performed a meta-analysis of ~1,200 single-nucleotide polymorphisms (SNPs) identified as the most significantly associated variants in GWAS published so far (ref. 5, accessed 2 March 2009). Using conservative parameters that tend to overestimate the size of linkage disequilibrium blocks, we found that in 40% of cases (472 of 1,170) no known exons overlap either the linked SNP or its associated haplotype block, suggesting that in more than one-third of cases non-coding sequence variation causally contributes to the traits under investigation. One possibility that could explain these GWAS hits is that the non-coding intervals contain enhancers, a category of gene regulatory sequence that can act over long distances. A simplified view of the current understanding of the role of enhancers in regulating genes is summarized in Fig. 1. The docking of RNA polymerase II to proximal promoter sequences and transcription initiation are fairly well characterized; by contrast, the mechanisms by which insulator and silencer elements buffer or repress gene regulation, respectively, are less well understood. Transcriptional enhancers are regulatory sequences that can be located upstream of, downstream of or within their target gene and can modulate expression independently of their orientation. In vertebrates, enhancer sequences are thought to comprise densely clustered aggregations of transcription-factor-binding sites. When appropriate occupancy of transcription-factor-binding sites is achieved, recruitment of transcriptional coactivators and chromatinremodelling proteins occurs. The resultant protein aggregates are thought to facilitate DNA looping and ultimately promoter-mediated gene activation (see page 199). In-depth studies of individual genes such as APOE or NKX2-5 (reviewed in ref. 9) have shown that many genes are regulated by complex arrays of enhancers, each driving distinct aspects of the messenger RNA expression pattern. These modular properties of mammalian enhancers are also supported by their additive regulatory activities in heterologous recombination experiments. The purely genetic evidence from GWAS does not allow any direct inferences regarding the underlying molecular mechanisms, but a number of in-depth studies of individual loci (see below) suggest that variation in distant-acting enhancer sequences and the resultant changes in their activities can contribute to human disorders. Although we anticipate a variety of other non-coding functional categories such as negative gene regulators or non-coding RNAs to have a role in human disease, in this Review we focus on the role of enhancers and on strategies to define their location and function throughout the genome.