Genomic views of distant-acting enhancers

Abstract

In contrast to protein-coding sequences, the significance of variation in non-coding DNA in human disease has been minimally explored. A great number of recent genome-wide association studies suggest that non-coding variation is a significant risk factor for common disorders, but the mechanisms by which this variation contributes to disease remain largely obscure. Distant-acting transcriptional enhancers--a major category of functional non-coding DNA--are involved in many developmental and disease-relevant processes. Genome-wide approaches to their discovery and functional characterization are now available and provide a growing knowledge base for the systematic exploration of their role in human biology and disease susceptibility.

Figure 1. Schematic overview of gene regulation by distant-acting enhancers

For many genes, the regulatory information embedded in the promoter is insufficient to drive the complex expression pattern observed at the mRNA level, suggesting that appropriate expression in time and space depends on additional distant-acting cis-regulatory sequences. Tissue-specific enhancers are thought to contain combinations of binding sites for different transcription factors. Only when all required transcription factors are present in a tissue, the enhancer becomes active: it binds to transcriptional co-activators, relocates into physical proximity of the gene promoter through a looping mechanism, and activates transcription by RNA polymerase II. In any given tissue, only a subset of enhancers is active, as shown here schematically for two separate enhancers with brain- and limb-specific activities. Insulator elements prevent enhancer-promoter interactions and can thus restrict the activity of enhancers to defined chromatin domains. In addition to activation by enhancers, negative regulatory elements including repressors and silencers can contribute to transcriptional regulation (not shown).

Figure 2. Consequences of deletion and mutation of the limb enhancer of Sonic hedgehog

A) The limb enhancer of the Sonic hedgehog gene is located approximately 1 megabase away from its target promoter in the intron of a neighboring gene (Lmbr1, exons not shown). In transgenic mouse reporter assays, this noncoding sequence targets gene expression to a posterior region of the developing limb bud . B) Mice with a targeted deletion of this enhancer have severely truncated limbs, which strikingly demonstrates its functional importance in development . C–E) Point mutations in the orthologous human enhancer sequence result in preaxial polydactyly, emphasizing the potential significance of variation in noncoding functional sequences in both rare and common human disorders . C, D) Hands of two different patients with point mutations in the Sonic hedgehog limb enhancer. E) Point mutations associated with preaxial polydactyly identified in four unrelated families.

Figure 3. Schematic overview of ChIP-chip and ChIP-seq methods

Both approaches depend on the cross-linking of protein to DNA by formaldehyde, either in cultured cells or in tissue samples. After cross-linking, chromatin is sheared and a suitable antibody is used to enrich for DNA fragments bound to a protein of interest. In many cases, antibodies binding to covalently modified proteins are used, e.g. recognizing methyl groups at defined amino acid residues of histones. Following immunoprecipitation and reversal of cross-links, the DNA libraries enriched in binding sites for the chromatin mark of interest are either analyzed by hybridization to microarrays (ChIP-chip) or by massively-parallel sequencing and alignment of the obtained sequence reads to the reference genome (ChIP-seq). See Text Boxes 1 and 2 for additional details about these techniques.