Functional autonomy of distant-acting human enhancers

Abstract

Many human genes are associated with dispersed arrays of transcriptional enhancers that regulate their expression in time and space. Studies in invertebrate model systems have suggested that these elements could function as discrete and independent regulatory units, but the in vivo combinatorial properties of vertebrate enhancers remain poorly understood. To explore the modularity and regulatory autonomy of human developmental enhancers, we experimentally concatenated up to four enhancers from different genes and used a transgenic mouse assay to compare the in vivo activity of these compound elements with that of the single modules. In all of the six different combinations of elements tested, the reporter gene activity patterns were additive without signs of interference between the individual modules, indicating that regulatory specificity was maintained despite the presence of closely-positioned heterologous enhancers. Even in cases where two elements drove expression in close anatomical proximity, such as within neighboring subregions of the developing limb bud, the compound patterns did not show signs of cross-inhibition between individual elements or novel expression sites. These data indicate that human developmental enhancers are highly modular and functionally autonomous and suggest that genomic enhancer shuffling may have contributed to the evolution of complex gene expression patterns in vertebrates.

Figure 1. Spatial additivity of tissue-specific enhancers fused from different genes

a) genomic environment of six conserved enhancers used in this study. A 50kb genomic interval bracketing each enhancer is shown, including intron/exon structure of overlapping genes (black) and conservation in 17 vertebrates (color shaded boxes; [26]). All enhancers included an ultraconserved core region (Suppl. Table 1; [27]). b) Single and compound enhancer constructs for in vivo testing. c) Enhancer activity of single elements at mouse embryonic day 11.5. d)-i) In vivo activity of heterologous compound enhancers. d) E1+E2, e) E2+E5, f) E3+E4, g) E1+E5, h) E5+E6, i) E1+E2+E5+E6. Only one representative embryo is shown for each single and compound pattern, see Suppl. Table 2 for reproducibility across independent transgenic animals.

Figure 2. Temporal and spatial additivity of individual enhancer activities within the developing limb

a)-l) dorsal surface view of forelimb buds of individual embryos transgenic for E3 (a-d), E4 (e–h) and the compound enhancer E3+E4 (i–l). For each construct, embryos were collected at e10.5 (a,e,i), e11.5 (b,f,j), and e12.5 (c,d,g,h,k,l) and representative limbs were stage-matched based on morphology. The transgenic status of the embryo shown in e) was confirmed by genotyping.