Conserved Cis-Acting Range Extender Element Mediates Extreme Long-Range Enhancer Activity in Mammals

Abstract

While most mammalian enhancers regulate their cognate promoters over moderate distances of tens of kilobases (kb), some enhancers act over distances in the megabase range. The sequence features enabling such extreme-distance enhancer-promoter interactions remain elusive. Here, we used in vivo enhancer replacement experiments in mice to show that short- and medium-range enhancers cannot initiate gene expression at extreme-distance range. We uncover a novel conserved cis-acting element, Range EXtender (REX), that confers extreme-distance regulatory activity and is located next to a long-range enhancer of Sall1. The REX element itself has no endogenous enhancer activity. However, addition of the REX to other short- and mid-range enhancers substantially increases their genomic interaction range. In the most extreme example observed, addition of the REX increased the range of an enhancer by an order of magnitude, from its native 71kb to 840kb. The REX element contains highly conserved [C/T]AATTA homeodomain motifs. These motifs are enriched around long-range limb enhancers genome-wide, including the ZRS, a benchmark long-range limb enhancer of Shh. Mutating the [C/T]AATTA motifs within the ZRS does not affect its limb-specific enhancer activity at short range, but selectively abolishes its long-range activity, resulting in severe limb reduction in knock-in mice. In summary, we identify a sequence signature globally associated with long-range enhancer-promoter interactions and describe a prototypical REX element that is necessary and sufficient to confer extreme-distance gene activation by remote enhancers.

Figure 1:. Transplanted Enhancers Lack Long-Range Limb Activity in Knock-in Mice.

(A) All selected limb enhancers (colored blocks) are marked by H3K27 acetylation and open chromatin and contain a conserved core sequence. For each enhancer, E11.5 forelimb H3K27ac ChIP-seq (Lex et al., 2020)), pseudobulk Shh+ ZPA scATAC-seq cluster and placental conservation tracks are shown. mHS72 and mHS1516 are mouse orthologous of HS72 and HS1516. (B) Corresponding enhancer activities in transgenic E11.5 mouse embryos. Zoomed in forelimb buds are shown on the right. The white dotted line demarcates the approximate ZPA region. (C) Corresponding genomic regions where curved lines indicate putative E–P interactions supported by capture Hi-C (Z. Chen et al., 2024), multiome analysis of E11.5 mouse limb buds (scATAC-seq + scRNA-seq correlation; this study) or matched enhancer activity and gene expression in limb buds of E11.5 or E10.5 embryos (Fig. S2D). H3K27ac ChIP-seq signal from E11.5 forelimbs is shown underneath each region (gray). (D-E) Limb phenotyping of knock-in mice with transplanted enhancers. The ZRS is located ~850 kb away from the promoter of Shh. (D) Schematic mouse Shh loci with the ZRS replaced by HS72, MM1564, HS1516, and MM1492 limb enhancers or a fragment of the lacZ sequence are shown. (E) Comparative Shh mRNA in situ hybridization analysis in wild type and knock-in mouse embryos during forelimb bud development (first column). The genotypes of the embryos are shown on the left. Corresponding skeletal preparations of E18.5 mouse embryos are shown in the second column: s, scapula; h, humerus; r, radius; u, ulna; a, autopod. The number of embryos that exhibited the representative limb phenotype over the total number of embryos with the genotype is indicated. See Fig. S2 for more details, including hindlimb bud analysis. The coordinates of enhancers used in B, D and E are: ZRS (chr5:29,314,497-29,315,844; mm10), HS72 (chr16:51,623,899-51,624,805; hg38), MM1564 (chr2:60,785,660-60,787,563; mm10), HS1516 (hg38_chr21:38,989,433-38,991,200; hg38) and MM1492 (chr4:154,707,415-154,711,162; mm10).

Figure 2:. Transplanted enhancers maintain open chromatin structure at the knock-in site and drive functional SHH expression in the limb when proximal to the Shh promoter.

(A) Top: allele-specific ZPA ATAC-seq signal at the transplanted HS1516 (green box) or HS72 (orange box) enhancers and the corresponding wild-type ZRS locus. Bottom: ATAC-seq profiles at endogenous mHS1516 (left; chr16:95,847,911-95,849,564) and mHS72 (right; chr8:89,454,508-89,455,383) mouse orthologous enhancers (green and orange striped boxes). (B) LacZ-stained transgenic E11.5 mouse embryo carrying an HS72 limb enhancer inserted upstream of the Shh promoter (light blue) and lacZ reporter gene (dark blue). The number of embryos with robust LacZ staining in limb buds over the total number of transgenic embryos screened is indicated.

Figure 3:. The REX element is necessary for long-range activation of Shh by a heterologous enhancer.

(A) An evolutionary conserved element of unknown function is located adjacent to the human HS72 enhancer. The HS72 enhancer region is shown together with evolutionary conservation tracks. (B) Replacement of the ZRS with an extended version of the HS72 enhancer (chr16:51,623,658-51,625,572; hg38) containing the REX element results in initiation of Shh expression in developing limb buds and full limb outgrowth in knock-in mice. *Extra digits (polydactyly) in ZRS^HS72+REX/ZRS^HS72+REX embryos. See Fig. S4 for hindlimb and E11.5 Shh expression analysis. (C) The REX element lacks classical enhancer activity in E11.5 transgenic embryos when placed upstream of the Shh promoter and lacZ reporter gene. The number of transgenic embryos with no LacZ activity in the limb over the total number of transgenic embryos screened is indicated.

Figure 4:. The REX element can convert a short-range enhancer into a long-range enhancer.

Replacement of the ZRS with a chimeric cis-regulatory element consisting of the short-range MM1492 enhancer and the REX element from the HS72 enhancer region results in the initiation of Shh expression in developing limb buds at E10.5 and full limb outgrowth at P0 in knock-in mice. *Extra digits (polydactyly) in ZRS^MM1492REX/ZRS^MM1492REX embryos. See Fig. S5B-C for hindlimb and E10.5 Shh expression analysis.

Figure 5:. [C/T]AATTA HD motifs are globally linked to long-range regulation.

(A) Position and evolutionary conservation of predicted TF motifs within the REX element. The conserved core of the REX element (chr16:51,624,707-51,624,984) is aligned with orthologous sequences from 12 mammalian species. Sequences matching TF binding preferences (below) are highlighted. (B) Schematic pipeline for genome-wide identification of putative enhancer-promoter interactions in the hindlimb. (C) Example of predicted E-P interaction between mHS919 limb enhancer and Rad21. The Rad21 locus (chr15:5000000-5300000) is shown with Hi-C data (top) (Jiang et al., 2017), pseudobulk chromatin accessibility tracks and a violin plot for Rad21 expression by cell type (bottom). Arches indicate hs919 enhancer-centric E–P interactions from enhancer capture Hi-C (red) or multiome (blue). (D-E) Table of top-most enriched limb-expressed TF motifs in long-range (400 kb - 2 Mb), as compared to short-range (10-200 kb) enhancers in developing limb buds for VISTA limb enhancers (D) and all predicted hindlimb enhancers (E) grouped by their similarity. [C/T]AATTA HD motifs are highlighted by red shading. False-discovery rate values are shown on the right. See Table S2 for a complete list of motifs. (F) Distribution of enhancer-promoter distances for VISTA limb enhancers with 0-1 or ≥2 conserved [C/T]AATTA HD motifs (P = 0.0053, Wilcoxon rank sum test). Observation counts for each group are displayed in the outlined boxes at the base of the plot. (G) Representative long-range limb enhancers containing [C/T]AATTA HD motifs.

Figure 6:. [C/T]AATTA HD motifs are required for long-range gene activation.

(A) Position of TF binding sites previously identified (Kvon et al., 2016; Lettice et al., 2017, 2012) within the mouse ZRS core (chr5:29,314,881-29,315,667). Sequences matching [C/T]AATTA motifs binding preferences (below) are highlighted. Bump-outs show evolutionary conservation of [C/T]AATTA motifs. Blue boxes demarcate the mutagenized regions. (B) A dual-enSERT transgenic construct containing wild type mouse ZRS driving mCherry (red) and ZRS allele with mutated [C/T]AATTA HD motifs (ZRSΔHD) driving eGFP (green) separated by a strong synthetic insulator. Hindlimb bud images from a representative transgenic embryo are shown below. The white dotted line encircles the quantified region. (C) Quantification of normalized mean intensity in embryos containing ZRS-mCherry/ZRSΔHD-eGFP and control ZRS-mCherry/Empty-eGFP constructs. (D) Skeletal forelimb preparation from E18.5 mice with three mutated [C/T]AATTA sites within the endogenous ZRS enhancer (ZRSΔHD) (E) Proposed model of extreme long-range enhancer activation in the developing limb buds.