Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions

Abstract

Mammalian genomes are organized into megabase-scale topologically associated domains (TADs). We demonstrate that disruption of TADs can rewire long-range regulatory architecture and result in pathogenic phenotypes. We show that distinct human limb malformations are caused by deletions, inversions, or duplications altering the structure of the TAD-spanning WNT6/IHH/EPHA4/PAX3 locus. Using CRISPR/Cas genome editing, we generated mice with corresponding rearrangements. Both in mouse limb tissue and patient-derived fibroblasts, disease-relevant structural changes cause ectopic interactions between promoters and non-coding DNA, and a cluster of limb enhancers normally associated with Epha4 is misplaced relative to TAD boundaries and drives ectopic limb expression of another gene in the locus. This rewiring occurred only if the variant disrupted a CTCF-associated boundary domain. Our results demonstrate the functional importance of TADs for orchestrating gene expression via genome architecture and indicate criteria for predicting the pathogenicity of human structural variants, particularly in non-coding regions of the human genome.

Figure 1. Different limb pathogenic structural variations in human and mouse map to the EPHA4 TAD

(A) Hi-C profile around the EPHA4 locus in human ES cells (Dixon et al., 2012). Dashed lines indicate the EPHA4 TAD and boundaries. Cen, centromeric; tel, telomeric. (B–D) Schematic of structural variants (left) and associated phenotypes (right). (B) Brachydactyly-associated deletions in families B1, B2 and B3. Note thumb and index finger shortening with partial webbing in a child (B1 patient) and adult (B2 patient). (C) F-syndrome-associated inversion in family F1 and duplication in family F2. Note similar phenotypes of index/thumb syndactyly. (D) Polydactyly-associated duplication (P1) and deletion in the doublefoot (Dbf) mouse mutant. Radiograph of patient hand and skeletal preparation of Dbf/+ mouse show similar 7-digit polydactyly. See also Figure S1.

Figure 2. TAD organization around the Wnt6/Ihh/Epha4/Pax3 locus

Hi-C profile derived from mouse ES cells (Dixon et al., 2012). Predicted TAD boundaries are indicated by vertical dashed lines. 4C-seq profiles from E11.5 mouse distal limbs with viewpoints on Wnt6, Ihh, Epha4, and Pax3. Y-axis indicates number of normalized reads. 4C-seq interactions sharply drop at the Hi-C-predicted boundaries. Percentage of contacts/Mb within TADs is shown on right. Orange bars represent contacts within corresponding TAD. Grey bars represent contacts located in the respective next two centromeric TADs and two telomeric TADs.

Figure 3. Ectopic interaction of Wnt6, Ihh and Pax3 with the Epha4 TAD and misexpression in mouse models

(A) Schematic of the locus. Dashed lines indicate Epha4 TAD and boundaries. In situ hybridization (right) shows Epha4 expression in distal limbs of an E11.5 wild-type embryo (white arrow). (B) Brachydactyly-like deletion generated by CRISPR/Cas (pink scissors). 4C-seq analysis with Pax3 as viewpoint in E11.5 wild-type and heterozygous mutant distal limbs shows ectopic interaction with the centromeric part of the Epha4 TAD in the mutant (red box). Pax3 shows ectopic distal limb expression in mutants (white arrow). (C) F-syndrome inversion generated by CRISPR/Cas (pink scissors). 4C-seq analysis with Wnt6 as viewpoint shows ectopic interaction with the centromeric part of the Epha4 TAD in the mutant (red box). Wnt6 shows ectopic distal limb expression in mutant limbs (white arrow). (D) Dbf deletion. 4C-seq analysis with Ihh as viewpoint shows ectopic interaction with most of the Epha4 TAD in mutants (red box). Ihh shows ectopic distal limb expression in mutants (white arrow). (E–F) Autopod and corresponding skeletal preparations of wild-type and mutant mice at postnatal day 3. Insets in (E) show digit II. Note reduction of digit II in the homozygous brachydactyly-like deletion and massive polydactyly in Dbf/+. See also Figures S2, S3 and S4.

Figure 4. 4C-seq analysis in patient-derived human adult fibroblasts

Schematic representation of the locus and the structural variations are shown on top. 4C-seq profiles from human adult fibroblasts from healthy controls and patients with heterozygous structural variations are shown below. Red boxes indicate ectopic interaction. The EPHA4 TAD is indicated by dashed lines. (A) Patient with brachydactyly- associated deletion. 4C-seq analysis with PAX3 as viewpoint shows extensive ectopic interaction with the EPHA4 TAD (red box). (B) Patient with the F-syndrome-associated duplication. 4C-seq profile with WNT6 as viewpoint shows ectopic interaction with the EPHA4 TAD (red box). (C) Patient with the polydactyly-associated duplication. 4C-seq profile with IHH as viewpoint shows ectopic interaction with the centromeric region of the EPHA4 TAD (red box).

Figure 5. A cluster of limb enhancers is located in the interacting region of the EphA4 TAD

(A) Color bars indicate the fragment of the Epha4 TAD showing increased interaction with target genes in different mouse mutant limbs or human patient fibroblasts. The 4C-seq profile of Epha4 in E11.5 distal mouse limbs is shown for comparison. Red box indicates region of minimal overlap analyzed for enhancer activity. (B) Close-up view of the chromosomal region analyzed. DNase HS, H3K27ac and p300 ChIP-seq from E11.5 limbs and conservation tracks were used to predict enhancers. Arrows indicate putative enhancer regions. Gray box indicates the location of a cluster of enhancers shown below (C). (C) Transgenic enhancer reporter (LacZ) staining at E11.5. (D) Pattern of forelimb expression of Pax3 (DelB), Wnt6 (InvF), and Ihh (Dbf) in mutants and endogenous pattern of expression of Epha4 by in situ hybridization at E11.5. Note the similarity of endogenous Epha4 and ectopic Pax3, Wnt6, and Ihh expression domains (D, white arrows) with the enhancer activity patterns (C, black arrows). See also Figures S5 and S6.

Figure 6. Boundary elements at both sides of the Epha4 TAD prevent ectopic expression of neighboring genes

(A) CTCF ChIP-seq track in E14.5 mouse limbs (ENCODE/LICR). Red boxes and octagons mark clusters of CTCF peaks located at the boundary of the Epha4 TAD. Grey box indicates Epha4 TAD. 4C-seq profiles were generated from distal limb buds at E11.5. All data was obtained from heterozygous animals. Aberrant interactions are indicated by red boxes. Pink scissors indicate CRISPR/Cas-induced breakpoints in each deletion. (B) A deletion (DelB^s) excluding the boundary region and CTCF cluster at the telomeric side of the Epha4 TAD (red octagon) was generated and compared to the brachydactyly-like deletion (DelB, including the CTCF cluster). Log2 ratio of the 4C-seq signal of DelB/DelB^S shows increased interaction with the Epha4 TAD in the DelB deletion when compared to DelB^s deletion (red box). Pax3 (right) shows normal expression of DelB^s /+ deletion mice, in contrast to Pax3 misexpression in DelB/+ mice (white arrow). (C) A deletion (Dbf^S) excluding the boundary region and CTCF cluster at the centromeric side of the Epha4 TAD (red octagon) was generated and compared to the doublefoot deletion (Dbf, including the CTCF cluster). Log2 ratio of the 4C-seq signal of Dbf/Dbf^S shows increased interaction with the Epha4 TAD in the Dbf deletion when compared to Dbf^S deletion (red box). Ihh (right) shows absence of limb expression in Dbf^s /+ deletion mice, in contrast to Ihh misexpression in Dbf/+ deletion mice (white arrow). See also Figure S7.

Figure 7. A model for pathogenicity of structural variations

Wild-type conformation shows the structure of two adjacent TADs. The activity of the enhancer (E) is restricted to gene 1 (G1) located inside the TAD. The TADs are separated by a boundary element (black). The black boxes represent the rearranged regions. The box with dotted lines represents the duplicated region. In the inversion, the enhancer is moved out of TADa and placed in the vicinity of gene 2 (G2). The boundary is now on the right side of E. This results in interaction of E with G2, but prevents its original interaction with G1. In the duplication, E is placed next to the duplicated G2’ resulting in interaction and misexpression. If the deletion removes the boundary and parts of TADb (black box), E is able to interact with G1 and G2, resulting in misexpression of G2. If the deletion leaves the boundary intact (Deletion^S, black box), the TAD structure remains intact and E interacts only with G1.