Functional characterization of tissue-specific enhancers in the DLX5/6 locus

Abstract

Disruption of distaless homeobox 5 and 6 (Dlx5/6) in mice results in brain, craniofacial, genital, ear and limb defects. In humans, chromosomal aberrations in the DLX5/6 region, some of which do not encompass DLX5/6, are associated with split hand/foot malformation 1 (SHFM1) as well as intellectual disability, craniofacial anomalies and hearing loss, suggesting that the disruption of DLX5/6 regulatory elements could lead to these abnormalities. Here, we characterized enhancers in the DLX5/6 locus whose tissue-specific expression and genomic location along with previously characterized enhancers correlate with phenotypes observed in individuals with chromosomal abnormalities. By analyzing chromosomal aberrations at 7q21, we refined the minimal SHFM1 critical region and used comparative genomics to select 26 evolutionary conserved non-coding sequences in this critical region for zebrafish enhancer assays. Eight of these sequences were shown to function as brain, olfactory bulb, branchial arch, otic vesicle and fin enhancers, recapitulating dlx5a/6a expression. Using a mouse enhancer assay, several of these zebrafish enhancers showed comparable expression patterns in the branchial arch, otic vesicle, forebrain and/or limb at embryonic day 11.5. Examination of the coordinates of various chromosomal rearrangements in conjunction with the genomic location of these tissue-specific enhancers showed a correlation with the observed clinical abnormalities. Our findings suggest that chromosomal abnormalities that disrupt the function of these tissue-specific enhancers could be the cause of SHFM1 and its associated phenotypes. In addition, they highlight specific enhancers in which mutations could lead to non-syndromic hearing loss, craniofacial defects or limb malformations.

Figure 1.

The SHFM1 critical region defined by 7q21-23 chromosomal abnormalities. (A) Chromosome 7q11-q22 deletions (chromosome 7: 74–100 Mb; hg18) found in humans with SHFM1 and myoclonus-dystonia syndrome (OMIM #159900) (,–,,,–26). (B) Two translocations that are not associated with SHFM1 were used to define the conservative SHFM1 critical region of 2.5 Mb (chr 7: 94 000 000–96 500 000 bp; hg18) (24,25), which contains 15 genes including DLX5/6. (C) The minimal SHFM1 critical region (chromosome 7: 95.3–96.5 Mb; hg18) was defined using the most proximal translocation breakpoint in an individual with isolated SHFM ∼900 kb away from DLX5/6 (23) and a microdeletion that does not include DLX5/6 (10) (both at the centromeric side). Gene orientation is depicted by black arrows and identified mouse tissue-specific enhancers using gray ovals. Black lines represent individuals with a split hand/foot phenotype and unfilled lines represent individuals without a split hand/foot phenotype. Lightning bolts represent translocation and inversion breakpoints and diamonds represent deletions.

Figure 2.

Functional enhancers in the minimal SHFM1 critical region characterized using zebrafish and mice. (A) A schematic of the minimal SHFM1 region. Black and red boxes represent coding exons and the orange ovals represent sequences that have enhancer activity. (B–F) Tissue-specific enhancers in zebrafish at 72 hpf. Tissue-specific enhancer expression is indicated by the red arrows: (B) pectoral fin, (D) otic vesicle and forebrain and (E and F) branchial arches. (B′–F″) Mouse enhancer expression at E11.5. (B′ and B″) DYNC1I1 eExon 15 shows the AER, limb mesenchyme and genital tubercle enhancer activity. (C′ and C″) DYNC1I1 eExon 17 shows the anterior limb bud mesenchyme enhancer activity. (D′ and D″) eDlx#23 shows the otic vesicle, forebrain, branchial arch and limb bud mesenchyme enhancer activity. (E′–F″) eDlx#18 and eDlx#19 show branchial arch enhancer activity. The red arrows highlight the tissue-specific expression and the numbers in the bottom right of the embryos indicate the number of embryos showing this expression pattern/total LacZ stained embryos. (G and H) Zebrafish whole-mount in situ hybridization of dlx6a and dlx5a at 72 hpf. (I and J) Mouse whole-mount in situ hybridization of Dlx6 and Dlx5. (K) Dlx5 expression in the genital tubercle. (L) Dlx5 is expressed in the limb, both in the AER and in the anterior limb bud (red arrow) similar to DYNC1I1 eExon 15 and 17 expression (B′ and C′), respectively. Images B′, C′, C′, I, J and L were taken with permission from Birnbaum et al. (15).