Coding exons function as tissue-specific enhancers of nearby genes

Abstract

Enhancers are essential gene regulatory elements whose alteration can lead to morphological differences between species, developmental abnormalities, and human disease. Current strategies to identify enhancers focus primarily on noncoding sequences and tend to exclude protein coding sequences. Here, we analyzed 25 available ChIP-seq data sets that identify enhancers in an unbiased manner (H3K4me1, H3K27ac, and EP300) for peaks that overlap exons. We find that, on average, 7% of all ChIP-seq peaks overlap coding exons (after excluding for peaks that overlap with first exons). By using mouse and zebrafish enhancer assays, we demonstrate that several of these exonic enhancer (eExons) candidates can function as enhancers of their neighboring genes and that the exonic sequence is necessary for enhancer activity. Using ChIP, 3C, and DNA FISH, we further show that one of these exonic limb enhancers, Dync1i1 exon 15, has active enhancer marks and physically interacts with Dlx5/6 promoter regions 900 kb away. In addition, its removal by chromosomal abnormalities in humans could cause split hand and foot malformation 1 (SHFM1), a disorder associated with DLX5/6. These results demonstrate that DNA sequences can have a dual function, operating as coding exons in one tissue and enhancers of nearby gene(s) in another tissue, suggesting that phenotypes resulting from coding mutations could be caused not only by protein alteration but also by disrupting the regulation of another gene.

Figure 1.

eExons within DYNC1I1 and HDAC9 characterized using a mouse transgenic enhancer assay. A schematic of the DYNC1I1-DLX5/6 (A) and HDAC9-TWIST1 (F) genomic regions. Black boxes and orange ovals represent coding exons and positive eExons, respectively. The black arrows point to the genes that are thought to be regulated by the eExons. (B–C′) Mouse enhancer assays of DYNC1I1 eExon 15 and 17 at embryonic day 11.5 (E11.5). (B,B′) DYNC1I1 eExon 15 shows apical ectodermal ridge (AER) and limb mesenchyme enhancer activity (red arrows), and (C,C′) DYNC1I1eExon 17 shows anterior limb bud mesenchyme enhancer activity (red arrow). (D,E) Mouse whole-mount in situ hybridization of Dlx6 and Dlx5. (D′,E′) Dlx6 and Dlx5 limb expression pattern is similar to DYNC1I1 eExon 15 enhancer activity. In addition, Dlx5 is also expressed in anterior limb bud as depicted by the red arrow (E′), similar to DYNC1I1 eExon 17 enhancer activity (C′). (G–H′) Mouse enhancer assays of HDAC9 eExons 18 and 19. (G,G′) HDAC9 eExon 18 shows anterior limb bud enhancer activity (red arrows), and (H,H′) HDAC9 eExon 19 shows posterior limb bud (red arrows) and branchial arch enhancer activity in E11.5 mice. (I) Mouse whole-mount in situ hybridization of Twist1 at E11.5. (I′) Twist1 limb expression pattern is similar to the HDAC9 eExon 18 anterior limb bud enhancer activity (G′) marked by red arrow and HDAC9 eExon 19 posterior limb bud enhancer activity (H′). For B, C, G, and H, the numbers in the bottom right corner indicate the number of embryos showing this limb expression pattern/total LacZ stained embryos.

Figure 2.

Segmental analysis of DYNC1I1 eExon 15 and HDAC9 eExon 19 enhancer function in zebrafish. (A) DYNC1I1 eExon 15 was divided into three overlapping segments: 5′ intron, exon, 3′ intron. (Below) The UCSC Genome Browser (http://genome.ucsc.edu) conservation track shows that only the 5′ intron and exon are conserved between human and fish. (B) Zebrafish enhancer assay results for the different DYNC1I1 eExon 15 segments. While the 5′ intron and exon show enhancer activity in the fins and somitic muscles, only the combination of both gives comparable enhancer expression to the 608-bp originally injected fragment of DYNC1I1 eExon 15. The 3′ intron segment did not show enhancer activity. (C) HDAC9 eExon 19 was divided into three overlapping segments: distal 5′ intron, proximal 5′ intron, and exon. (Below) The UCSC Genome Browser conservation track shows that the proximal 5′ intron and exon are conserved between human and fish. (D) Zebrafish enhancer assay results for the different HDAC9 eExon 19 segments. While the proximal 5′ intron and exon show enhancer activity in the pectoral fin and branchial arches, only the combination of both gave comparable enhancer expression to the previously injected 1098-bp HDAC9 eExon 19 sequence. The distal 5′ intron segment did not show enhancer activity. Enhancer function is plotted as percentage of GFP expression/total live embryos. Each of these segments was injected into at least 100 zebrafish embryos.

Figure 3.

Histone modification signatures of the Dync1i1 eExon 15 in the mouse E11.5 limb bud. (A) Schematic representation of the Dync1i1-Dlx5/6 locus, showing the relative positions of primer sets used for ChIP-qPCR analyses: Dync1i1 exon 6 and eExon 15; Dlx6 promoter (pro.) and exon 2; Dlx5 exon 2 and promoter (pro.). (B) ChIP–qPCR analyses of H3K4me1, an enhancer histone mark. (C) ChIP-qPCR analyses of H3K27ac, an active enhancer histone mark. (D) ChIP-qPCR analyses of H3K4me3, a promoter histone mark. (E) ChIP-qPCR analyses of H3K36me3, a transcribed gene histone mark. (X-axis) Primer pairs; (y-axis) percentage of input recovery. (Error bars) SE from three technical replicates of a representative experiment.

Figure 4.

3C and DNA-FISH show a physical interaction between Dync1i1 eExon 15 and Dlx5/6 promoter regions in the mouse E11.5 limb. (A) Schematic of the Dync1i1-Dlx5/6 locus, showing the relative location of the primers used for 3C and the BAC probes used for DNA-FISH. (B) Chromatin looping events detected using 3C between Dync1i1 eExon 15 (orange oval) and promoters within the Dync1i1-Dlx5/6 locus. The closest HindIII restriction sites (RS1 and RS2) of each promoter were used to analyze the interaction frequencies to Dync1i1 eExon 15 (anchoring point). In the limb, the interaction frequencies between Dync1i1eExon 15 and Dlx6 and Dlx5 promoter regions were significantly higher compared to the heart negative control (more than 10- and 15-fold, respectively). No significant interaction differences were found between Dync1i1eExon 15 and the Dync1i1 promoter, the closest tested site to the anchoring point, or the two control regions (∼900 kb away from the Dync1i1-Dlx5/6 locus) in limb versus heart tissues. (Error bars) SE of the average of three independent PCR reactions. (C–L) DNA-FISH results with BAC probe RP23-430G21, which covers the Dync1i1eExon 15 region (red), and BAC probe RP23-77O3, which covers the Dlx5/6 gene regions (green). (C) E11.5 limb section with the dotted line highlighting the AER, as depicted by p63 staining in the nucleus. (D) BAC probes and DAPI staining of E11.5 limbs. (Squares) Magnified regions in E and F that highlight the colocalization of Dync1i1eExon 15 and Dlx5/6 signals. (G) E11.5 heart section shows p63 staining in the cytoplasm. (H) BAC probes and DAPI staining of E11.5 heart. (Squares) Magnified regions in I and J that show a separation of Dync1i1eExon 15 and Dlx5/6 signals. The white scale bars represent 5 μm length. (K,L) Calculated frequencies for every 0.2 μm distance interval in mouse E11.5 AER (K) and heart (L) tissues. (Black columns) Fraction of colocalized signals (0–0.2 mm). The number (n) of loci observed in this experiment indicates a significant difference between the frequencies of the colocalized signals in the AER and heart tissues (**P < 0.01; Student's t-test).

Figure 5.

Chromosomal abnormalities at chromosome 7q21-23 associated with SHFM1. A schematic representation of the genomic positions of breakpoints from chromosomal rearrangements in individuals with SHFM1 mapped to human genome assembly 18 (hg18) and compared to the location of DYNC1I1 eExon 15 and 17. An 880-kb microdeletion in an individual with a split foot phenotype was found to be at 95.39–96.27 Mb (Kouwenhoven et al. 2010). In the GK family, the 46,XY,t(7;20)(q22;p13) translocation breakpoints mapped to chr 7: 96.2–96.47 Mb. In the K6200 family, the chromosomal inversion breakpoints mapped to chr 7: 96,219,611 and 109,486,136. The breakpoint coordinates of a 7:46, XY, inv(7) (p22q21.3) with SHFM1 and pervasive developmental disorder-not otherwise specified (PDD-NOS) was found to be at chr 7: 95.53–95.72 Mb (van Silfhout et al. 2009). All of these chromosomal abnormalities overlap with DYNC1I1 eExon 15 and 17 (orange ovals). Two of these chromosomal aberrations do not overlap with the BS1 AER enhancer (white oval). (Lightning bolts) Translocation and inversion breakpoints; (diamonds) deletion.