Multi-species comparative analysis of the equine ACE gene identifies a highly conserved potential transcription factor binding site in intron 16

Abstract

Angiotensin converting enzyme (ACE) is essential for control of blood pressure. The human ACE gene contains an intronic Alu indel (I/D) polymorphism that has been associated with variation in serum enzyme levels, although the functional mechanism has not been identified. The polymorphism has also been associated with cardiovascular disease, type II diabetes, renal disease and elite athleticism. We have characterized the ACE gene in horses of breeds selected for differing physical abilities. The equine gene has a similar structure to that of all known mammalian ACE genes. Nine common single nucleotide polymorphisms (SNPs) discovered in pooled DNA were found to be inherited in nine haplotypes. Three of these SNPs were located in intron 16, homologous to that containing the Alu polymorphism in the human. A highly conserved 18 bp sequence, also within that intron, was identified as being a potential binding site for the transcription factors Oct-1, HFH-1 and HNF-3β, and lies within a larger area of higher than normal homology. This putative regulatory element may contribute to regulation of the documented inter-individual variation in human circulating enzyme levels, for which a functional mechanism is yet to be defined. Two equine SNPs occurred within the conserved area in intron 16, although neither of them disrupted the putative binding site. We propose a possible regulatory mechanism of the ACE gene in mammalian species which was previously unknown. This advance will allow further analysis leading to a better understanding of the mechanisms underpinning the associations seen between the human Alu polymorphism and enzyme levels, cardiovascular disease states and elite athleticism.

Figure 1. Schematic representation of the equine ACE gene structure.

The genomic structure (a) depicts exons (boxes), introns (bars) and the locations of polymorphisms identified in this study. The broken bars indicate where sequencing was not completed through introns. The grey bar beneath the gene indicates areas screened in the pools for polymorphisms. Common polymorphisms used for haplotype analysis are indicated in blue font, and the active sites and transmembrane domain are indicated. The predicted testicular (b) and endothelial (c) transcripts, including exon sizes (bp), are shown. The duplicated areas of the gene (regions 1 and 2) are shaded in light grey and the predicted UTR’s are shaded dark grey.

Figure 2. Maximum likelihood consensus tree of ACE cDNA sequence across 11 species.

The horse, dog, cat, dolphin, cow, rabbit, elephant, human, chimpanzee, rat and mouse orthologous ACE genes were included, with the elephant designated as the outgroup [EnSembl: ENSECAG00000012910, ENSCAFG00000012998, ENSFCAG00000002078, ENSTTRG00000001667, ENSBTAG00000024950, ENSOCUT00000001559, ENSLAFG00000006295, ENST00000290866, ENSPTRT00000049041, ENSRNOT00000010627 and ENSMUST00000001963 respectively]. The GTR model of nucleotide substitution was applied and bootstrap branch supports are shown (100). Transition/transversion ratio, number of invariable sites and gamma distribution parameters were estimated from the data.

Figure 3. Sequence conservation across intron 16 of multi species ACE genes.

(a) Plot of conservation spanning exons 15 to 18 between the developed equine ACE sequence and the reference sequences from the dog, human, elephant, rabbit and mouse. Pink coloured regions are >70% conserved between the horse and query sequence; while the dark blue regions are annotated exons. Pink conserved peaks are clearly visible in intron 16. The positions of common equine SNPs are marked with black arrows, and the human I/D variant is noted in blue. The human intron sequence analysed here was of the deletion allele. (b) Aligned intron sequence from six species showing the conserved nucleotides highlighted in yellow. The position of SNP rs4338 that occurs in the middle of the human CNE is highlighted in green. (c) Results of the rVISTA scan showing likely motif binding sites (green bars) for the three identified transcription factors.

Figure 4. Haplotypes identified in the equine ACE gene.

(a) Haplotype representation, with blue boxes showing major alleles, red boxes minor alleles and the yellow box shows the third allele of SNP 6. (b) Haplotype distributions across the whole population and divided into the different breeds examined: Thoroughbred (TB), Arabian (AR), Standardbred (SB) and heavy horses (HH, including Clydesdales and Shires).