A family history of DUX4: phylogenetic analysis of DUXA, B, C and Duxbl reveals the ancestral DUX gene

Abstract

Background: DUX4 is causally involved in the molecular pathogenesis of the neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD). It has previously been proposed to have arisen by retrotransposition of DUXC, one of four known intron-containing DUX genes. Here, we investigate the evolutionary history of this multi-member double-homeobox gene family in eutherian mammals.

Results: Our analysis of the DUX family shows the distribution of different homologues across the mammalian class, including events of secondary loss. Phylogenetic comparison, analysis of gene structures and information from syntenic regions confirm the paralogous relationship of Duxbl and DUXB and characterize their relationship with DUXA and DUXC. We further identify Duxbl pseudogene orthologues in primates. A survey of non-mammalian genomes identified a single-homeobox gene (sDUX) as a likely representative homologue of the mammalian DUX ancestor before the homeobox duplication. Based on the gene structure maps, we suggest a possible mechanism for the generation of the DUX gene structure.

Conclusions: Our study underlines how secondary loss of orthologues can obscure the true ancestry of individual gene family members. Their relationships should be considered when interpreting the relevance of functional data from DUX4 homologues such as Dux and Duxbl to FSHD.

Figure 1

DUX family overview. Representative structures of the four intron-containing and two intronless DUX genes are shown. Open reading frame in coloured boxes, homeobox sequences in dark blue. Conserved C-terminal domain of unknown function in yellow. Dotted boxes indicate UTR with uncertainty about transcriptional start and end sites. Note that introns and exons are drawn to different scales.

Figure 2

Mammalian DUX distribution. Summary of our mammalian DUX catalogue. Presumed functional genes (with intact ORF across all four homeobox exons, first exon may be unidentified) in green. DUX sequences with stop codons or deleted/missing exons in well sequenced regions in red. Putative DUX homologues with unclear functional status (mainly due to gaps in assembly) in yellow. Unless marked with ?, synteny was confirmed with anchor genes. Phylogenetic relationship between species according to references[28,44].

Figure 3

Anchor genes. Main anchor genes used to identify syntenic regions across species. DUX genes coloured yellow, anchor genes in black. Cphx as a marker of Duxbl/DUXB duplication shown in green. Gene sizes and intergenic spacing not drawn to scale. ENSEMBL access information: Human Cphx 10q processed transcript (ENSG00000230091); Human Cphx-like 16q protein coding (ENSG00000232078); Mouse CJ057 (ENSG00000021867); Human CJ057 (ENSG00000133678).

Figure 4

Conserved C-terminal domain. Amino acid alignment of conserved C-terminal domain found in DUXC, DUX4 and rodent Dux. All residues up to the stop codon are shown. Shading intensity indicates % of sequences that agree with consensus at that residue. Dark grey = >80%, grey = >60%, light grey = >40%, white = 40% or fewer. Figure produced with JalView 2.5.

Figure 5

Duxbl genes and pseudogenes. a) Overview of Duxbl pseudogenes and intact rodent Duxbl. Intact genes in colour, pseudogenes in grey. Dotted lines contain small gaps/contig holes in assembly. Asterisk next to species name indicates lack of synteny information. Star above exon = stop codon. b) Mapping of partial primate Duxbl deletion. Genomic nucleic acid alignment based on LAGAN in mVISTA of orangutan, human and chimpanzee Duxbl pseudogene loci. Conserved sequences shown as peaks, with Duxbl exons marked by dark bars. Gorilla was not included due to incomplete assembly.

Figure 6

Maximum Likelihood trees. a) Tree based on concatenated homeodomain sequences (120 amino acids). b) Tree based on individual homeodomains (60 amino acids). Bootstrap values shown at internal nodes. Number after DUX name denotes homeodomain 1 or 2. Number in brackets indicates sDUX copy number.

Figure 7

Single-homeobox sDUX. a) Schematics of identified sDUX exons. Asterisk denotes missing synteny information. Number in brackets corresponds to copy number in diagram below. b) Map of sDUX genes relative to anchors. All locations, genes sizes and distances drawn to scale. Red arrows = two sDUX exons including intron. Orientation of arrow indicates strand orientation. Distance between lizard Anxa11 and CJ057 too large to show to scale. c) Amino acid alignment of homeodomain. Duxbl homeodomains 1 and 2 included for comparison. Arrow marks splice junction. Conserved residues shaded as in Figure 4.

Figure 8

Relationship of sDUX to known human PRD class homeodomains. Maximum Likelihood tree based on single homeodomains. Apart from sDUX, all homeodomains are from the human orthologues (data extracted from HomeoDB[23]). Number in brackets indicates sDUX copy number. sDUX homeodomains cluster with DUX.

Figure 9

Homeobox duplication in DUX genes. Hypothetical scenario of homeobox duplication at the sDUX locus. As an illustrative example, the two tandem wallaby sDUX copies are shown with all sizes and distances to scale. A deletion could join the two genes leaving a single intact DUX ancestral gene. New intron redrawn at 1/8^th size (intron scale). Subsequent splicing changes could result in intron gain. Mouse Duxbl is shown for comparison.