Evolutionary genomics implies a specific function of Ant4 in mammalian and anole lizard male germ cells

Abstract

Most vertebrates have three paralogous genes with identical intron-exon structures and a high degree of sequence identity that encode mitochondrial adenine nucleotide translocase (Ant) proteins, Ant1 (Slc25a4), Ant2 (Slc25a5) and Ant3 (Slc25a6). Recently, we and others identified a fourth mammalian Ant paralog, Ant4 (Slc25a31), with a distinct intron-exon structure and a lower degree of sequence identity. Ant4 was expressed selectively in testis and sperm in adult mammals and was indeed essential for mouse spermatogenesis, but it was absent in birds, fish and frogs. Since Ant2 is X-linked in mammalian genomes, we hypothesized that the autosomal Ant4 gene may compensate for the loss of Ant2 gene expression during male meiosis in mammals. Here we report that the Ant4 ortholog is conserved in green anole lizard (Anolis carolinensis) and demonstrate that it is expressed in the anole testis. Further, a degenerate DNA fragment of putative Ant4 gene was identified in syntenic regions of avian genomes, indicating that Ant4 was present in the common amniote ancestor. Phylogenetic analyses suggest an even more ancient origin of the Ant4 gene. Although anole lizards are presumed male (XY) heterogametic, like mammals, copy numbers of the Ant2 as well as its neighboring gene were similar between male and female anole genomes, indicating that the anole Ant2 gene is either autosomal or located in the pseudoautosomal region of the sex chromosomes, in contrast to the case to mammals. These results imply the conservation of Ant4 is not likely simply driven by the sex chromosomal localization of the Ant2 gene and its subsequent inactivation during male meiosis. Taken together with the fact that Ant4 protein has a uniquely conserved structure when compared to other somatic Ant1, 2 and 3, there may be a specific advantage for mammals and lizards to express Ant4 in their male germ cells.

Figure 1. Anole lizard has orthologs of the Ant1, 2, 3, and 4 genes.

(A) The schematic diagram from the Ensemble database shows the configuration of genes encoding Ant proteins in human and anole lizard. Exons and introns are shown as black boxes and lines, respectively. Numbers represent the amount of amino acid sequence identity that the products of each Ant gene in the human and anole lizard exhibit. (B) Alignment of the inferred amino acid sequences of the products of the Ant1, 2, 3, and 4 genes in mammals (human, cow, mouse) and reptile (anole lizard). Ant amino acid sequences of the selected species were aligned using ClustalW2. Amino acids sequence surrounding a signature motif in the Ant4 protein (RRRMMM) and the N- and C-terminal parts of the proteins are shown. Amino acid residues are numbered from the initiation codon (M) to the termination codon in each Ant protein.

Figure 2. The maximum-likelihood (ML) estimate of Ant phylogeny supports an ancient origin of Ant4.

(A) Large scale phylogeny of animal Ant homologs obtained by ML analysis of amino acid sequences using the LG+Γ+F model of evolution. Support based upon 500 bootstrap replicates is shown as a percentage adjacent to the relevant branches when it exceeds 50%. This phylogeny included a fungal (Saccharomyces cerevisiae) outgroup, and the root of the tree was assumed to lie between fungi and the animal-choanoflagellate clade (the latter group is represented by Monosiga brevicollis). This phylogeny suggests a number of ancient gene duplications, one of which led to a “core group” that includes the vertebrate Ant homologs. (B) Phylogeney of the “core group” obtained using the same model. Support based upon 500 bootstrap replicates is shown as a percentage adjacent to the relevant branches when it exceeds 50%; support for some clades within well-supported groups (e.g. branches within teleost fish) was omitted in the interest of simplicity. Analyses using this more limited taxon sample were conducted to test the possibility that the position of Ant4 was influenced by the inclusion of divergent sequences. The position of Ant4 was robust to the taxon set analyzed; in fact, the bootstrap support for an ancient origin (the Ant4-tunicate clade) increased for the smaller taxon sample.

Figure 3. Anole Ant4 is expressed in testis.

Ant1, 2, 3 and 4 gene expression in heart, liver, and testis of anole lizard was examined by qRT-PCR. Ant mRNA amounts were normalized based upon the expression of the β-actin mRNA. Error bars indicate standard deviations of triplicate samples.

Figure 4. Degenerate DNA fragment of putative Ant4 gene in the syntenic region of chicken genome.

(A) The syntenic region that includes Ant4 in the human, anole lizard and chicken genomes. The schematic diagram shows Ant4 (red boxes) with chromosomal location flanked by neighboring genes (blue boxes). Translated amino acid sequence of degenerate DNA regions of the putative Ant4 gene loci in the chicken genome was aligned with amino acid sequence of Ant4 of human and anole lizard. The asterisk in the chicken sequence is a stop codon; additional inactivating mutations include a 2-bp frameshift between nucleotide that would encode the D and K at the end of the chicken sequence and a stop codon immediately after the K. (B) Vertebrate phylogeny showing approximate divergence times (in millions of years before present) and the expected changes in ω (see text for definition) for the early and late inactivation models. (C) Phylogeny with branch lengths reflecting numbers of synonymous and non-synonymous substitutions. The light gray lines are included to make it easier to identify the taxon associated with each terminal, they have no biological significance.

Figure 5. Gene dosage analysis in female and male animals.

Relative copy number ratio of Ant1, 2, 3 and 4 genes and Slc25a43 gene in male and female animals were examined by quantitative PCR analysis of genomic DNA of anole lizard and mouse. PCR amplification was normalized to the control β-actin gene. Error bars indicate standard deviations of three independent experiments. (A) Relative gene dosages of the Ant 1, 2, 3 and 4 between female and male in anole lizard and mouse. The result was confirmed with two different sets of primers for anole Ant2 and Ant4 genes. (B) Relative gene dosage of the Slc25a43 between female and male in anole lizard and mouse. The Slc25a43 gene localizes adjacent to the Ant2 gene in both anole and mouse genome (left panel).

Figure 6. CpG methylation of the anole and mouse Ant2 genes.

The CpG methylation of the Ant2 gene promoter regions in female and male anole lizard and mouse was examined by bisulfite sequencing analysis. The schematic diagrams indicate CpG islands in the Ant2 gene promoter regions examined in anole lizard and mouse. Each row of the circles represents an individual clone of a PCR amplicon in bisulfate sequencing analysis.