Missed, not missing: Phylogenomic evidence for the existence of Avian FoxP3

The Forkhead box transcription factor FoxP3 is pivotal to the development and function of regulatory T cells (Tregs), which make a major contribution to peripheral tolerance. FoxP3 is believed to perform a regulatory role in all the vertebrate species in which it has been detected. The prevailing view is that FoxP3 is absent in birds and that avian Tregs rely on alternative developmental and suppressive pathways. Prompted by the automated annotation of foxp3 in the ground tit (Parus humilis) genome, we have questioned this assumption. Our analysis of all available avian genomes has revealed that the foxp3 locus is missing, incomplete or of poor quality in the relevant genomic assemblies for nearly all avian species. Nevertheless, in two species, the peregrine falcon (Falco peregrinus) and the saker falcon (F. cherrug), there is compelling evidence for the existence of exons showing synteny with foxp3 in the ground tit. A broader phylogenomic analysis has shown that FoxP3 sequences from these three species are similar to crocodilian sequences, the closest living relatives of birds. In both birds and crocodilians, we have also identified a highly proline-enriched region at the N terminus of FoxP3, a region previously identified only in mammals

The genomic conservation of <i>foxp3</i> with its downstream flanking genes, <i>cacna1f</i> and <i>ccdc22</i>.

<p>The genomic conservation of <i>foxp3</i> with its downstream flanking genes, <i>cacna1f</i> and <i>ccdc22</i>.</p

A summary of the structural domains and regions of FoxP3 in different clades of the Animal Kingdom.

<p>A summary of the structural domains and regions of FoxP3 in different clades of the Animal Kingdom.</p

The genomic context of avian <i>foxp3</i>.

<p>(A) The gene neighbourhood of <i>foxp3 in</i> a number of archosaurs showing the significant masked repeats of the American crow between <i>hsd17b10</i> and <i>cacna1f</i> (gaps in the line indicate intergenic repeat regions), the approximate location of the <i>foxp3</i> locus in the saker and peregrine falcon (marked by a pink arrow with a dashed line), and the low nucleotide sequence identity of <i>foxp3</i> between the American alligator and the ground tit. In the displayed region the overall masked repeats are as follows: 54.7% in the American crow, 28.0% in the saker falcon, 31.4% in the peregrine falcon, 6.8% in the ground tit and 0.02% in the American alligator. (B) The <i>foxp3</i> gene neighbourhood of the mouse, American alligator and ground tit, showing the gene variation upstream of <i>foxp3</i> in mammals, birds and crocodilians and the low nucleotide sequence identity of murine <i>foxp3</i> versus the American alligator and ground tit. (C) A comparison of the approximate <i>foxp3</i> loci in both falcons with the ground tit <i>foxp3 locus</i>, showing masked repeats (gaps in the line) and predicted exons (rectangles). All subfigures were produced using Easyfig [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150988#pone.0150988.ref047" target="_blank">47</a>] with BLASTN identity comparisons indicated by the scales on each subfigure.</p

Alignment of the forkhead domain of FoxP3 and FoxP3-like proteins, highlighting mammalian and avian signature residues.

<p>The consistent exon boundaries in the ground tit and peregrine falcon were used to curate the gap within the sequence of the saker falcon. The mouse sequence is used as the baseline sequence and only amino acids in other sequences that differ are shown.</p

Avian genome quality and its relationship to the annotation and assembly of the <i>foxp3</i> gene neighbourhood.

<p>Avian genome quality and its relationship to the annotation and assembly of the <i>foxp3</i> gene neighbourhood.</p

Ground tit <i>foxp3</i> transcriptome coverage.

<p>The 11 numbered exons of the putative ground tit <i>foxp3</i> sequence annotated with the sequence regions that encode ground tit FoxP3 functional regions. A collection of reads with 100% identity (shown in green) were found with a BLASTN search of ground tit RNA-Seq data from the muscle in the SRA (SRX246872; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150988#pone.0150988.s002" target="_blank">S2 File</a>).</p

The distinct forkhead domain of FoxP3.

<p>The FoxP3 forkhead domain of the mouse, alligator and ground tit, aligned to FoxP1, FoxP2 and FoxP4 paralogues in the same species, demonstrating its divergent evolution and unique signature residues. The mouse FoxP3 sequence is used as the baseline sequence and only amino acids in other sequences that differ are shown.</p