Expanding Duplication of Free Fatty Acid Receptor-2 (GPR43) Genes in the Chicken Genome

Abstract

Free fatty acid receptors (FFAR) belong to a family of five G-protein coupled receptors that are involved in the regulation of lipid metabolism, so that their loss of function increases the risk of obesity. The aim of this study was to determine the expansion of genes encoding paralogs of FFAR2 in the chicken, considered as a model organism for developmental biology and biomedical research. By estimating the gene copy number using quantitative polymerase chain reaction, genomic DNA resequencing, and RNA sequencing data, we showed the existence of 23 ± 1.5 genes encoding FFAR2 paralogs in the chicken genome. The FFAR2 paralogs shared an identity from 87.2% up to 99%. Extensive gene conversion was responsible for this high degree of sequence similarities between these genes, and this concerned especially the four amino acids known to be critical for ligand binding. Moreover, elevated nonsynonymous/synonymous substitution ratios on some amino acids within or in close-vicinity of the ligand-binding groove suggest that positive selection may have reduced the effective rate of gene conversion in this region, thus contributing to diversify the function of some FFAR2 paralogs. All the FFAR2 paralogs were located on a microchromosome in a same linkage group. FFAR2 genes were expressed in different tissues and cells such as spleen, peripheral blood mononuclear cells, abdominal adipose tissue, intestine, and lung, with the highest rate of expression in testis. Further investigations are needed to determine whether these chicken-specific events along evolution are the consequence of domestication and may play a role in regulating lipid metabolism in this species.

Keywords: FFAR; chicken; duplication; evolution; gene conversion; positive selection.

Fig. 1.—

FFAR2 has numerous copies in European broiler and in ancestral chicken genome. qPCR on genomic DNA shows clear difference in ΔCq between FFAR2 genes and the genes carrying only one copy per genome (four genes in European lines and three genes in Ancestral lines). qPCR was performed using “universal” primers able to amplify the 26 sequences of FFAR2 (see supplementary data S1, Supplementary Material online). FFAR2 error bars indicate standard deviation between two individual chickens. For the single copy genes, error bars represent standard deviation between the Cq measures for the three or four genes from two individual chickens.

Fig. 2.—

Phylogenetic tree of FFAR2 paralogous genes. The phylogenetic tree was reconstructed using PhyML (Guindon and Gascuel 2003). Bootstrap values are given when nodes are strongly supported (>70%). The scale represents the substitution rate. The sequence with an * has a specific insertion of eight amino acids (DNGSEADG) at the following positions: ENSGALP00000034780 (163-170), ENSGALP00000028197 (152-159), and ENSGALP00000019806 (158-165).

Fig. 3.—

Amino acids that are critical for ligand binding are conserved within the FFAR gene family. Amino acid sequences of one of the chicken FFAR2 paralogs, and FFAR1, -2 and -3 human sequences were aligned. The well-conserved amino acids that are critical of ligand binding are indicated with colored stars. Sequence identities are reported white on a black background, whereas similarities are boxed. Secondary structures, as deduced from our modeling (supplementary data S7, Supplementary Material online, and fig. 8), are reported above the sequences.

Fig. 4.—

Localization on human chromosome 19 of genes from the FFAR2 region. (A) Fragment of human chromosome 19 from 35.9 to 48.6 Mb and (B) FFAR2 region on human chromosome 19. Maps are given in Mb, from assembly version GRCh37/hg19. At the left of each map are shown blocks of conserved synteny between HSA19 and the chicken genome, obtained through RH mapping. Genes with an asterisk are not found in the current version of the chicken genome (Galgal4).

Fig. 5.—

FFAR2 mRNA expression in different tissues in chicken (A), in chicken Sertoli cells, seminiferous tubules, and whole testis (B), in adipose tissue of lean and fat divergent lines (C). Relative tissue abundances of FFAR2 genes were examined by RT-qPCR using “universal” primers able to amplify the 26 sequences of FFAR2. The ribosomal subunit 18 S, RPL15, and GAPDH were used as reference genes. Y axis indicates relative level of mRNA expressed in comparison to the lowest level observed (Uropygial gland [A], seminiferous tubules [B], and fat chickens [C]). Error bars indicate standard deviation between three biological replicates (A), three independent experiments performed in triplicate (B), and ten animals (C). Different letters indicate significant expression difference (P < 0.05).

Fig. 6.—

FFAR expression in chicken and pig using RNA-Seq in adipose tissue (A) and liver (B). Values correspond to the mean of eight individual expression counts (four males and four females). Chicken FFAR2 reads are the sum of all the paralog expression counts. Errors bars indicate standard deviation between individual reads.

Fig. 7.—

Gene conversion events in the FFAR2 cluster. Each line represents a gene and each ribbon represents a gene conversion event between two genes of the FFAR2 cluster. The thickness of the ribbon represents the significance of each identified fragment. The biggest, medium and smallest ribbons represent the fragments identified with three, two and one significant P values, respectively. For clarity, Ensembl gene IDs are shortened. The correspondence between reduced gene ID, gene ID, and protein ID can be found in the supplementary data S1, Supplementary Material online. The coordinates of each fragment can be found in the supplementary data S9, Supplementary Material online. Note that only the 24 genes involved in at least one gene conversion event are represented.

Fig. 8.—

Ribbon representation of the 3D model of FFAR2. The model was made by homology with the 3D structure of the proteinase-activated receptor 1 (pdb 3vw7). This last structure was obtained in complex with the antagonist vorapaxar, whose binding site is shown here light gray shaded. The four critical amino acids (corresponding to H140, R180, H242, and R255 in fig. 3 after correction of the alignment) are presented in yellow, whereas amino acids under positive selection, as reported in table 2, are indicated in orange, red, and purple.