A frame-shift mutation in COMTD1 is associated with impaired pheomelanin pigmentation in chicken

Author summaryVertebrates possess two types of melanin, red/yellow pheomelanin and black/brown eumelanin. In this study, we report that the recessive Inhibitor of gold phenotype in chicken, which causes a severe defect in pheomelanin pigmentation, is associated with a mutation that most likely inactivates the COMTD1 gene. This gene encodes an O-methyltransferase enzyme and is present throughout vertebrate evolution, but is one of the many genes in vertebrate genomes for which the biological function is still poorly understood. This is the first report of a COMTD1 mutation associated with a phenotypic effect. We show that the COMTD1 protein is present in mitochondria in pigment cells. Furthermore, inactivation of the gene in a mouse pigment cell line results in a significant reduction in metabolites that are important for the synthesis of pheomelanin. We hypothesize that COMTD1 activity protects pigment cells from oxidative stress and that inactivation of this function impairs the production of pheomelanin. It is likely that COMTD1 has a similar function in other cell types. This study establishes this chicken mutation as a model for further studies of COMTD1 function.The biochemical pathway regulating the synthesis of yellow/red pheomelanin is less well characterized than the synthesis of black/brown eumelanin. Inhibitor of gold (IG phenotype) is a plumage colour variant in chicken that provides an opportunity to further explore this pathway since the recessive allele (IG) at this locus is associated with a defect in the production of pheomelanin. IG/IG homozygotes display a marked dilution of red pheomelanin pigmentation, whilst black pigmentation (eumelanin) is only slightly affected. Here we show that a 2-base pair insertion (frame-shift mutation) in the 5(th) exon of the Catechol-O-methyltransferase containing domain 1 gene (COMTD1), expected to cause a complete or partial loss-of-function of the COMTD1 enzyme, shows complete concordance with the IG phenotype within and across breeds. We show that the COMTD1 protein is localized to mitochondria in pigment cells. Knockout of Comtd1 in a mouse melanocytic cell line results in a reduction in pheomelanin metabolites and significant alterations in metabolites of glutamate/glutathione, riboflavin, and the tricarboxylic acid cycle. Furthermore, COMTD1 overexpression enhanced cellular proliferation following chemical-induced transfection, a potential inducer of oxidative stress. These observations suggest that COMTD1 plays a protective role for melanocytes against oxidative stress and that this supports their ability to produce pheomelanin

Strong signatures of selection in the domestic pig genome

Domestication of wild boar (Sus scrofa) and subsequent selection have resulted in dramatic phenotypic changes in domestic pigs for a number of traits, including behavior, body composition, reproduction, and coat color. Here we have used whole-genome resequencing to reveal some of the loci that underlie phenotypic evolution in European domestic pigs. Selective sweep analyses revealed strong signatures of selection at three loci harboring quantitative trait loci that explain a considerable part of one of the most characteristic morphological changes in the domestic pig—the elongation of the back and an increased number of vertebrae. The three loci were associated with the NR6A1, PLAG1, and LCORL genes. The latter two have repeatedly been associated with loci controlling stature in other domestic animals and in humans. Most European domestic pigs are homozygous for the same haplotype at these three loci. We found an excess of derived nonsynonymous substitutions in domestic pigs, most likely reflecting both positive selection and relaxed purifying selection after domestication. Our analysis of structural variation revealed four duplications at the KIT locus that were exclusively present in white or white-spotted pigs, carrying the Dominant white, Patch, or Belt alleles. This discovery illustrates how structural changes have contributed to rapid phenotypic evolution in domestic animals and how alleles in domestic animals may evolve by the accumulation of multiple causative mutations as a response to strong directional selection

A frame-shift mutation in COMTD1 is associated with impaired pheomelanin pigmentation in chicken.

The biochemical pathway regulating the synthesis of yellow/red pheomelanin is less well characterized than the synthesis of black/brown eumelanin. Inhibitor of gold (IG phenotype) is a plumage colour variant in chicken that provides an opportunity to further explore this pathway since the recessive allele (IG) at this locus is associated with a defect in the production of pheomelanin. IG/IG homozygotes display a marked dilution of red pheomelanin pigmentation, whilst black pigmentation (eumelanin) is only slightly affected. Here we show that a 2-base pair insertion (frame-shift mutation) in the 5th exon of the Catechol-O-methyltransferase containing domain 1 gene (COMTD1), expected to cause a complete or partial loss-of-function of the COMTD1 enzyme, shows complete concordance with the IG phenotype within and across breeds. We show that the COMTD1 protein is localized to mitochondria in pigment cells. Knockout of Comtd1 in a mouse melanocytic cell line results in a reduction in pheomelanin metabolites and significant alterations in metabolites of glutamate/glutathione, riboflavin, and the tricarboxylic acid cycle. Furthermore, COMTD1 overexpression enhanced cellular proliferation following chemical-induced transfection, a potential inducer of oxidative stress. These observations suggest that COMTD1 plays a protective role for melanocytes against oxidative stress and that this supports their ability to produce pheomelanin

Genotype data at SNP positions of the <i>IG</i> locus used for identical-by-descent mapping.

Genotype data at SNP positions of the IG locus used for identical-by-descent mapping.</p

CRISPR/Cas9 mediated inactivation of <i>Comtd1</i> in B16F10 cells.

(A) Schematic description of CRISPR/Cas9 mediated inactivation of murine Comtd1. Black bars indicate the target sites of two gRNAs in the exons of Comtd1. Yellow arrow indicates a 236bp deletion introduced by paired sgRNAs. The primer pair indicated by red arrows is used for amplifying genomic DNA. (B) Six colonies retrieved from Comtd1 knockout in B16F10 cells. The PAM sequence is in red. Dash (-) indicate deleted nucleotides. The top one is wild type and the others are KO clones, of which, three lines (KO1, KO2, KO3) were generated using sgRNA1, one line was generated using sgRNA2, and the last two lines carrying 236bp deletion were generated by the sgRNA pair. (C) Western blot analysis of whole-cell lysates prepared from six COMTD1 knockout clones and two WT cell lines using antibodies against COMTD1 and the control β-actin. (D) Quantitative RT-PCR analysis of Comtd1 expression in KO and WT cell lines. Date are presented as mean ± SD (n = 3 biological replicates). (E) Cell growth curve of KO (grey) and WT (black) were recorded by the Incucyte Zoom live-cell imaging system and data are expressed as cell confluence (%; mean ± SEM, n = 6 in KO, n = 3 in WT).</p

Sequence variants (non-reference alleles) in the coding region of <i>LRMDA</i>, <i>ZNF503</i>, and <i>COMTD1</i> detected by sequencing one homozygous <i>IG/IG</i> bird and the corresponding allele frequencies among non-IG <i>(N/-</i>) birds.

Sequence variants (non-reference alleles) in the coding region of LRMDA, ZNF503, and COMTD1 detected by sequencing one homozygous IG/IG bird and the corresponding allele frequencies among non-IG (N/-) birds.</p

Genotype distribution of the 2-bp insertion in <i>COMTD1</i> associated with the Inhibitor of Gold (IG) phenotype in different populations sorted by phenotype.

Genotype distribution of the 2-bp insertion in COMTD1 associated with the Inhibitor of Gold (IG) phenotype in different populations sorted by phenotype.</p