Functional characterization of the V9D and R10C substitutions in chicken ARF.

<p>(A) Far-UV circular dichroism (CD) is measured in mDeg and monitors protein secondary structure. Spectra of MDM2_204-298, ARF_1-14^WT, and complexes of ARF_1-14^WT/MDM2_204-298, ARF_1-14^V9D/MDM2_204-298, and ARF_1-14^R10C/MDM2_204-298. MDM2_204-298 was used at 10 μM and all peptides at 64 μM final concentrations. Spectra of all peptides in free form and at 25 μM concentration in complex with MDM2_204-298 are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006665#pgen.1006665.s008" target="_blank">S5 Fig</a>. (B) Isothermal titration calorimetry (ITC) experiments in which the heat (enthalpy) associated with binding is recorded as μcal/sec for each titration point (top panel) and integrated and normalized against the concentrations of ARF peptide and MDM2_204-298 to obtain a binding isotherm (expressed as kcal mol^-1 versus molar ratio; bottom panel). In the experiment, ARF peptides (WT, V9D, and R10C, respectively) were titrated into 100 μM MDM2_204-298 with the peptide concentration increasing by approximately 10 μM in each titration point. Peaks (top) and integrated energies (bottom) corrected for heats of dilution are shown. Raw data are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006665#pgen.1006665.s009" target="_blank">S6 Fig</a>. (C) Assessment of the effect of the two coding mutations on the interaction between ARF and MDM2 based on a luciferase assay. Reduced luciferase activity implies weaker interaction between ARF and MDM2, monitored as a decreased ability of ARF to protect the transcription factor p53 from degradation (Student’s t-test; *<i>P</i><0.05, ***<i>P</i><0.001).</p

Alleles and phenotypes at the <i>Sex-linked barring</i> (<i>CDKN2A</i>) locus in chicken.

<p>(A) Female and male Coucou de Rennes chicken with separately depicted feather illustrating the iconic Sex-linked barring phenotype caused by the <i>B1</i> allele. (B) <i>Sex-linked barring</i> alleles and associated sequence variants. SNP1 and SNP2 are non-coding while SNP3 and SNP4 constitute non-synonymous changes in the region encoding the MDM2 binding domain. (C) Sex-linked dilution phenotype caused by the <i>B2</i> allele. Note how the homozygous male has an almost white appearance whereas the hemizygous female as well as the heterozygous male show a Sex-linked barring pattern. (D) Phenotype with individual feathers from <i>N/N</i>, <i>B2/N</i> and <i>B0/N</i> chicken. Photo credits: (A) Hervé Ronné, Ecomusée du pays de Rennes, (C) Susanne Kerje, (D) Dominic Wright and Doreen Schwochow-Thalmann.</p

Differential expression of <i>CDKN2A</i> in feathers.

<p>(A) Relative expression of <i>CDKN2A</i> in Sex-linked barred chickens carrying the <i>B0</i> or <i>B2</i> allele and non-barred control feathers. Expression data was normalized using <i>EEF2</i> and <i>UB</i>. (B) Allele-specific expression of <i>CDKN2A</i> in <i>B2/N</i> feathers, skin and liver. Left panel: cDNA data using tissue samples from four <i>B2/N</i> chickens. Right panel: Genomic DNA from the different genotypes was used as control. The relative expression of the two alleles was determined by pyrosequencing. (Student’s t-test; * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001).</p

Characterization of the expression of MITF, MART1, <i>TYR</i> and <i>CDKN2A</i> in melanocyte progenitors and differentiated pigment cells in feathers from different genotypes at the <i>Sex-linked barring</i> locus.

<p>(A) Anatomy of growing feather, papilla ectoderm (PE), lower bulge (LB), middle bulge (MB), upper bulge (UB), ramogenic zone (RGZ), and barb (BA). (B) Distribution of cells from the melanocyte lineage across different parts of the feather in different genotypes detected by immunohistochemistry (MITF and MART) and <i>in-situ</i> hybridization (<i>TYR</i> and <i>CDKN2A</i>). (C) Average number of MITF+, MART1+, <i>TYR+</i> and <i>CDKN2A+</i> positive cells in the barbs of chickens with different genotypes. Significant differences are indicated by stars (One-way ANOVA, Tukey’s multi-comparison post-hoc test; * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001).</p

Metabolomics analysis of <i>Comtd1</i>-KO and wild-type B16F10 cells.

Principal component analysis (PCA) and Partial Least-Squares Discriminant Analysis (PLS-DA) with model validation results. PCA is an unsupervised multivariate analysis, which provides an unbiased overview of the metabolite features due to unawareness of the two groups compared (WT and KO in this case). PLS-DA is a multivariate analysis which considers data from the two groups and select the most discriminating metabolites that separates the two groups. This is the reason why PLS-DA show a better separation between WT and KO groups. (A) PCA in MS positive mode detection. (B) PLS-DA in MS positive mode detection. (C) PCA in MS negative mode detection. (D) PLS-DA in MS negative mode detection. (E) Significantly altered pathways impacted by Comtd1 knockout. (PDF)</p

HA-tagged COMTD1 localizes to mitochondria in immortalized mouse melanocytes.

(A-D) Immortalized melan-Ink4a cells from Ink4a-deficient C57BL/6J mice were transiently transfected to express COMTD1 fused with the HA11 epitope at either the N-terminus (HA-COMTD1; A, C) or C-terminus (COMTD1-HA; B, D). Two days later, cells were fixed and analyzed by bright field (BF) and immunofluorescence microscopy for HA and either the mitochondrial resident protein MAVS (A, B) or the ER resident protein calnexin (CNX; C, D). Individual images of labelled cells or the bright field image are shown in addition to an overlay of HA (green) with MAVS (red; HA/ MAVS), CNX (red; HA/ CNX), or the pseudocolored bright field image (magenta; HA/BF). Insets show a 5-fold magnified image of the boxed region to emphasize overlap or lack thereof. Main scale bar, 10 μm; inset scale bar, 2 μm. (E) Quantification of the degree of overlap of COMTD1-HA or HA-COMTD1, as indicated, with markers of the ER (CNX; N = 29 for COMTD1-HA, N = 17 for HA-COMTD1), mitochondria (MAVS; N = 25 for COMTD1-HA, N = 16 for HA-COMTD1), mature melanosomes (TYRP1; N = 16), immature melanosomes (PMEL; N = 17), late endosomes/ lysosomes (LAMP2; N = 15), or early endosomes (STX13; N = 21). Data from 4–5 individual experiments are presented as a box and whiskers plot in which the area of overlap is shown relative to the total area occupied by HA (e.g., CNX vs. HA) or by the indicated marker (e.g., HA vs. CNX). See S2 Fig for examples of the data for TYRP1, PMEL, LAMP2 and STX13. Statistical significance was determined by ordinary one-way ANOVA with Tukey’s tests for multiple comparisons; ****, P < 0.0001.</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

Primer sequences used in this paper.

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.</div

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

Chemical characterization of feather melanin.

(A) depicts levels of total melanin in wild-type birds (R+ and R-) and in IG birds analyzed by Soluene-350 solubilization. (B) depicts A650/A500 ratios analyzed by Soluene-350 solubilization. (C) depicts eumelanin (EM), benzothiazine-pheomelanin (BT-PM), and benzothiazole-pheomelanin (BZ-PM) analyzed as PTCA, 4-AHP, and TTCA, respectively. Feather samples were obtained from neck regions from 3 males and 3 females. Results are shown with the means ± SEM of 6 birds. *** and ** indicate P P = 0.0001–0.01 between R- and IG birds and between R- and R+ birds (Student’s t test).</p