Epistatic and Combinatorial Effects of Pigmentary Gene Mutations in the Domestic Pigeon

SummaryUnderstanding the molecular basis of phenotypic diversity is a critical challenge in biology, yet we know little about the mechanistic effects of different mutations and epistatic relationships among loci that contribute to complex traits. Pigmentation genetics offers a powerful model for identifying mutations underlying diversity and for determining how additional complexity emerges from interactions among loci. Centuries of artificial selection in domestic rock pigeons (Columba livia) have cultivated tremendous variation in plumage pigmentation through the combined effects of dozens of loci. The dominance and epistatic hierarchies of key loci governing this diversity are known through classical genetic studies [1–6], but their molecular identities and the mechanisms of their genetic interactions remain unknown. Here we identify protein-coding and cis-regulatory mutations in Tyrp1, Sox10, and Slc45a2 that underlie classical color phenotypes of pigeons and present a mechanistic explanation of their dominance and epistatic relationships. We also find unanticipated allelic heterogeneity at Tyrp1 and Sox10, indicating that color variants evolved repeatedly though mutations in the same genes. These results demonstrate how a spectrum of coding and regulatory mutations in a small number of genes can interact to generate substantial phenotypic diversity in a classic Darwinian model of evolution [7]

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.This work was supported by funding from the Medical Research Council and the European Research Council (ERC, 281847) (A.P.J.), the Lister Institute for Preventative Medicine (A.P.J. and G.S.S.), Medical Research Scotland (L.S.B.), German Federal Ministry of Education and Research (BMBF, 01GM1404) and E-RARE network EuroMicro (B.W), Wellcome Trust (M. Hurles), CMMC (P.N.), Cancer Research UK (C17183/A13030) (G.S.S. and M.R.H), Swiss National Science Foundation (P2ZHP3_158709) (O.M.), AIRC (12710) and ERC/EU FP7 (CIG_303806) (S.S.), Cancer Research UK (C6/A11224) and ERC/EU FP7 (HEALTH-F2- 2010-259893) (A.N.B. and S.P.J.).This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ng.345

A Val85Met mutation in melanocortin-1 receptor is associated with reductions in eumelanic pigmentation and cell surface expression in domestic rock pigeons (Columba livia).

Variation in the melanocortin-1 receptor (Mc1r) is associated with pigmentation diversity in wild and domesticated populations of vertebrates, including several species of birds. Among domestic bird species, pigmentation variation in the rock pigeon (Columbalivia) is particularly diverse. To determine the potential contribution of Mc1r variants to pigment diversity in pigeons, we sequenced Mc1r in a wide range of pigeon breeds and identified several single nucleotide polymorphisms, including a variant that codes for an amino acid substitution (Val85Met). In contrast to the association between Val85Met and eumelanism in other avian species, this change was associated with pheomelanism in pigeons. In vitro cAMP accumulation and protein expression assays revealed that Val85Met leads to decreased receptor function and reduced cell surface expression of the mutant protein. The reduced in vitro function is consistent with the observed association with reduced eumelanic pigmentation. Comparative genetic and cellular studies provide important insights about the range of mechanisms underlying diversity among vertebrates, including different phenotypic associations with similar mutations in different species

Molecular Signatures of Placentation and Secretion Uncovered in Poeciliopsis Maternal Follicles

Placentation evolved many times independently in vertebrates. Although the core functions of all placentas are similar, we know less about how this similarity extends to the molecular level. Here, we study Poeciliopsis, a unique genus of live-bearing fish that have independently evolved complex placental structures at least three times. The maternal follicle is a key component of these structures. It envelops yolk-rich eggs and is morphologically simple in lecithotrophic species but has elaborate villous structures in matrotrophic species. Through sequencing, the follicle transcriptome of a matrotrophic, Poeciliopsis retropinna, and lecithotrophic, P. turrubarensis, species we found genes known to be critical for placenta function expressed in both species despite their difference in complexity. Additionally, when we compare the transcriptome of different river populations of P. retropinna, known to vary in maternal provisioning, we find differential expression of secretory genes expressed specifically in the top layer of villi cells in the maternal follicle. This provides some of the first evidence that the placental structures of Poeciliopsis function using a secretory mechanism rather than direct contact with maternal circulation. Finally, when we look at the expression of placenta proteins at the maternal-fetal interface of a larger sampling of Poeciliopsis species, we find expression of key maternal and fetal placenta proteins in their cognate tissue types of all species, but follicle expression of prolactin is restricted to only matrotrophic species. Taken together, we suggest that all Poeciliopsis follicles are poised for placenta function but require expression of key genes to form secretory villi.</p

Met85 alleles of Mc1r are associated with pheomelanism in the domestic rock pigeon.

<p><b>A</b>. Genotype frequencies across all sampled breeds, with birds categorized as eumelanic or pheomelanic. Met85 genotypes are significantly enriched in pheomelanic birds (p < 0.03, Chi-square test). <b>B</b>. Met85 genotypes are not significantly enriched in pheomelanic <i>recessive red</i> birds (p = 0.21, Chi-square test). <b>C</b>. Met85 genotypes are significantly enriched in pheomelanic standard and Indian fantails (p < 0.04, Chi-square test).</p

Functional differences between Val85 and Met85 alleles of Mc1r.

<p><b>A</b>. cAMP production in response to αMSH stimulation is reduced in cells transfected with the Met85 allele relative to the Val85 allele (p < 0.02, paired t-test). Error bars, ± SEM. <b>B</b>. Total cellular protein expression of HA/FLAG-tagged Mc1r is equivalent in COS-7 cells transfected with pigeon Val85 and Met85 alleles. The non-specific OD_{492/620 nm} value (GFP) was 0.008 ± 0.001 (0% set point) and the OD_{492/620 nm} value of the Val85 allele was 0.415 ± 0.070 (100% set point). <b>C</b>. Surface protein expression of the Met85 allele is reduced relative to Val85 allele (p < 0.007, one-sample t-test). Non-specific OD_{492/620 nm} value (GFP) was 0.053 ± 0.050 (0%) and the OD_{492/620 nm} value of the Val85 allele was 0.501 ± 0.049 (100%). </p

Examples of plumage pigment variation in domestic rock pigeons.

<p>All birds are show homer breed unless otherwise noted. <b>A</b>–<b>C</b>. Eumelanic phenotypes: <b>A</b>, black check; <b>B</b>, blue bar (same color as <b>A</b>, but different pattern); <b>C</b>, brown (Mookee). <b>D</b>–<b>G</b>. Pheomelanic phenotypes: <b>D</b>, ash-red check; <b>E</b>, ash-red bar (same color as <b>D</b>, but different pattern); <b>F</b>, yellow check (dilute form of phenotype in <b>D</b>); <b>G</b>, <i>recessive red</i> (Chinese owl). <b>H</b>. White (white carneau). <b>I</b>. Fantail, a breed examined in subset of association tests. Panels <b>H</b> and <b>I</b> modified after [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074475#B27" target="_blank">27</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074475#B48" target="_blank">48</a>], respectively; photos courtesy of Eric Domyan (<b>A</b>–<b>F</b>) and Sydney Stringham (<b>H</b>).</p

Amino acid sequence variation among avian orthologs of Mc1r.

<p>Several variants have been implicated in pigment variation within avian species. The Val85Met mutation found in domestic pigeons is associated with eumelanism in the lesser snow goose and red-footed booby. Functional studies of Mc1r protein variants have been conducted for chicken [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074475#B14" target="_blank">14</a>] and pigeon (this study). Additional mutations in chicken have been identified on the E92K background [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074475#B13" target="_blank">13</a>] but are not shown here. Figure based on previous review of avian Mc1r diversity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074475#B2" target="_blank">2</a>].</p