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1.  Dominant Red Coat Color in Holstein Cattle Is Associated with a Missense Mutation in the Coatomer Protein Complex, Subunit Alpha (COPA) Gene 
PLoS ONE  2015;10(6):e0128969.
Coat color in Holstein dairy cattle is primarily controlled by the melanocortin 1 receptor (MC1R) gene, a central determinant of black (eumelanin) vs. red/brown pheomelanin synthesis across animal species. The major MC1R alleles in Holsteins are Dominant Black (MC1RD) and Recessive Red (MC1Re). A novel form of dominant red coat color was first observed in an animal born in 1980. The mutation underlying this phenotype was named Dominant Red and is epistatic to the constitutively activated MC1RD. Here we show that a missense mutation in the coatomer protein complex, subunit alpha (COPA), a gene with previously no known role in pigmentation synthesis, is completely associated with Dominant Red in Holstein dairy cattle. The mutation results in an arginine to cysteine substitution at an amino acid residue completely conserved across eukaryotes. Despite this high level of conservation we show that both heterozygotes and homozygotes are healthy and viable. Analysis of hair pigment composition shows that the Dominant Red phenotype is similar to the MC1R Recessive Red phenotype, although less effective at reducing eumelanin synthesis. RNA-seq data similarly show that Dominant Red animals achieve predominantly pheomelanin synthesis by downregulating genes normally required for eumelanin synthesis. COPA is a component of the coat protein I seven subunit complex that is involved with retrograde and cis-Golgi intracellular coated vesicle transport of both protein and RNA cargo. This suggests that Dominant Red may be caused by aberrant MC1R protein or mRNA trafficking within the highly compartmentalized melanocyte, mimicking the effect of the Recessive Red loss of function MC1R allele.
PMCID: PMC4456281  PMID: 26042826
2.  Recurrent Evolution of Melanism in South American Felids 
PLoS Genetics  2015;11(2):e1004892.
Morphological variation in natural populations is a genomic test bed for studying the interface between molecular evolution and population genetics, but some of the most interesting questions involve non-model organisms that lack well annotated reference genomes. Many felid species exhibit polymorphism for melanism but the relative roles played by genetic drift, natural selection, and interspecies hybridization remain uncertain. We identify mutations of Agouti signaling protein (ASIP) or the Melanocortin 1 receptor (MC1R) as independent causes of melanism in three closely related South American species: the pampas cat (Leopardus colocolo), the kodkod (Leopardus guigna), and Geoffroy’s cat (Leopardus geoffroyi). To assess population level variation in the regions surrounding the causative mutations we apply genomic resources from the domestic cat to carry out clone-based capture and targeted resequencing of 299 kb and 251 kb segments that contain ASIP and MC1R, respectively, from 54 individuals (13–21 per species), achieving enrichment of ~500–2500-fold and ~150x coverage. Our analysis points to unique evolutionary histories for each of the three species, with a strong selective sweep in the pampas cat, a distinctive but short melanism-specific haplotype in the Geoffroy’s cat, and reduced nucleotide diversity for both ancestral and melanism-bearing chromosomes in the kodkod. These results reveal an important role for natural selection in a trait of longstanding interest to ecologists, geneticists, and the lay community, and provide a platform for comparative studies of morphological variation in other natural populations.
Author Summary
Color polymorphism in closely related animal species provides an opportunity to study how the balance between natural selection and genetic drift shapes the evolution of appearance and form. The cat family, Felidae, is especially interesting; 13 of 37 extant species exhibit polymorphism for melanism, but evidence for any adaptive role is lacking, in part because the potential benefits of melanism to felid predators are not clear, and in part because the tools for genomic analysis of natural populations are limited. We identify the mutations responsible for melanism in three closely related South American wild felids, the pampas cat, the kodkod, and Geoffroy’s cat, then adapt a new approach for targeted genome sequencing to characterize molecular variation in the region surrounding each melanism mutation. We find that each mutation has developed independently, with strong evidence for natural selection in the black pampas cat, and reduced genetic variation in the entire population of kodkods. Our results demonstrate that some “black cats” are black not by chance, but by selection for a mutation that provides increased fitness.
PMCID: PMC4335015  PMID: 25695801
3.  Enhanced stability and polyadenylation of select mRNAs support rapid thermogenesis in the brown fat of a hibernator 
eLife  null;4:e04517.
During hibernation, animals cycle between torpor and arousal. These cycles involve dramatic but poorly understood mechanisms of dynamic physiological regulation at the level of gene expression. Each cycle, Brown Adipose Tissue (BAT) drives periodic arousal from torpor by generating essential heat. We applied digital transcriptome analysis to precisely timed samples to identify molecular pathways that underlie the intense activity cycles of hibernator BAT. A cohort of transcripts increased during torpor, paradoxical because transcription effectively ceases at these low temperatures. We show that this increase occurs not by elevated transcription but rather by enhanced stabilization associated with maintenance and/or extension of long poly(A) tails. Mathematical modeling further supports a temperature-sensitive mechanism to protect a subset of transcripts from ongoing bulk degradation instead of increased transcription. This subset was enriched in a C-rich motif and genes required for BAT activation, suggesting a model and mechanism to prioritize translation of key proteins for thermogenesis.
eLife digest
Many mammals hibernate to avoid food scarcity and harsh conditions during winter. Hibernation involves entering a state called torpor, which drastically reduces the amount of energy used by the body. During torpor, body temperature also decreases. This is particularly exemplified in ground squirrels, whose body temperature can hover at just above or even below the point of freezing. However, hibernating mammals cannot remain in this state continuously over the months of hibernation but instead cycle between bouts of torpor lasting for 1–3 weeks and brief periods of ‘arousal’ lasting between 12–24 hr, during which their body rapidly warms up.
The heat required to start warming up the hibernator is generated from a specialized form of fat called brown adipose tissue. Normally, the bursts of metabolic activity that are required to create this heat depend on certain proteins being produced. Making a protein involves ‘translating’ its sequence from template molecules called messenger RNA (mRNA), which are ‘transcribed’ from the gene that encodes the protein. During the low body temperatures experienced during torpor, both of these processes stop. So how is the hibernator able to quickly and efficiently heat itself up during the arousal periods of hibernation?
Grabek et al. investigated this by analyzing the relative levels of mRNA in the brown adipose tissue of hibernating 13-lined ground squirrels. Using a special technique to sample and sequence small fragments of mRNA taken from brown adipose tissue, Grabek et al. compiled a profile of the mRNA molecules present at different points in the torpor–arousal cycle and compared this with a similar profile taken from squirrels that were not hibernating.
From this analysis, Grabek et al. detected that a particular group of mRNA molecules that are required for producing heat increase in abundance during torpor, even though body temperature is low enough to stop gene transcription. This increased abundance does not occur because more of the mRNA molecules are made; instead, the mRNA molecules are modified to become more stable and long lasting. Once the animal warms up during arousal, gene transcription is reactivated and more new mRNA molecules are made.
Grabek et al. suggest that the key mRNAs required for brown adipose tissue function are selectively stabilized during torpor through a temperature-dependent protective mechanism. These mRNAs are then preferentially translated into proteins during arousal to rapidly and efficiently heat the hibernator. Most other mRNA molecules degrade throughout torpor, and so their numbers decline as replacements are not transcribed until body temperature briefly recovers during arousal. Whether this protective mechanism is also used in other tissues during torpor remains a question for future work.
PMCID: PMC4383249  PMID: 25626169
Ictidomys tridecemlineatus; non-shivering thermogenesis; biological oscillation; digital transcriptome; other
4.  Molecular and Functional Analysis of Human β-Defensin 3 Action at Melanocortin Receptors 
Chemistry & biology  2013;20(6):784-795.
The β-defensins are a class of small, cationic proteins first recognized as antimicrobial components of the innate and adaptive immune system. More recently, one of the major β-defensins produced in skin, β-defensin 3, has been discovered to function as a melanocortin receptor ligand in vivo and in vitro, but its biophysical and pharmacological basis of action has been enigmatic. Here we report functional and biochemical studies focused on human β-defensin 3 (HBD3) and melanocortin receptors 1 and 4. Genetic and pharmacologic studies indicate that HBD3 acts as a neutral melanocortin receptor antagonist, capable of blocking the action of either stimulatory agonists, such as α-melanocyte stimulating hormone, or inhibitory inverse agonists such as Agouti signaling protein (Asip) and Agouti-related protein (Agrp). A comprehensive structure-function analysis demonstrates that two patches of positively charged residues, located on opposite poles of HBD3 and spatially organized by the compact β-defensin fold, are primarily responsible for high affinity binding to melanocortin receptors. These findings identify a distinct mode of melanocortin receptor-ligand interactions based primarily on electrostatic complementarity, with implications for designing ligands that target melanocortin and potentially other seven transmembrane receptors.
PMCID: PMC3714013  PMID: 23790489
5.  Ribosomal mutations cause p53-mediated dark skin and pleiotropic effects 
Nature genetics  2008;40(8):963-970.
Mutations in genes encoding ribosomal proteins cause the Minute phenotype in Drosophila and mice, and Diamond-Blackfan syndrome in humans. Here we report two mouse dark skin (Dsk) loci caused by mutations in Rps19 (ribosomal protein S19) and Rps20 (ribosomal protein S20). We identify a common pathophysiologic program in which p53 stabilization stimulates Kit ligand expression, and, consequently, epidermal melanocytosis via a paracrine mechanism. Accumulation of p53 also causes reduced body size and erythrocyte count. These results provide a mechanistic explanation for the diverse collection of phenotypes that accompany reduced dosage of genes encoding ribosomal proteins, and have implications for understanding normal human variation and human disease.
PMCID: PMC3979291  PMID: 18641651
6.  Modeling 3D Facial Shape from DNA 
PLoS Genetics  2014;10(3):e1004224.
Human facial diversity is substantial, complex, and largely scientifically unexplained. We used spatially dense quasi-landmarks to measure face shape in population samples with mixed West African and European ancestry from three locations (United States, Brazil, and Cape Verde). Using bootstrapped response-based imputation modeling (BRIM), we uncover the relationships between facial variation and the effects of sex, genomic ancestry, and a subset of craniofacial candidate genes. The facial effects of these variables are summarized as response-based imputed predictor (RIP) variables, which are validated using self-reported sex, genomic ancestry, and observer-based facial ratings (femininity and proportional ancestry) and judgments (sex and population group). By jointly modeling sex, genomic ancestry, and genotype, the independent effects of particular alleles on facial features can be uncovered. Results on a set of 20 genes showing significant effects on facial features provide support for this approach as a novel means to identify genes affecting normal-range facial features and for approximating the appearance of a face from genetic markers.
Author Summary
The face is perhaps the most inherently fascinating and aesthetic feature of the human body. It is a principle subject of art throughout human history and across cultures and populations. It provides the most significant means by which we communicate our emotions and intentions in addition to health, sex, and age. And yet features such as the strength of the brow ridge, the spacing between the eyes, the width of the nose, and the shape of the philtrum are largely scientifically unexplained. Here, we use a novel method to measure face shape in population samples with mixed West African and European ancestry from three locations (United States, Brazil, and Cape Verde). We show that facial variation with regard to sex, ancestry, and genes can be systematically studied with our methods, allowing us to lay the foundation for predictive modeling of faces. Such predictive modeling could be forensically useful; for example, DNA left at crime scenes could be tested and faces predicted in order to help to narrow the pool of potential suspects. Further, our methods could be used to predict the facial features of descendants, deceased ancestors, and even extinct human species. In addition, these methods could prove to be useful diagnostic tools.
PMCID: PMC3961191  PMID: 24651127
7.  Coordinated Regulation of Hepatic Energy Stores by Leptin and Hypothalamic Agouti-Related Protein 
The Journal of Neuroscience  2013;33(29):11972-11985.
Like obesity, prolonged food deprivation induces severe hepatic steatosis; however, the functional significance of this phenomenon is not well understood. In this study, we show that the fall in plasma leptin concentration during fasting is required for the development of hepatic steatosis in mice. Removal of leptin receptors from AGRP neurons diminishes fasting-induced hepatic steatosis. Furthermore, the suppressive effects of leptin on fasting-induced hepatic steatosis are absent in mice lacking the gene encoding agouti-related protein (Agrp), suggesting that this function of leptin is mediated by AGRP. Prolonged fasting leads to suppression of hepatic sympathetic activity, increased expression of acyl CoA:diacylglycerol acyltransferase-2 in the liver, and elevation of hepatic triglyceride content and all of these effects are blunted in the absence of AGRP. AGRP deficiency, despite having no effects on feeding or body adiposity in the free-fed state, impairs triglyceride and ketone body release from the liver during prolonged fasting. Furthermore, reducing CNS Agrp expression in wild-type mice by RNAi protected against the development of hepatic steatosis not only during starvation, but also in response to consumption of a high-fat diet. These findings identify the leptin-AGRP circuit as a critical modulator of hepatic triglyceride stores in starvation and suggest a vital role for this circuit in sustaining the supply of energy from the liver to extrahepatic tissues during periods of prolonged food deprivation.
PMCID: PMC3713731  PMID: 23864684
8.  Specifying and Sustaining Pigmentation Patterns in Domestic and Wild Cats 
Science (New York, N.Y.)  2012;337(6101):1536-1541.
Color markings among felid species display both a remarkable diversity and a common underlying periodicity. A similar range of patterns in domestic cats suggests a conserved mechanism whose appearance can be altered by selection. We identified the gene responsible for tabby pattern variation in domestic cats as Transmembrane aminopeptidase Q (Taqpep), which encodes a membrane-bound metalloprotease. Analyzing 31 other felid species, we identified Taqpep as the cause of the rare king cheetah phenotype, in which spots coalesce into blotches and stripes. Histologic, genomic expression, and transgenic mouse studies indicate that paracrine expression of Endothelin3 (Edn3) coordinates localized color differences. We propose a two-stage model in which Taqpep helps to establish a periodic pre-pattern during skin development that is later implemented by differential expression of Edn3.
PMCID: PMC3709578  PMID: 22997338
9.  Genetic Architecture of Skin and Eye Color in an African-European Admixed Population 
PLoS Genetics  2013;9(3):e1003372.
Variation in human skin and eye color is substantial and especially apparent in admixed populations, yet the underlying genetic architecture is poorly understood because most genome-wide studies are based on individuals of European ancestry. We study pigmentary variation in 699 individuals from Cape Verde, where extensive West African/European admixture has given rise to a broad range in trait values and genomic ancestry proportions. We develop and apply a new approach for measuring eye color, and identify two major loci (HERC2[OCA2] P = 2.3×10−62, SLC24A5 P = 9.6×10−9) that account for both blue versus brown eye color and varying intensities of brown eye color. We identify four major loci (SLC24A5 P = 5.4×10−27, TYR P = 1.1×10−9, APBA2[OCA2] P = 1.5×10−8, SLC45A2 P = 6×10−9) for skin color that together account for 35% of the total variance, but the genetic component with the largest effect (∼44%) is average genomic ancestry. Our results suggest that adjacent cis-acting regulatory loci for OCA2 explain the relationship between skin and eye color, and point to an underlying genetic architecture in which several genes of moderate effect act together with many genes of small effect to explain ∼70% of the estimated heritability.
Author Summary
Differences in skin and eye color are some of the most obvious traits that underlie human diversity, yet most of our knowledge regarding the genetic basis for these traits is based on the limited range of variation represented by individuals of European ancestry. We have studied a unique population in Cape Verde, an archipelago located off the West African coast, in which extensive mixing between individuals of Portuguese and West African ancestry has given rise to a broad range of phenotypes and ancestral genome proportions. Our results help to explain how genes work together to control the full range of pigmentary phenotypic diversity, provide new insight into the evolution of these traits, and provide a model for understanding other types of quantitative variation in admixed populations.
PMCID: PMC3605137  PMID: 23555287
10.  Levels of the Mahogunin Ring Finger 1 E3 Ubiquitin Ligase Do Not Influence Prion Disease 
PLoS ONE  2013;8(1):e55575.
Prion diseases are rare but invariably fatal neurodegenerative disorders. They are associated with spongiform encephalopathy, a histopathology characterized by the presence of large, membrane-bound vacuolar structures in the neuropil of the brain. While the primary cause is recognized as conversion of the normal form of prion protein (PrPC) to a conformationally distinct, pathogenic form (PrPSc), the cellular pathways and mechanisms that lead to spongiform change, neuronal dysfunction and death are not known. Mice lacking the Mahogunin Ring Finger 1 (MGRN1) E3 ubiquitin ligase develop spongiform encephalopathy by 9 months of age but do not become ill. In cell culture, PrP aberrantly present in the cytosol was reported to interact with and sequester MGRN1. This caused endo-lysosomal trafficking defects similar to those observed when Mgrn1 expression is knocked down, implicating disrupted MGRN1-dependent trafficking in the pathogenesis of prion disease. As these defects were rescued by over-expression of MGRN1, we investigated whether reduced or elevated Mgrn1 expression influences the onset, progression or pathology of disease in mice inoculated with PrPSc. No differences were observed, indicating that disruption of MGRN1-dependent pathways does not play a significant role in the pathogenesis of transmissible spongiform encephalopathy.
PMCID: PMC3559536  PMID: 23383230
11.  How the Leopard Hides Its Spots: ASIP Mutations and Melanism in Wild Cats 
PLoS ONE  2012;7(12):e50386.
The occurrence of melanism (darkening of the background coloration) is documented in 13 felid species, in some cases reaching high frequencies at the population level. Recent analyses have indicated that it arose multiple times in the Felidae, with three different species exhibiting unique mutations associated with this trait. The causative mutations in the remaining species have so far not been identified, precluding a broader assessment of the evolutionary dynamics of melanism in the Felidae. Among these, the leopard (Panthera pardus) is a particularly important target for research, given the iconic status of the ‘black panther’ and the extremely high frequency of melanism observed in some Asian populations. Another felid species from the same region, the Asian golden cat (Pardofelis temminckii), also exhibits frequent records of melanism in some areas. We have sequenced the coding region of the Agouti Signaling Protein (ASIP) gene in multiple leopard and Asian golden cat individuals, and identified distinct mutations strongly associated with melanism in each of them. The single nucleotide polymorphism (SNP) detected among the P. pardus individuals was caused by a nonsense mutation predicted to completely ablate ASIP function. A different SNP was identified in P. temminckii, causing a predicted amino acid change that should also induce loss of function. Our results reveal two additional cases of species-specific mutations implicated in melanism in the Felidae, and indicate that ASIP mutations may play an important role in naturally-occurring coloration polymorphism.
PMCID: PMC3520955  PMID: 23251368
12.  Genome-Wide Association Studies of Quantitatively Measured Skin, Hair, and Eye Pigmentation in Four European Populations 
PLoS ONE  2012;7(10):e48294.
Pigmentation of the skin, hair, and eyes varies both within and between human populations. Identifying the genes and alleles underlying this variation has been the goal of many candidate gene and several genome-wide association studies (GWAS). Most GWAS for pigmentary traits to date have been based on subjective phenotypes using categorical scales. But skin, hair, and eye pigmentation vary continuously. Here, we seek to characterize quantitative variation in these traits objectively and accurately and to determine their genetic basis. Objective and quantitative measures of skin, hair, and eye color were made using reflectance or digital spectroscopy in Europeans from Ireland, Poland, Italy, and Portugal. A GWAS was conducted for the three quantitative pigmentation phenotypes in 176 women across 313,763 SNP loci, and replication of the most significant associations was attempted in a sample of 294 European men and women from the same countries. We find that the pigmentation phenotypes are highly stratified along axes of European genetic differentiation. The country of sampling explains approximately 35% of the variation in skin pigmentation, 31% of the variation in hair pigmentation, and 40% of the variation in eye pigmentation. All three quantitative phenotypes are correlated with each other. In our two-stage association study, we reproduce the association of rs1667394 at the OCA2/HERC2 locus with eye color but we do not identify new genetic determinants of skin and hair pigmentation supporting the lack of major genes affecting skin and hair color variation within Europe and suggesting that not only careful phenotyping but also larger cohorts are required to understand the genetic architecture of these complex quantitative traits. Interestingly, we also see that in each of these four populations, men are more lightly pigmented in the unexposed skin of the inner arm than women, a fact that is underappreciated and may vary across the world.
PMCID: PMC3485197  PMID: 23118974
13.  Guidelines for Genome-Wide Association Studies 
PLoS Genetics  2012;8(7):e1002812.
PMCID: PMC3390399  PMID: 22792080
14.  Ancestral Components of Admixed Genomes in a Mexican Cohort 
PLoS Genetics  2011;7(12):e1002410.
For most of the world, human genome structure at a population level is shaped by interplay between ancient geographic isolation and more recent demographic shifts, factors that are captured by the concepts of biogeographic ancestry and admixture, respectively. The ancestry of non-admixed individuals can often be traced to a specific population in a precise region, but current approaches for studying admixed individuals generally yield coarse information in which genome ancestry proportions are identified according to continent of origin. Here we introduce a new analytic strategy for this problem that allows fine-grained characterization of admixed individuals with respect to both geographic and genomic coordinates. Ancestry segments from different continents, identified with a probabilistic model, are used to construct and study “virtual genomes” of admixed individuals. We apply this approach to a cohort of 492 parent–offspring trios from Mexico City. The relative contributions from the three continental-level ancestral populations—Africa, Europe, and America—vary substantially between individuals, and the distribution of haplotype block length suggests an admixing time of 10–15 generations. The European and Indigenous American virtual genomes of each Mexican individual can be traced to precise regions within each continent, and they reveal a gradient of Amerindian ancestry between indigenous people of southwestern Mexico and Mayans of the Yucatan Peninsula. This contrasts sharply with the African roots of African Americans, which have been characterized by a uniform mixing of multiple West African populations. We also use the virtual European and Indigenous American genomes to search for the signatures of selection in the ancestral populations, and we identify previously known targets of selection in other populations, as well as new candidate loci. The ability to infer precise ancestral components of admixed genomes will facilitate studies of disease-related phenotypes and will allow new insight into the adaptive and demographic history of indigenous people.
Author Summary
Admixed individuals, such as African Americans and Latinos, arise from mating between individuals from different continents. Detailed knowledge about the ancestral origin of an admixed population not only provides insight regarding the history of the population itself, but also affords opportunities to study the evolutionary biology of the ancestral populations. Applying novel statistical methods, we analyzed the high-density genotype data of nearly 1,500 Mexican individuals from Mexico City, who are admixed among Indigenous Americans, Europeans, and Africans. The relative contributions from the three continental-level ancestral populations vary substantially between individuals. The European ancestors of these Mexican individuals genetically resemble Southern Europeans, such as the Spaniard and the Portuguese. The Indigenous American ancestry of the Mexicans in our study is largely attributed to the indigenous groups residing in the southwestern region of Mexico, although some individuals have inherited varying degrees of ancestry from the Mayans of the Yucatan Peninsula and other indigenous American populations. A search for signatures of selection, focusing on the parts of the genomes derived from an ancestral population (e.g. Indigenous American), identifies regions in which a genetic variant may have been favored by natural selection in that ancestral population.
PMCID: PMC3240599  PMID: 22194699
15.  Loop Swapped Chimeras of the Agouti-related Protein (AgRP) and the Agouti Signaling Protein (ASIP) Identify Contacts Required for Melanocortin 1 Receptor (MC1R) Selectivity and Antagonism 
Journal of molecular biology  2010;404(1):45-55.
Agouti related protein (AgRP) and agouti signaling protein (ASIP) are homologs that play critical roles in energy balance and pigmentation, respectively, by functioning as antagonistic ligands at their cognate melanocortin receptors (MCRs). Signaling specificity is mediated in part through receptor binding selectivity brought about by alterations in the cysteine-rich carboxy-terminal domains of the ligands. AgRP binds with high affinity to the melanocortin 3 and melanocortin 4 receptors (MC3R and MC4R), but not to the MC1R, whereas ASIP binds with high affinity to all three receptors. This work explores the structural basis for receptor selectivity by studying chimeric proteins developed by interchanging loops between the cysteine-rich domains of ASIP and AgRP. Binding data demonstrate that MC4R responds to all chimeras, and is therefore highly tolerant of gross loop changes. By contrast, MC1R responds primarily to those chimeras with sequence close to wild type ASIP. Further analysis of binding and functional data suggests that the ASIP C-terminal loop – a six amino acid segment closed by the final disulfide bond – is essential for high affinity MC1R binding and inverse agonism. Comparison with previously published molecular models suggests that this loop makes contact to the first extracellular loop (EC1) of MC1R through a series of key hydrophobic interactions.
PMCID: PMC2972358  PMID: 20831872
16.  A Nervous Origin for Fish Stripes 
PLoS Genetics  2011;7(5):e1002081.
PMCID: PMC3098193  PMID: 21625563
17.  PDK1-Foxo1 in Agouti-Related Peptide Neurons Regulates Energy Homeostasis by Modulating Food Intake and Energy Expenditure 
PLoS ONE  2011;6(4):e18324.
Insulin and leptin intracellular signaling pathways converge and act synergistically on the hypothalamic phosphatidylinositol-3-OH kinase/3-phosphoinositide-dependent protein kinase 1 (PDK1). However, little is known about whether PDK1 in agouti-related peptide (AGRP) neurons contributes to energy homeostasis. We generated AGRP neuron-specific PDK1 knockout (AGRPPdk1−/−) mice and mice with selective expression of transactivation-defective Foxo1 (Δ256Foxo1AGRPPdk1−/−). The AGRPPdk1−/− mice showed reductions in food intake, body length, and body weight. The Δ256Foxo1AGRPPdk1−/− mice showed increased body weight, food intake, and reduced locomotor activity. After four weeks of calorie-restricted feeding, oxygen consumption and locomotor activity were elevated in AGRPPdk1−/− mice and reduced in Δ256Foxo1AGRPPdk1−/− mice. In vitro, ghrelin-induced changes in [Ca2+]i and inhibition of ghrelin by leptin were significantly attenuated in AGRPPdk1−/− neurons compared to control neurons. However, ghrelin-induced [Ca2+]i changes and leptin inhibition were restored in Δ256Foxo1AGRPPdk1−/− mice. These results suggested that PDK1 and Foxo1 signaling pathways play important roles in the control of energy homeostasis through AGRP-independent mechanisms.
PMCID: PMC3072380  PMID: 21694754
18.  Structural and Molecular Evolutionary Analysis of Agouti and Agouti-Related Proteins 
Chemistry & biology  2006;13(12):1297-1305.
Agouti (ASIP) and Agouti-related protein (AgRP) are endogenous antagonists of melanocortin receptors that play critical roles in the regulation of pigmentation and energy balance, respectively, and which arose from a common ancestral gene early in vertebrate evolution. The N-terminal domain of ASIP facilitates antagonism by binding to an accessory receptor, but here we show that the N-terminal domain of AgRP has the opposite effect and acts as a prodomain that negatively regulates antagonist function. Computational analysis reveals similar patterns of evolutionary constraint in the ASIP and AgRP C-terminal domains, but fundamental differences between the N-terminal domains. These studies shed light on the relationships between regulation of pigmentation and body weight, and they illustrate how evolutionary structure function analysis can reveal both unique and common mechanisms of action for paralogous gene products.
PMCID: PMC2907901  PMID: 17185225
19.  A β-Defensin Mutation Causes Black Coat Color in Domestic Dogs 
Science (New York, N.Y.)  2007;318(5855):1418-1423.
Genetic analysis of mammalian color variation has provided fundamental insight into human biology and disease. In most vertebrates, two key genes, Agouti and Melanocortin 1 receptor (Mc1r), encode a ligand-receptor system that controls pigment type-switching, but in domestic dogs, a third gene is implicated, the K locus, whose genetic characteristics predict a previously unrecognized component of the melanocortin pathway. We identify the K locus as β-defensin 103 (CBD103) and show that its protein product binds with high affinity to the Mc1r and has a simple and strong effect on pigment type-switching in domestic dogs and transgenic mice. These results expand the functional role of β-defensins, a protein family previously implicated in innate immunity, and identify an additional class of ligands for signaling through melanocortin receptors.
PMCID: PMC2906624  PMID: 17947548
20.  Molecular and Evolutionary History of Melanism in North American Gray Wolves 
Science (New York, N.Y.)  2009;323(5919):1339-1343.
Morphological diversity within closely related species is an essential aspect of evolution and adaptation. Mutations in the Melanocortin 1 receptor (Mc1r) gene contribute to pigmentary diversity in natural populations of fish, birds, and many mammals. However, melanism in the gray wolf, Canis lupus, is caused by a different melanocortin pathway component, the K locus, that encodes a beta-defensin protein that acts as an alternative ligand for Mc1r. We show that the melanistic K locus mutation in North American wolves derives from past hybridization with domestic dogs, has risen to high frequency in forested habitats, and exhibits a molecular signature of positive selection. The same mutation also causes melanism in the coyote, Canis latrans, and in Italian gray wolves, and hence our results demonstrate how traits selected in domesticated species can influence the morphological diversity of their wild relatives.
PMCID: PMC2903542  PMID: 19197024
21.  Dominant Role of the p110β Isoform of PI3K over p110α in Energy Homeostasis Regulation by POMC and AgRP Neurons 
Cell Metabolism  2009;10(5):343-354.
PI3K signaling is thought to mediate leptin and insulin action in hypothalamic pro-opiomelanocortin (POMC) and agouti-related protein (AgRP) neurons, key regulators of energy homeostasis, through largely unknown mechanisms. We inactivated either p110α or p110β PI3K catalytic subunits in these neurons and demonstrate a dominant role for the latter in energy homeostasis regulation. In POMC neurons, p110β inactivation prevented insulin- and leptin-stimulated electrophysiological responses. POMCp110β null mice exhibited central leptin resistance, increased adiposity, and diet-induced obesity. In contrast, the response to leptin was not blocked in p110α-deficient POMC neurons. Accordingly, POMCp110α null mice displayed minimal energy homeostasis abnormalities. Similarly, in AgRP neurons, p110β had a more important role than p110α. AgRPp110α null mice displayed normal energy homeostasis regulation, whereas AgRPp110β null mice were lean, with increased leptin sensitivity and resistance to diet-induced obesity. These results demonstrate distinct metabolic roles for the p110α and p110β isoforms of PI3K in hypothalamic energy regulation.
PMCID: PMC2806524  PMID: 19883613
22.  Scientists←Editors←Scientists: The Past, Present, and Future of PLoS Genetics 
PLoS Genetics  2009;5(7):e1000580.
PMCID: PMC2712074  PMID: 19649314
23.  Frequent somatic mutations of GNAQ in uveal melanoma and blue nevi 
Nature  2008;457(7229):599-602.
BRAF and NRAS are common targets for somatic mutations in benign and malignant neoplasms that arise from melanocytes situated in epithelial structures and lead to constitutive activation of the MAP-kinase pathway1, 2. However, BRAF and NRAS mutations are absent in a number of other melanocytic neoplasms in which the equivalent oncogenic events are currently unknown3. We report frequent somatic mutations in the heterotrimeric G protein alpha subunit, GNAQ, in blue nevi (83%) and ocular melanoma of the uvea (46%). The mutations occur exclusively in codon 209 in the ras-like domain and result in constitutive activation, turning GNAQ into a dominant acting oncogene. Our results demonstrate an alternative route to MAP-kinase activation in melanocytic neoplasia providing new opportunities for therapeutic intervention.
PMCID: PMC2696133  PMID: 19078957
24.  PLoS Genetics Turns Three: Looking Back, Looking Ahead 
PLoS Genetics  2008;4(7):e1000135.
PMCID: PMC2444048  PMID: 18654620
25.  The PIAS-Like Protein Zimp10 Is Essential for Embryonic Viability and Proper Vascular Development▿  
Molecular and Cellular Biology  2007;28(1):282-292.
Members of the PIAS (for protein inhibitor of activated STAT) family play critical roles in modulating the activity of a variety of transcriptional regulators. Zimp10, a novel PIAS-like protein, is a transcriptional coregulator and may be involved in the modification of chromatin through interactions with the SWI/SNF chromatin-remodeling complexes. Here, we investigate the biological role of Zimp10 in zimp10-deficient mice. Homozygosity for the Zimp10-targeted allele resulted in developmental arrest at approximately embryonic day 10.5. Analysis of knockout embryos revealed severe defects in the reorganization of the yolk sac vascular plexus. No significant abnormality in hematopoietic potential was observed in zimp10 null mice. Microarray and quantified reverse transcription-PCR analyses showed that the expression of the Fos family member Fra-1, which is involved in extraembryonic vascular development, was reduced in yolk sac tissues of zimp10 null embryos. Using fra-1 promoter/reporter constructs, we further demonstrate the regulatory role of Zimp10 on the transcription of Fra-1. This study provides evidence to demonstrate a crucial role for Zimp10 in vasculogenesis.
PMCID: PMC2223308  PMID: 17967885

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