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1.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34518374

RESUMO

Most endangered species exist today in small populations, many of which are isolated. Evolution in such populations is largely governed by genetic drift. Empirical evidence for drift affecting striking phenotypes based on substantial genetic data are rare. Approximately 37% of tigers (Panthera tigris) in the Similipal Tiger Reserve (in eastern India) are pseudomelanistic, characterized by wide, merged stripes. Camera trap data across the tiger range revealed the presence of pseudomelanistic tigers only in Similipal. We investigated the genetic basis for pseudomelanism and examined the role of drift in driving this phenotype's frequency. Whole-genome data and pedigree-based association analyses from captive tigers revealed that pseudomelanism cosegregates with a conserved and functionally important coding alteration in Transmembrane Aminopeptidase Q (Taqpep), a gene responsible for similar traits in other felid species. Noninvasive sampling of tigers revealed a high frequency of the Taqpep p.H454Y mutation in Similipal (12 individuals, allele frequency = 0.58) and absence from all other tiger populations (395 individuals). Population genetic analyses confirmed few (minimal number) tigers in Similipal, and its genetic isolation, with poor geneflow. Pairwise FST (0.33) at the mutation site was high but not an outlier. Similipal tigers had low diversity at 81 single nucleotide polymorphisms (mean heterozygosity = 0.28, SD = 0.27). Simulations were consistent with founding events and drift as possible drivers for the observed stark difference of allele frequency. Our results highlight the role of stochastic processes in the evolution of rare phenotypes. We highlight an unusual evolutionary trajectory in a small and isolated population of an endangered species.


Assuntos
Evolução Biológica , Deriva Genética , Variação Genética , Genética Populacional , Melanose/genética , Fenótipo , Tigres/fisiologia , Sequência de Aminoácidos , Animais , Conservação dos Recursos Naturais , Espécies em Perigo de Extinção , Genoma , Genótipo , Índia , Repetições de Microssatélites , Homologia de Sequência , Tigres/genética
2.
Nat Commun ; 12(1): 5127, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34493721

RESUMO

Intricate color patterns are a defining aspect of morphological diversity in the Felidae. We applied morphological and single-cell gene expression analysis to fetal skin of domestic cats to identify when, where, and how, during fetal development, felid color patterns are established. Early in development, we identify stripe-like alterations in epidermal thickness preceded by a gene expression pre-pattern. The secreted Wnt inhibitor encoded by Dickkopf 4 plays a central role in this process, and is mutated in cats with the Ticked pattern type. Our results bring molecular understanding to how the leopard got its spots, suggest that similar mechanisms underlie periodic color pattern and periodic hair follicle spacing, and identify targets for diverse pattern variation in other mammals.


Assuntos
Gatos/genética , Regulação da Expressão Gênica no Desenvolvimento , Pigmentação/genética , Animais , Animais Domésticos , Gatos/crescimento & desenvolvimento , Epiderme/crescimento & desenvolvimento , Epiderme/metabolismo , Genótipo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Queratinócitos/metabolismo , Mutação , Fenótipo , Análise de Célula Única , Pele/anatomia & histologia , Pele/crescimento & desenvolvimento , Pele/metabolismo , Via de Sinalização Wnt
3.
Nat Ecol Evol ; 5(10): 1415-1423, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34385618

RESUMO

Distinctive colour patterns in dogs are an integral component of canine diversity. Colour pattern differences are thought to have arisen from mutation and artificial selection during and after domestication from wolves but important gaps remain in understanding how these patterns evolved and are genetically controlled. In other mammals, variation at the ASIP gene controls both the temporal and spatial distribution of yellow and black pigments. Here, we identify independent regulatory modules for ventral and hair cycle ASIP expression, and we characterize their action and evolutionary origin. Structural variants define multiple alleles for each regulatory module and are combined in different ways to explain five distinctive dog colour patterns. Phylogenetic analysis reveals that the haplotype combination for one of these patterns is shared with Arctic white wolves and that its hair cycle-specific module probably originated from an extinct canid that diverged from grey wolves more than 2 million years ago. Natural selection for a lighter coat during the Pleistocene provided the genetic framework for widespread colour variation in dogs and wolves.

4.
Mol Ecol ; 30(2): 379-390, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33174253

RESUMO

One of the most iconic wild equids, the plains zebra occupies a broad region of sub-Saharan Africa and exhibits a wide range of phenotypic diversity in stripe patterns that have been used to classify multiple subspecies. After decades of relative stability, albeit with a loss of at least one recognized subspecies, the total population of plains zebras has undergone an approximate 25% decline since 2002. Individuals with abnormal stripe patterns have been recognized in recent years but the extent to which their appearance is related to demography and/or genetics is unclear. Investigating population genetic health and genetic structure are essential for developing effective strategies for plains zebra conservation. We collected DNA from 140 plains zebra, including seven with abnormal stripe patterns, from nine locations across the range of plains zebra, and analyzed data from restriction site-associated and whole genome sequencing (RAD-seq, WGS) libraries to better understand the relationships between population structure, genetic diversity, inbreeding, and abnormal phenotypes. We found that genetic structure did not coincide with described subspecific variation, but did distinguish geographic regions in which anthropogenic habitat fragmentation is associated with reduced gene flow and increased evidence of inbreeding, especially in certain parts of East Africa. Further, zebras with abnormal striping exhibited increased levels of inbreeding relative to normally striped individuals from the same populations. Our results point to a genetic cause of stripe pattern abnormalities, and dramatic evidence of the consequences of habitat fragmentation.


Assuntos
Equidae , Endogamia , África Oriental , Animais , Sequência de Bases , Equidae/genética , Variação Genética
5.
PLoS Genet ; 16(12): e1008671, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33290415

RESUMO

Cerebral cortical size and organization are critical features of neurodevelopment and human evolution, for which genetic investigation in model organisms can provide insight into developmental mechanisms and the causes of cerebral malformations. However, some abnormalities in cerebral cortical proliferation and folding are challenging to study in laboratory mice due to the absence of gyri and sulci in rodents. We report an autosomal recessive allele in domestic cats associated with impaired cerebral cortical expansion and folding, giving rise to a smooth, lissencephalic brain, and that appears to be caused by homozygosity for a frameshift in PEA15 (phosphoprotein expressed in astrocytes-15). Notably, previous studies of a Pea15 targeted mutation in mice did not reveal structural brain abnormalities. Affected cats, however, present with a non-progressive hypermetric gait and tremors, develop dissociative behavioral defects and aggression with age, and exhibit profound malformation of the cerebrum, with a 45% average decrease in overall brain weight, and reduction or absence of the ectosylvian, sylvian and anterior cingulate gyrus. Histologically, the cerebral cortical layers are disorganized, there is substantial loss of white matter in tracts such as the corona radiata and internal capsule, but the cerebellum is relatively spared. RNA-seq and immunohistochemical analysis reveal astrocytosis. Fibroblasts cultured from affected cats exhibit increased TNFα-mediated apoptosis, and increased FGFb-induced proliferation, consistent with previous studies implicating PEA15 as an intracellular adapter protein, and suggesting an underlying pathophysiology in which increased death of neurons accompanied by increased proliferation of astrocytes gives rise to abnormal organization of neuronal layers and loss of white matter. Taken together, our work points to a new role for PEA15 in development of a complex cerebral cortex that is only apparent in gyrencephalic species.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Encefalopatias/veterinária , Doenças do Gato/genética , Córtex Cerebral/metabolismo , Mutação com Perda de Função , Fosfoproteínas/genética , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Astrócitos/citologia , Astrócitos/metabolismo , Encefalopatias/genética , Encefalopatias/patologia , Doenças do Gato/patologia , Gatos , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Neurogênese , Fosfoproteínas/metabolismo
6.
BMC Biol ; 18(1): 3, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31915011

RESUMO

BACKGROUND: The lion (Panthera leo) is one of the most popular and iconic feline species on the planet, yet in spite of its popularity, the last century has seen massive declines for lion populations worldwide. Genomic resources for endangered species represent an important way forward for the field of conservation, enabling high-resolution studies of demography, disease, and population dynamics. Here, we present a chromosome-level assembly from a captive African lion from the Exotic Feline Rescue Center (Center Point, IN) as a resource for current and subsequent genetic work of the sole social species of the Panthera clade. RESULTS: Our assembly is composed of 10x Genomics Chromium data, Dovetail Hi-C, and Oxford Nanopore long-read data. Synteny is highly conserved between the lion, other Panthera genomes, and the domestic cat. We find variability in the length of runs of homozygosity across lion genomes, indicating contrasting histories of recent and possibly intense inbreeding and bottleneck events. Demographic analyses reveal similar ancient histories across all individuals during the Pleistocene except the Asiatic lion, which shows a more rapid decline in population size. We show a substantial influence on the reference genome choice in the inference of demographic history and heterozygosity. CONCLUSIONS: We demonstrate that the choice of reference genome is important when comparing heterozygosity estimates across species and those inferred from different references should not be compared to each other. In addition, estimates of heterozygosity or the amount or length of runs of homozygosity should not be taken as reflective of a species, as these can differ substantially among individuals. This high-quality genome will greatly aid in the continuing research and conservation efforts for the lion, which is rapidly moving towards becoming a species in danger of extinction.


Assuntos
Genoma , Leões/genética , Animais , Feminino , Leões/classificação , Sintenia
7.
Biophys J ; 109(9): 1946-58, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26536271

RESUMO

The ß-defensins are a class of small cationic proteins that serve as components of numerous systems in vertebrate biology, including the immune and melanocortin systems. Human ß-defensin 3 (HBD3), which is produced in the skin, has been found to bind to melanocortin receptors 1 and 4 through complementary electrostatics, a unique mechanism of ligand-receptor interaction. This finding indicates that electrostatics alone, and not specific amino acid contact points, could be sufficient for function in this ligand-receptor system, and further suggests that other small peptide ligands could interact with these receptors in a similar fashion. Here, we conducted molecular-similarity analyses and functional studies of additional members of the human ß-defensin family, examining their potential as ligands of melanocortin-1 receptor, through selection based on their electrostatic similarity to HBD3. Using Poisson-Boltzmann electrostatic calculations and molecular-similarity analysis, we identified members of the human ß-defensin family that are both similar and dissimilar to HBD3 in terms of electrostatic potential. Synthesis and functional testing of a subset of these ß-defensins showed that peptides with an HBD3-like electrostatic character bound to melanocortin receptors with high affinity, whereas those that were anticorrelated to HBD3 showed no binding affinity. These findings expand on the central role of electrostatics in the control of this ligand-receptor system and further demonstrate the utility of employing molecular-similarity analysis. Additionally, we identified several new potential ligands of melanocortin-1 receptor, which may have implications for our understanding of the role defensins play in melanocortin physiology.


Assuntos
Receptor Tipo 1 de Melanocortina/química , Eletricidade Estática , beta-Defensinas/química , Sequência de Aminoácidos , Ligação Competitiva , Bases de Dados de Proteínas , Humanos , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Dobramento de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , beta-Defensinas/genética
8.
J Neurosci ; 33(29): 11972-85, 2013 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-23864684

RESUMO

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.


Assuntos
Proteína Relacionada com Agouti/genética , Metabolismo Energético/fisiologia , Hipotálamo/metabolismo , Leptina/metabolismo , Fígado/metabolismo , Receptores para Leptina/genética , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Proteína Relacionada com Agouti/metabolismo , Animais , Composição Corporal/efeitos dos fármacos , Composição Corporal/fisiologia , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/fisiologia , Metabolismo Energético/efeitos dos fármacos , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Privação de Alimentos/fisiologia , Hipotálamo/efeitos dos fármacos , Leptina/farmacologia , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Norepinefrina/metabolismo , Obesidade/genética , Obesidade/metabolismo , Receptores para Leptina/metabolismo
9.
Chem Biol ; 20(6): 784-95, 2013 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-23790489

RESUMO

The ß-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 ß-defensins produced in skin, ß-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 ß-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 ß-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.


Assuntos
Receptor Tipo 1 de Melanocortina/metabolismo , beta-Defensinas/metabolismo , Proteína Agouti Sinalizadora/agonistas , Proteína Agouti Sinalizadora/genética , Proteína Agouti Sinalizadora/metabolismo , Proteína Relacionada com Agouti/agonistas , Proteína Relacionada com Agouti/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Receptor Tipo 1 de Melanocortina/antagonistas & inibidores , Receptor Tipo 1 de Melanocortina/genética , Receptor Tipo 4 de Melanocortina/química , Receptor Tipo 4 de Melanocortina/genética , Receptor Tipo 4 de Melanocortina/metabolismo , Eletricidade Estática , beta-Defensinas/genética
10.
Annu Rev Anim Biosci ; 1: 125-56, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25387014

RESUMO

Color variation in companion animals has long been of interest to the breeding and scientific communities. Simple traits, like black versus brown or yellow versus black, have helped to explain principles of transmission genetics and continue to serve as models for studying gene action and interaction. We present a molecular genetic review of pigmentary variation in dogs and cats using a nomenclature and logical framework established by early leaders in the field. For most loci in which molecular variants have been identified (nine in dogs and seven in cats), homologous mutations exist in laboratory mice and/or humans. Exceptions include the K locus in dogs and the Tabby locus in cats, which give rise to alternating stripes or marks of different color, and which illustrate the continued potential of coat color genetics to provide insight into areas that transcend pigment cell biology.


Assuntos
Gatos/fisiologia , Cães/fisiologia , Cor de Cabelo/genética , Pigmentos Biológicos/metabolismo , Animais , Gatos/genética , Cães/genética , Pigmentos Biológicos/genética
11.
Science ; 337(6101): 1536-41, 2012 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-22997338

RESUMO

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.


Assuntos
Aminopeptidases/genética , Gatos/genética , Endotelina-3/genética , Felidae/genética , Cor de Cabelo/genética , Metaloproteases/genética , Pele/metabolismo , Acinonyx/genética , Acinonyx/metabolismo , Alelos , Aminopeptidases/química , Aminopeptidases/metabolismo , Animais , Gatos/embriologia , Gatos/crescimento & desenvolvimento , Gatos/metabolismo , Endotelina-3/metabolismo , Epistasia Genética , Felidae/crescimento & desenvolvimento , Felidae/metabolismo , Regulação da Expressão Gênica , Frequência do Gene , Variação Genética , Cabelo/embriologia , Cabelo/crescimento & desenvolvimento , Folículo Piloso/embriologia , Haplótipos , Metaloproteases/química , Metaloproteases/metabolismo , Camundongos , Camundongos Transgênicos , Panthera/genética , Panthera/metabolismo , Fenótipo , Polimorfismo de Nucleotídeo Único , Pele/anatomia & histologia , Pele/embriologia , Especificidade da Espécie
12.
J Mol Biol ; 404(1): 45-55, 2010 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-20831872

RESUMO

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. 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 receptor and the melanocortin 4 receptor, but not to the melanocortin 1 receptor (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 domain of ASIP and the cysteine-rich domain of AgRP. Binding data demonstrate that melanocortin 4 receptor responds to all chimeras and is therefore highly tolerant of gross loop changes. By contrast, MC1R responds primarily to those chimeras with a sequence close to that of 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 with the first extracellular loop of MC1R through a series of key hydrophobic interactions.


Assuntos
Proteína Agouti Sinalizadora/genética , Proteína Agouti Sinalizadora/metabolismo , Proteína Relacionada com Agouti/genética , Proteína Relacionada com Agouti/metabolismo , Mapeamento de Interação de Proteínas , Receptor Tipo 1 de Melanocortina/antagonistas & inibidores , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Receptor Tipo 3 de Melanocortina/antagonistas & inibidores , Receptor Tipo 4 de Melanocortina/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
13.
Genetics ; 181(4): 1427-36, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19189957

RESUMO

Alternating patches of black and yellow pigment are a ubiquitous feature of mammalian color variation that contributes to camouflage, species recognition, and morphologic diversity. X-linked determinants of this pattern--recognized by variegation in females but not in males--have been described in the domestic cat as Orange, and in the Syrian hamster as Sex-linked yellow (Sly), but are curiously absent from other vertebrate species. Using a comparative genomic approach, we develop molecular markers and a linkage map for the euchromatic region of the Syrian hamster X chromosome that places Sly in a region homologous to the centromere-proximal region of human Xp. Comparison to analogous work carried out for Orange in domestic cats indicates, surprisingly, that the cat and hamster mutations lie in nonhomologous regions of the X chromosome. We also identify the molecular cause of recessively inherited black coat color in hamsters (historically referred to as nonagouti) as a Cys115Tyr mutation in the Agouti gene. Animals doubly mutant for Sly and nonagouti exhibit a Sly phenotype. Our results indicate that Sly represents a melanocortin pathway component that acts similarly to, but is genetically distinct from, Mc1r and that has implications for understanding both the evolutionary history and the mutational mechanisms of pigment-type switching.


Assuntos
Ligação Genética , Cor de Cabelo/genética , Mesocricetus/genética , Cromossomo X/genética , Proteína Agouti Sinalizadora/genética , Animais , Cricetinae , Epistasia Genética/fisiologia , Feminino , Variação Genética , Genética Populacional , Masculino , Mesocricetus/fisiologia , Modelos Biológicos , Linhagem , Fenótipo , Receptor Tipo 1 de Melanocortina/genética
14.
Science ; 318(5855): 1418-23, 2007 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-17947548

RESUMO

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 beta-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 beta-defensins, a protein family previously implicated in innate immunity, and identify an additional class of ligands for signaling through melanocortin receptors.


Assuntos
Cães/genética , Cor de Cabelo/genética , Receptor Tipo 1 de Melanocortina/metabolismo , beta-Defensinas/genética , beta-Defensinas/metabolismo , Proteína Agouti Sinalizadora/genética , Proteína Agouti Sinalizadora/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Mapeamento Cromossômico , Cães/metabolismo , Feminino , Haplótipos , Humanos , Queratinócitos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Polimorfismo Genético , Análise de Sequência de DNA , Deleção de Sequência , Transdução de Sinais , Pele/metabolismo , beta-Defensinas/química
15.
PLoS One ; 2(8): e702, 2007 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-17684549

RESUMO

Agouti-related protein encodes a neuropeptide that stimulates food intake. Agrp expression in the brain is restricted to neurons in the arcuate nucleus of the hypothalamus and is elevated by states of negative energy balance. The molecular mechanisms underlying Agrp regulation, however, remain poorly defined. Using a combination of transgenic and comparative sequence analysis, we have previously identified a 760 bp conserved region upstream of Agrp which contains STAT binding elements that participate in Agrp transcriptional regulation. In this study, we attempt to improve the specificity for detecting conserved elements in this region by comparing genomic sequences from 10 mammalian species. Our analysis reveals a symmetrical organization of conserved sequences upstream of Agrp, which cluster into two inverted repeat elements. Conserved sequences within these elements suggest a role for homeodomain proteins in the regulation of Agrp and provide additional targets for functional evaluation.


Assuntos
Proteína Relacionada com Agouti/genética , Mamíferos/genética , Análise de Sequência de DNA , Animais , Animais Geneticamente Modificados , Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/metabolismo , Sequência de Bases , Sequência Conservada/genética , Evolução Molecular , Regulação da Expressão Gênica , Humanos , Sequências Repetidas Invertidas , Dados de Sequência Molecular , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Alinhamento de Sequência
16.
Endocrinology ; 148(1): 72-80, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17023536

RESUMO

Leptin is an adipocyte-derived hormone that signals body energy status to the brain by acting on multiple neuronal subgroups in the hypothalamus, including those that express proopiomelanocortin (Pomc) and agouti-related protein (Agrp). Signal transducer and activator of transcription 3 (Stat3) is an important intracellular signaling molecule activated by leptin, and previous studies have shown that mice carrying a mutated leptin receptor that abolished Stat3 binding are grossly obese. To determine the extent to which Stat3 signaling in Pomc neurons was responsible for these effects, we constructed Pomc-specific Stat3 mutants using a Cre recombinase transgene driven by the Pomc promoter. We find that Pomc expression is diminished in the mutant mice, suggesting that Stat3 is required for Pomc transcription. Pomc-specific Stat3 female mutant mice exhibit a 2-fold increase in fat pad mass but only a slight increase in total body weight. Mutant mice remain responsive to leptin-induced hypophagia and are not hypersensitive to a high-fat diet; however, mutant mice fail to mount a normal compensatory refeeding response. These results demonstrate a requirement for Stat3 in transcriptional regulation of Pomc but indicate that this circuit is only one of several components that underlie the neuronal response to leptin and the role of Stat3 in that response.


Assuntos
Comportamento Alimentar/fisiologia , Obesidade/metabolismo , Obesidade/fisiopatologia , Pró-Opiomelanocortina/genética , Fator de Transcrição STAT3/metabolismo , Animais , Gorduras na Dieta/farmacologia , Metabolismo Energético/fisiologia , Feminino , Homeostase/fisiologia , Leptina/metabolismo , Leptina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Pró-Opiomelanocortina/metabolismo , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/genética , Transcrição Genética/fisiologia , Ganho de Peso/fisiologia
17.
Mol Endocrinol ; 20(10): 2591-602, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16709597

RESUMO

Energy homeostasis depends on the regulation of hypothalamic neurons by leptin, an adipocyte hormone whose circulating levels communicate body energy stores. Leptin activates the transcription factor signal transducer and activator of transcription 3 (Stat3) in hypothalamic neurons, including neuronal subtypes producing Agouti-related protein (Agrp), a neuropeptide that stimulates feeding. Previous studies have suggested a model in which high levels of Agrp transcription during fasting represent a default state that is actively repressed by phospho-Stat3 induced by leptin signaling in the fed state. We identify putative Stat3 binding elements in the Agrp promoter that have been highly conserved during vertebrate evolution. Using a reporter assay in transgenic mice that faithfully recapitulates normal regulation of Agrp, we show that these sites are required, but in a way opposite to that predicted by the existing model: mutation of the sites leads to a default state characterized by a low level of Agrp transcription and insensitivity to fasting. We also find that removing activatable Stat3 from Agrp neurons has no detectable effect on steady-state levels of Agrp mRNA in the fed or fasted state. These results suggest a new model for transcriptional regulation of orexigenic neuropeptides in which the default level of expression is low in the fed state, and transcriptional activation in response to fasting is mediated by factors other than Stat3.


Assuntos
Jejum , Regulação da Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neuropeptídeos/biossíntese , RNA Mensageiro/metabolismo , Transcrição Genética/genética , Proteína Relacionada com Agouti , Animais , Sequência de Bases , Sítios de Ligação/genética , Cromossomos Artificiais Bacterianos , Hipotálamo/citologia , Imuno-Histoquímica , Hibridização In Situ , Leptina/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutagênese , Neurônios/metabolismo , RNA Mensageiro/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Análise de Sequência de DNA
18.
PLoS Biol ; 3(12): e415, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16296893

RESUMO

Normal aging in humans and rodents is accompanied by a progressive increase in adiposity. To investigate the role of hypothalamic neuronal circuits in this process, we used a Cre-lox strategy to create mice with specific and progressive degeneration of hypothalamic neurons that express agouti-related protein (Agrp) or proopiomelanocortin (Pomc), neuropeptides that promote positive or negative energy balance, respectively, through their opposing effects on melanocortin receptor signaling. In previous studies, Pomc mutant mice became obese, but Agrp mutant mice were surprisingly normal, suggesting potential compensation by neuronal circuits or genetic redundancy. Here we find that Pomc-ablation mice develop obesity similar to that described for Pomc knockout mice, but also exhibit defects in compensatory hyperphagia similar to what occurs during normal aging. Agrp-ablation female mice exhibit reduced adiposity with normal compensatory hyperphagia, while animals ablated for both Pomc and Agrp neurons exhibit an additive interaction phenotype. These findings provide new insight into the roles of hypothalamic neurons in energy balance regulation, and provide a model for understanding defects in human energy balance associated with neurodegeneration and aging.


Assuntos
Metabolismo Energético , Hipotálamo/patologia , Hipotálamo/fisiopatologia , Degeneração Neural/fisiopatologia , Proteína Relacionada com Agouti , Animais , Peso Corporal/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ingestão de Alimentos , Deleção de Genes , Genes Reporter/genética , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Hipotálamo/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Degeneração Neural/genética , Degeneração Neural/metabolismo , Pró-Opiomelanocortina/deficiência , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/metabolismo , RNA Mensageiro/genética
19.
J Clin Invest ; 115(4): 951-8, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15761497

RESUMO

Central control of energy balance depends on the ability of proopiomelanocortin (POMC) or agouti-related protein (Agrp) hypothalamic neurons to sense and respond to changes in peripheral energy stores. Leptin and insulin have been implicated as circulating indicators of adiposity, but it is not clear how changes in their levels are perceived or integrated by individual neuronal subtypes. We developed mice in which a fluorescent reporter for PI3K activity is targeted to either Agrp or POMC neurons and used 2-photon microscopy to measure dynamic regulation of PI3K by insulin and leptin in brain slices. We show that leptin and insulin act in parallel to stimulate PI3K in POMC neurons but in opposite ways on Agrp neurons. These results suggest a new view of hypothalamic circuitry, in which the effects of leptin and insulin are integrated by anorexigenic but not by orexigenic neurons.


Assuntos
Metabolismo Energético , Hipotálamo/citologia , Insulina/metabolismo , Leptina/metabolismo , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Relacionada com Agouti , Animais , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Genes Reporter , Homeostase , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Neurônios/citologia , Pró-Opiomelanocortina/metabolismo , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transmissão Sináptica/fisiologia
20.
Endocrinology ; 145(12): 5798-806, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15345681

RESUMO

Agouti-related protein (Agrp) encodes a hypothalamic neuropeptide that promotes positive energy balance by stimulating food intake and reducing energy expenditure. Agrp expression in the brain is restricted to neurons within the arcuate nucleus of the hypothalamus, and expression levels are elevated as a consequence of food deprivation. We tested a series of bacterial artificial chromosome reporter constructs with varying amounts of sequence flanking the Agrp transcription unit in transgenic mice to identify and refine a region of DNA capable of recapitulating characteristics of Agrp expression. We report that a 42.5-kb region upstream of Agrp, containing three distinct regions that are evolutionarily conserved between mouse and human, is necessary and sufficient to consistently drive reporter expression specifically within AgRP neurons in a fasting-responsive manner. In addition, we demonstrate that this region allows for the stable expression of Cre recombinase in transgenic mice, providing a genetic tool for studying anabolic neural circuits that control energy balance.


Assuntos
Proteínas/genética , Transcrição Genética/fisiologia , Proteína Relacionada com Agouti , Animais , Mapeamento Cromossômico , Sequência Conservada , Regulação da Expressão Gênica/fisiologia , Biblioteca Gênica , Genes Reporter , Integrases/genética , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Sequências Reguladoras de Ácido Nucleico , Transgenes/genética , Proteínas Virais/genética
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