Agouti Gene Research Articles

Countershading in zebrafish results from an Asip1 controlled dorsoventral gradient of pigment cell differentiation

Dorso-ventral (DV) countershading is a highly-conserved pigmentary adaptation in vertebrates. In mammals, spatially regulated expression of agouti-signaling protein (ASIP) generates the difference in shading by driving a switch between the production of chemically-distinct melanins in melanocytes in dorsal and ventral regions. In contrast, fish countershading seemed to result from a patterned DV distribution of differently-coloured cell-types (chromatophores). Despite the cellular differences in the basis for counter-shading, previous observations suggested that Agouti signaling likely played a role in this patterning process in fish. To test the hypotheses that Agouti regulated counter-shading in fish, and that this depended upon spatial regulation of the numbers of each chromatophore type, we engineered asip1 homozygous knockout mutant zebrafish. We show that loss-of-function asip1 mutants lose DV countershading, and that this results from changed numbers of multiple pigment cell-types in the skin and on scales. Our findings identify asip1 as key in the establishment of DV countershading in fish, but show that the cellular mechanism for translating a conserved signaling gradient into a conserved pigmentary phenotype has been radically altered in the course of evolution.

Synergy between MC1R and ASIP for coat color in horses (Equus caballus)1.

Through domestication and human selection, horses have acquired various coat colors, including seven phenotypes: black, brown, dark bay, bay, chestnut, white, and gray. Here we determined the genotypes for melanocortin-1 receptor (MC1R) and agouti signaling protein (ASIP) in 709 horses from 15 breeds. We found that the EEEE genotype frequency at MC1R decreased from dark to light colors (black = 64.5%, brown = 67.5%, dark bay = 47.0%, bay = 16.5%, and chestnut = 0.0%), whereas the AAAA genotype frequency at ASIP increased as coat color lightened (black = 0.0%, brown = 22.9%, dark bay = 69.2%, and bay = 83.0%). When combined genotypes at MC1R and ASIP were examined, different advantage genotype combinations were found for each color: black EEEE-AaAa (64.5%), brown EEEE-AAAa (47.0%), dark bay EEEE-AAAA, and EEEe-AAAA (36.2% and 33.0%, totally 69.2%), bay EEEe-AAAA (69.6%), and chestnut EeEe-AAAA (62.6%). The χ2 test showed that the phenotypes of horse coat colors were significantly related with the genotypes of MC1R and ASIP (p < 0.001). Furthermore, in contrast to a previous study where AaAa was only found in black, chestnut, and gray horses, we also found this allele in brown, dark bay, bay, and white horses. These results indicated that MC1R and ASIP may synergistically affect the levels of melanin in equine coat colors and that additional genes are likely involved in regulating coat colors, especially for white and gray colors. Our research provides new data for further studies on the synergetic actions of MC1R and ASIP in coat color of horses.

How natural selection affects mouse coat color

Genetic Evolution Evolution, at its core, involves changes in the frequency of alleles subject to natural selection. But identifying the target of selection can be difficult. Barrett et al. investigated how allele frequencies affecting pigmentation change over time (see the Perspective by Pelletier). Wild-caught mice ( Peromyscus maniculatus ) were exposed to avian predators against naturally occurring dark or light backgrounds. Natural selection yielded shifts in coloration owing to genetic variants in the mouse coat color Agouti gene. Science , this issue p. [499][1]; see also p. [452][2] [1]: /lookup/doi/10.1126/science.aav3824 [2]: /lookup/doi/10.1126/science.aaw3097

TBX3 and ASIP genotypes reveal discrepancies in officially recorded coat colors of Hucul horses

Although only a few specific pigmentation types are allowed within the Hucul horse registry, accurate determination of particular coat colors can be uncertain due to the presence of variation in color shades and segregation of multiple dun dilution variants. Herein, we genotyped the previously identified polymorphisms within two coat color loci TBX3 (T-box 3) and ASIP (Agouti Signaling Protein) in 462 Hucul individuals and compared the genotype predicted phenotypes with observed pigmentation types provided in the Polish Horse Breeders Association database. We identified disagreement between the predicted and recorded coat color in 157 horses (34%). The most common error was misclassification of horses with the nd1/nd1 and nd1/nd2 genotypes, what may be related with the occurrence of some ‘intermediate’ dilution phenotypes in such individuals. We have also proven that the frequency of the dominant dun dilution allele (D) (0.30) is higher than previously predicted by available studbooks. The D allele(s) is easily ‘hidden’ in various phenotypic groups including dark bay and black, therefore we hypothesized that the dun dilution effect itself is not as strongly epistatic in the Hucul horse as described in other horse breeds. This may be the result of an additional genetic modifier suppressing D allele phenotypic effect.

On association of the lethal yellow (AY) mutation in the agouti gene with the alterations in mouse brain and behavior

Lethal yellow (AY) mutation causes obesity and type-2 diabetes in mice. Here we studied the effect of the AY mutation on the brain and behavior. The experiments were carried out on adult (11–12 weeks old) males of AY/a mice and their wild-type littermates (a/a). Mice of AY/a and a/a genotypes did not differ in their home cage activity, sleep, food and water consumption, learning ability in the Morris water maze, anxiety in the open field and elevated plus-maze, as well as in the level of monoamines, metabolites and some genes expression in the brain. At the same time, the fat mass, depressive-like immobility in the forced swim and tail suspension tests were significantly increased in AY/a mice compared with a/a ones. Magnetic resonance imaging revealed a significant reduction of cortex volume in AY/a mice. The level of mRNA of Ptpn5 gene encoding striatal enriched tyrosine phosphatase in the frontal cortex of AY/a mice was significantly elevated compared with their wild-type littermates. This is the first report on the alterations in the brain and behavior in the AY/a mouse line. It is tempting to speculate that this mouse line can serve as a new and useful preclinical model to study neurobehavioral complications associated with obesity and type-2 diabetes.

ASIP disruption via CRISPR/Cas9 system induces black patches dispersion in Oujiang color common carp

Pigment pattern formation in economical fish remains elusive to date. Oujiang color common carp (Cyprinus carpio var. color) is a variant of common carp with different pigmentation patterns and important farm fish in China. Agouti signaling protein (ASIP) is an endogenous antagonist of melanocortin in the melanogenesis pathway. In this study, two ASIP genes (ASIP 1 and ASIP 2) were first identified in color common carp. Their expression was higher in the pale belly than in the dark dorsal skin, which was consistent with the typical dorsal–ventral expression pattern such as that of wild common carp. In comparison, an unusually higher expression was detected in the non-melanophore side skin than in the melanophore side skin of Oujiang color common carp. Thus, CRISPR/Cas9 was used to disrupt the two ASIP genes in Oujiang color common carp of red with big black patches (RB) pattern. Consequently, the black patches disappeared and the melanophores dispersed along the dorsal skin in the F0 mosaic RB individuals. The findings of this study indicate that the ASIP gene is involved in regulating melanin aggregation and distribution during black patch formation in Oujiang color common carp.

The Serine Protease Activity of Corin Is Required for Normal Pigment Type Switching

The Serine Protease Activity of Corin Is Required for Normal Pigment Type Switching

Lethal yellow (AY) mutation in the agouti gene causes the depressive-like alterations in the mouse brain and behavior

Lethal yellow (AY) mutation in the agouti gene causes the depressive-like alterations in the mouse brain and behavior

Epigenetic Responses to Low Dose Ionizing Radiation

Two epigenetically regulated subsets of genes that potentially link environmental exposures early in development to adult diseases are imprinted genes and those with metastable epialleles. Genes with metastable epialleles have highly variable expressions because of stochastic allelic modifications in the epigenome. Genomic imprinting is an unusual epigenetic form of gene regulation that results in monoallelic expression in a parent-of-origin dependent manner. The viable yellow agouti (Avy) mouse harbors a metastable Agouti gene because of an upstream insertion of a transposable element. We previously used this animal model to demonstrate that nutritional and chemical toxicant exposures during early development induce persistent epigenetic changes at the Avy locus that result in alterations in coat color and adult disease susceptibility. We also showed that low doses of ionizing radiation (

Polymorphisms in MC1R and ASIP Genes are Associated with Coat Color Variation in the Arabian Camel.

Pigmentation in mammals is primarily determined by the distribution of eumelanin and pheomelanin, the ratio of which is mostly controlled by the activity of melanocortin 1 receptor (MC1R) and agouti signaling protein (ASIP) genes. Using 91 animals from 10 Arabian camel populations, that included the 4 predominant coat color phenotypes observed in the dromedary (light brown, dark brown, black, and white), we investigated the effects of the MC1R and ASIP sequence variants and identified candidate polymorphisms associated with coat color variation. In particular, we identified a single nucleotide polymorphism (SNP), found in the coding region of MC1R (901C/T), linked to the white coat color, whereas a 1-bp deletion (23delT/T) and a SNP (25G/A) in exon 2 of ASIP are associated with both black and dark-brown coat colors. Our results also indicate support that the light-brown coat color is likely the ancestral coat color for the dromedary. These sequence variations at the MC1R and ASIP genes represent the first documented evidence of candidate polymorphisms associated with Mendelian traits in the dromedary.

Genetic background of coat colour in sheep

Abstract. The coat colour of animals is an extremely important trait that affects their behaviour and is decisive for survival in the natural environment. In farm animal breeding, as a result of the selection of a certain coat colour type, animals are characterized by a much greater variety of coat types. This makes them an appropriate model in research in this field. A very important aspect of the coat colour types of farm animals is distinguishing between breeds and varieties based on this trait. Furthermore, for the sheep breeds which are kept for skins and wool, coat/skin colour is an important economic trait. Until now the study of coat colour inheritance in sheep proved the dominance of white colour over pigmented/black coat or skin and of black over brown. Due to the current knowledge of the molecular basis of ovine coat colour inheritance, there is no molecular test to distinguish coat colour types in sheep although some are available for other species, such as cattle, dogs, and horses. Understanding the genetic background of variation in one of the most important phenotypic traits in livestock would help to identify new genes which have a great effect on the coat colour type. Considering that coat colour variation is a crucial trait for discriminating between breeds (including sheep), it is important to broaden our knowledge of the genetic background of pigmentation. The results may be used in the future to determine the genetic pattern of a breed. Until now, identified candidate genes that have a significant impact on colour type in mammals mainly code for factors located in melanocytes. The proposed candidate genes code for the melanocortin 1 receptor (MC1R), agouti signaling protein (ASIP), tyrosinase-related protein 1 (TYRP1), microphthalmia-associated transcription factor MITF, and v-kit Hardy–Zuckerman 4 feline sarcoma viral oncogene homologue (KIT). However, there is still no conclusive evidence of established polymorphisms for specific coat colour types in sheep.

Molecular Study of the Extension Locus in Association with Coat Colour Variation of Iranian Indigenous Sheep Breeds

A tremendous variety is observed in coat colouration of the Iranian indigenous sheep, whereas a few molecular based studies have been carried out to identify the responsible genes in this geographical area. Function and distribution of melanin, categorized as phaeomelanin and eumelanin determine the coat colour in vertebrates, which are primarily regulated by interactions of the two main loci, agouti and extension, currently termed as Agouti Signalling Protein and Melanocortin 1 Receptor, respectively. In current study, the upstream region and a part of the coding sequence of the MC1R gene were assessed in three Iranian sheep breeds, Zandi, Baluchi and Zel, through molecular methods and in silico predictions. PCR-SSCP results of the 5' UTR flanking region showed a clear banding pattern in which the Eab was most frequent (67 per cent), observed in the individuals with light coloured phenotypes like white Baluchi and light grey and cream Zandi sheep, while the two homozygous patterns, Eaa and Ebb were rare in the populations. Direct sequencing the fragments revealed that the pattern is consistent with the–206G>A polymorphic site in the upstream region and four putative transcription binding sites close to this site were defined and characterized. The 12A>G and 51G>A transitions were also detected and verified as silent mutations in the 5' end of the coding region of the MC1R gene, with synonymous effect on amino acid sequence. Semi quantitative RT-PCR of various phenotypes resulted in acquisition of the highest expression in the dark grey phenotype of the Zel sheep.

Left-right pigmentation pattern of Japanese flounder corresponds to expression levels of melanocortin receptors (MC1R and MC5R), but not to agouti signaling protein 1 (ASIP1) expression

Body coloration in flatfish is one of the most distinctive asymmetries in the animal kingdom, although the fundamental molecular mechanism of the pigmentation is unclear. In the dorso-ventral coloration (countershading) of other teleost fishes, ventral-specific expression of agouti signaling protein 1 (ASIP1), an endogenous antagonist of melanocortin 1 receptor (MC1R), has been reported to play a pivotal role. Contribution of ASIP1 is also suggested in the asymmetrical pigmentation of flatfish. In order to confirm the contribution of ASIP1 and further examine receptor function in the body coloration of Japanese flounder, expression levels of asip1, mc1r, melanocortin 5 receptor (mc5r), and melanin-concentrating hormone receptor 2 (mchr2) were measured in the normally pigmented area of the left side, the normally non-pigmented area of the right side, and the abnormally pigmented (exhibiting hypermelanosis) area of the right side. Measurement was also carried out under conditions of hypermelanosis stimulated by cortisol and during the transition from non-pigmentation to pigmentation in areas of hypermelanosis. Contrary to our expectations, no difference was detected in asip1 expression between pigmented and non-pigmented areas. There was also no difference between normal and hormonally stimulated pigmented conditions in areas of hypermelanosis or during the transition process. Instead, the expression levels of mc1r, mc5r, and mchr2 were consistently higher in pigmented areas, and were especially increased under hormonally stimulated conditions. In addition, expressions of these receptor genes increased prior to pigmentation in areas of future hypermelanosis. Our results suggest that MC1Rand MC5R, but not necessarily ASIP1, contribute to pigmentation and hypermelanosis in Japanese flounder. We propose a yet unknown molecular mechanism for asymmetrical pigmentation in flatfish that is distinct from that of countershading in other vertebrates.

Agouti Signaling Protein and Its Receptors as Potential Molecular Markers for Intramuscular and Body Fat Deposition in Cattle

Transcriptome analyses of bovine muscle tissue differing in intramuscular fat (IMF) content identified agouti signaling protein (ASIP) as a promising candidate gene for fat deposition. The protein is secreted from adipocytes and may serve as a signaling molecule in cross-talk between adipocytes and muscle fibers or other cells. Known receptors for ASIP are the melanocortin receptors (e.g., MC4R) and attractin (ATRN). The present study was conducted to determine relationships between the expression of ASIP and its receptors in different bovine tissues with fat deposition. Adipose tissues, liver, and longissimus muscle tissue were collected from 246 F2-generation bulls (Charolais × Holstein cross) and gene expression was measured with RT-qPCR. During analysis of subcutaneous fat (SCF) of all bulls, 17 animals were identified with a transposon-derived transcript (Exon2C) inserted in the ASIP gene and dramatically increased ASIP mRNA levels. Significant correlations between normalized mRNA values of SCF and phenotypic traits related to fat deposition were found in bulls without Exon2C. Three retrospectively assigned groups [Exon2C, n = 17; high carcass fat (HCF), n = 20; low carcass fat (LCF), n = 20] were further analyzed to verify expression differences and elucidate molecular reasons. Expression of ASIP could be detected in isolated muscle fibers and adipocytes of Exon2C bulls in contrast to HCF and LCF bulls, indicating ectopic ASIP expression if the transposon is present. Among adipose tissues, highest ASIP mRNA levels were measured in SCF with significantly higher values in HCF compared to LCF bulls (1.6-fold, P < 0.05). However, the protein abundance was below the detection limit in all bulls. Potential ASIP receptors were detected in most investigated tissues. The expression of MC4R was higher and of ATRN was lower in several tissues of LCF compared to HCF bulls, whereas MC1R was not differentially expressed. Bulls of the Exon2C group had lower ATRN mRNA values than HCF and LCF bulls in perirenal fat (PF), but higher (P < 0.05) values in muscle. Receptors were also expressed in tissues where ASIP mRNA was not detected. Consequently, those tissues could be targets for ASIP if it circulates.

Molecular analyses of the agouti allele in the Japanese house mouse identify a novel variant of the agouti gene.

It has been thought that the Japanese house mouse carries the Aw allele at the agouti locus causing light-colored bellies, but they do not always show this coloration. Thus, the presence of the Aw allele seems to be doubtful in them. To ascertain whether the Aw allele is present, a two-pronged approach was used. First, we compared lengths of DNA fragments obtained from three PCRs conducted on them to the known fragment sizes generated from mouse strains exhibiting homozygosities of either a/a, A/A, or Aw/Aw. PCR I, PCR II, and PCR III amplify only in the A and Aw alleles, the a and Aw alleles, and the a allele, respectively, and we detected amplifications in strains with A/A and Aw/Aw by PCR I, in those with a/a and the Japanese house mouse by PCR II, and in those with a/a by PCR III. Second, we sequenced the exon 1A region of the agouti gene and obtained sequences corresponding to the above strains and the Japanese house mouse, but their sequences were similar to those of the a allele. We concluded that their agouti allele is not identical to the Aw allele and seems to be a novel type similar to the a allele.

Protein-Protein Interaction of Mutated Agouti Signaling Protein (ASIP) to Melanocortin Receptor 1 (MC1R) in Melanoma Skin Cancer: An Insilico Study

Melanoma is a type of skin cancer which is rare and most serious. It is developed due to the defect in skin pigmentation pathway associated with the major receptor called melanocortin receptor 1(MC1R). Agouti signaling protein (ASIP) on binding to MC1R induced the melanoma by producing pheomelanin. In this study, our aim is to prevent the binding of ASIP to MC1R by mutating ASIP with various amino acids computationally.The MC1R protein sequence (Q01726) was modelled using MODELLER 9.18. Mutation study was performed with SPDBV software. Protein-protein interaction was conducted with HADDOCK 2.2 server (High Ambiguity Driven DOCKing). Mutation was carried out in the active residue of ASIP-R117 with A, C, K, S based on their physiochemical property with wide variety. Docking result shows that, the parameters viz., haddock score, RMSD, electrostatic energy and z-score of MC1R-ASIP (R117A) were found to have -97.9±2.7, -9.5±0.2 A, -61.0±25.4 kcal/mol and -2.0 respectively which was higher than that of the standard values given in the literature. Whereas the mutant R117K shows RMSD (0.8±0.5 A), electrostatic energy (-193.0±25.8 kcal/mol) and z-score (-2.3) along with haddock score (-131.2±13.1)which was very low when compared with native form. Hence, the mutant MC1R-ASIP (R117A) exhibited to have poor binding affinity. Moreover, MC1R-ASIP (R117A) complex does not show any hydrogen bond interaction. Therefore, the mutant ASIP (R117A) might be the probable choice to prevent the MC1R-ASIP complex formation. However, more detailed analysis need to be carried out to have an in-depth understanding on the in vivo significance of this bi-molecular interaction.

Correlation of single nucleotide polymorphisms in the agouti signaling protein (Asip) and a type iii receptor Protein-Tyrosine Kinase (KIT) loci with colour in the bactrian camel (Camelus bactrianus)

Correlation of single nucleotide polymorphisms in the agouti signaling protein (Asip) and a type iii receptor Protein-Tyrosine Kinase (KIT) loci with colour in the bactrian camel (Camelus bactrianus)

Potential Causative Mutation for Melanism in Rats Identified in the Agouti Signaling Protein Gene (Asip) of the Rattus rattus Species Complex on Okinawa Island, Japan.

The occurrence of black fur, or melanism, in many mammalian species is known to be linked to DNA sequence variation in the agouti signaling protein (Asip) gene, which is a major determinant of eumelanin and pheomelanin pigments in coat color. We investigated 38 agouti (i.e., banded wildtype) and four melanistic Rattus rattus species complex (RrC) lineage II specimens from Okinawa Island, Ryukyu Islands, Japan, for genetic variation in three exons and associated flanking regions in the Asip gene. On Okinawa, a predicted loss-of-function mutation caused by a cysteine to serine amino acid change at p.124C>S (c.370T>A) in the highly conserved functional domain of Asip was found in melanistic rats, but was absent in agouti specimens, suggesting that the p.124C>S mutation is responsible for the observed melanism. Phylogeographic analysis found that Asip sequences from Okinawan RrC lineage II, including both agouti and melanistic specimens, differed from: 1) both agouti and melanistic RrC lineage I from Otaru, Hokkaido, Japan, and 2) agouti RrC lineages I and II from South Australia. This suggests the possibility of in-situ mutation of the Asip gene, either within the RrC lineage II population on Okinawa or in an unsampled RrC lineage II population with biogeographic links to Okinawa, although incomplete lineage sorting could not be ruled out.

Structure-Activity Relationship Studies on a Macrocyclic Agouti-Related Protein (AGRP) Scaffold Reveal Agouti Signaling Protein (ASP) Residue Substitutions Maintain Melanocortin-4 Receptor Antagonist Potency and Result in Inverse Agonist Pharmacology at the Melanocortin-5 Receptor.

The melanocortin system consists of five reported receptors, agonists from the proopiomelanocortin gene transcript, and two antagonists, agouti-signaling protein (ASP) and agouti-related protein (AGRP). For both ASP and AGRP, the hypothesized Arg-Phe-Phe pharmacophores are on exposed β-hairpin loops. In this study, the Asn and Ala positions of a reported AGRP macrocyclic scaffold (c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-DPro]) were explored with 14-compound and 8-compound libraries, respectively, to generate more potent, selective melanocortin receptor antagonists. Substituting diaminopropionic acid (Dap), DDap, and His at the Asn position yielded potent MC4R ligands, while replacing Ala with Ser maintained MC4R potency. Since these substitutions correlate to ASP loop residues, an additional Phe to Ala substitution was synthesized and observed to maintain MC4R potency. Seventeen compounds also possessed inverse agonist activity at the MC5R, the first report of this pharmacology. These findings are useful in developing molecular probes to study negative energy balance conditions and unidentified functions of the MC5R.

RETRACTED ARTICLE: The melanocortin signaling cAMP axis accelerates repair and reduces mutagenesis of platinum-induced DNA damage

Using primary melanocytes and HEK293 cells, we found that cAMP signaling accelerates repair of bi- and mono-functional platinum-induced DNA damage. Elevating cAMP signaling either by the agonistic MC1R ligand melanocyte stimulating hormone (MSH) or by pharmacologic cAMP induction by forskolin enhanced clearance of intrastrand cisplatin-adducts in melanocytes or MC1R-transfected HEK293 cells. MC1R antagonists human beta-defensin 3 and agouti signaling protein blocked MSH- but not forskolin-mediated enhancement of platinum-induced DNA damage. cAMP-enhanced repair of cisplatin-induced DNA damage was dependent on PKA-mediated phosphorylation of ATR on S435 which promoted ATR’s interaction with the key NER factor xeroderma pigmentosum A (XPA) and facilitated recruitment of an XPA-ATR-pS435 complex to sites of cisplatin DNA damage. Moreover, we developed an oligonucleotide retrieval immunoprecipitation (ORiP) assay using a novel platinated-DNA substrate to establish kinetics of ATR-pS435 and XPA’s associations with cisplatin-damaged DNA. Expression of a non-phosphorylatable ATR-S435A construct or deletion of A kinase-anchoring protein 12 (AKAP12) impeded platinum adduct clearance and prevented cAMP-mediated enhancement of ATR and XPA’s associations with cisplatin-damaged DNA, indicating that ATR phosphorylation at S435 is necessary for cAMP-enhanced repair of platinum-induced damage and protection against cisplatin-induced mutagenesis. These data implicate cAMP signaling as a critical regulator of genomic stability against platinum-induced mutagenesis.