The mouse mahoganoid coat Rapid Publication color mutation disrupts a novel C3HC4 RING domain protein Loan K. Phan,1,2 Feng Lin,1 Charles A. LeDuc,1 Wendy K. Chung,1,3 and Rudolph L. Leibel1,2,3 1Division of Molecular Genetics, Department of Pediatrics, 2Institute of Human Nutrition, and 3Naomi Berrie Diabetes Center, Columbia University, New York, New York, USA The mouse coat color mutant mahoganoid (md) darkens coat color and to MC3R and MC4R, resulting in hyper- decreases the obesity of Ay mice that ectopically overexpress agouti-sig- phagia, increased body fat, and disor- naling protein. The phenotypic effects of md are similar to those of the dered insulin homeostasis (8). recently identified coat color mutant mahogany (Atrnmg). We report the Two other mutations, mahogany positional cloning of mahoganoid, encoding a novel 494–amino acid (Atrnmg, Chr 2) and mahoganoid (md, Chr protein containing a C3HC4 RING (really interesting new gene) 16), affect coat color and body weight. domain that may function as an E3 ubiquitin ligase. The mutations in Both mutations are unique in their the mahoganoid allelic series (md, md2J, md5J) are all due to large retrovi- ability to suppress the obesity and ral insertions. In md and md2J, the result is minimal expression of the darken the coat (umbrous effect) normal size transcripts in all tissues examined. Unlike Atrnmg/Atrnmg ani- caused by mutations resulting in the mals, we observe no evidence of neurological deficit or neuropatholo- ectopic overexpression of ASP. There gy in md/md mice. Body weight and body mass index (a surrogate for are three reported alleles of mahogany, adiposity) measurements of B6.C3H-md-Amd/+ and md/md animals on Atrnmg, Atrnmg-L, and Atrnmg-3J, that are 9% and 45% kcal fat diets indicate that mahoganoid does not suppress coisogenic on mouse strains LDJ/Le, body weight in B6.C3H animals in a gene dose-dependent fashion. C3H/HeJ, and C3HeB/FeJ, respectively Mahoganoid effects on energy homeostasis are, therefore, most evident (9, 10). Atrnmg and Atrnmg-L are each the in the circumstances of epistasis to hypothalamic overexpression of result of approximately 5 kb retrovirus ASP in Ay and possibly other obesity-causing mutations. insertions in introns 26 and 27, respec- This article was published online in advance of the print edition. tively, that disrupt the splicing of Atrn. The date of publication is available from the JCI website, http://www.jci.org. Atrnmg-3J has a 5-bp deletion at J. Clin. Invest. 110:1449–1459 (2002). doi:10.1172/JCI200216131. nucleotide 2,809, introducing a stop codon that results in a severely trun- cated protein. Attractin (Atrn) encodes a Introduction agouti-signaling protein (ASP) that is single-pass transmembrane, approxi- Molecular cloning of the five extant normally secreted only in the follicular mately 210-kDa protein containing monogenic forms of rodent obesity cells of the dermal papilla of the skin three EGF domains, two laminin- identified critical molecules in the path- (2–4). Mutations of agouti (e.g., Ay and like EGF repeats, a CUB domain, ways regulating energy homeostasis in Avy) that cause ectopic overexpression of two plexinlike repeats, a C-type lectin, animals and humans (1). The agouti (A) ASP in the hypothalamus and skin and seven consecutive Kelch repeats gene encodes a 131–amino acid peptide result in a pleiotropic syndrome that (9, 10). The predicted structure of includes increased lean and adipose tis- ATTRACTIN protein suggests that it sue, yellow pelage (coat), hyperinsuline- functions as a receptor or receptor-like Received for publication June 10, 2002, and mia, hyperphagia, and hyperglycemia protein. The Atrnmg mutation does not accepted in revised form September 12, 2002. (3–5). ASP antagonizes the binding of suppress the obese phenotype of Mc4r- Address correspondence to: Rudolph L. α-melanocortin stimulating hormone null mice or that of several monogenic Leibel, 1150 St. Nicholas Avenue, Room 620, (α-MSH) to the melanocortin 1 receptor obese models (Leprdb, Lepob, tub, Cpefat) New York, New York 10032, USA. Phone: (MC1R), causing melanogenesis to (9, 10), but does suppress diet-induced (212) 851-5315; Fax: (212) 851-5306; E-mail: [email protected]. switch from the production of obesity (10). Homozygosity for LDJ/Le Conflict of interest: The authors have black/brown pigment (eumelanin) to a Atrnmg backcrossed for six to eight gen- declared that no conflict of interest exists. yellow/red pigment (pheomelanin) (see erations onto a C57BL/6J background Nonstandard abbreviations used: agouti- Figure 1) (2). In the hypothalamus, suppresses Ay- induced weight gain by signaling protein (ASP); α-melanocortin stimulating hormone (α-MSH); melano- agouti-related protein (AgRP) is the nat- increasing basal metabolic rate (11). In cortin 1 receptor (MC1R); agouti-related ural antagonist of MC3R and MC4R. animals doubly mutant for mg and Ay, protein (AgRP); expressed sequence tags AgRP also acts as an inverse agonist at food intake is not reduced relative to (ESTs); National Center for Biotechnology MC4R in both the human and mouse controls (homozygous wild-type Ay/a (NCBI); intracisternal type A particle (IAP); y body mass index (BMI); really interesting (6, 7). In A mice, ectopically produced and mg/mg a/a), but body weight is new gene (RING). ASP competes with α-MSH for binding reduced due to higher metabolic rate The Journal of Clinical Investigation | November 2002 | Volume 110 | Number 10 1449 (11). Mahogany mice are not hyper- mouse mahoganoid through a combi- nasoanal body length were measured. phagic on a C3H/HeJ background (12). nation of genetic mapping and bioin- The kidneys were removed and imme- Homozygous Atrnmg animals develop formatic approaches. diately frozen at –80°C for sub- abnormal myelination and vacuoliza- sequent isolation of genomic DNA. ob tion throughout the brain and spinal Methods Other mice used were B6.V-Lep (N30), y cord in association with severe tremors Animals. N7F14 B6.C3H-md-A (md/md B6.Cg-A (N66), C57BL/6J (F217), 2J and flaccid paresis (13). The tremors and md/+) mice were obtained from C3H/HeJ (F243), and C3H/HeJ-md may account for the increased meta- The Jackson Laboratory. The coat color (N2). All were obtained from The Jack- bolic rate. The neuropathological effect of the mutation is easiest to son Laboratory. changes characteristic of Atrnmg ani- detect on an agouti (A) coat (Figure 2), Microsatellite genotyping. Genomic mals and the specificity of binding of hence these animals have been selected DNA was made from tail tips clipped ASP but not AgRP to ATRN (14) make and bred for phenotypes at two loci on at weaning using QIAamp Tissue Kit it unlikely that ATRN plays a specific different chromosomes (md and A). (QIAGEN Inc, Valencia, California, role in hypothalamic control of energy Progeny for genetic mapping of the USA). Mice genotypes were deter- homeostasis, aside from its effects on region around md were generated by mined for Whitehead Institute muscle motor activity. mating md/md A/?× md/+ A/? animals microsatellites in the region of md Md has effects on coat color and of this congenic strain. The fact that (http://www-genome.wi.mit.edu/cgi- obesity in Ay mice that are analogous this line was maintained by coat color bin/mouse/gmap) by PCR amplifica- to Atrnmg. The md mutation originally selection offered the opportunity to tion. D16Mit182, D16Mit107, arose spontaneously in the C3H/HeJ map and reduce by meiotic recombina- D16Mit154, D16Mit130, D16Mit129, strain at The Jackson Laboratory (Bar tion the C3H genetic interval contain- D16Mit54, D16Mit159, D16Mit122, Harbor, Maine, USA) in the early ing the md locus. Mice were housed in and D16Mit81 were scored using 1960s. Subsequently, four additional a barrier facility under pathogen-free primers obtained from Research spontaneous mutations (md2J, md4J, conditions with a 12-hour light/dark Genetics (Huntsville, Alabama, USA). md5J, and md6J) have been documented cycle. Mice were weaned at 21 days and PCR conditions consisted of 40 cycles at this locus (http://www.informat- were then given ad libitum access to 9% of denaturation at 94°C for 30 sec- ics.jax.org/searches/allele_report.cgi? kcal fat Picolab Rodent Chow 20 (Puri- onds, annealing at 55°C for 30 sec- _Marker_key=11177). Like Atrn mg, na Mills Inc., Richmond, Indiana, USA) onds, and extension at 72°C for 45 mahoganoid darkens the back, ears, or to 45% kcal fat D12451 (Research seconds. PCR fragments were resolved and tail of nonalbino mice (15, 16). Diets Inc., New Brunswick, New Jersey, on 6% denaturing polyacrylamide gels. This darkening effect is described as USA). Mice were fasted for 2 hours Genetic and physical mapping. One an “umbrous” coat. Md suppresses prior to sacrifice by CO2 asphyxiation hundred forty-nine progeny of an y A -induced yellow pigmentation and at 105–120 days of age. Weight and N7F14 B6.C3H-md/md A/? × N7F14 Ay-induced obesity in a gene dose- dependent manner (17). Similar to Atrnmg on the C57BL/6J background, homozygosity for md on the C3H/HeJ background causes hyperphagia (11). The ability of md to induce hyperpha- gia suggests that the md gene product has effects on energy homeostasis dis- tinct from epistatic effects in the con- text of overexpression of ASP (Ay). Genetic studies have positioned md, functionally, at the same level or upstream of MC1R and downstream of ASP based on findings that the Mc1re mutation (extension, resulting in a yellow coat) suppressed the coat color effect of md and that md sup- Figure 1 pressed both the yellow and obese Schematic of melanocortin-signaling pathways, specifically melanocortin signaling in the hair phenotypes of Ay mice (17) (Figure 1). follicle and hypothalamus.