Genetic suppression of the circadian Clock mutation by the melatonin biosynthesis pathway

Abstract

Most laboratory mouse strains including C57BL/6J do not produce detectable levels of pineal melatonin owing to deficits in enzymatic activity of arylalkylamine N-acetyltransferase (AANAT) and N-acetylserotonin O-methyl transferase (ASMT), two enzymes necessary for melatonin biosynthesis. Here we report that alleles segregating at these two loci in C3H/HeJ mice, an inbred strain producing melatonin, suppress the circadian period-lengthening effect of the Clock mutation. Through a functional mapping approach, we localize mouse Asmt to chromosome X and show that it, and the Aanat locus on chromosome 11, are significantly associated with pineal melatonin levels. Treatment of suprachiasmatic nucleus (SCN) explant cultures from Period2(Luciferase) (Per2(Luc)) Clock/+ reporter mice with melatonin, or the melatonin agonist, ramelteon, phenocopies the genetic suppression of the Clock mutant phenotype observed in living animals. These results demonstrate that melatonin suppresses the Clock/+ mutant phenotype and interacts with Clock to affect the mammalian circadian system.

Fig. 1.

Effect of genetic background on Clock/+ phenotype. (A) Representative activity records for suppression of Clock mutant circadian behavior in C3H/HeJ background. Locomotor activity records from wild-type (Left) and Clock/+ mice (Right). The genetic backgrounds represented are C57BL/6J (B6) coisogenic animals (Upper, two records) and (C3H/HeJ × C57BL/6J)F₁ hybrids (Lower, four records). The period suppression is observed only in Clock/+ mice. (B) Effect of genetic background on free-running period of locomotor activity rhythm. Both strain background and Clock genotype significantly affect circadian period. A two-way ANOVA was highly significant for the Clock genotype [F(1,32) = 56.24, P = 3.00 × 10⁻⁸], strain background [F(1,32) = 30.05, P = 6.65 × 10⁻⁶] and interaction [F(1,32) = 23.86, P = 3.49 × 10⁻⁵]. Each data point represents data from 7 to 25 mice. Error bars represent the mean ± SD. B6: C57BL/6J and F₁: (C3H/HeJ × C57BL/6J)F_1. (C) Distribution of circadian period for Clock/+ mice of different genetic backgrounds. C57BL/6J (24.3 ± 0.13 h, n = 13; Top), (C3H/HeJ × C57BL/6J)F₁ (23.6 ± 0.22 h, n = 7; Middle), and [(C3H/HeJ × C57BL/6J)F₁ × C57BL/6J]N₂ (24.2 ± 0.27 h, n = 96; Bottom). C3H/HeJ animals carry dominant suppressors of the Clock mutation that segregate in the [(C3H/HeJ × C57BL/6J)F₁ × C57BL/6J]N₂ genetic background.

Fig. 2.

Genetic mapping of Clock suppressors. (A) Results of QTL analysis (single marker regression) with 55 [(C3H/HeJ × C57BL/6J)F₁ × C57BL/6J]N₂ animals. A significant association was detected with markers at the distal end of chromosome 11 (LOD = 4.7). A weak association was detected on chromosome X (LOD = 2.3). A significance threshold of LOD = 3.20 for P = 0.05 was determined by analysis of 1,000 permutation tests for this data set. (B) Examination of 41 additional Clock/+ N₂ progeny confirms association of Clock suppression and loci on chromosomes 11 and X. Data shown represent 96 total N₂ Clock/+ progeny. Peak associations occur with SSLP markers D11Mit12 on chromosome 11 (~113 Mb) and DXMit223 on chromosome X (~163 Mb). (C) Allelic effect of chromosome 11 (Left) and chromosome X (Right) on free-running period in 96 backcross Clock/+ mice. Data shown are mean ± SEM. A one-way ANOVA was significant for genotype at D11Mit12 on chromosome 11 [~113 Mb; ANOVA F(1,95) = 26.24, P = 1.67 × 10⁻⁶] and DXMit223 on chromosome X [~163 Mb; ANOVA F(1,95) = 8.33, P = 0.004). For chromosome 11, B stands for B6/B6 and H stands for B6/C3H genotypes. For chromosome X, B stands for either B6/B6 (female) or B6 (male) and H stands for either B6/C3H (female) or C3H (male).

Fig. 3.

Effect of melatonin on the circadian phenotype of Clock/+ mice. (A) Histogram of pineal melatonin content in N₂ hybrid mice. Data are from 61 [(C3H/HeJ × C57BL/6J)F₁ × C57BL/6J]N₂ and 66 [(C3H/HeJ × BALB/cJ)F₁ × BALB/cJ]N₂ hybrid mice. A horizontal bar indicates those animals scored as melatonin positive. (B) Loci on the distal ends of chromosomes 11 and X are tightly linked to melatonin content. A total of 9 mice (4 from a C3H/HeJ × BALB/cJ cross and 5 from a C3H/HeJ × C57BL/6J cross) were genotyped with a custom SNP panel described previously (29). Two candidate suppressor loci were identified on these chromosomes: Aanat on 11 and Asmt on X. A two-way ANOVA was significant in the C57BL/6J × C3H/HeJ cross for Aanat genotype [F(1, 57) = 11.65, P = 0.001), Asmt genotype [F(1,57) = 9.37, P = 0.0033] and their interaction [F(1,57) = 5.07, P = 0.028]. In the BALB/cJ × C3H/HeJ cross, a two-way ANOVA was significant for Aanat genotype [F(1,61) = 7.16, P = 0.0095] and Asmt genotype [F(1,61) = 15.85, P = 0.00018]. Their interaction was not significant [F(1,61) = 1.88, P = 0.17]. Blue indicates BALB/cJ or C57BL/6J genome and pink indicates C3H/HeJ genome. (C) Effect of strain background on pineal melatonin content in Clock/+ [(C3H/HeJ × C57BL/6J)F₁ × C57BL/6J]N₂ mice. A total of 105 progeny were genotyped for the Aanat and Asmt loci. Mice carrying C3H alleles at both the Aanat and Asmt loci had significantly higher pineal melatonin content than animals with one or more B6 alleles. A two-way ANOVA was significant for Aanat genotype [F(1,101) = 21.7, P = 9.6 × 10⁻⁶], Asmt genotype [F(1,101) = 34.4, P = 6.0 × 10⁻⁸], and their interaction [F(1,101) = 12.4, P = 6.4 × 10⁻⁴]. For the Aanat locus, B stands for B6/B6 and H stands for B6/C3H genotypes. For the Asmt locus, B stands for either B6/B6 (female) or B6 (male). H stands for either B6/C3H (female) or C3H (male). (D) Effect of genotype of Aanat and Asmt on circadian free-running period in 157 [(C3H/HeJ × C57BL/6J)F₁ × C57BL/6J]N₂ mice. Mice carrying C3H alleles at Aanat and Asmt expressed shorter free-running periods than those carrying B6 alleles at either locus. A two-way ANOVA was significant for Aanat genotype [F(1,153) = 15.35, P = 1.3 × 10⁻⁴] and Asmt genotype [F(1,153) = 9.25, P = 2.7 × 10⁻³]. The interaction between Aanat and Asmt was not significant [F(1,153) = 0.31, P = 0.57]. Abbreviations of genotype are the same as in Fig. 3C.

Fig. 4.

Effect of melatonin and ramelteon on the period of PER2::LUC bioluminescence rhythms from Clock/+ SCN explants. (A) Representative records of bioluminescence rhythms from SCN explants of Clock/+ mice treated with melatonin (Left) or ramelteon (Right). In each panel, red lines represent treatment groups (10⁻⁹ M melatonin or 10⁻¹² M ramelteon) and green lines represent vehicle controls. (B) Period analysis of bioluminescence rhythms of cultured Per2^Luc SCN explants. For each culture, a single dose of melatonin or ramelteon was administered at the indicated concentration at the beginning of the experiment. Each data point represents the mean ± SEM of 3–14 samples. Nonlinear curve fitting was performed using GraphPad Prism 5.