Reversible DNA methylation regulates seasonal photoperiodic time measurement

Abstract

In seasonally breeding vertebrates, changes in day length induce categorically distinct behavioral and reproductive phenotypes via thyroid hormone-dependent mechanisms. Winter photoperiods inhibit reproductive neuroendocrine function but cannot sustain this inhibition beyond 6 mo, ensuring vernal reproductive recrudescence. This genomic plasticity suggests a role for epigenetics in the establishment of seasonal reproductive phenotypes. Here, we report that DNA methylation of the proximal promoter for the type III deiodinase (dio3) gene in the hamster hypothalamus is reversible and critical for photoperiodic time measurement. Short photoperiods and winter-like melatonin inhibited hypothalamic DNA methyltransferase expression and reduced dio3 promoter DNA methylation, which up-regulated dio3 expression and induced gonadal regression. Hypermethylation attenuated reproductive responses to short photoperiods. Vernal refractoriness to short photoperiods reestablished summer-like methylation of the dio3 promoter, dio3 expression, and reproductive competence, revealing a dynamic and reversible mechanism of DNA methylation in the mammalian brain that plays a central role in physiological orientation in time.

Keywords: biological rhythms; photoperiodism.

Fig. 1.

Short photoperiods inhibit reproduction and activate hypothalamic mRNA expression via epigenetic mechanisms. Acute transfer from LD to SD photoperiods caused gonadal regression (A), increased hypothalamic dio3 mRNA expression (B), and decreased hypothalamic dnmt1 and dnmt3b mRNA expression (C) after 8 wk. (D) Immunocytochemical localization of DNMT3b (DNMT3b-ir) in the hamster mediobasal hypothalamus (MBH). DNMT3b-ir was evident throughout the MBH and in the ependymal cell (EC) layer along the third ventricle (III). (E) Transfer from LD to SD reduced DNA methylation in the dio3 proximal promoter, as measured using an MSRE assay. (F) Proportion of LD and SD hamsters in which methylation was present in each of 11 CpG sites in the dio3 proximal promoter, as measured by direct sequencing of sodium bisulfite-treated DNA. The abscissa (not to scale) depicts the 11 CpG sites in the −490 to −210-bp region of the dio3 proximal promoter. All data are mean ± SEM. *P < 0.05; ***P < 0.005 vs. LD value.

Fig. 2.

MEL mediates SD-induced epigenetic events. Hamsters housed in an LD photoperiod were treated with MEL (50 μg administered s.c.) in the late afternoon (for 2 d, 1 wk, or 4 wk) to extend the endogenous plasma MEL profile. (A) MEL treatments mimicked effects of SD on the reproductive system. MEL treatments up-regulated hypothalamic dio3 mRNA expression and decreased hypothalamic dnmt3b mRNA expression (B) and decreased methylation of the dio3 proximal promoter (C). All data are mean ± SEM. *P < 0.05; ***P < 0.005 vs. LD value.

Fig. 3.

Neuroendocrine refractoriness to SD reverses patterns of DNA methylation induced by acute SD exposure. (A) Acute (10 wk, SD) exposure to SD induced gonadal regression, whereas prolonged exposure (42 wk, SD-R) triggered neuroendocrine refractoriness and gonadal recrudescence. Refractoriness in SD-R hamsters was characterized by a complete reversal of hypothalamic dio3 and dnmt3b mRNA expression (B and C) and by remethylation of DNA in the dio3 proximal promoter (D). (E) Proportion of LD, SD, and SD-R hamsters in which methylation was present in each of 11 CpG sites in the dio3 proximal promoter, as measured by sodium bisulfite DNA sequencing. The abscissa (not to scale) depicts the 11 CpG sites in the −490 to −210-bp region of the dio3 proximal promoter. All data are mean ± SEM. *P < 0.05; ***P < 0.005 vs. LD value.