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Circadian behavior is light-reprogrammed by plastic DNA methylation

Abstract

The timing of daily circadian behavior can be highly variable among different individuals, and twin studies have suggested that about half of this variability is environmentally controlled. Similar plasticity can be seen in mice exposed to an altered lighting environment, for example, 22-h instead of 24-h, which stably alters the genetically determined period of circadian behavior for months. The mechanisms mediating these environmental influences are unknown. We found that transient exposure of mice to such lighting stably altered global transcription in the suprachiasmatic nucleus (SCN) of the hypothalamus (the master clock tissue regulating circadian behavior in mammals). In parallel, genome-wide methylation profiling revealed global alterations in promoter DNA methylation in the SCN that correlated with these changes. Behavioral, transcriptional and DNA methylation changes were reversible after prolonged re-entrainment to 24-h d. Notably, infusion of a methyltransferase inhibitor to the SCN suppressed period changes. We conclude that the SCN utilizes DNA methylation as a mechanism to drive circadian clock plasticity.

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Figure 1: Altered day length in young mice alters the FRP and gene expression in the SCN.
Figure 2: The SCN global transcriptome is altered by day length.
Figure 3: Chromatin-modifying enzymes, histone methyltransferases and their regulators show altered expression during aftereffects of T-cycle exposure.
Figure 4: Altered day length induces global changes in DNA methylation in the SCN.
Figure 5: Zebularine suppresses period length changes during entrainment to altered day lengths.
Figure 6: DNMT3a protein expression decreases with aging.

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Acknowledgements

This work was supported by the Velux Foundation, the Swiss National Science Foundation and the Zürich Clinical Research Program 'Sleep and Health'. A.A. and S.A.B. are members of the Zürich Neurozentrum graduate program, and S.A.B. is also a member of the Molecular Life Sciences program. R.D. is a Feodor Lynen Fellow of the Humboldt Foundation.

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A.A. and A.C. performed all experiments, with assistance from R.D., H.R. and A.P. Knockdown experiments in U2OS cells were performed and analyzed by B.M. with assistance from A.K. Data analysis was conducted by A.A., R.D., H.R., B.M. and A.P. The paper was written by A.A., R.D., B.M., A.K. and S.A.B.

Corresponding author

Correspondence to Steven A Brown.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Basic experimental design used to test for aftereffects.

All mice were raised under normal day lengths of 24 hours during the first four weeks after birth. They were then transferred to individual cages and exposed to constant darkness (DD) for 7 days to record their free running period (FRP). After 7 days they received an additional 6 weeks of altered environment — exposed to short day length (ST) (Light:Dark 11:11), normal day length (NT) (L:D: 12:12), or long day length (LT) (L:D 13:13). After entrainment, all mice were then transferred to DD for an additional 10 days to record their FRP.

Supplementary Figure 2 Entrainment to different day lengths (T-cycles) is stable for months in constant conditions.

Representative double-plotted actograms of mice entrained to ST and LT. Four-week-old mice were exposed to DD for 6 days to record their FRP, then entrained to ST and NT-cycles (22 hours or 26 hours) for 4 weeks, and released into DD conditions for 2 months.

Supplementary Figure 3 Old mice neither entrained to short-day ST cycles nor showed aftereffects.

Wheel running behavior assay of mice before, during and after entrainment to ST cycles. Three-week-old mice and 9-month-old mice were exposed to DD for 7 days and then they were entrained to 11:11hours light:dark for 1 month. After entrainment they were exposed to DD during 10 days. (a–b) Representative actograms of (a) young and (b) old mice. (c) average FRP before and after entrainment to ST cycle of old mice (n = 9 mice/group, p = 0.8072, mean ±s.e.m. shown).

Supplementary Figure 4 Circadian clock entrainment is necessary for aftereffects.

Wheel running behavior of mice before, during and after entrainment to LD 11:11 or 10:10. Representative actograms of 4-week-old mice (a, b) exposed to DD for 7 days and then entrained to LD 11:11 or 10:10 LD cycles for 3 weeks. Whereas 11:11 mice could entrain to the assigned photoperiod, 10:10 mice could not. After entrainment, mice were released into DD for 7 days. (c) Average FRP before and after entrainment (n = 5 mice per group, t-test LD 11:11 ** p = 0.0010 and LD 10:10 p = 0.6062, mean ±s.e.m. shown).

Supplementary Figure 5 Clock gene expression during aftereffects of T cycle exposure.

(a and b) Autoradiographs showing coronal brain sections hybridized with circadian gene probes. One month-old mice were entrained to ST and LT cycle for one month and then released into DD. To collect brains at indicated circadian timepoints, FRP was measured after 7 days in DD individually for each mouse. CT0 was defined as one half-cycle after activity onset, and CT12 as activity onset. (c) Quantification of data from mice entrained to ST and LT cycles, and harvested at CT8 (n = 3 mice/group). T-test * p < 0.05, mean ±s.e.m. shown. Quantification of part A is presented in the main text.

Supplementary Figure 6 DNA methylation changes in the SCN require long-term exposure to altered day length.

Methylation levels at Sap30l and Avpr2 promoters for mice entrained to ST and NT for either (a) 2 weeks (2W) or (b) 4 weeks (4W), and then analyzed by methylation-dependent restriction enzyme digestion followed by qPCR. Data are normalized to the maximal methylation observed for each gene. n = 5 mice/group, T-test p< 0.05, mean ±s.e.m. shown.

Supplementary Figure 7 Alterations in methylation observed in the SCN under short T-cycles did not occur in the cortex.

Methylation at the promoters of the E2F8, Golim4, and Vav3 genes in SCN and cortex, determined by MeDIP-qPCR, mean ±s.e.m. shown.

Supplementary Figure 8 Control experiment to demonstrate third ventricle cannulation.

(a) Infusion of TGFα into the third ventricle is known to alter the locomotor activity of mice (Kramer et al., Science (2001) 294:2511-15). Bar graph showing average normalized 3-day activity before, during and after 7 days of infusion with aCSF or TGFα. Four-week-old mice were exposed to DD for 10 days to record their FRP and then infused with aCSF or TGFα for 7 days in DD via osmotic minipump, mean ±s.e.m. shown. (b) Coronal brain section from representative cannulated mice, showing the needle track towards the third ventricle.

Supplementary Figure 9 Infusion of zebularine alone has no effect on the FRP.

(a) Representative actograms of behavioral activity before and during infusion of aCSF or zebularine. (b) Bar graph showing the FRP of mice before and during infusion of aCSF and zebularine in constant darkness. Four-week-old mice were exposed to DD for 10 days to record their FRP and then infused with aCSF or zebularine during 7 days in DD. Red stars indicate the day of pump and cannula implantation. (n = 3 mice per group, no effect of drug in repeated measures ANOVA, p = 0.348, mean ±s.e.m. shown).

Supplementary Figure 10 Experimental design used to examine reversibility of aftereffects.

All mice were raised until 4 weeks of age under a normal light/dark cycle of 24 hours, (NT) (L:D 12:12). They were then transferred to individual cages and exposed to constant darkness (DD) for 7 days to record their free running period (FRP). After 7 days they were exposed for an additional 4 weeks to NT lighting conditions, or exposed to short T (ST) (L:D 11:11). Subsequently, all mice were then transferred to DD for additional 7 days to record their FRP. Finally, all mice from both groups were entrained to NT for 4 weeks, and FRP was recorded under DD conditions.

Supplementary Figure 11 Multiple loci affected by T-cycles are necessary for normal circadian clock function.

Left, Z-scored circadian period of U2OS cells infected with a lentivirus expressing an RNAi hairpin silencing each gene whose transcription was significantly affected by short-day T cycles. Right, Identical graph for genes whose methylation was affected by T cycles. Dotted lines, mean period and its standard deviation.

Supplementary Figure 12 Changes in expression of DNA methyltransferases and tet2–3 enzymes in response to a light pulse.

The bar graph shows the fold changes in transcript levels for the indicated genes following 2 hours of light exposure in mice during the subjective night compared to sham-exposed controls. Six-week-old mice were raised under normal day lengths (NT) and then transferred to constant darkness for two consecutive days. They were then exposed to a 2 hour light pulse (normal room light) or a dark pulse (sham) between circadian time CT12 and CT14. Brains were then collected and sliced immediately to isolate SCNs for RNA analysis. Bars represent mean ±s.e.m. Light pulse vs. sham control unpaired t-test DNMT3a p = 0.011; DNMT3b p = 0.011; Tet2 p = 0.069; Tet3 p = 0.004 Per1 p = 0.002.

Supplementary Figure 13 Single injection of zebularine did not change shortening in FRP after ST cycle entrainment.

(a) Wheel running behavior of mice before, during and after entrainment to ST cycle. At the end of the ST-cycle (4 weeks) mice received a single injection of 500 nl of either aCSF or zebularine (500 uM). Red stars indicate the day of injection. (b) Average FRP before and after entrainment to ST-cycle (n = 4 mice per group, aCSF before vs. after paired t-test ** p = 0.0003, zebularine before vs. after t-test ** p = 0.0005, mean ±s.e.m. shown).

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Azzi, A., Dallmann, R., Casserly, A. et al. Circadian behavior is light-reprogrammed by plastic DNA methylation. Nat Neurosci 17, 377–382 (2014). https://doi.org/10.1038/nn.3651

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