Maged1 has a global impact on circadian stability. (A) Actograms from wild-type and Maged1 KO mice that were subjected first to LD cycle, followed by a 4 h light phase advance. Red line indicates the onset of activity. (B) Re-entrainment traces from an average of wild type (green, n=19) and Maged1 KO (red, n=17) mice. ^*P<0.05; ^**P<0.01; ^***P<0.001, unpaired two-tailed Student's t-test (see Supplementary Table S2 for each recovery data). (C) Representative bioluminescence waveforms emitted by lung (upper panels), adrenal glands (middle panels) and testis (bottom panels) from wild-type/mPer2^Luc mice (green) and Maged1KO/mPer2^Luc mice (red). (D, E) Waveform alignments at the first peak after 50% serum shock in wild-type/mPer2^Luc MEFs (green) and Maged1KO/mPer2^Luc MEFs (red) (D), and overexpression MAGED1 rat Per1-Luc fibroblasts (E) (different colours indicate independent expressing lines) and control cells (green).

Activation of Bmal1 transcription by Rors and Maged1. Bar graphs depict relative luciferase activities mean±s.d. of three replicates from a single assay. The results shown are representative of three independent experiments. (A) Effect of Maged1 expression on the Bmal1-Luc promoter. (B) Effects of Maged1 expression on the Bmal1-RORE mutant-Luc promoter. (C) Q–PCR analysis of endogenous Bmal1 expression after overexpression of MAGED1 and/or RORα in NIH3T3 fibroblasts. (D) Co-immunoprecipitation assays of HEK 293T cells using epitope-tagged MAGED1 and RORα proteins as indicated. Each blot shows a representative example from three independent replicates. (E) Confirmation of interaction between RORα and MAGED1 in liver tissues. Liver tissues were collected at indicated times. IP was performed with anti-RORα. Immunoprecipitated proteins were further analysed by western blotting with anti-MAGED1 antibody. (^**P<0.01, ^***P<0.001, unpaired two-tailed Student's t-test).

Identification of other circadian genes targeted by MAGED1. (A) Q–PCR analysis of endogenous Rev-erbα and E4bp4 expression in WT and Maged1 KO liver tissues. The relative levels of RNA were estimated by Q–PCR and normalized to Gapdh. Data represent mean±s.d. (n=3) and show a representative from three independent replicates. (B) Overexpression of MAGED1 and RORα inhibits the Rev-erbα promoter activity in HEK 293T cells. Bar graphs depict relative luciferase activities mean±s.d. of three replicates from a single assay. The results shown are representative of three independent experiments. (C) Q–PCR analysis of endogenous Rev-erbα expression after overexpression of MAGED1 in NIH3T3 fibroblasts. (D) Effect of Maged1 expression on the E4bp4-Luc promoter in HEK 293T cells. (E) Q–PCR analysis of endogenous E4bp4 expression after overexpression of MAGED1 in NIH3T3 fibroblasts. (F) ChIP assay with MAGED1 antibody or control (IgG) in WT and Maged1 KO hepatocytes. PCR was used to amplify a fragment flanking the proximal RORE on the indicated genes. (^*P<0.05, ^***P<0.001, unpaired two-tailed Student's t-test).

Locomotor activity of wild type and Maged1 KO mice. (A) Voluntary locomotor activity was recorded as wheel-running activity from Maged1 KO mice (23.19±0.21 h, n=42, male) and wild-type siblings (23.72±0.17 h, n=40, male). (Mean period±s.d.) (P<0.001, unpaired two-tailed Student's t-test). (B) Histograms for distributions of period length from the wheel-running recordings. (C–F) CLAMS was used to monitor rest–activity behaviour in wild type (black bars) and Maged1 KO (white bars) mice. See ‘Materials and methods' for detailed description. (C) Rest bouts of 40 s to 2 min per LD cycle. Maged1 KO mice showed significantly increased percentages of short-duration rest episodes (40 s–2 min) compared with their wild-type littermates in the light phase. (D, E) Rest bouts of long duration in the light phase (D) and dark phase (E) per LD cycle. Maged1 KO mice showed significant decreases in long-lasting rest episodes (5–6 and 7–8 min). (F) Total activity was reduced in Maged1 KO mice compared to their wild-type siblings. (n=4, mean period±s.d. ^*P<0.05; ^**P<0.01; ^***P<0.001, unpaired two-tailed Student's t-test). Three independent experiments were carried out.

Expression levels of clock genes in Maged1 KO mice. (A) Q–PCR analysis of expression of clock genes in liver tissues. All tissues were collected at 4 h intervals over the first day in DD for total 44 h. The relative levels of RNA were estimated by Q RT–PCR and normalized by Gapdh. Data represent mean±s.d. (n=3). (B) In situ hybridization showing Bmal1 expression in the SCN of Maged1 KO and wild-type mice. The expression level of Bmal1 was severely reduced at both CT 6 and CT 18 in the SCN of Maged1 KO mice. Two independent experiments were performed. (C, D) Representative protein oscillation profiles of clock genes from nuclear extracts at the indicated CTs over the first day in DD in wild type and Maged1 KO lung (C) and liver (D) tissues. Three independent experiments, each with a time point from at least three mice, gave similar results for both mRNA and protein.

Characterizing MAGED1 functional domain. (A) Construct strategies for truncated HA-tagged MAGED1. (B) Effects of truncated MAGED1 on Bmal1-Luc activity in HEK 293T cells. MAGED1 lacking the MAGE/necdin domain (MAGED1ΔC) retains the ability to activate Bmal1 promoter. (^***P<0.001, unpaired two-tailed Student's t-test). (C) Co-immunoprecipitation assays of HEK 293T cells using Myc-tagged RORα and HA-tagged truncated MAGED1 as indicated. Deletion with the unique hexapeptide repeat domain or the C-terminal domain abolished the interaction between MAGED1 and RORα. Stars represent non-specific signals.

Expression of Maged1 mRNA and protein. (A) Upper panel: temporal Maged1 mRNA abundance in liver and SCN at indicated CTs. Bottom panel: protein expression profiles of MAGED1 in total liver and SCN area lysates at indicated CTs using MAGED1 antibody. (B) MAGED1 nuclear protein levels at the indicated CTs from liver tissues. (C–E) Q–PCR assays of Maged1 mRNA expression at CT 6 and CT 18 in circadian mutant mice as indicated at the bottom. Right panel shows Bmal1 expression in C57BL/6J mice as control (n=3 for each genotype). (F) Comparison of Maged1 mRNA level from WT MEFs after serum shock or dexamethasone treatment at indicated time points, (^***P<0.001, unpaired two-tailed Student's t-test). Three independent experiments were carried out. (G) A model for Maged1 regulation in circadian rhythm. The complex of MAGED1 and ROR proteins regulates the amplitude of Bmal1 by activating RORE in Bmal1 promoter. MAGED1 may also participate in the inhibition of Rev-erbα and activation of E4bp4 and thereby affect output pathway. The existence of other undefined transcriptional factors may contribute to the regulation preference and specificity of Maged1. The clock is thought to send an increasingly strong wake-promoting signal during the day, allowing wakefulness to be maintained. Similarly, during sleep, the clock may send a strong sleep-promoting signal, allowing sleep to be maintained. When the robustness of the circadian clock is impaired such as Maged1 knockout or serum shock resulting in Maged1 downregulation, the endogenous clock is entrained easily and increases sensitivity to respond to external cues.