Circadian clocks are cell-autonomous oscillators regulating daily rhythms in a wide range of physiological, metabolic and behavioral processes. Feedback of metabolic signals, such as redox state, NAD+/NADH and AMP/ADP ratios, or heme, modulate circadian rhythms and thereby optimize energy utilization across the 24-h cycle. We show that rhythmic heme degradation, which generates the signaling molecule carbon monoxide (CO), is required for normal circadian rhythms as well as circadian metabolic outputs. CO suppresses circadian transcription by attenuating CLOCK–BMAL1 binding to target promoters. Pharmacological inhibition or genetic depletion of CO-producing heme oxygenases abrogates normal daily cycles in mammalian cells and Drosophila. In mouse hepatocytes, suppression of CO production leads to a global upregulation of CLOCK–BMAL1-dependent circadian gene expression and dysregulated glucose metabolism. Together, our findings show that CO metabolism is an important link between the basic circadian-clock machinery, metabolism and behavior.
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We thank A. Grudziecki, B. Koller and U. Ungethüm for excellent technical support. We also thank M. Rauer and H. Herzel for bioinformatics help as well as A. Zenclusen (Otto von Guericke University Magdeburg), M. Brunner (Ruprecht-Karls-University Heidelberg), S. Taketani (Insect Biomedical Research Center, Kyoto Institute of Technology) and the NIG-Fly Stock Center (Genetic Strain Research Center, National Institute of Genetics Mishima) for materials. This work was supported by the BBSRC (grant BB/J018589/1 to R.S.) and the German Research foundation (Emmy Noether grant SCHU 2546/1-1 to M.S. and SFB 618/A4 and SFB 740/D2 to A.K.).
The authors declare no competing financial interests.
Integrated supplementary information
(a) Ho-1 mRNA levels oscillate in mouse peritoneal macrophages. These data are taken from our experiments published in Keller, M. et al. Proc. Natl. Acad. Sci. USA. 106, 21407-21412 (2009). Cells harvested every 4 h via peritoneal lavage from four C57BL/6 mice kept in constant darkness were magnetically purified for CD11b surface expression. Three individual RNA samples of each time were pooled and subjected to global gene transcription measurement by using Affymetrix microarrays. Circadian oscillation of Ho-1 transcript is significant (p < 0.005 CircWave). (b) Ho-1 mRNA levels do not oscillate in primary hepatocytes from Bmal1-/- mice. Ho-1 mRNA levels in hemin and CoPPIX treated (30 μM each) primary hepatocytes from Bmal1-/- mice. Cells were dexamethasone-synchronized and harvested after 24 hour and 40 hours, which correspond to trough and peak of Ho-1 mRNA levels in wild-type cells (see Fig. 2b). Data are normalized to Gapdh expression and presented relative to mean expression in untreated cells. Given are means ± sd of three independent samples. (c) Conserved E-box (blue) in mouse, rat and human Ho-1 promoters close the transcription start site (yellow) and start of coding region (red). (d) Ho-1 mRNA levels are reduced in unsynchronized primary hepatocytes from Bmal1-/- mice compared to hepatocytes from wild-type littermates (wt). Data are normalized to Gapdh expression and presented relative to mean expression in wild-type cells. Given is mean ± sd of three independent samples.
Supplementary Figure 2 The heme-degradation product CO modulates circadian transcription: additional data.
Upper left: Transcript levels of Rev-Erbα and Dbp in CO or N2 (6%) treated primary fibroblasts of Ho-1-/- mice or wild-type littermates harvested 24 hours after dexamethasone synchronization. Other panels: Transcript levels of Dbp in embryonic fibroblasts from Ho-1-/- mice (Ho1KO) or wild-type littermates (Ho1WT) 24 hours after dexamethasone synchronization, which were treated for 1 hour (or 3 x 1 hour; lower right) with 100 μM CO-releasing molecules (CORM) or inactive control molecules (iCORM) before harvesting. Data are normalized to Gapdh expression and presented relative to mean expression in wild-type control cells. Given is mean ± sem of three independent samples or – upper left – mean (bars) of two independent samples (small symbols).
(a) Clock gene mRNA and pre-mRNA levels of primary fibroblasts of Ho-1-/- mice (KO) or wild-type littermates (WT) harvested 24 hours after dexamethasone synchronization. Data are normalized to Gapdh expression and presented relative to mean expression in wild-type control cells. Given is mean ± sd of three independent samples. (b) Pre-mRNA levels of Dbp in embryonic fibroblasts from Ho-1-/- mice (KO) or wild-type littermates (WT) 24 hours after dexamethasone synchronization, which were treated for 1 hour with 100 μM CO-releasing molecules (CORM) or inactive control molecules (iCORM) before harvesting. Data are normalized to Gapdh expression and presented relative to mean expression in wild-type control cells. Given is mean ± sd of three independent samples. (c) CO does not acutely alter BMAL1 protein level. BMAL1 protein levels in U2-OS cells 24 hours after dexamethasone synchronization, which were treated for 1 hour with 100 μM CO-releasing molecules (CORM) or inactive control molecules (iCORM) before harvesting. Shown are four independent samples.
Supplementary Figure 4 Ho-1 knockout alone has no effect on circadian dynamics, probably because of residual activity from HO-2.
(a) Circadian oscillation dynamics of synchronized primary fibroblasts of Ho-1-/- mice (Ho1KO) or wild-type littermates (Ho1WT) lentivirally transduced with a Bmal1 promoter-luciferase reporter construct. Shown are two representative examples of raw data (upper panel) and detrended (lower panel) time-series for each genotype. Note, the absolute light levels slightly decrease upon Ho-1 knockout consistent with the overall slightly lower Bmal1 transcript levels (compare Fig. 3a). (b) Heme oxygenase activity in fibroblasts with specific depletion/knockout of heme oxygenase isoforms. Lysates of primary fibroblasts of indicated Ho-1 genotype with or without additional RNAi-mediated depletion of Ho-2 were analyzed for total heme oxygenase activity.
Supplementary Figure 5 Heme oxygenase–derived CO is essential for normal circadian dynamics in mammalian cells: additional data.
(a) Circadian dynamics of synchronized primary fibroblasts from Ho-1-/- mice lentivirally transduced with (i) shRNA constructs targeting Ho-2 or a non-silencing (ns) control and (ii) a Bmal1 promoter-luciferase reporter construct. Shown are representative examples of raw data time-series. For detrended time series and period quantification see Fig. 5a. Note, that the absolute light levels decrease upon Ho‑2 knockdown consistent with the overall increase in Rev-Erbα levels and thus putatively lower Bmal1 transcript levels (compare Fig. 5b). (b) CO partly rescues the long period oscillations in heme oxygenase depleted cells. Circadian oscillation dynamics of synchronized primary fibroblasts from Ho-1-/- mice lentivirally transduced with (i) shRNA constructs targeting Ho-2 and (ii) a Bmal1 promoter-luciferase reporter construct. Cells were continuously treated with 6% CO or N2. Shown are representative examples of raw data time-series. For detrended time series see Fig. 5c. (c) Exogenous CO treatment has no effect on oscillation dynamics of wild-type cells. Circadian oscillation dynamics of synchronized primary fibroblasts from wild-type mice lentivirally transduced with a Bmal1 promoter-luciferase reporter construct, which were continuously treated with 6% CO or N2. Shown is a representative example of raw data (upper panel) and detrended time-series (lower panel). Note, since CO can only shorten the circadian period in Ho-depleted cells (Fig. 5c) but not in wild-type cells, period lengthening in Ho-depleted cells is very likely specifically due to the lack of endogenous CO rather than to other functions of heme oxygenases.
Supplementary Figure 6 Heme oxygenases modulate metabolic gene expression in primary hepatocytes at the transcriptional level.
Pre-mRNA levels of Pck1, G6pc, Lpl and Cyp7a1 in primary hepatocytes (24 hours after dexamethasone synchronization) of Ho‑1-/- or wild-type littermate mice with or without additional Ho-2 depletion by RNAi. Data are normalized to Gapdh expression and presented relative to mean expression in wild-type cells transduced with the non-silencing control. Shown are mean levels (bars) of two independent samples (small symbols).
Supplementary Figure 7 Heme oxygenase (dHo) is essential for normal daily activity patterns in Drosophila: additional data.
(a) Mean locomotor activity of Drosophila males analysed in 12 hr light: 12 hr dark conditions for 5-8 days. A different dHo-RNAi construct inserted either on chromosome 3 (UAS-dHo-RNAi:R1) or chromosome 2 (UAS-dHo-RNAi:R3) was either driven in all clock cells: timeless-gal4 (tim >); PDF-positive clock neurons: Pdf-gal4 (Pdf >); all neurons: elav-gal4 (elav >), or all glia cells: repo-gal4 (repo>). Note the phase advance caused by dHo knockdown in clock cells and neurons, whereas glia-specific knockdown or ‘driver-only’ controls results in normal behaviour. At least 16 flies were tested for each genotype. Bars show mean activity within in 30 min; dark bars: ‘lights-off’, white bars: ‘lights on’, dots indicate sem. (b) Real-time luciferase recordings of flies expressing different period-luciferase fusion genes. Male flies were recorded in LD and DD as indicated by the bars below the plots (white and black bars indicate ‘lights-on’ and ‘lights-off’ respectively). Left: Flies expressing luciferase under control of the period promoter in all clock cells (including peripheral clocks) encoded by the plo transgene29: grey circles—tim-gal4:27/+;plo:86-6/UAS-dHo-RNAi:21-1 (n=16); black circles—tim-gal4:27/UAS-GFP;plo:86:6/+ (n=12). Middle: Flies expressing a PERIOD-LUCIFERASE fusion protein in all clock cells (including peripheral clocks) encoded by the XLG-luc transgene30: grey circles—tim-gal4:27/+;XLG-luc/UAS-dHo-RNAi:21-1 (n=8); black circles—tim-gal4:27/UAS-GFP;XLG-luc/+ (n=8); open circles—tim-gal4:27/UAS-attP-51C;XLG-luc/+ (n=4). Note that in the left and middle panels no significant differences are observable between dHo-RNAi and control flies. Similar results were obtained in two independent experiments including the dHoRNAi:21-8 line. Right: Flies expressing a PERIOD-LUCIFERASE fusion protein in dorsal clock neurons encoded by the promoter-less 8.0-luc transgene30. Exact genotypes: grey circles—8.0-luc/+;tim-gal4:67/UAS-dHo-RNAi:21-8 (n=8); black circles—8.0-luc/UAS-GFP;tim-gal4:67/+ (n=10). Note the higher peak levels in LD and DD, and the increased amplitude of PER-LUC oscillations in dHo-RNAi flies.
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Klemz, R., Reischl, S., Wallach, T. et al. Reciprocal regulation of carbon monoxide metabolism and the circadian clock. Nat Struct Mol Biol 24, 15–22 (2017). https://doi.org/10.1038/nsmb.3331
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