Nuclear receptor corepressor and histone deacetylase 3 govern circadian metabolic physiology

Abstract

Rhythmic changes in histone acetylation at circadian clock genes suggest that temporal modulation of gene expression is regulated by chromatin modifications. Furthermore, recent studies demonstrate a critical relationship between circadian and metabolic physiology. The nuclear receptor corepressor 1 (Ncor1) functions as an activating subunit for the chromatin modifying enzyme histone deacetylase 3 (Hdac3). Lack of Ncor1 is incompatible with life, and hence it is unknown whether Ncor1, and particularly its regulation of Hdac3, is critical for adult mammalian physiology. Here we show that specific, genetic disruption of the Ncor1-Hdac3 interaction in mice causes aberrant regulation of clock genes and results in abnormal circadian behaviour. These mice are also leaner and more insulin-sensitive owing to increased energy expenditure. Unexpectedly, loss of a functional Ncor1-Hdac3 complex in vivo does not lead to sustained increases in known catabolic genes, but instead significantly alters the oscillatory patterns of several metabolic genes, demonstrating that circadian regulation of metabolism is critical for normal energy balance. These findings indicate that activation of Hdac3 by Ncor1 is a nodal point in the epigenetic regulation of circadian and metabolic physiology.

Figure 1. NCoR-HDAC3 regulates peripheral clock and circadian physiology

(a) Bmal1 expression in WT and DADm livers during ZT 7–9 (WT: n=6, DADm: n=4). (b) ChIP for acetylated histone H4 from WT and DADm livers at the Bmal1 RORE (n=3). (c) Bmal1 expression in immortalized mouse embryonic fibroblasts (MEFs) (n=3). (d) Bmal1 expression in MEFS after treatment with MS-275 (n=3). (e) Effect of Rev-erbα knockdown on MEF Rev-erbα and Bmal1 expression (n=3). (f) Bmal1 expression in MEFs following cell synchronization (fold difference relative to WT, time 0; n=3 per time point). (g) ChIP of acetylated histone H4 at the Bmal1 RORE following 50% serum shock. Mean +/− s.e.m of duplicate samples. Independent experiments gave similar results. (h) Voluntary locomotor wheel running activity, double plotted in each panel. (i) Average free running period (n=3). Three independent experiments gave similar results. Data are presented as mean +/− s.e.m. *p<0.05, **p<.01.

Figure 2. DADm mice exhibit increased energy expenditure

(a) Body weight of 24 week old male WT and DADm mice (n=10) (b) Representative abdominal images of WT and DADm mice. (c) Perigonadal fat pads harvested from mice in (b). (d) % Body fat composition measured by nuclear magnetic resonance for cohort presented in (a). (e–i) Effect of DADm on (e) locomotor activity measured by photobeam breaks, (f) food intake, (g,h) oxygen consumption (VO2), and (i) heat. Average VO2 for 3 consecutive measurements taken every 27 minutes is shown in (h) (n=4). Data is presented as mean +/− s.e.m. *p<0.05.

Figure 3. DADm mice are resistant to diet-induced obesity

(a) Hyperinsulinemic-euglycemic clamp measurements for 16 week old male WT and DADm mice fed a normal chow diet (n=4). (b) Weight curve for age matched WT and DADm mice started on 60% high fat diet (HFD) at 8 weeks. (c) Insulin tolerance test for mice in (b) at ZT8 after 15 weeks on HFD (WT, n=13 , DADm, n=12). Data is presented as mean +/− s.e.m. *p<0.05, **p<0.01.

Figure 4. Activation of HDAC3 by NCoR regulates circadian metabolic gene expression in the liver

(a) Diurnal expression of lipid metabolic genes (WT, open diamonds; DADm, black squares relative to WT, ZT0. Lights on and off at ZT0 and ZT12, respectively. (n=2–3 per genotype per ZT). (b) ChIP for HDAC3 from WT and DADm MEFs (n=3). (c, d) ChIP for acetylated histone H4 from livers at (c) CPT1a TRE and (d) MCAD NRRE (n=3 per genotype per ZT). qPCR results are relative to WT, ZT5. Data is presented as mean +/− s.e.m. *p<0.05 relative to ZT5, ^p<0.05 relative to WT, #p<.05 relative to ZT10.