Significance: Circadian clocks orchestrate daily rhythms in behavior and physiology using temporal regulation of gene expression to control core clock genes and rhythmic output programs. Although transcription regulation was shown to drive extensive diurnal mRNA rhythms, less is known about the proteins. Here, we provide a proteome-wide study of rhythmic protein accumulation in mouse liver, showing that proteins preferentially accumulate in the morning and during the night. About one-half of these rhythmic proteins could not be explained by rhythmic mRNAs, suggesting that translational or posttranslational regulation plays an important role. Moreover, such rhythms involved many secreted proteins and were clock-independent. Hence, these findings indicate that feeding behavior might determine the rhythmic functions of circulating proteins in the blood.

Diurnal oscillations of gene expression controlled by the circadian clock and its connected feeding rhythm enable organisms to coordinate their physiologies with daily environmental cycles. While available techniques yielded crucial insights into regulation at the transcriptional level, much less is known about temporally controlled functions within the nucleus and their regulation at the protein level. Here, we quantified the temporal nuclear accumulation of proteins and phosphoproteins from mouse liver by SILAC proteomics. We identified around 5,000 nuclear proteins, over 500 of which showed a diurnal accumulation. Parallel analysis of the nuclear phosphoproteome enabled the inference of the temporal activity of kinases accounting for rhythmic phosphorylation. Many identified rhythmic proteins were parts of nuclear complexes involved in transcriptional regulation, ribosome biogenesis, DNA repair, and the cell cycle and its potentially associated diurnal rhythm of hepatocyte polyploidy. Taken together, these findings provide unprecedented insights into the diurnal regulatory landscape of the mouse liver nucleus.

The circadian clock in animals orchestrates widespread oscillatory gene expression programs, which underlie 24-h rhythms in behavior and physiology. Several studies have shown the possible roles of transcription factors and chromatin marks in controlling cyclic gene expression. However, how daily active enhancers modulate rhythmic gene transcription in mammalian tissues is not known. Using circular chromosome conformation capture (4C) combined with sequencing (4C-seq), we discovered oscillatory promoter–enhancer interactions along the 24-h cycle in the mouse liver and kidney. Rhythms in chromatin interactions were abolished in arrhythmic Bmal1 knockout mice. Deleting a contacted intronic enhancer element in the Cryptochrome 1 (Cry1) gene was sufficient to compromise the rhythmic chromatin contacts in tissues. Moreover, the deletion reduced the daily dynamics of Cry1 transcriptional burst frequency and, remarkably, shortened the circadian period of locomotor activity rhythms. Our results establish oscillating and clock-controlled promoter–enhancer looping as a regulatory layer underlying circadian transcription and behavior.

Lysine acetylation is involved in various biological processes and is considered a key reversible post-translational modification in the regulation of gene expression, enzyme activity, and subcellular localization. This post-translational modification is therefore highly relevant in the context of circadian biology, but its characterization on the proteome-wide scale and its circadian clock dependence are still poorly described. Here, we provide a comprehensive and rhythmic acetylome map of the mouse liver. Rhythmic acetylated proteins showed subcellular localization-specific phases that correlated with the related metabolites in the regulated pathways. Mitochondrial proteins were over-represented among the rhythmically acetylated proteins and were highly correlated with SIRT3-dependent deacetylation. SIRT3 activity being nicotinamide adenine dinucleotide (NAD)+ level-dependent, we show that NAD+ is orchestrated by both feeding rhythms and the circadian clock through the NAD+ salvage pathway but also via the nicotinamide riboside pathway. Hence, the diurnal acetylome relies on a functional circadian clock and affects important diurnal metabolic pathways in the mouse liver.