The mammalian liver is a central hub for systemic metabolic homeostasis. Liver tissue is spatially structured, with hepatocytes operating in repeating lobules, and sub-lobule zones performing distinct functions. The liver is also subject to extensive temporal regulation, orchestrated by the interplay of the circadian clock, systemic signals and feeding rhythms. However, liver zonation was previously analyzed as a static phenomenon, and liver chronobiology at tissue level resolution. Here, we use single-cell RNA-seq to investigate the interplay between gene regulation in space and time. Using mixed-effect models of mRNA expression and smFISH validations, we find that many genes in the liver are both zonated and rhythmic, most of them showing multiplicative space-time effects. Such dually regulated genes cover key hepatic functions such as lipid, carbohydrate and amino acid metabolism, but also previously unassociated genes, such as protein chaperones. Our data also suggest that rhythmic and localized expression of Wnt targets could be explained by rhythmically expressed Wnt ligands from non-parenchymal cells near the central vein. Core circadian clock genes are expressed in a non-zonated manner, indicating that the liver clock is robust to zonation. Together, our scRNA-seq analysis reveals how liver function is compartmentalized spatio-temporally at the sub-lobular scale.

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 knockout mice. Deleting a contacted intronic enhancer element in the () gene was sufficient to compromise the rhythmic chromatin contacts in tissues. Moreover, the deletion reduced the daily dynamics of 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.

20The mammalian liver performs key physiological functions for maintaining energy and 21 metabolic homeostasis. Liver tissue is both spatially structured and temporally 22 orchestrated. Hepatocytes operate in repeating anatomical units termed lobules and 23 different lobule zones perform distinct functions. The liver is also subject to extensive 24 temporal regulation, orchestrated by the interplay of the circadian clock, systemic signals 25 and feeding rhythms. Liver zonation was previously analyzed as a static phenomenon and 26 liver chronobiology at the tissue level. Here, we use single-cell RNA-seq to investigate 27 the interplay between gene regulation in space and time. Categorizing mRNA expression 28 profiles using mixed-effect models and smFISH validations, we find that many genes in 29 the liver are both zonated and rhythmic, most of them showing multiplicative space-time 30 effects. Such dually regulated genes cover key hepatic functions such as lipid, 31 carbohydrate and amino acid metabolism. In particular, our data suggest that rhythmic 32 and localized expression of Wnt targets may be explained by rhythmic Wnt signaling 33 from endothelial cells near the central vein. Core circadian clock genes are expressed in 34 a non-zonated manner, indicating that the liver clock is robust to zonation. Together, our 35 comprehensive data reveal how liver function is compartmentalized spatio-temporally at 36 the sub-lobular scale. 37Recently, we combined single-cell RNA-sequencing (scRNA-seq) of dissociated 53 hepatocytes and single-molecule RNA fluorescence in situ hybridization (smFISH) to 54 reconstruct spatial mRNA expression profiles along the porto-central axis 7 . This analysis 55 revealed an unexpected breadth of spatial heterogeneity, with ~50% of genes showing 56 spatially non-uniform patterns. Among them, functions related to ammonia clearance, 57 carbohydrate catabolic and anabolic processes, xenobiotics detoxification, bile acid and 58 cholesterol synthesis, fatty acid metabolism, targets of the Wnt and Ras pathways, and 59 hypoxia-induced genes were strongly zonated. 60In addition to its spatial heterogeneity, the liver is also highly dynamic 61 temporally. Chronobiology studies showed that temporally gated physiological and 62 metabolic programs in the liver result from the complex interplay between the 63 endogenous circadian liver oscillator, rhythmic systemic signals, and feeding/fasting 64 cycles 8,9,10 . An intact circadian clock has repeatedly been demonstrated as key for healthy 65 metabolism, also in humans 11 . Temporal compartmentalization can prevent two opposite 66 and incompatible processes from simultaneously occurring, for example, glucose is 67 stored as glycogen following a meal and is later released into the blood circulation during 68 fasting period to maintain homeostasis in plasma glucose levels. Functional genomics 69 studies of the circadian liver were typically performed on bulk liver tissue 12 . 70In particular, we and others showed how both the circadian clock and the fee...