Circadian Rhythms in Anticancer Immunity: Mechanisms and Treatment Opportunities.

Circadian rhythms of approximately 24 h have emerged as important modulators of the immune system. These oscillations are important for mounting short-term, innate immune responses, but surprisingly also long-term, adaptive immune responses. Recent data indicate that they play a central role in antitumor immunity, in both mice and humans. In this review, we discuss the evolving literature on circadian antitumor immune responses and the underlying mechanisms that control them. We further provide an overview of circadian treatment regimens-chrono-immunotherapies-that harness time-of-day differences in immunity for optimal efficacy. Our aim is to provide an overview for researchers and clinicians alike, for a better understanding of the circadian immune system and how to best harness it for chronotherapeutic interventions. This knowledge is important for a better understanding of immune responses per se and could revolutionize the way we approach the treatment of cancer and a range of other diseases, ultimately improving clinical practice.

Cell maturation: Hallmarks, triggers, and manipulation.

How cells become specialized, or "mature," is important for cell and developmental biology. While maturity is usually deemed a terminal fate, it may be more helpful to consider maturation not as a switch but as a dynamic continuum of adaptive phenotypic states set by genetic and environment programing. The hallmarks of maturity comprise changes in anatomy (form, gene circuitry, and interconnectivity) and physiology (function, rhythms, and proliferation) that confer adaptive behavior. We discuss efforts to harness their chemical (nutrients, oxygen, and growth factors) and physical (mechanical, spatial, and electrical) triggers in vitro and in vivo and how maturation strategies may support disease research and regenerative medicine.

Atlas of Circadian Metabolism Reveals System-wide Coordination and Communication between Clocks.

Metabolic diseases are often characterized by circadian misalignment in different tissues, yet how altered coordination and communication among tissue clocks relate to specific pathogenic mechanisms remains largely unknown. Applying an integrated systems biology approach, we performed 24-hr metabolomics profiling of eight mouse tissues simultaneously. We present a temporal and spatial atlas of circadian metabolism in the context of systemic energy balance and under chronic nutrient stress (high-fat diet [HFD]). Comparative analysis reveals how the repertoires of tissue metabolism are linked and gated to specific temporal windows and how this highly specialized communication and coherence among tissue clocks is rewired by nutrient challenge. Overall, we illustrate how dynamic metabolic relationships can be reconstructed across time and space and how integration of circadian metabolomics data from multiple tissues can improve our understanding of health and disease.

Diet-Induced Circadian Enhancer Remodeling Synchronizes Opposing Hepatic Lipid Metabolic Processes.

Overnutrition disrupts circadian metabolic rhythms by mechanisms that are not well understood. Here, we show that diet-induced obesity (DIO) causes massive remodeling of circadian enhancer activity in mouse liver, triggering synchronous high-amplitude circadian rhythms of both fatty acid (FA) synthesis and oxidation. SREBP expression was rhythmically induced by DIO, leading to circadian FA synthesis and, surprisingly, FA oxidation (FAO). DIO similarly caused a high-amplitude circadian rhythm of PPARα, which was also required for FAO. Provision of a pharmacological activator of PPARα abrogated the requirement of SREBP for FAO (but not FA synthesis), suggesting that SREBP indirectly controls FAO via production of endogenous PPARα ligands. The high-amplitude rhythm of PPARα imparted time-of-day-dependent responsiveness to lipid-lowering drugs. Thus, acquisition of rhythmicity for non-core clock components PPARα and SREBP1 remodels metabolic gene transcription in response to overnutrition and enables a chronopharmacological approach to metabolic disorders.

Light Affects Mood and Learning through Distinct Retina-Brain Pathways.

Light exerts a range of powerful biological effects beyond image vision, including mood and learning regulation. While the source of photic information affecting mood and cognitive functions is well established, viz. intrinsically photosensitive retinal ganglion cells (ipRGCs), the central mediators are unknown. Here, we reveal that the direct effects of light on learning and mood utilize distinct ipRGC output streams. ipRGCs that project to the suprachiasmatic nucleus (SCN) mediate the effects of light on learning, independently of the SCN's pacemaker function. Mood regulation by light, on the other hand, requires an SCN-independent pathway linking ipRGCs to a previously unrecognized thalamic region, termed perihabenular nucleus (PHb). The PHb is integrated in a distinctive circuitry with mood-regulating centers and is both necessary and sufficient for driving the effects of light on affective behavior. Together, these results provide new insights into the neural basis required for light to influence mood and learning.

Aged Stem Cells Reprogram Their Daily Rhythmic Functions to Adapt to Stress.

Normal homeostatic functions of adult stem cells have rhythmic daily oscillations that are believed to become arrhythmic during aging. Unexpectedly, we find that aged mice remain behaviorally circadian and that their epidermal and muscle stem cells retain a robustly rhythmic core circadian machinery. However, the oscillating transcriptome is extensively reprogrammed in aged stem cells, switching from genes involved in homeostasis to those involved in tissue-specific stresses, such as DNA damage or inefficient autophagy. Importantly, deletion of circadian clock components did not reproduce the hallmarks of this reprogramming, underscoring that rewiring, rather than arrhythmia, is associated with physiological aging. While age-associated rewiring of the oscillatory diurnal transcriptome is not recapitulated by a high-fat diet in young adult mice, it is significantly prevented by long-term caloric restriction in aged mice. Thus, stem cells rewire their diurnal timed functions to adapt to metabolic cues and to tissue-specific age-related traits.

PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period.

PERIOD (PER) and Casein Kinase 1δ regulate circadian rhythms through a phosphoswitch that controls PER stability and repressive activity in the molecular clock. CK1δ phosphorylation of the familial advanced sleep phase (FASP) serine cluster embedded within the Casein Kinase 1 binding domain (CK1BD) of mammalian PER1/2 inhibits its activity on phosphodegrons to stabilize PER and extend circadian period. Here, we show that the phosphorylated FASP region (pFASP) of PER2 directly interacts with and inhibits CK1δ. Co-crystal structures in conjunction with molecular dynamics simulations reveal how pFASP phosphoserines dock into conserved anion binding sites near the active site of CK1δ. Limiting phosphorylation of the FASP serine cluster reduces product inhibition, decreasing PER2 stability and shortening circadian period in human cells. We found that Drosophila PER also regulates CK1δ via feedback inhibition through the phosphorylated PER-Short domain, revealing a conserved mechanism by which PER phosphorylation near the CK1BD regulates CK1 kinase activity.

Circadian mechanisms in adipose tissue bioenergetics and plasticity.

The circadian clock plays an essential role in coordinating feeding and metabolic rhythms with the light/dark cycle. Disruption of clocks is associated with increased adiposity and metabolic disorders, whereas aligning feeding time with cell-autonomous rhythms in metabolism improves health. Here, we provide a comprehensive overview of recent literature in adipose tissue biology as well as our understanding of molecular mechanisms underlying the circadian regulation of transcription, metabolism, and inflammation in adipose tissue. We highlight recent efforts to uncover the mechanistic links between clocks and adipocyte metabolism, as well as its application to dietary and behavioral interventions to improve health and mitigate obesity.

Butt-seq: a new method for facile profiling of transcription.

A wide range of sequencing methods has been developed to assess nascent RNA transcription and resolve the single-nucleotide position of RNA polymerase genome-wide. These techniques are often burdened with high input material requirements and lengthy protocols. We leveraged the template-switching properties of thermostable group II intron reverse transcriptase (TGIRT) and developed Butt-seq (bulk analysis of nascent transcript termini sequencing), which can produce libraries from purified nascent RNA in 6 h and from as few as 10,000 cells-an improvement of at least 10-fold over existing techniques. Butt-seq shows that inhibition of the superelongation complex (SEC) causes promoter-proximal pausing to move upstream in a fashion correlated with subnucleosomal fragments. To address transcriptional regulation in a tissue, Butt-seq was used to measure the circadian regulation of transcription from fly heads. All the results indicate that Butt-seq is a simple and powerful technique to analyze transcription at a high level of resolution.

The multiple roles of salt-inducible kinases in regulating physiology.

Salt-inducible kinases (SIKs), which comprise a family of three homologous serine-threonine kinases, were first described for their role in sodium sensing but have since been shown to regulate multiple aspects of physiology. These kinases are activated or deactivated in response to extracellular signals that are cell surface receptor mediated and go on to phosphorylate multiple targets including the transcription cofactors CRTC1-3 and the class IIa histone deacetylases (HDACs). Thus, the SIK family conveys signals about the cellular environment to reprogram transcriptional and posttranscriptional processes in response. In this manner, SIKs have been shown to regulate metabolic responses to feeding/fasting, cell division and oncogenesis, inflammation, immune responses, and most recently, sleep and circadian rhythms. Sleep and circadian rhythms are master regulators of physiology and are exquisitely sensitive to regulation by environmental light and physiological signals such as the need for sleep. Salt-inducible kinases have been shown to be central to the molecular regulation of both these processes. Here, we summarize the molecular mechanisms by which SIKs control these different domains of physiology and highlight where there is mechanistic overlap with sleep/circadian rhythm control.