The PER2:BRCA1:POU2F1(OCT-1) ternary complex represents a multi-component scaffold model for circadian gene regulation

Many mammalian peripheral tissues have circadian clocks; endogenous oscillators that generate transcriptional rhythms thought to be important for the daily timing of physiological processes. The extent of circadian gene regulation in peripheral tissues is unclear, and to what degree circadian regulation in different tissues involves common or specialized pathways is unknown. Here we report a comparative analysis of circadian gene expression in vivo in mouse liver and heart using oligonucleotide arrays representing 12,488 genes. We find that peripheral circadian gene regulation is extensive (> or = 8-10% of the genes expressed in each tissue), that the distributions of circadian phases in the two tissues are markedly different, and that very few genes show circadian regulation in both tissues. This specificity of circadian regulation cannot be accounted for by tissue-specific gene expression. Despite this divergence, the clock-regulated genes in liver and heart participate in overlapping, extremely diverse processes. A core set of 37 genes with similar circadian regulation in both tissues includes candidates for new clock genes and output genes, and it contains genes responsive to circulating factors with circadian or diurnal rhythms.

When the ancestors of modern Eurasians migrated out of Africa and interbred with Eurasian archaic hominins, namely, Neanderthals and Denisovans, DNA of archaic ancestry integrated into the genomes of anatomically modern humans. This process potentially accelerated adaptation to Eurasian environmental factors, including reduced ultraviolet radiation and increased variation in seasonal dynamics. However, whether these groups differed substantially in circadian biology and whether archaic introgression adaptively contributed to human chronotypes remain unknown. Here, we traced the evolution of chronotype based on genomes from archaic hominins and present-day humans. First, we inferred differences in circadian gene sequences, splicing, and regulation between archaic hominins and modern humans. We identified 28 circadian genes containing variants with potential to alter splicing in archaics (e.g., CLOCK, PER2, RORB, and RORC) and 16 circadian genes likely divergently regulated between present-day humans and archaic hominins, including RORA. These differences suggest the potential for introgression to modify circadian gene expression. Testing this hypothesis, we found that introgressed variants are enriched among expression quantitative trait loci for circadian genes. Supporting the functional relevance of these regulatory effects, we found that many introgressed alleles have associations with chronotype. Strikingly, the strongest introgressed effects on chronotype increase morningness, consistent with adaptations to high latitude in other species. Finally, we identified several circadian loci with evidence of adaptive introgression or latitudinal clines in allele frequency. These findings identify differences in circadian gene regulation between modern humans and archaic hominins and support the contribution of introgression via coordinated effects on variation in human chronotype.

Tuberculosis (TB) remains a global public health challenge, with the current BCG vaccine providing limited efficacy in adults, and available treatments being lengthy and debilitating. To overcome these challenges, we have previously developed a recombinant BCG strain expressing the detoxified E. coli Heat-Labile Toxin (LTAK63), providing increased protection in mouse models and reduced lung pathology. Here, using systems biology and RNA sequencing of lung tissues in a murine model, we uncover the molecular mechanisms underlying rBCG-LTAK63’s increased protection. Immunization triggered early activation of cAMP-related pathways, leading to hypoxia, autophagy, and circadian rhythm gene regulation. These processes were associated with an enhanced innate immunity and promoted long-lasting Th1/Th17 adaptive responses. Upon challenge, mice immunized with rBCG-LTAK63 exhibited an earlier onset of interferon-gamma response, reduced bacterial burden, and improved lung histopathology. Notably, circadian rhythm regulation was directly linked to a controlled inflammatory response and reduced migration of infection-susceptible cells, resulting in decreased immunopathology. Our findings demonstrate that rBCG-LTAK63 orchestrates protection through the integration of metabolic and temporal immune pathways. This work provides mechanistic insights into how rational vaccine design can reprogram host immunity to enhance protection and reduce pathology, supporting rBCG-LTAK63 as a promising next-generation TB vaccine candidate.

Circadian regulatory system is an evolutionarily ancient biological system. Its prevalence in life kingdoms suggests it has fundamental role in life processes. Although genomic scale of circadian gene expression has been found in various species from cyanobacteria to mammalians, transcriptional patterns and mechanisms of global circadian gene regulation have not yet been revealed. Using high resolution temporal profiling of mouse circadian gene expression, we show that contrary with previously demonstrated clustering tendency of functionally related genes in mammalian genomes, circadian regulated genes display anti-clustering propensity in mouse liver. This unique property does not conform to the notion of domain-wide coordinated gene regulation dictated by acetyl modifications, which is recently identified as a hallmark of circadian regulation. These results suggest that global circadian regulation in mouse liver might involve other structural chromosome interactions irrelevant with clustering regulation.

Adult bones are continuously remodeled by the balance between bone resorption by osteoclasts and subsequent bone formation by osteoblasts. Many studies have provided molecular evidence that bone remodeling is under the control of circadian rhythms. Circadian fluctuations have been reported in the serum and urine levels of bone turnover markers, such as digested collagen fragments and bone alkaline phosphatase. Additionally, the expressions of over a quarter of all transcripts in bones show circadian rhythmicity, including the genes encoding master transcription factors for osteoblastogenesis and osteoclastogenesis, osteogenic cytokines, and signaling pathway proteins. Serum levels of calcium, phosphate, parathyroid hormone, and calcitonin also display circadian rhythmicity. Finally, osteoblast- and osteoclast-specific knockout mice targeting the core circadian regulator gene Bmal1 show disrupted bone remodeling, although the results have not always been consistent. Despite these studies, however, establishing a direct link between circadian rhythms and bone remodeling in vivo remains a major challenge. It is nearly impossible to repeatedly collect bone materials from human subjects while following circadian changes. In addition, the differences in circadian gene regulation between diurnal humans and nocturnal mice, the main model organism, remain unclear. Filling the knowledge gap in the circadian regulation of bone remodeling could reveal novel regulatory mechanisms underlying many bone disorders including osteoporosis, genetic diseases, and fracture healing. This is also an important question for the basic understanding of how cell differentiation progresses under the influence of cyclically fluctuating environments.

P96. New laboratory method to mirror individual circadian rhythms and sleep preference in idiopathic hypersomnia

Boolean function network analysis of time course liver transcriptome data to reveal novel circadian transcriptional regulators in mammals

Many of the gene regulatory processes of Plasmodium falciparum, the deadliest malaria parasite, remain poorly understood. To develop a comprehensive guide for exploring this organism's gene regulatory network, we generated a systems-level model of P. falciparum gene regulation using a well-validated, machine-learning approach for predicting interactions between transcription regulators and their targets. The resulting network accurately predicts expression levels of transcriptionally coherent gene regulatory programs in independent transcriptomic data sets from parasites collected by different research groups in diverse laboratory and field settings. Thus, our results indicate that our gene regulatory model has predictive power and utility as a hypothesis-generating tool for illuminating clinically relevant gene regulatory mechanisms within P. falciparum. Using the set of regulatory programs we identified, we also investigated correlates of artemisinin resistance based on gene expression coherence. We report that resistance is associated with incoherent expression across many regulatory programs, including those controlling genes associated with erythrocyte-host engagement. These results suggest that parasite populations with reduced artemisinin sensitivity are more transcriptionally heterogenous. This pattern is consistent with a model where the parasite utilizes bet-hedging strategies to diversify the population, rendering a subpopulation more able to navigate drug treatment.

Pinealectomy leads to melatonin deficiency, which is known to disrupt circadian clock regulation and may increase vulnerability of the hippocampus to oxidative stress and neuroinflammatory processes. The objective of this study was to examine the gene expression levels of circadian locomotor output cycles kaput (CLOCK), brain and muscle ARNT-like 1 (BMAL1), period circadian regulator 1 (PER1), cryptochrome circadian regulator 1 (CRY1), brain-derived neurotrophic factor (BDNF), and interleukin-6 (IL-6) in the hippocampus to elucidate the impact of pinealectomy-induced circadian dysregulation on these gene expressions and to assess its association with hippocampal alterations. A total of 30 Wistar rats were randomly divided into three groups: Control, Sham, and Pinealectomy (PNX) (n = 10 per group). Gene expression levels were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical analysis was performed to assess caspase-3 and glial fibrillary acidic protein (GFAP) immunoreactivity. In addition, oxidative stress parameters, including malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), as well as the inflammatory marker tumor necrosis factor-alpha (TNF-α), were measured. The pinealectomy group showed a significant downregulation of BMAL1, BDNF, CLOCK, CRY1, and PER1 gene expression levels, with decreases ranging from approximately 60% to 83% compared with the sham and control groups, whereas IL-6 expression was significantly increased by approximately 185% (p < 0.05). Immunohistochemical analysis demonstrated significantly increased caspase-3 and GFAP immunoreactivity in the PNX group. Furthermore, pinealectomy resulted in a significant increase in MDA and TNF-α levels, accompanied by marked decreases in SOD, CAT, and GSH levels (p < 0.05). In conclusion, pinealectomy is associated with significant disruption of hippocampal circadian clock gene expression, accompanied by oxidative stress, neuroinflammation, and histopathological alterations. These findings highlight the critical role of circadian regulation in maintaining hippocampal cellular integrity.

The circadian rhythm has profound effect on the body, exerting effects on diverse events like sleep-wake patterns, eating behavior and hepatic detoxification. The cytochrome p450 s (Cyps) is the main group of enzymes responsible for detoxification. However, the underlying mechanisms behind circadian regulation of the Cyps are currently not fully clarified. Therefore, the aim of the present study was to investigate the requirement of hepatic peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) for the circadian regulation of the hepatic expression of Cyp1−4 using liver-specific PGC-1α knockout (LKO) mice and littermate controls. The circadian regulator genes Bmal1 and Clock displayed decreased mRNA content at zeitgeber time (ZT) 12, compared to ZT-2 and the mRNA content of Cyp2a4 and Cyp2e1 was higher at ZT-12 than at ZT-2. Moreover, the increase in Cyp2e1 mRNA content was not observed in the PGC-1α LKO mice and hepatic PGC-1α deficiency tended to blunt the rhythmic expression of Clock and Bmal1. However, no circadian regulation was evident at the protein level for the investigated Cyps except for a change in Cyp2e1 protein content in the LKO mice. Of the measured transcription factors, only, the mRNA content of peroxisome proliferator-activated receptor α, showed rhythmic expression. To further analyze the difference between the control and LKO mice, principal component analysis were executed on the mRNA data. This demonstrated a clear separation of the experimental groups with respect to ZT and genotype. Our finding provides novel insight into the role of hepatic PGC-1α for basic and circadian expression of Cyps in mouse liver. This is important for our understanding of the molecular events behind circadian Cyp regulation and hence circadian regulation of hepatic detoxification capacity.

Circadian rhythm exerts its influence on animal physiology and behavior by regulating gene expression at various levels. Here we systematically explored circadian long non-coding RNAs (lncRNAs) in mouse liver and examined their circadian regulation. We found that a significant proportion of circadian lncRNAs are expressed at enhancer regions, mostly bound by two key circadian transcription factors, BMAL1 and REV-ERBα. These circadian lncRNAs showed similar circadian phases with their nearby genes. The extent of their nuclear localization is higher than protein coding genes but less than enhancer RNAs. The association between enhancer and circadian lncRNAs is also observed in tissues other than liver. Comparative analysis between mouse and rat circadian liver transcriptomes showed that circadian transcription at lncRNA loci tends to be conserved despite of low sequence conservation of lncRNAs. One such circadian lncRNA termed lnc-Crot led us to identify a super-enhancer region interacting with a cluster of genes involved in circadian regulation of metabolism through long-range interactions. Further experiments showed that lnc-Crot locus has enhancer function independent of lnc-Crot's transcription. Our results suggest that the enhancer-associated circadian lncRNAs mark the genomic loci modulating long-range circadian gene regulation and shed new lights on the evolutionary origin of lncRNAs.

Emerging evidence suggests that tumors produce molecular signals to subvert and perturb circadian-regulated immune system functions. Elucidating the mechanisms of tumor-mediated circadian disruption will promote the development of new therapies targeting tumor remote regulatory abilities. Claudins, the structural backbone of tight junctions (TJs), play a pivotal role in tumor progression, metastasis, stemness, chemotherapy resistance, and crosstalk with signaling pathways in breast cancer, making them potential biomarkers for detection, diagnosis, and treatment. To explore how tumors remotely govern immune responses across tissues in a circadian manner in triple-negative breast cancer (TNBC), we generate a diurnal transcriptome in high (4T1) and low (E0771) claudin TNBC mouse models. We generated 528 transcriptomes from liver, spleen, thymus, bone marrow, PBMC, adjacent fat pad, distal fat pad, and control mammary fat pad, at four time points within a 24-hour cycle to understand tissue-wide and cell-type-specific responses to tumor stress. We found that tumor stress impacts gene expression across tissues. Key pathways are dysregulated in response to tumor stress irrespective of claudin levels, with upregulation in immune response and cell cycle regulation, and downregulation in metabolic pathways (DNA, lipid, monocarboxylic acid, and amino acid metabolism) across multiple tissues and tumor models. Rhythmicity analysis revealed that tumor stress-responsive tissues such as the spleen in 4T1 models and the thymus in E0771 models, exhibited shifts in circadian gene expression in tumor-bearing groups, with an increase in circadian-enhanced genes during the night. Our results suggest that tumor stress alters circadian gene regulation across tissues, triggering remote, cross-tissue interactions that disrupt tissue-specific functions in a coordinated, circadian-dependent manner. Citation Format: Chuqian Liang, Zunpeng Liu, Tao Zhang, Qi Zhang, Bingqiu Xiu, Gongwei Wu, Stuart Benjamin Fass, Li-Lun Ho, Manolis Kellis, Myles A. Brown. Decoding systemic circadian regulation across multi-tissues in response to tumor stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB083.

Novel insights into the circadian modulation of lipid metabolism in chicken livers revealed by RNA sequencing and weighted gene co-expression network analysis

Background The prevalence of UC in older individuals (i.e., aged &amp;gt; 60 years) is increasing globally because of an ageing population and a subset of population with elderly-onset (i.e., onset aged ≥ 60 years) UC, which causes high burden on healthcare. Circadian rhythms are 24-h oscillations that control various biological processes in the living system and have been recognized to be associated with the development of autoimmune diseases and aging. However, the implication of circadian rhythm in elderly UC remains largely unknown. Methods We downloaded datasets from the Gene Expression Omnibus (GEO) database. The differential expression of circadian regulators between early adult (15-25 years old) and elderly (≥ 60 years old) UC patients was identified. We constructed and compared random forest (RF) and support vector machine (SVM) models. Feature genes were selected to develop a nomogram model with the superior model. The receiver operator characteristic (ROC) curves were used to validate the performances of predictive models and signature genes. The infiltration of 22 kinds of immune cells in elderly UC was analyzed by ssGSEA, and the relationship between feature circadian genes and the proportion of immune infiltration was studied. We identified circadian subtypes based on significantly differentially expressed circadian regulatory genes. Comprehensive evaluation of the two circadian patterns was performed, including immune characteristics and potential functions. Results The expression levels of circadian regulatory genes were extracted and a total of 44 differentially expressed circadian regulators were identified between early adult and elderly UC patients. Five feature circadian regulatory genes, AGT, EGF, NR3C2, PPY, and PYY were determined (p &amp;lt; 0.05) to establish a nomogram model that can predict the incidence of elderly UC with the RF model. The signature circadian regulators were found to be correlated with activated memory CD4+ T cells, gamma delta T cells, and macrophages. We identified two circadian subtypes based on the five significant circadian regulatory genes. Cluster A had a higher immune infiltration of activated CD4+T cells, CD8+ T cells and B cells than cluster B (p &amp;lt; 0.01). The signature circadian genes can also be used to predict the risk of elderly UC and have good diagnostic efficacy. Conclusion The circadian regulatory genes play non-negligible roles in elderly UC. A nomogram model developed by five feature circadian regulatory genes could be used to predict the incidence of elderly UC. Two circadian patterns were identified and evaluated comprehensively, which may provide insights into the classification of elderly UC patients and guide treatment.

The rotation of the Earth around its axis generates 24 h cycles of environmental change, such as daily rhythms of light and temperature. Circadian clocks, cellular biological oscillators that generate 24 h rhythms of gene expression and metabolism, are thought to synchronize the functioning of organisms with these daily environmental changes. Circadian regulation enables organisms to anticipate environmental changes such as dawn and dusk and co-ordinate their metabolism, physiology and behaviour with daily changes in the environment. This is particularly important for plants, which cannot move to escape environmental challenges. In the experimental model Arabidopsis thaliana (Arabidopsis), correct circadian regulation increases photosynthesis, biomass accumulation, survival, seed number and viability (Green, Tingay, Wang, & Tobin 2002; Dodd et al. 2005). It is estimated that almost 90% of Arabidopsis transcripts can oscillate in abundance over the 24 h cycle, with about 30% of transcripts being circadian-regulated (Michael et al. 2008). This multitude of genes under circadian control highlights the pervasiveness of circadian regulation in co-ordinating the functioning of plants with their rhythmic environment. Because photosynthetic light harvesting can only occur during the day and stored carbohydrate reserves require mobilization at night to supply respiration and growth, plant metabolism is intimately associated with cycles of day and night. Building upon extensive underpinning research into the molecular genetics of circadian oscillators, the interactions between metabolism, signalling and circadian regulation have become an important growth area in plant circadian biology. For example, breakthroughs have demonstrated that the rate of nocturnal starch breakdown is intricately timed so that plants do not starve at night (Graf, Schlereth, Stitt, & Smith 2010), sugars produced by photosynthesis can entrain the circadian clock (Haydon, Mielczarek, Robertson, Hubbard, & Webb 2013) and the concentrations of ions such as Ca2+ and Mg2+ are regulated by, and can regulate, the circadian oscillator (Dodd et al. 2007; Feeney et al. 2016). In this issue of Plant, Cell & Environment, Shin et al. 2017 identified another potential connection between metabolism and circadian regulation. The authors established that an energy-sensing protein complex can influence circadian rhythms. AKIN10 (known also as KIN10 or SnRK1.1) is a catalytic α-subunit of Snf1 (sucrose non-fermenting1)-related kinase 1 (SnRK1), which is an evolutionarily conserved energy sensor. SnRK1 controls metabolic enzymes through protein phosphorylation (Sugden, Donaghy, Halford, & Hardie 1999) and also regulates >1000 transcripts in response to starvation by controlling transcription factor activity (Baena-González, Rolland, Thevelein, & Sheen 2007; Mair et al. 2015). SnRK1 plays such a fundamental role in energy metabolism that AKIN10 knockouts are lethal (Baena-González et al. 2007). By overexpressing AKIN10 with a chemically inducible promoter, the authors explored the role of AKIN10 in circadian regulation. They found that inducing very high levels of AKIN10 expression caused the circadian clock to assume a long period, of up to 5 h longer than controls, when plants were under conditions of continuous light. Interestingly, the long circadian period caused by AKIN10 overexpression disappeared in experiments performed under continuous darkness, such that AKIN10 overexpressing plants had the same circadian period as the controls. When AKIN10 overexpressors were in constant darkness, supplementing the growth media with sugars did not restore the long circadian period that occurred in the light. The authors interpret this to indicate that starvation does not cause the insensitivity of circadian period to AKIN10 overexpression in darkness. Instead, Shin et al. (2017) propose that the influence of AKIN10 upon circadian period forms a response to the light environment. The study also found that under both light/dark cycles and constant light, AKIN10 overexpression caused a delay in the peak of expression of transcripts encoding the evening-expressed circadian oscillator component GIGANTEA (GI). This is interesting because gi-11 mutants are insensitive to a long-term effect of sucrose upon the circadian oscillator (Dalchau et al. 2011). Additionally, the authors found that the period of plants harbouring the tic-2 mutation in the circadian oscillator gene TIME FOR COFFEE (TIC) had reduced sensitivity to the effects of AKIN10 overexpression, suggesting a role for TIC in the regulation of circadian period by AKIN10. It is intriguing that AKIN10, a key player in the regulation of energy metabolism of Arabidopsis, can influence circadian rhythms. The work of Shin et al. (2017) builds on studies demonstrating bidirectional regulatory interactions between circadian regulation and metabolism (Fig. 1). For example, the environmental cycles of day and night dictate when photosynthesis can occur, and photosynthesis is also regulated extensively by the circadian oscillator (Dodd, Kusakina, Hall, Gould, & Hanaoka 2014). Importantly, the products of photosynthesis can, in turn, entrain the circadian oscillator (Haydon et al. 2013). Each morning, the up-regulation of photosynthesis causes an accumulation of sugars, which alters circadian oscillator gene expression and can adjust the circadian phase (Haydon et al. 2013). Similarly, the circadian oscillator controls the rate of nocturnal starch consumption (Graf et al. 2010), with one mathematical model for the regulation of nocturnal starch degradation assuming the presence of a sugar sensing mechanism (Feugier & Satake 2013). In this way, the environment affects metabolism, metabolism regulates the circadian oscillator and the circadian oscillator regulates metabolism (Fig. 1). By demonstrating that a subunit of the central energy sensor SnRK1 affects the functioning of the circadian oscillator, Shin et al. (2017) have identified a mechanism that has the potential to couple metabolism with circadian regulation. This adds to the evidence that reciprocal regulation between the circadian oscillator and energy metabolism exists across several Kingdoms of life. For example, in mammals, there are circadian rhythms of NAD+ and ATP synthesis and feeding can reset the circadian oscillator, and in both plants and cyanobacteria, the availability of energy can regulate circadian rhythms (Rust, Golden, & O'Shea 2011; Bass 2012; Haydon et al. 2013). Therefore, the long circadian period caused by AKIN10 overexpression (Shin et al. 2017) could point to a role for AKIN10 in interfacing the circadian oscillator with both metabolism and environment, given the extensive influence of environmental conditions upon the metabolic state of plants. In future, it will be informative to determine the function and position of SnRK1 within the circadian system, to understand how a sensor of cellular energy status contributes to the responses of plants to the daily changes that occur in the environment.