CRY1-CBS binding regulates circadian clock function and metabolism.

Circadian disruption influences metabolic health. Metabolism modulates circadian function. However, the mechanisms coupling circadian rhythms and metabolism remain poorly understood. Here, we report that cystathionine ß-synthase (CBS), a central enzyme in one-carbon metabolism, functionally interacts with the core circadian protein cryptochrome 1 (CRY1). In cells, CBS augments CRY1-mediated repression of the CLOCK/BMAL1 complex and shortens circadian period. Notably, we find that mutant CBS-I278T protein, the most common cause of homocystinuria, does not bind CRY1 or regulate its repressor activity. Transgenic CbsZn/Zn  mice, while maintaining circadian locomotor activity period, exhibit reduced circadian power and increased expression of E-BOX outputs. CBS function is reciprocally influenced by CRY1 binding. CRY1 modulates enzymatic activity of the CBS. Liver extracts from Cry1-/- mice show reduced CBS activity that normalizes after the addition of exogenous wild-type (WT) CRY1. Metabolomic analysis of WT, CbsZn/Zn , Cry1-/- , and Cry2-/- samples highlights the metabolic importance of endogenous CRY1. We observed temporal variation in one-carbon and transsulfuration pathways attributable to CRY1-induced CBS activation. CBS-CRY1 binding provides a post-translational switch to modulate cellular circadian physiology and metabolic control.

An in-depth neurobehavioral characterization shows anxiety-like traits, impaired habituation behavior, and restlessness in male Cryptochrome-deficient mice.

Many psychiatric disorders, for example, anxiety, are accompanied by disturbances of circadian rhythms, including disturbed sleep/wake cycles, changes in locomotor activity, and abnormal endocrine function. Conversely, alternations of circadian rhythms are a risk factor for the development of psychiatric disorders. This assumption is supported by animals with clock gene mutations which often display behaviors that resemble human psychiatric disorders. In this study, we performed an in-depth behavioral analysis with male mice lacking the central clock genes Cryptochrome 1 and 2 (Cry1/2-/- ), which are thus unable to express endogenous circadian rhythms. With wild-type and Cry1/2-/- mice, we performed an extensive behavioral analysis to study their cognitive abilities, social behavior, and their expression of depression-like and anxiety-like behavior. While Cry1/2-/- mice showed only mild abnormalities at cognitive and social behavioral levels, they were consistently more anxious than wildtype mice. Anxiety-like behavior was particularly evident in reduced mobility in new environments, altered ability to habituate, compensatory behavior, and consistent restless behavior across many behavioral tests. In line with their anxiety-like behavioral phenotype, Cry1/2-/- mice have higher c-Fos activity in the amygdala after exposure to an anxiogenic stressor than wild-type mice. In our study, we identified Cry1/2-/- mice as animals that qualify as a translational mouse model for anxiety disorder in humans because of its consistent behavior of restlessness, increased immobility, and dysfunctional habituation in new environments.

Cryptochrome deficiency enhances transcription but reduces protein levels of pineal Aanat

Cryptochrome (Cry) 1 and 2 are essential for circadian rhythm generation, not only in the suprachiasmatic nucleus, the site of the mammalian master circadian clock, but also in peripheral organs throughout the body. CRY is also known as a repressor of arylalkylamine-N-acetyltransferase (Aanat) transcription; therefore, Cry deficiency is expected to induce constantly high pineal melatonin content. Nevertheless, we previously found that the content was consistently low in melatonin-proficient Cry1 and Cry2 double-deficient mice (Cry1−/−/Cry2−/−) on C3H background. This study aims to clarify the mechanism underlying this discrepancy. In the Cry1−/−/Cry2−/− pineal, expression levels of Aanat and clock gene Per1 were consistently high with no circadian fluctuation on the first day in constant darkness, demonstrating that CRY acts in vivo as a repressor of the pineal circadian clock and AANAT. In contrast, the enzyme activity and protein levels of AANAT remained low throughout the day, supporting our previous observation of continuously low melatonin. Thus, effects of Cry deficiency on the responses of ß-adrenergic receptors were examined in cultured pineal glands. Isoproterenol, a ß-adrenergic stimulant, significantly increased melatonin content, although the increase was smaller in Cry1−/−/Cry2−/− than in WT mice, during both the day and night. However, the increase in cAMP in response to forskolin was similar in both genotypes, indicating that CRY deficiency does not affect the pathway downstream of the ß-adrenergic receptor. These results suggest that a lack of circadian adrenergic input due to CRY deficiency decreases ß-receptor activity and cAMP levels, resulting in consistently low AANAT levels despite abundant Aanat mRNA.

Long-term in vivo recording of circadian rhythms in brains of freely moving mice.

Endogenous circadian clocks control 24-h physiological and behavioral rhythms in mammals. Here, we report a real-time in vivo fluorescence recording system that enables long-term monitoring of circadian rhythms in the brains of freely moving mice. With a designed reporter of circadian clock gene expression, we tracked robust Cry1 transcription reporter rhythms in the suprachiasmatic nucleus (SCN) of WT, Cry1-/- , and Cry2-/- mice in LD (12 h light, 12 h dark) and DD (constant darkness) conditions and verified that signals remained stable for over 6 mo. Further, we recorded Cry1 transcriptional rhythms in the subparaventricular zone (SPZ) and hippocampal CA1/2 regions of WT mice housed under LD and DD conditions. By using a Cre-loxP system, we recorded Per2 and Cry1 transcription rhythms specifically in vasoactive intestinal peptide (VIP) neurons of the SCN. Finally, we demonstrated the dynamics of Per2 and Cry1 transcriptional rhythms in SCN VIP neurons following an 8-h phase advance in the light/dark cycle.

Mouse Models of Primary Aldosteronism: From Physiology to Pathophysiology.

Primary aldosteronism (PA) is a common form of endocrine hypertension that is characterized by the excessive production of aldosterone relative to suppressed plasma renin levels. PA is usually caused by either a unilateral aldosterone-producing adenoma or bilateral adrenal hyperplasia. Somatic mutations have been identified in several genes that encode ion pumps and channels that may explain the aldosterone excess in over half of aldosterone-producing adenomas, whereas the pathophysiology of bilateral adrenal hyperplasia is largely unknown. A number of mouse models of hyperaldosteronism have been described that recreate some features of the human disorder, although none replicate the genetic basis of human PA. Animal models that reproduce the genotype-phenotype associations of human PA are required to establish the functional mechanisms that underlie the endocrine autonomy and deregulated cell growth of the affected adrenal and for preclinical studies of novel therapeutics. Herein, we discuss the differences in adrenal physiology across species and describe the genetically modified mouse models of PA that have been developed to date.

Different Roles of Negative and Positive Components of the Circadian Clock in Oncogene-induced Neoplastic Transformation.

In mammals, circadian rhythms in physiological function are generated by a molecular oscillator driven by transcriptional-translational feedback loop consisting of negative and positive regulators. Disruption of this circadian clock machinery is thought to increase the risk of cancer development, but the potential contributions of each component of circadian clock to oncogenesis have been little explored. Here we reported that negative and positive transcriptional regulators of circadian feedback loop had different roles in oncogene-induced neoplastic transformation. Mouse embryonic fibroblasts prepared from animals deficient in negative circadian clock regulators, Period2 (Per2) or Cryptochrome1/2 (Cry1/2), were prone to transformation induced by co-expression of H-ras(V12) and SV40 large T antigen (SV40LT). In contrast, mouse embryonic fibroblasts prepared from mice deficient in positive circadian clock regulators, Bmal1 or Clock, showed resistance to oncogene-induced transformation. In Per2 mutant and Cry1/2-null cells, the introduction of oncogenes induced expression of ATF4, a potent repressor of cell senescence-associated proteins p16INK4a and p19ARF. Elevated levels of ATF4 were sufficient to suppress expression of these proteins and drive oncogenic transformation. Conversely, in Bmal1-null and Clock mutant cells, the expression of ATF4 was not induced by oncogene introduction, which allowed constitutive expression of p16INK4a and p19ARF triggering cellular senescence. Although genetic ablation of either negative or positive transcriptional regulators of the circadian clock leads to disrupted rhythms in physiological functions, our findings define their different contributions to neoplastic cellular transformation.

Circadian rhythm of RNA N6-methyladenosine and the role of cryptochrome.

Methylation of RNA N(6)-methyladenosine has fundamental cellular functions, including translation regulation, RNA export, and stem cells renewal. However, the regulation of RNA N(6)-methyladenosine methylation is poorly understood. Here, we observed a robust circadian rhythm in N(6)-methyladenosine modifications of RNA. Deficiency of core mammalian clock genes, cryptochromes, decreased the levels of N(6)-methyladenosine in RNA. Cryptochrome1/2 knockout mice had significantly lower N(6)-methyladenosine methylation of RNA and lost the circadian rhythm of N(6)-methyladenosine levels in RNA. Global analysis of the circadian methylomes of N(6)-methyladenosine in RNA revealed that gene transcription, translation regulation, and RNA metabolism were highly correlated with N(6)-methyladenosine oscillation. Our findings extended a fundamental link between the circadian rhythm and N(6)-methyladenosine modification of RNA and suggested that this link is critical in controlling post-transcriptional gene expression and RNA metabolism.

4-Hydroxychalcone attenuates hyperaldosteronism, inflammation, and renal injury in cryptochrome-null mice.

In the present study, we aimed to investigate the preventive effects of 4-hydroxychalcone (4HCH) on resistant hypertension. We used cryptochrome-null mice, which characteristically show high plasma aldosterone levels, inflammation, and renal injury. The cryptochrome-null mice received high-salt treatment and were treated orally with 4HCH 10 mg/kg, 4HCH 20 mg/kg, and 4HCH 40 mg/kg, respectively. The salt administration in cryptochrome-null mice is able to induce an increase in systolic pressure which is associated with hyperaldosteronism, inflammation, and kidney injury. Treatment with 40 mg/kg 4HCH reduced systolic hypertension, serum IL-1ß, and TNF-α levels and suppressed the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and renal injury. The impact of 4HCH on the hyperaldosteronism, inflammation, and kidney injury provides new insights for future development of therapeutic strategies in resistant hypertension.

Opposing actions of Per1 and Cry2 in the regulation of Per1 target gene expression in the liver and kidney.

Mounting evidence suggests that the circadian clock plays an integral role in the regulation of many physiological processes including blood pressure, renal function, and metabolism. The canonical molecular clock functions via activation of circadian target genes by Clock/Bmal1 and repression of Clock/Bmal1 activity by Per1-3 and Cry1/2. However, we have previously shown that Per1 activates genes important for renal sodium reabsorption, which contradicts the canonical role of Per1 as a repressor. Moreover, Per1 knockout (KO) mice exhibit a lowered blood pressure and heavier body weight phenotype similar to Clock KO mice, and opposite that of Cry1/2 KO mice. Recent work has highlighted the potential role of Per1 in repression of Cry2. Therefore, we postulated that Per1 potentially activates target genes through a Cry2-Clock/Bmal1-dependent mechanism, in which Per1 antagonizes Cry2, preventing its repression of Clock/Bmal1. This hypothesis was tested in vitro and in vivo. The Per1 target genes αENaC and Fxyd5 were identified as Clock targets in mpkCCDc14 cells, a model of the renal cortical collecting duct. We identified PPARα and DEC1 as novel Per1 targets in the mouse hepatocyte cell line, AML12, and in the liver in vivo. Per1 knockdown resulted in upregulation of Cry2 in vitro, and this result was confirmed in vivo in mice with reduced expression of Per1. Importantly, siRNA-mediated knockdown of Cry2 and Per1 demonstrated opposing actions for Cry2 and Per1 on Per1 target genes, supporting the potential Cry2-Clock/Bmal1-dependent mechanism underlying Per1 action in the liver and kidney.

Histone methyltransferase MLL3 contributes to genome-scale circadian transcription.

Daily cyclical expression of thousands of genes in tissues such as the liver is orchestrated by the molecular circadian clock, the disruption of which is implicated in metabolic disorders and cancer. Although we understand much about the circadian transcription factors that can switch gene expression on and off, it is still unclear how global changes in rhythmic transcription are controlled at the genomic level. Here, we demonstrate circadian modification of an activating histone mark at a significant proportion of gene loci that undergo daily transcription, implicating widespread epigenetic modification as a key node regulated by the clockwork. Furthermore, we identify the histone-remodelling enzyme mixed lineage leukemia (MLL)3 as a clock-controlled factor that is able to directly and indirectly modulate over a hundred epigenetically targeted circadian "output" genes in the liver. Importantly, catalytic inactivation of the histone methyltransferase activity of MLL3 also severely compromises the oscillation of "core" clock gene promoters, including Bmal1, mCry1, mPer2, and Rev-erbα, suggesting that rhythmic histone methylation is vital for robust transcriptional oscillator function. This highlights a pathway by which the clockwork exerts genome-wide control over transcription, which is critical for sustaining temporal programming of tissue physiology.

A diversity of paracrine signals sustains molecular circadian cycling in suprachiasmatic nucleus circuits.

The suprachiasmatic nucleus (SCN) is the principal circadian pacemaker of mammals, coordinating daily rhythms of behavior and metabolism. Circadian timekeeping in SCN neurons revolves around transcriptional/posttranslational feedback loops, in which Period (Per) and Cryptochrome (Cry) genes are negatively regulated by their protein products. Recent studies have revealed, however, that these "core loops" also rely upon cytosolic and circuit-level properties for sustained oscillation. To characterize interneuronal signals responsible for robust pacemaking in SCN cells and circuits, we have developed a unique coculture technique using wild-type (WT) "graft" SCN to drive pacemaking (reported by PER2::LUCIFERASE bioluminescence) in "host" SCN deficient either in elements of neuropeptidergic signaling or in elements of the core feedback loop. We demonstrate that paracrine signaling is sufficient to restore cellular synchrony and amplitude of pacemaking in SCN circuits lacking vasoactive intestinal peptide (VIP). By using grafts with mutant circadian periods we show that pacemaking in the host SCN is specified by the genotype of the graft, confirming graft-derived factors as determinants of the host rhythm. By combining pharmacological with genetic manipulations, we show that a hierarchy of neuropeptidergic signals underpins this paracrine regulation, with a preeminent role for VIP augmented by contributions from arginine vasopressin (AVP) and gastrin-releasing peptide (GRP). Finally, we show that interneuronal signaling is sufficiently powerful to maintain circadian pacemaking in arrhythmic Cry-null SCN, deficient in essential elements of the transcriptional negative feedback loops. Thus, a hierarchy of paracrine neuropeptidergic signals determines cell- and circuit-level circadian pacemaking in the SCN.

Mammalian clock gene Cryptochrome regulates arthritis via proinflammatory cytokine TNF-alpha.

The mammalian clock genes, Period and Cryptochrome (Cry), regulate circadian rhythm. We show that circadian rhythmicity and rhythmic expression of Period in the nuclei of inflammatory synovial cells and spleen cells are disturbed in mouse models of experimental arthritis. Expressions of other clock genes, Bmal1 and Dbp, are also disturbed in spleen cells by arthritis induction. Deletion of Cry1 and Cry2 results in an increase in the number of activated CD3(+) CD69(+) T cells and a higher production of TNF-alpha from spleen cells. When arthritis is induced, Cry1(-/-)Cry2(-/-) mice develop maximal exacerbation of joint swelling, and upregulation of essential mediators of arthritis, including TNF-alpha, IL-1beta and IL-6, and matrix metalloproteinase-3. Wee-1 kinase is solely upregulated in Cry1(-/-)Cry2(-/-) mice, in line with upregulation of c-Fos and Wee-1 kinase in human rheumatoid arthritis. The treatment with anti-TNF-alpha Ab significantly reduced the severity and halted the progression of the arthritis of Cry1(-/-)Cry2(-/-) mice and vice versa, ectopic expression of Cry1 in the mouse embryonic fibroblast from Cry1(-/-)Cry2(-/-) mice significantly reduced the trans activation of TNF-alpha gene. Thus, the biological clock and arthritis influence each other, and this interplay can influence human health and disease.