Lint-O cooperates with L(3)mbt in target gene suppression to maintain homeostasis in fly ovary and brain.

Loss-of-function mutations in Drosophila lethal(3)malignant brain tumor [l(3)mbt] cause ectopic expression of germline genes and brain tumors. Loss of L(3)mbt function in ovarian somatic cells (OSCs) aberrantly activates germ-specific piRNA amplification and leads to infertility. However, the underlying mechanism remains unclear. Here, ChIP-seq for L(3)mbt in cultured OSCs and RNA-seq before and after L(3)mbt depletion shows that L(3)mbt genomic binding is not necessarily linked to gene regulation and that L(3)mbt controls piRNA pathway genes in multiple ways. Lack of known L(3)mbt co-repressors, such as Lint-1, has little effect on the levels of piRNA amplifiers. Identification of L(3)mbt interactors in OSCs and subsequent analysis reveals CG2662 as a novel co-regulator of L(3)mbt, termed "L(3)mbt interactor in OSCs" (Lint-O). Most of the L(3)mbt-bound piRNA amplifier genes are also bound by Lint-O in a similar fashion. Loss of Lint-O impacts the levels of piRNA amplifiers, similar to the lack of L(3)mbt. The lint-O-deficient flies exhibit female sterility and tumorous brains. Thus, L(3)mbt and its novel co-suppressor Lint-O cooperate in suppressing target genes to maintain homeostasis in the ovary and brain.

mTOR-AKT Signaling in Cellular Clock Resetting Triggered by Osmotic Stress.

Aims: The circadian clock oscillates in a cell-autonomous manner with a period of ∼24 h, and the phase is regulated by various time cues such as light and temperature through multiple clock input pathways. We previously found that osmotic and oxidative stress strongly affected the circadian period and phase of cellular rhythms, and triple knockout of apoptosis signal-regulating kinase (ASK) family members, Ask1, Ask2, and Ask3, abolished the phase shift (clock resetting) induced by hyperosmotic pulse treatment. We aimed at exploring a key molecule(s) and signaling events in the clock input pathway dependent on ASK kinases. Results: The phase shift of the cellular clock induced by the hyperosmotic pulse treatment was significantly reduced by combined deficiencies of the clock(-related) genes, Dec1, Dec2, and E4 promoter-binding protein 4 (also known as Nfil3) (E4bp4). In addition, liquid chromatography mass/mass spectrometry (LC-MS/MS)-based proteomic analysis identified hyperosmotic pulse-induced phosphorylation of circadian locomotor output cycles caput (CLOCK) Ser845 in an AKT-dependent manner. We found that AKT kinase was phosphorylated at Ser473 (i.e., activated) in response to the hyperosmotic pulse experiments. Inhibition of mechanistic target of rapamycin (mTOR) kinase by Torin 1 treatment completely abolished the AKT activation, suppressed the phosphorylation of CLOCK Ser845, and blocked the clock resetting induced by the hyperosmotic pulse treatment. Innovation and Conclusions: We conclude that mTOR-AKT signaling is indispensable for the CLOCK Ser845 phosphorylation, which correlates with the clock resetting induced by the hyperosmotic pulse treatment. Immediate early induction of the clock(-related) genes and CLOCK carboxyl-terminal (C-terminal) region containing Ser845 also play important roles in the clock input pathway through redox-sensitive ASK kinases. Antioxid. Redox Signal. 37, 631-646.

Post-transcriptional repression of circadian component CLOCK regulates cancer-stemness in murine breast cancer cells.

Disruption of the circadian clock machinery in cancer cells is implicated in tumor malignancy. Studies on cancer therapy reveal the presence of heterogeneous cells, including breast cancer stem-like cells (BCSCs), in breast tumors. BCSCs are often characterized by high aldehyde dehydrogenase (ALDH) activity, associated with the malignancy of cancers. In this study, we demonstrated the negative regulation of ALDH activity by the major circadian component CLOCK in murine breast cancer 4T1 cells. The expression of CLOCK was repressed in high-ALDH-activity 4T1, and enhancement of CLOCK expression abrogated their stemness properties, such as tumorigenicity and invasive potential. Furthermore, reduced expression of CLOCK in high-ALDH-activity 4T1 was post-transcriptionally regulated by microRNA: miR-182. Knockout of miR-182 restored the expression of CLOCK, resulted in preventing tumor growth. Our findings suggest that increased expression of CLOCK in BCSCs by targeting post-transcriptional regulation overcame stemness-related malignancy and may be a novel strategy for breast cancer treatments.

A-to-I RNA editing enzyme ADAR2 regulates light-induced circadian phase-shift.

In mammals, the central circadian clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus and it orchestrates peripheral clocks in the whole body to organize physiological and behavioral rhythms. Light-induced phase-shift of the SCN clock enables synchronization of the circadian clock system with 24-h environmental light/dark cycle. We previously found that adenosine deaminase acting on RNA 2 (Adar2), an A-to-I RNA editing enzyme catalyzing rhythmic A-to-I RNA editing, governs a wide range of mRNA rhythms in the mouse liver and regulates the circadian behavior. In brain, ADAR2-mediated A-to-I RNA editing was reported to occur in various transcripts encoding ion channels and neurotransmitter receptors, which could influence neuronal function of the SCN. Here we show that ADAR2 plays a crucial role for light-induced phase-shift of the circadian clock. Intriguingly, exposure of Adar2-knockout mice to a light pulse at late night caused an aberrant phase-advance of the locomotor rhythms. By monitoring the bioluminescence rhythms of the mutant SCN slices, we found that a phase-advance induced by treatment with pituitary adenylyl cyclase-activating polypeptide (PACAP) was markedly attenuated. The present study suggests that A-to-I RNA editing in the SCN regulates a proper phase response to light in the mouse circadian system.

Stereotaxic Surgery for Suprachiasmatic Nucleus Lesions in Mice.

Site-specific lesions are invaluable methods for investigating the function of brain regions within the central nervous system and can be used to study neural mechanisms of behaviors. Precise stereotaxic surgery is required to lesion small regions of the brain such as the suprachiasmatic nucleus (SCN), which harbors the master circadian clock. In this protocol, we describe stereotaxic surgery optimized for bilateral lesion of the mouse SCN by loading electric current. Success of the SCN lesion is verified histologically and behaviorally by monitoring arrhythmic locomotor activity. The SCN-lesioned mouse allows for the evaluation of behavioral, biochemical, and physiological consequences of ablation of the master circadian clock.

ADARB1 catalyzes circadian A-to-I editing and regulates RNA rhythm.

It has been proposed that the CLOCK-ARNTL (BMAL1) complex drives circadian transcription of thousands of genes, including Per and Cry family genes that encode suppressors of CLOCK-ARNTL-dependent transcription. However, recent studies demonstrated that 70-80% of circadian-oscillating mRNAs have no obvious rhythms in their de novo transcription, indicating the potential importance of post-transcriptional regulation. Our CLOCK-ChIP-seq analysis identified rhythmic expression of adenosine deaminase, RNA-specific, B1 (Adarb1, also known as Adar2), an adenosine-to-inosine (A-to-I) RNA-editing enzyme. RNA-seq showed circadian rhythms of ADARB1-mediated A-to-I editing in a variety of transcripts. In Adarb1-knockout mice, rhythms of large populations of mRNA were attenuated, indicating a profound impact of ADARB1-mediated A-to-I editing on RNA rhythms. Furthermore, Adarb1-knockout mice exhibited short-period rhythms in locomotor activity and gene expression. These phenotypes were associated with abnormal accumulation of CRY2. The present study identifies A-to-I RNA editing as a key mechanism of post-transcriptional regulation in the circadian clockwork.

The E3 ubiquitin ligase UBE3A is an integral component of the molecular circadian clock through regulating the BMAL1 transcription factor.

Post-translational modifications (such as ubiquitination) of clock proteins are critical in maintaining the precision and robustness of the evolutionarily conserved circadian clock. Ubiquitination of the core clock transcription factor BMAL1 (brain and muscle Arnt-like 1) has recently been reported. However, it remains unknown whether BMAL1 ubiquitination affects circadian pacemaking and what ubiquitin ligase(s) is involved. Here, we show that activating UBE3A (by expressing viral oncogenes E6/E7) disrupts circadian oscillations in mouse embryonic fibroblasts, measured using PER2::Luc dynamics, and rhythms in endogenous messenger ribonucleic acid and protein levels of BMAL1. Over-expression of E6/E7 reduced the level of BMAL1, increasing its ubiquitination and proteasomal degradation. UBE3A could bind to and degrade BMAL1 in a ubiquitin ligase-dependent manner. This occurred both in the presence and absence of E6/E7. We provide in vitro (knockdown/over-expression in mammalian cells) and in vivo (genetic manipulation in Drosophila) evidence for an endogenous role of UBE3A in regulating circadian dynamics and rhythmic locomotor behaviour. Together, our data reveal an essential and conserved role of UBE3A in the regulation of the circadian system in mammals and flies and identify a novel mechanistic link between oncogene E6/E7-mediated cell transformation and circadian (BMAL1) disruption.

CLOCK-controlled polyphonic regulation of circadian rhythms through canonical and noncanonical E-boxes.

In mammalian circadian clockwork, the CLOCK-BMAL1 complex binds to DNA enhancers of target genes and drives circadian oscillation of transcription. Here we identified 7,978 CLOCK-binding sites in mouse liver by chromatin immunoprecipitation-sequencing (ChIP-Seq), and a newly developed bioinformatics method, motif centrality analysis of ChIP-Seq (MOCCS), revealed a genome-wide distribution of previously unappreciated noncanonical E-boxes targeted by CLOCK. In vitro promoter assays showed that CACGNG, CACGTT, and CATG(T/C)G are functional CLOCK-binding motifs. Furthermore, we extensively revealed rhythmically expressed genes by poly(A)-tailed RNA-Seq and identified 1,629 CLOCK target genes within 11,926 genes expressed in the liver. Our analysis also revealed rhythmically expressed genes that have no apparent CLOCK-binding site, indicating the importance of indirect transcriptional and posttranscriptional regulations. Indirect transcriptional regulation is represented by rhythmic expression of CLOCK-regulated transcription factors, such as Krüppel-like factors (KLFs). Indirect posttranscriptional regulation involves rhythmic microRNAs that were identified by small-RNA-Seq. Collectively, CLOCK-dependent direct transactivation through multiple E-boxes and indirect regulations polyphonically orchestrate dynamic circadian outputs.

The circadian clock regulates rhythmic activation of the NRF2/glutathione-mediated antioxidant defense pathway to modulate pulmonary fibrosis.

The disruption of the NRF2 (nuclear factor erythroid-derived 2-like 2)/glutathione-mediated antioxidant defense pathway is a critical step in the pathogenesis of several chronic pulmonary diseases and cancer. While the mechanism of NRF2 activation upon oxidative stress has been widely investigated, little is known about the endogenous signals that regulate the NRF2 pathway in lung physiology and pathology. Here we show that an E-box-mediated circadian rhythm of NRF2 protein is essential in regulating the rhythmic expression of antioxidant genes involved in glutathione redox homeostasis in the mouse lung. Using an in vivo bleomycin-induced lung fibrosis model, we reveal a clock "gated" pulmonary response to oxidative injury, with a more severe fibrotic effect when bleomycin was applied at a circadian nadir in NRF2 levels. Timed administration of sulforaphane, an NRF2 activator, significantly blocked this phenotype. Moreover, in the lungs of the arrhythmic Clock(Δ19) mice, the levels of NRF2 and the reduced glutathione are constitutively low, associated with increased protein oxidative damage and a spontaneous fibrotic-like pulmonary phenotype. Our findings reveal a pivotal role for the circadian control of the NRF2/glutathione pathway in combating oxidative/fibrotic lung damage, which might prompt new chronotherapeutic strategies for the treatment of human lung diseases, including idiopathic pulmonary fibrosis.

Interacting targets of the farnesyl of transducin gamma-subunit.

G protein gamma-subunits are isoprenylated and carboxyl methylated at the C-terminal cysteine residue, and the set of the posttranslational modifications is indispensable for the function of the photoreceptor G protein transducin (Talpha/Tbetagamma). To explore farnesyl-mediated molecular interactions, we investigated molecular targets of a Tbetagamma analogue that was engineered to have a photoreactive farnesyl analogue, (3-azidophenoxy)geranyl (POG), covalently bound to the C-terminal cysteine of Tgamma. POG-modified Tbetagamma was further subjected to modification by methylation at the C-terminal carboxyl group, which copies a complete set of the known posttranscriptional modifications of Tbetagamma. Photoaffinity labeling experiment with the photoreactive Tbetagamma analogue in its free form indicated that the POG moiety of Tgamma interacted with Tbeta. In the trimeric Talpha/Tbetagamma complex, the POG moiety was cross-linked with Talpha in addition to concurrent affinity labeling of Tbeta. When photoreactive Tbetagamma was reconstituted with Talpha and light-activated rhodopsin (Rh*) in rod outer segment (ROS) membranes, the POG moiety interacted with not only Talpha and Tbeta but also Rh* and membrane phospholipids. The cross-linked phospholipid species was analyzed by ELISA employing a variety of lipid-binding probes, which revealed phosphatidylethanolamine (PE) and phosphatidylserine (PS) as selective phospholipids for POG interaction in the ROS membranes. These results demonstrate that the modifying group of Tgamma plays an active role in protein-protein and protein-membrane interactions and suggest that the farnesyl-PE/PS interaction may support the efficient formation of the signaling ternary complex between transducin and Rh*.

Top-down analysis of protein isoprenylation by electrospray ionization hybrid quadrupole time-of-flight tandem mass spectrometry; the mouse Tgamma protein.

Protein isoprenylation, an important post-translational modification with a lipid, involves the selective attachment of two types of isoprenoids, farnesyl (C15) and geranylgeranyl (C20). The isoprenoid is linked via a thioether bond to the C-terminal cysteine residue of a variety of cellular proteins, including the heterotrimeric G protein gamma-subunits. One member of the G protein family, transducin (Talpha/Tbetagamma), plays a central role in visual transduction, and the structure-function relationship has been extensively studied with purified proteins, predominantly with bovine transducin that was shown to be farnesylated at the C-terminal cysteine residue of the gamma-subunit (Tgamma). We report here the structure of the C-terminal modification of mouse Tgamma, which has not yet been elucidated owing to the low amount of protein that can be isolated from the mouse retina. Electrospray ionization mass spectrometry (ESI-MS) of the high-performance liquid chromatography (HPLC)-purified Tgamma was in good agreement with the calculated mass of the farnesylated and methylated form of mouse Tgamma (Pro1-Cys70). A 'top-down' analysis of intact Tgamma using an ESI hybrid quadrupole time-of-flight (TOF) tandem mass spectrometer provided isoprenyl-specific ions that were observed to produce ions separated by 204 Da from the conventional (unmodified) precursor ion or the C-terminal sequence ions. Such characteristic fragmentation on an isoprenoid observed in top-down analysis could be useful in general for determining the type of isoprenylation as well as probing the site of modification in the protein sequence.

Negative control of circadian clock regulator E4BP4 by casein kinase Iepsilon-mediated phosphorylation.

Light-dependent transcriptional regulation of clock genes is a crucial step in the entrainment of the circadian clock. E4bp4 is a light-inducible gene in the chick pineal gland, and it encodes a bZIP protein that represses transcription of cPer2, a chick pineal clock gene. Here, we demonstrate that prolonged light period-dependent accumulation of E4BP4 protein is temporally coordinated with a delay of the rising phase of cPer2 in the morning. E4BP4 was phosphorylated progressively and then disappeared in parallel with induced cPer2 expression. Characterization of E4BP4 revealed Ser182, a phosphoacceptor site located at the amino-terminal border of the Ser/Thr cluster, which forms the phosphorylation motifs for casein kinase 1epsilon (CK1epsilon). CK1epsilon physically associated with E4BP4 and phosphorylated it. CK1epsilon-catalyzed phosphorylation of E4BP4 resulted in proteasomal proteolysis-dependent decrease of E4BP4 levels, while E4BP4 nuclear accumulation was attenuated by CK1epsilon in a kinase activity-independent manner. CK1epsilon-mediated posttranslational regulation was accompanied by reduction of the transcriptional repression executed by E4BP4. These results not only demonstrate a phosphorylation-dependent regulatory mechanism for E4BP4 function but also highlight the role of CK1epsilon as a negative regulator for E4BP4-mediated repression of cPer2.

Effects of gonadal steroids on pineal morphogenesis and cell differentiation of the embryonic quail studied under cell culture conditions.

Receptors for gonadal steroid hormones have been localized in the pineal glands of several vertebrate species. No studies, however, have reported on pineal morphogenesis and cell differentiation following hormonal application in vitro during avian embryonic development. Hormonal regulation of embryonic development is crucial in all vertebrate classes. Although gonadal hormones are known to affect organogenesis in avian embryos and chicks, we wanted to investigate whether gonadal steroids (testosterone and estradiol) have any effect on the morphogenesis and cell differentiation of the avian pineal gland. The steroid hormones had a stimulatory influence on pineal morphogenesis in vitro as evidenced from the radial arrangement of colony-forming cells and the subsequent formation of a follicular-like structure under dispersed-cell culture condition. Administration of testosterone in culture medium significantly promoted the numbers of cells that were positively stained for arginine vasopressin and tyrosine hydroxylase, while estradiol showed only a slight effect. Both of the two steroid hormones significantly decreased the numbers of cells positively stained for serotonin and melatonin. Melatonin released in the culture medium decreased in content within the 24 h following steroid treatment (supported by low immunoreactivity in cultured cells and low level released to the medium). These results clearly suggest active roles of gonadal steroid hormones on embryonic pineal morphogenesis and cell differentiation and its physiological activity as they do in adult animals.

Suprachiasmatic nucleus circadian oscillatory protein, a novel binding partner of K-Ras in the membrane rafts, negatively regulates MAPK pathway.

Suprachiasmatic nucleus circadian oscillatory protein (SCOP) is a member of the leucine-rich repeat (LRR)-containing protein family. In addition to circadian expression in the rat hypothalamic suprachiasmatic nucleus, SCOP is constitutively expressed in neurons throughout the rat brain. Here we found that a substantial amount of SCOP was localized in the brain membrane rafts, in which only K-Ras was abundant among Ras isoforms. SCOP interacted directly through its LRR domain with a subset of K-Ras in the guanine nucleotide-free form that was present in the raft fraction. This interaction interfered with the binding of added guanine nucleotide to K-Ras in vitro. A negative regulatory role of SCOP for K-Ras function was examined in PC12 cell lines stably overexpressing SCOP or its deletion mutants. Overexpression of full-length SCOP markedly down-regulated ERK1/ERK2 activation induced by depolarization or phorbol ester stimulation, and this inhibitory effect of overexpressed SCOP was dependent on its LRR domain. These results strongly suggest that SCOP negatively regulates K-Ras signaling in the membrane rafts, identifying a novel mechanism for regulation of the Ras-MAPK pathway.