Project description:Circadian rhythms regulate cell proliferation and differentiation; however, little is known about their roles in myogenic differentiation. Our synchronized differentiation studies demonstrate that myoblast proliferation and subsequent myotube formation by cell fusion occur in circadian manners. We found that one of the core regulators of circadian rhythms Cry2, but not Cry1, is critical for the circadian patterns of these two critical steps in myogenic differentiation. This is achieved through the specific interaction between Cry2 and Bclaf1, which stabilizes mRNAs encoding cyclin D1, a G1/S phase transition regulator, and Tmem176b, a transmembrane regulator for myogenic cell fusion. Myoblasts lacking Cry2 display premature cell cycle exit and form short myotubes due to inefficient cell fusion. Consistently, muscle regeneration is impaired in Cry2-/- mice. Bclaf1 knockdown recapitulated the phenotypes of Cry2 knockdown: early cell cycle exit and inefficient cell fusion. This study uncovers a post-transcriptional regulation of myogenic differentiation by circadian rhythms.
Project description:Cyclin D1 is an important cell cycle regulator but in cancer its overexpression also increases cellular migration mediated by p27KIP1 stabilization and RhoA inhibition. Recently, a common polymorphism at the exon 4-intron 4 boundary of the human cyclin D1 gene within a splice donor region was associated with an altered risk of developing cancer. Altered RNA splicing caused by this polymorphism gives rise to a variant cyclin D1 isoform termed cyclin D1b, which has the same N-terminus as the canonical cyclin D1a isoform but a distinct C-terminus. Analysis was performed of mouse cyclin D1 knockout 3T3 cells infected with splice variants of cyclin D1. 3T3 cells transduced with retroviral vectors expressing each cyclin D1 isoform were processed for expression analysis. Keywords: Cancer associated risk factor
Project description:Cyclin D1 is an important cell cycle regulator but in cancer its overexpression also increases cellular migration mediated by p27KIP1 stabilization and RhoA inhibition. Recently, a common polymorphism at the exon 4-intron 4 boundary of the human cyclin D1 gene within a splice donor region was associated with an altered risk of developing cancer. Altered RNA splicing caused by this polymorphism gives rise to a variant cyclin D1 isoform termed cyclin D1b, which has the same N-terminus as the canonical cyclin D1a isoform but a distinct C-terminus. Analysis was performed of mouse cyclin D1 knockout 3T3 cells infected with splice variants of cyclin D1. 3T3 cells transduced with retroviral vectors expressing each cyclin D1 isoform were processed for expression analysis. Experiment Overall Design: Three Mouse Embryonic Fibroblasts cell lines obtained from littermate cyclin D1 knockout E14 embyos were serial passaged to obtain cyclin D1 knockout (D1KO) 3T3 cells. Each of the D1KO 3T3 cell lines were infected using the Murine Stem Cell Virus (MSCV) expressing splice variants of cyclin D1; cyclin D1a/ cyclin D1b or GFP control to give triplicate sample sets. 7 days post infection total RNA from each sample was extracted using Trizol and further purified using Qiagenâs RNeasy Kit. Preparation of biotinylated cRNA and hybridization to oligonucleotide arrays (Affymetrix mouse genome genechip 430 2.0) were performed in conjuction with Pestell lab and the Nucleic Acid Core Facility at Thomas Jefferson University. Mouse 430 2.0 genechip contains 39,000 transcripts. Gene chips were scanned and analyzed using Robust Multi-array Average (RMA) algorithm.
Project description:N6-methyladenosine (m6A) modification is the major post-transcriptional modification present in mammalian mRNA. m6A controls fundamental biological processes including cell proliferation, but the molecular mechanism remains unclear. Herein, we demonstrate that the m6A demethylase fat mass and obesity-associated (FTO) controls the cell cycle by targeting cyclin D1, the key regulator required for G1 phase progression. FTO silencing suppressed cyclin D1 expression and induced G1 arrest. FTO depletion upregulated cyclin D1 m6A modification, which in turn accelerated the degradation of cyclin D1 mRNA. Importantly, m6A modification of cyclin D1 oscillates in a cell cycle-dependent manner; m6A levels were suppressed during the G1 phase and enhanced during other phases. Low m6A levels during G1 were associated with nuclear translocation of FTO from the cytosol. Furthermore, nucleocytoplasmic shuttling of FTO is regulated by Casein Kinase II-mediated phosphorylation at Thr 150 of FTO. Our results highlight the role of m6A in regulating cyclin D1 mRNA stability, and add a new layer of complexity to cell cycle regulation.
Project description:OBJECTIVES: Abnormal chondrocyte gene expression promotes osteoarthritis (OA) pathogenesis. RNA-sequencing revealed that circadian rhythm pathway and expression of core clock protein cryptochrome 2 (Cry2) are dysregulated in human OA cartilage. Here we determined expression patterns and function Cry1 and Cry2. METHODS: Cry mRNA and protein expression was analyzed in normal and OA human and mouse cartilage. Mice with deletion of Cry1 or Cry2 were analyzed for severity of experimental OA and to determine genes and pathways that are regulated by CRY. RESULTS: In human OA cartilage, CRY2 but not CRY1 staining and mRNA expression was significantly decreased. Cry2 was also suppressed in mice with surgical or aging-related OA. Cry2 KO but not Cry1 KO mice with experimental OA showed significantly increased severity of histopathological changes in cartilage, subchondral bone and synovium. In OA chondrocytes, the levels of Cry1 and Cry2 and the amplitude of circadian fluctuation were significantly lower. RNA-seq on knee articular cartilage of wild-type and Cry2 KO mice identified 53 differentially expressed genes, including known CRY2 target circadian genes Nr1d1, Nr1d2, Dbp and Tef. Pathway analysis indicated that circadian rhythm and extracellular matrix remodeling were dysregulated in Cry2 KO mice. CONCLUSIONS: These results show an active role of the circadian clock in general, and of CRY2 in particular, in maintaining ECM homeostasis in cartilage. This cell autonomous network of circadian rhythm genes is disrupted in OA chondrocytes. Targeting CRY2 has potential to correct abnormal gene expression patterns and reduce the severity of OA.
Project description:Circadian pace is modulated by light intensity, known as the Aschoff’s rule, with largely unrevealed mechanisms. Here we report that photoreceptor CRY2 mediates blue light input to circadian clock by directly interacting with clock core component PRR9 in blue light dependent manner. This physical interaction dually blocks the accessibility of PRR9 protein to its co-repressor TPL/TPRs and the resulting kinase PPKs. Notably, phosphorylation of PRR9 by PPKs is critical for its DNA binding and repressive activity, hence to ensure proper circadian speed. Given the labile nature of CRY2 in strong blue light, our findings provide a mechanistic explanation for Aschoff’s rule in plants, i.e., blue light triggers CRY2 turnover in proportional to its intensity, which accordingly releasing PRR9 to fine tune circadian speed. Our findings not only reveal a novel network mediating light input into circadian clock, but also unmask a mechanism by which Arabidopsis circadian clock sensing light intensity.
Project description:Disruption of circadian rhythms increases the risk of several types of cancer. Mammalian cryptochromes (CRY1 and CRY2) are circadian transcriptional repressors that are related to DNA repair enzymes. While CRYs lack DNA repair activity, they modulate the transcriptional response to DNA damage, and CRY2 can promote SCFFBXL3-mediated ubiquitination of c-MYC and other targets. Here, we characterize five mutations in CRY2 observed in human cancers in The Cancer Genome Atlas. We demonstrate that two orthologous mutations of mouse CRY2 (D325H and S510L) accelerate the growth of primary mouse fibroblasts expressing high levels of c-MYC. Neither mutant affects steady state levels of overexpressed c-MYC, and they have divergent impacts on circadian rhythms and on the ability of CRY2 to interact with SCFFBXL3. Unexpectedly, stable expression of either CRY2 D325H or of CRY2 S510L robustly suppresses P53 target gene expression, suggesting that this is the primary mechanism by which they influence cell growth.
Project description:Circadian clocks are the time-keeping cellular apparatuses that are photoentrained according to the day-night photocycles on Earth 1,2. Cryptochromes (CRYs) are photoreceptors mediating photoentrainment of the circadian clock in plants and animals but how CRYs mediate light regulation of the molecular clock remains unclear 1,3. Here we show that CRYs mediate photoresponses of the circadian clock by regulating the activity of N6-methyladenosine (m6A) RNA methyltransferase and photoresponses of the epitranscriptome. In contrast to the presently known CRY complexome, the CRY2-methyltransferase complex exbibits multivalent interactions and photoresponsive condensation in response to blue light. We show that photoexcited CRY2 undergoes phosphorylation-assisted demixing to form photobodies with the properties of condensed liquid phase, which facilitates assembly of the CRY2-methyltransferase complex in the condensed liquid phase of CRY2 in vivo. The mta mutant impaired in m6A methyltransferase and the cry1cry2 mutant missing CRY photoreceptors share common defects of lengthened circadian period, reduced m6A RNA methylation, and accelerated degradation of mRNAs encoding core components of the molecular clock. These results argue for a photoentrainment mechanism by which blue light elicits liquid-liquid phase separation of the CRY2-methyltransferase complex to regulate m6A mRNA methylation, consequently suppressing mRNA degradation and period lengthening of the circadian clock
Project description:Circadian clocks are the time-keeping cellular apparatuses that are photoentrained according to the day-night photocycles on Earth 1,2. Cryptochromes (CRYs) are photoreceptors mediating photoentrainment of the circadian clock in plants and animals but how CRYs mediate light regulation of the molecular clock remains unclear 1,3. Here we show that CRYs mediate photoresponses of the circadian clock by regulating the activity of N6-methyladenosine (m6A) RNA methyltransferase and photoresponses of the epitranscriptome. In contrast to the presently known CRY complexome, the CRY2-methyltransferase complex exbibits multivalent interactions and photoresponsive condensation in response to blue light. We show that photoexcited CRY2 undergoes phosphorylation-assisted demixing to form photobodies with the properties of condensed liquid phase, which facilitates assembly of the CRY2-methyltransferase complex in the condensed liquid phase of CRY2 in vivo. The mta mutant impaired in m6A methyltransferase and the cry1cry2 mutant missing CRY photoreceptors share common defects of lengthened circadian period, reduced m6A RNA methylation, and accelerated degradation of mRNAs encoding core components of the molecular clock. These results argue for a photoentrainment mechanism by which blue light elicits liquid-liquid phase separation of the CRY2-methyltransferase complex to regulate m6A mRNA methylation, consequently suppressing mRNA degradation and period lengthening of the circadian clock
Project description:Interferons have been ascribed to mediate antitumor effects. IRF-1 is a major target gene of interferons. It inhibits cell proliferation and oncogenic transformation. Here we show that 60% of all mRNAs deregulated by oncogenic transformation mediated by c-myc and H-ras are reverted to the expression levels of non-transformed cells by IRF-1. These include cell cycle regulating genes. Activation of IRF-1 decreases cyclin D1 expression and CDK4 kinase activity concomitant with dephosphorylation of pRb. These effects of IRF-1 are mediated by inhibition of the MEK-ERK pathway and a transcriptional repression of cyclin D1. IRF-1 mediated effects on cell cycle progression were found to be overridden by ectopic expression of cyclin D1. Ablation of cyclin D1 by RNA interference experiments prevents transformation and tumor growth in nude mice. The data demonstrate that cyclin D1 is a key target for IRF-1 mediated tumor suppressive effects. Keywords: NIH3T3 cells, myc, ras, IRF-1, proliferation, transformation, G1/S cell cycle ceckpoint