TDP-43, a novel rhythmic protein, regulates the ubiquitination degradation of BMAL1, cellular glucose utilization and energy synthesis
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE260737 | GEO | 2024/03/07
REPOSITORIES: GEO
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Project description:Circadian rhythms are a series of endogenous autonomous 24-hour oscillations generated by the circadian clock. At the molecular level, the circadian clock is generated by a transcription-translation feedback loop, where BMAL1 and CLOCK transcription factors of the positive arm activate the expression of CRYPTOCHROME and PERIOD (PER) genes of the negative arm as well as the circadian clock-regulated genes. In this project, we aimed at finding the interactome of PER2 protein in human U2OS osteosarcoma cell line using proximity-dependent biotin identification (BioID) technique. U2OS clones overexpressing PER2-BioID2 or BioID2 were treated with dexamethasone in order to reset the circadian rhythm, and cells were then incubated in biotin-containing media for 12 hours to label the proteins in close proximity of PER2-BioID2. Samples were collected after 36 and 48 hours of the resetting to identify the labeled proteins by mass spectrometry. In addition to known interactors such as CRY1 and CRY2, many novel interactors were identified. In summary, we obtained a network of PER2 interactome and confirmed some of the novel interactions using classical the co-immunoprecipitation method.
2022-04-22 | PXD025179 | Pride
Project description:The circadian clock is intricately connected with metabolism, however the precise details of these connections are incomplete. Here we used high temporal resolution metabolite profiling to determine circadian regulation of mouse liver and cell autonomous metabolism. In mouse liver, we found ~50% of metabolites were circadian, with strong enrichment of the nucleotide, amino acid, and methylation pathways. In U2OS cells, 27% of metabolites were circadian, including amino acids and NAD biosynthesis, also clock controlled in liver. To assess whether cell autonomous metabolite rhythms were clock-dependent, we used RNAi to perturb Bmal1, Cry1, and Cry2. Bmal1 knockdown eliminated most metabolite rhythms, while Cry1 generally shortened and Cry2 lengthened rhythms. Surprisingly, we found Cry1 knockdown induced 8 hr rhythms in amino acid, methylation, and vitamin metabolites, decoupling metabolite and transcriptional rhythms. These results provide the first comprehensive views of circadian liver and cell autonomous metabolism.
2017-04-05 | MTBLS292 | MetaboLights
Project description:The mammalian circadian clock is a molecular oscillator composed of a feedback loop that involves transcriptional activators CLOCK and BMAL1, and repressors Cryptochrome (CRY) and Period (PER). Here we show that a direct CLOCK-BMAL1 target gene, Gm129, is a novel regulator of the feedback loop. ChIP analysis revealed that the CLOCK:BMAL1:CRY1 complex strongly occupies the promoter region of Gm129. Both mRNA and protein levels of GM129 exhibit high amplitude circadian oscillations in mouse liver, and Gm129 gene encodes a nuclear-localized protein that directly interacts with BMAL1 and represses CLOCK:BMAL1 activity. In vitro and in vivo protein-DNA interaction results demonstrate that, like CRY1, GM129 functions as a repressor by binding to the CLOCK:BMAL1 complex on DNA. Although Gm129-/- or Cry1-/- Gm129-/- mice retain a robust circadian rhythm, the peaks of Nr1d1 and Dbp mRNAs in liver exhibit significant phase delay compared to control. Our results suggest that, in addition to CRYs and PERs, GM129 protein contributes to the transcriptional feedback loop by modulating CLOCK:BMAL1 activity as a transcriptional repressor. Examination of 3 transcriptional regulators in mouse liver
2014-01-07 | E-GEOD-53828 | biostudies-arrayexpress
Project description:The human osteosarcoma cell line U2OS contains a functional circadian clock but expresses only a few rhythmic genes. We identified by ChIP-seq analysis 3040 binding sites of the circadian transcription factors BMAL1, CLOCK, and CRY1 in the genome of U2OS cells, comparable to the number found in highly rhythmic tissues like liver. ChIP was performed as described previously (Rey et al, 2011; doi:10.1371/journal.pbio.1000595) using confluent desynchronized U2OS cells and BMAL1, CLOCK, and CRY1-antibodies. Immunoprecipitated chromatin (10 ng DNA of 8 independent BMAL1 ChIPs with enrichment levels > 80-fold, 9 ng DNA of a CRY1 ChIP with 10-fold enrichment, and 8 ng DNA of a CLOCK ChIP with 40-fold enrichment) was used for library preparation. Three lanes of the BMAL1 library, and one lane each of the CRY1 and CLOCK library were sequenced on the Illumina Genome Analyzer IIx using Single-Read Cluster Generation Kit and 36 Cycle Sequencing Kit v2 (Lausanne Genomics Technologies Facility).
2016-02-11 | E-GEOD-44236 | biostudies-arrayexpress
Project description:Implantation is dependent on synchronized interactions between the conceptus and surrounding decidual cells but the involvement of clock genes in this process is not well understood. Circadian oscillations are predicated on transcriptional-translational feedback loops, which balance the activities of the transcriptional activators CLOCK and BMAL1 and repressors encoded by PER and CRY genes. Here we show that loss of PER2 expression silences circadian oscillations in decidualizing human endometrial stromal cells (HESCs). Downregulation was preceded by reduced CLOCK binding to a noncanonical E-box enhancer in the PER2 promoter and occurred between 12 - 24 h after exposure to a deciduogenic stimulus. RNA sequencing revealed that premature inhibition of PER2 by siRNA knockdown leads to a grossly disorganised decidual response. Gene ontology analysis highlighted a preponderance of cell cycle regulators amongst the 1,121 genes perturbed upon PER2 knockdown. Congruently, PER2 inhibition abrogated mitotic expansion of differentiating HESCs by inducing cell cycle block at G2/M. Analysis of mid-luteal endometrial biopsies revealed an inverse correlation between PER2 transcript levels and the number of miscarriages in women suffering reproductive failure. Thus, PER2 synchronizes mitotic expansion of HESCs with a periodic decidual gene expression; uncoupling of these events may cause persistent pregnancy failure. Endometrial mRNA profiles of paired control (siRNA-NT) and siRNA-PER2 were generated by deep sequencing, in triplicate using Illumina
2015-09-16 | E-GEOD-62854 | biostudies-arrayexpress
Project description:Cryptochromes are negative transcriptional regulators of the circadian clock in mammals. It is not clear how reducing the level of endogenous CRY1 in mammals will affect circadian rhythm and the relation of such a decrease with apoptosis. Here, we discovered a molecule (M47) that destabilizes Cryptochrome 1 (CRY1) both in vitro and in vivo. The M47 selectively enhanced the degradation rate of CRY1 by increasing its ubiquitination and resulted in increasing the circadian period length of U2OS Bmal1-dLuc cells. In addition, subcellular fractionation studies from mice liver indicated that M47 increased degradation of the CRY1 in the nucleus. Furthermore, M47-mediated CRY1 reduction enhanced cisplatin-induced apoptosis in Ras-transformed p53 null fibroblast cells. Systemic repetitive administration of M47 increased the median lifespan of p53-/- mice by ~25%. Collectively our data suggest that M47 is a very promising molecule to treat forms of cancer depending on the p53 mutation.
2022-11-07 | PXD037311 | Pride
Project description:In mammals, the circadian clock consists of transcriptional and translational feedback loops through DNA cis-elements such as E-box and RRE. In this study, we established mutant mice deficient for rhythmic transcription of Bmal1 gene by deleting its upstream RRE elements. We observed apparently normal circadian rhythms in the mutant, but the circadian period and amplitude of the mutants were more susceptible to disturbance of CRY1 protein rhythm. Our findings demonstrate that the RRE-mediated feedback regulation of Bmal1 underpins the E-box-mediated rhythm in cooperation with CRY1-dependent posttranslational regulation of BMAL1 protein, thereby conferring the perturbation-resistant oscillation and chronologically organized output of the circadian clock.
2022-05-17 | GSE199061 | GEO
Project description:The mammalian circadian clock is a molecular oscillator composed of a feedback loop that involves transcriptional activators CLOCK and BMAL1, and repressors Cryptochrome (CRY) and Period (PER). Here we show that a direct CLOCK-BMAL1 target gene, Gm129, is a novel regulator of the feedback loop. ChIP analysis revealed that the CLOCK:BMAL1:CRY1 complex strongly occupies the promoter region of Gm129. Both mRNA and protein levels of GM129 exhibit high amplitude circadian oscillations in mouse liver, and Gm129 gene encodes a nuclear-localized protein that directly interacts with BMAL1 and represses CLOCK:BMAL1 activity. In vitro and in vivo protein-DNA interaction results demonstrate that, like CRY1, GM129 functions as a repressor by binding to the CLOCK:BMAL1 complex on DNA. Although Gm129-/- or Cry1-/- Gm129-/- mice retain a robust circadian rhythm, the peaks of Nr1d1 and Dbp mRNAs in liver exhibit significant phase delay compared to control. Our results suggest that, in addition to CRYs and PERs, GM129 protein contributes to the transcriptional feedback loop by modulating CLOCK:BMAL1 activity as a transcriptional repressor.
2014-01-07 | GSE53828 | GEO
Project description:To screen for specific circadian outputs that may distinguish the pacemaker in the mammalian suprachiasmatic nucleus (SCN) from peripheral-type oscillators in which the canonical clockworks are similarly regulated in a circadian manner, the rhythmic behavior of the transcriptome in forskolin-stimulated NIH/3T3 fibroblasts was analyzed and compared to that found in the rat SCN in vivo and SCN2.2 cells in vitro. Similar to the scope of circadian gene expression in SCN2.2 cells and the rat SCN, NIH/3T3 fibroblasts exhibited circadian fluctuations in the expression of the core clock genes, Per2, Bmal1 (Mop3), and Cry1 and 323 functionally diverse transcripts (2.6%), many of which were involved in cell communication. Overlap in rhythmically-expressed transcripts among NIH/3T3 fibroblasts, SCN2.2 cells and the rat SCN was limited to these clock genes and four other genes that mediate fatty acid and lipid metabolism or function as nuclear factors. Compared to NIH/3T3 cells, circadian gene expression in SCN oscillators was more prevalent among cellular pathways mediating glucose metabolism and neurotransmission. Coupled with evidence for the rhythmic regulation of the inducible isoform of nitric oxide synthase, the enzyme responsible for the production of nitric oxide, in SCN2.2 cells and the rat SCN but not in fibroblasts, studies examining the effects of a NOS inhibitor on metabolic rhythms in co-cultures containing SCN2.2 cells and untreated NIH/3T3 cells suggest that this gaseous neurotransmitter may play a key role in SCN pacemaker function. Thus, this comparative analysis of circadian gene expression in SCN and non-SCN cells may have important implications in the selective identification of circadian signals involved in the coupling of SCN oscillators and the regulation of rhythmicity in downstream cells or tissues. Experiment Overall Design: Circadian profiling of the NIH/3T3 fibroblast transcriptome entailed the treatment of NIH/3T3 cells with a 15uM forskolin pulse, subsequent washout of the drug, and collection of total RNA immediately after washout and every 6 hours across two circadian cycles for each of three experiments. Timepoint values reflect the average of three samples from these biological replicates.
2008-06-14 | E-GEOD-5810 | biostudies-arrayexpress
Project description:To screen for specific circadian outputs that may distinguish the pacemaker in the mammalian suprachiasmatic nucleus (SCN) from peripheral-type oscillators in which the canonical clockworks are similarly regulated in a circadian manner, the rhythmic behavior of the transcriptome in forskolin-stimulated NIH/3T3 fibroblasts was analyzed and compared to that found in the rat SCN in vivo and SCN2.2 cells in vitro. Similar to the scope of circadian gene expression in SCN2.2 cells and the rat SCN, NIH/3T3 fibroblasts exhibited circadian fluctuations in the expression of the core clock genes, Per2, Bmal1 (Mop3), and Cry1 and 323 functionally diverse transcripts (2.6%), many of which were involved in cell communication. Overlap in rhythmically-expressed transcripts among NIH/3T3 fibroblasts, SCN2.2 cells and the rat SCN was limited to these clock genes and four other genes that mediate fatty acid and lipid metabolism or function as nuclear factors. Compared to NIH/3T3 cells, circadian gene expression in SCN oscillators was more prevalent among cellular pathways mediating glucose metabolism and neurotransmission. Coupled with evidence for the rhythmic regulation of the inducible isoform of nitric oxide synthase, the enzyme responsible for the production of nitric oxide, in SCN2.2 cells and the rat SCN but not in fibroblasts, studies examining the effects of a NOS inhibitor on metabolic rhythms in co-cultures containing SCN2.2 cells and untreated NIH/3T3 cells suggest that this gaseous neurotransmitter may play a key role in SCN pacemaker function. Thus, this comparative analysis of circadian gene expression in SCN and non-SCN cells may have important implications in the selective identification of circadian signals involved in the coupling of SCN oscillators and the regulation of rhythmicity in downstream cells or tissues. Keywords: Circadian time course
2006-10-31 | GSE5810 | GEO