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Chromosome conformation capture-on-chip of dexamethasone synchronized cells at several circadian times

ABSTRACT: The spatial organization of genes in the interphase nucleus plays an important role in establishment and regulation of gene expression. The circadian expressed and clock controlled genes represent about 10% of the transcripts in the mammalian cells, however the role of genome topology in the expression of genes in a circadian manner remains to be elucidated. In this study we investigated the characteristics and dynamics of the genomic loci that contact the clock controlled gene Dbp during the circadian cycle. To do so, we combined genome-wide interaction profiling by chromosome conformation capture-on-chip (4C) technology with analyses of circadian gene expression in synchronized mouse embryonic fibroblasts (MEFs). This approach allowed us to elucidate the three-dimensional organization of the genome during the circadian cycle around the clock controlled gene Dbp, paralleling its circadian expression. We found that the Dbp genomic environment remains largely similar during the circadian cycle. However, specific DNA regions change their frequency of interaction with Dbp as the circadian cycle progresses, delineating a Dbp circadian interactome. These specific changes are not present in Bmal1-/- MEF, suggesting that the clock machinery is implicated in shaping the genomic landscape around the Dbp locus. The Dbp interactome is enriched in DNA elements related to the clock system (E boxes). We found a spatial clustering of functionally related genes. The Dbp circadian interactome contains a subset of circadian genes whose expression closely parallels that for Dbp. Taken together, our results indicate that the Dbp subnuclear environment is organized to privilege the clock directed gene expression program.

ORGANISM(S): Mus musculus

PROVIDER: GSE49582 | GEO | 2013/08/08

SECONDARY ACCESSION(S): PRJNA214557

REPOSITORIES: GEO

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Project description:The spatial organization of genes in the interphase nucleus plays an important role in establishment and regulation of gene expression. The circadian expressed and clock controlled genes represent about 10% of the transcripts in the mammalian cells, however the role of genome topology in the expression of genes in a circadian manner remains to be elucidated. In this study we investigated the characteristics and dynamics of the genomic loci that contact the clock controlled gene Dbp during the circadian cycle. To do so, we combined genome-wide interaction profiling by chromosome conformation capture-on-chip (4C) technology with analyses of circadian gene expression in synchronized mouse embryonic fibroblasts (MEFs). This approach allowed us to elucidate the three-dimensional organization of the genome during the circadian cycle around the clock controlled gene Dbp, paralleling its circadian expression. We found that the Dbp genomic environment remains largely similar during the circadian cycle. However, specific DNA regions change their frequency of interaction with Dbp as the circadian cycle progresses, delineating a Dbp circadian interactome. These specific changes are not present in Bmal1-/- MEF, suggesting that the clock machinery is implicated in shaping the genomic landscape around the Dbp locus. The Dbp interactome is enriched in DNA elements related to the clock system (E boxes). We found a spatial clustering of functionally related genes. The Dbp circadian interactome contains a subset of circadian genes whose expression which closely parallel that for Dbp. Taken together, our results indicate that the Dbp subnuclear environment is organized to privilege the clock directed gene expression program. Wild type mouse embryonic fibroblasts at the circadian times (CT) 22, 26, 30, 34 and 46. Bmal1-/- mouse embrionic fibroblasts at CT 22 and CT 34 Biological replicate using an independent line of wild type mouse embrionic fibroblast at CT 22 and CT 34 This submission represents the chromosome conformation capture-on-chip component of the overall study

Project description:Circadian rhythms are daily oscillations in metabolism and physiology and are generated by the circadian clock. In fruit fly Drosophila, the circadian clock is generated by a transcription-translation feedback loop in which the positive arm components Clock and Cycle activate the expression of the Period and Timeless genes of negative arm, as well as other circadian clock-regulated genes. After being retained in the cytoplasm, the Period and Timeless proteins then migrate to the nucleus to inhibit the Clock/Cycle transactivity by protein-protein interactions (PPIs). The endogenous circadian clock is synchronized with the geological (solar) clock via photoreceptors. Drosophila Cryptochrome protein functions as a circadian photoreceptor. In the early morning, exposure of Cryptochrome to light causes a conformational change in it which results in the formation of new PPIs. Light-activated Cryptochrome interacts with the core clock protein Timeless and the E3 ubiquitin ligase-substrate adaptor protein Jetlag, which results in the ubiquitylation of Timeless by Jetlag-E3 ligase complex and then degradation of Timeless within minutes by proteasome system. Rapid degradation of Timeless and then its partner protein Period, because of its instability in the absence of Timeless, relieves the inhibition on the Clock/Cycle transcription factors suddenly. Therefore, Clock/Cycle-driven expression of circadian clock-regulated genes are induced again, which is the restart of the circadian oscillation or the resetting of the clock. Following Timeless degradation, Cryptochrome is also degraded so the photoreceptor mechanism does not start a new resetting signal until all the required factors are re-synthesized in a circadian manner. Light-dependent degradation of Drosophila Cryptochrome can be observed in Drosophila S2 cell line in culture. In this project, we aimed at finding the interactome of Cryptochrome protein in Drosophila S2 cell line under light and in the dark using proximity labeling method. Because of the fast kinetics of Cryptochrome degradation, we chose the enzymes that can saturate in less than one hour. TurboID (TID) and APEX2 enzymes label proteins with biotin in the proximity even though they work with different mechanisms. They were fused to Cryptochrome protein, and proximity labeling was performed in the dark or under light. We have identified novel light-dependent or -independent interactors of Drosophila Cryptochrome and confirmed some of them using classical coimmunoprecipitation technique.

Project description:Renal excretion of water and major electrolytes exhibits a significant circadian rhythm. This functional periodicity is believed to result, at least in part, from circadian changes in secretion/reabsorption capacities of the distal nephron and collecting ducts. Here, we studied the molecular mechanisms underlying circadian rhythms in the distal nephron segments, i.e. distal convoluted tubule (DCT) and connecting tubule (CNT) and, the cortical collecting duct (CCD). Temporal expression analysis performed on microdissected mouse DCT/CNT or CCD revealed a marked circadian rhythmicity in the expression of a large number of genes crucially involved in various homeostatic functions of the kidney. This analysis also revealed that both DCT/CNT and CCD possess an intrinsic circadian timing system characterized by robust oscillations in the expression of circadian core clock genes (clock, bma11, npas2, per, cry, nr1d1) and clock-controlled Par bZip transcriptional factors dbp, hlf and tef. The clock knockout mice or mice devoid of dbp/hlf/tef (triple knockout) exhibit significant changes in renal expression of several key regulators of water or sodium balance (vasopressin V2 receptor, aquaporin-2, aquaporin-4, M-oM-^AM-!ENaC). Functionally, the loss of clock leads to a complex phenotype characterized by partial diabetes insipidus, dysregulation of sodium excretion rhythms and a significant decrease in blood pressure. Collectively, this study uncovers a major role of molecular clock in renal function. Experiment Overall Design: We examined the temporal profiles of gene expression in mouse distal nephron segments and collecting ducts. The RNA was extracted from microdissected distal convoluted tubules and connecting tubules (DCT/CNT samples) or, cortical collecting ducts (CCD samples). Animals were sacrificed for microdissection every 4 hours, i.e. at ZT0, ZT4, ZT8, ZT12, ZT16 and ZT20 (ZT M-bM-^@M-^S Zeitgeber (circadian) time, indicates time of light-on as ZT0 and time of light-off as ZT12). The microarray hybridization was performed in duplicates on two pools of RNA composed of equivalent amounts of RNA prepared from five animals at each ZT time-point.

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Chromosome conformation capture-on-chip of dexamethasone synchronized cells at several circadian times

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