Circadian Rhythm of Gene Expression Patterns in Liver of Mouse
ABSTRACT: Genes encoding the circadian pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) of mammals have recently been identified, but the molecubasis of circadian timing in peripheral tissue is not well understood. We used a bead-based microarray to identify mouse liver transcripts that show circadian cycles of abundance under constant conditions. Keywords: Time course Overall design: microarray-based expression profiling of mRNA with triplicates in mouse liver for 48 hours at 4-hour intervals
Project description:Genes encoding the circadian pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) of mammals have recently been identified, but the molecubasis of circadian timing in peripheral tissue is not well understood. We used a bead-based microarray to identify mouse liver transcripts that show circadian cycles of abundance under constant conditions. Keywords: Time course microarray-based expression profiling of mRNA with triplicates in mouse liver for 48 hours at 4-hour intervals
Project description:The mammalian circadian timing system consists of a master pacemaker in the suprachiasmatic nucleus (SCN) that synchronizes self-sustained oscillators in most peripheral cells. Rhythmic gene expression in peripheral tissues can be driven by cyclic systemic cues emanating from the SCN or by local oscillators. To discriminate between these two mechanisms, we engineered a mouse strain with a conditionally active liver clock. Transcriptome profiling revealed that the circadian transcription of most genes depends on functional hepatocyte clocks. However, the expression of 31 genes, including mPer2, oscillates robustly in clock-arrested hepatocytes. Such genes may be implicated in the synchronization of liver oscillators
Project description:Background: Identifying the gene regulatory networks governing physiological signal integration remains an important challenge in circadian biology. Epidermal growth factor receptor (EGFR) has been implicated in circadian function and EGFR is expressed in the suprachiasmatic nucleus (SCN), the core circadian pacemaker. The transcription networks downstream of EGFR in the SCN are unknown, but by analogy to other SCN inputs we expect the response to EGFR activation to depend on circadian timing and thus be “circadian context–dependent”. Results: We have undertaken a systems level analysis of EGFR circadian context–dependent signaling in the SCN. We collected gene expression profiles to study how the SCN response to EGFR activation depends on circadian timing. Mixed–model analysis of variance (ANOVA) was employed to identify genes with circadian context–dependent EGFR regulation. The expression data was integrated with transcription factor (TF) binding predictions through gene group enrichment analyses to generate robust hypotheses about TFs responsible for the circadian phase–dependent EGFR responses. Conclusions: The analysis results suggest that the transcriptional response to EGFR signaling in the SCN may be partly mediated by established EGFR signaling regulated TFs (AP1, Ets1), TFs involved in circadian clock entrainment (CREB), and by core clock TFs (Rorα). qRT-PCR measurements of several TF expression levels support a model in which circadian context-dependent EGFR responses are partly achieved by circadian regulation of upstream signaling components. Our study suggests an important role for EGFR signaling in SCN function and provides an example for gaining physiological insights through systems-level analysis. Keywords: dose response; repeat sample Overall design: A 2X2 factorial experimental design was used to investigate differences between "day" (8 hours after lights on) and "night" (2 hours after lights off) SCN gene expression responses to EGFR activation induced by EGF treatment (20 nM, 1 hr). Two SCN were obtained from each rat and served as EGF–treated and vehicle-treated samples. Pairing control and treated samples from the same rat permitted detection of EGF effects in the presence of substantial animal-to-animal variability. SCN from two rats were experimentally treated at each circadian time, yielding a total of eight biological samples. Since our goal was a preliminary characterization of EGFR response clock phase dependency, samples were hybridized to one microarray each. A universal reference design was used for the microarrays themselves.
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.
Project description:The timing of daily “circadian” behavior can be highly variable among different individuals, and twin studies suggest that about half of this variability is environmentally controlled. Similar plasticity can be seen in mice exposed to an altered lighting environment – for example, 22-hour days instead of 24-hour ones – which stably alters the genetically determined period of circadian behavior for months. The mechanisms mediating these environmental influences are unknown. Here, we show that transient exposure of mice to such lighting stably alters global transcription in the suprachiasmatic nucleus of the hypothalamus (the SCN, the “master clock” tissue determining circadian behavior in mammals). We have also showed that, these changes in transcription are due to change in DNA methylation in the SCN. Indeed, genome-wide methylation profiling revealed global alterations in promoter DNA methylation in the SCN. Importantly, infusion of a methyltransferase inhibitor to the SCN during 22-hour days suppressed period changes. We also found that these behavioral and DNA methylation changes are reversible upon entrainment to 24-hours days. We conclude that the SCN utilizes DNA methylation as a mechanism to drive circadian clock plasticity. MeDIP array of profiling, demonstrated that genomicDNA methylation changes in mice entrained to short-T cycle. comparison of methylation profile in the suprachiasmatic nuclei of mice entrained to normal T-cycle and short T-cycle
Project description:The suprachiasmatic nucleus (SCN) acts as the central clock to coordinate circadian oscillations in mammalian behavior, physiology and gene expression. Despite our knowledge of the circadian transcriptome of the SCN, how it impacts genome-wide protein expression is not well understood. Here, we interrogated the murine SCN proteome across the circadian cycle using SILAC-based quantitative mass spectrometry.
Project description:We performed a circadian RNA expression profile of the mammalian biological clock, the suprachiasmatic nucleus (SCN) in C57/BL6 mice, at 2-hour resolution using microarrays, and at 6-hour resolution using RNA-seq. 8 samples total covering 8 time points, with no replicates. SCN samples from mouse brains collected every 6 hours for 2 days (8 samples total).
Project description:We performed a circadian RNA expression profile of the mammalian biological clock, the suprachiasmatic nucleus (SCN) in C57/BL6 mice, at 2-hour resolution using microarrays, and at 6-hour resolution using RNA-seq. 24 samples total covering 24 time points, with no replicates. SCN samples from mouse brains collected every 2 hours for 2 days (24 samples total).
Project description:This the single cell model from the article:
A multiscale model to investigate circadian rhythmicity of pacemaker neurons in the suprachiasmatic nucleus.
Vasalou C, Henson MA. PLoS Comput Biol
2010 Mar 12;6(3):e1000706.
, DOI: 10.1371/journal.pcbi.1000706
The suprachiasmatic nucleus (SCN) of the hypothalamus is a multicellular system that drives daily rhythms in mammalian behavior and physiology. Although the gene regulatory network that produces daily oscillations within individual neurons is well characterized, less is known about the electrophysiology of the SCN cells and how firing rate correlates with circadian gene expression. We developed a firing rate code model to incorporate known electrophysiological properties of SCN pacemaker cells, including circadian dependent changes in membrane voltage and ion conductances. Calcium dynamics were included in the model as the putative link between electrical firing and gene expression. Individual ion currents exhibited oscillatory patterns matching experimental data both in current levels and phase relationships. VIP and GABA neurotransmitters, which encode synaptic signals across the SCN, were found to play critical roles in daily oscillations of membrane excitability and gene expression. Blocking various mechanisms of intracellular calcium accumulation by simulated pharmacological agents (nimodipine, IP3- and ryanodine-blockers) reproduced experimentally observed trends in firing rate dynamics and core-clock gene transcription. The intracellular calcium concentration was shown to regulate diverse circadian processes such as firing frequency, gene expression and system periodicity. The model predicted a direct relationship between firing frequency and gene expression amplitudes, demonstrated the importance of intracellular pathways for single cell behavior and provided a novel multiscale framework which captured characteristics of the SCN at both the electrophysiological and gene regulatory levels.
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Project description:Circadian rhythms are oscillations with a periodicity of 24 hours that are controlled by an endogenous clock and are observed in virtually all aspects of mammalian function from expression of genes to complex physiological processes. The master clock is present in the suprachiasmatic nucleus (SCN) in the anterior part of the hypothalamus and controls peripheral clocks present in other parts of the body . Although much is known about the mechanism of the central clock in the SCN, the regulation of clocks present in peripheral tissues is still unclear. This study is designed to examine fluctuations in gene expression in lungs within the 24 hour circadian cycle in normal animals. The objectives of this study is to identify and analyze circadian oscillation in gene expression in lungs, and to identify the role of circadian regulation in coordinating the functioning of this dynamic organ. Overall design: Fifty-four male normal Wistar rats (250-350 g body weight) were housed in a strictly controlled stress free environment with light:dark cycles of 12 hr:12hr. Three animals were sacrificed at each of 18 selected time points within the 24 hour cycle. RNA was prepared from lungs for gene arrays. Time point designations reflect time after lights on in hours.