RNA-seq study on time of day specific Glucocorticoid action in mouse liver and lung tissues
ABSTRACT: Glucocorticoids are critical regulators of energy metabolism and immunity and major drug targets in inflammatory disease. It is also known that the circadian clock regulates both metabolism and immunity. This experiment is part of wider study to understand the coupling between circadian clock components and Glucocorticoid receptor in multiple mouse tissues. Here, RNA-seq analysis was performed in vehicle and dexamethasone (a synthetic Glucocorticoid) treated WT mouse liver and lung tissues at two circadian times, to understand time of the day variation in Glucocorticoid regulated genes. Mice were treated at ZT6 (6 hours after lights on, 1:30pm) or at ZT18 (6 hours after lights off, 1:30am) with dexamethasone (1 mg/kg intraperitoneal) or vehicle (methylcyclodextrin 1mg/kg intraperitoneal) for 2 hours before sacrifice by cervical dislocation. Lung and liver tissue were lysed and total RNA prepared using SV Total RNA Isolation System (Promega). Quality and integrity of total RNA samples were assessed by 2100 Bioanalyzer or a 2200 TapeStation (Agilent Technologies) according to the manufacturer’s instructions. RNA sequencing (RNA-seq) libraries were generated using the TruSeq® Stranded mRNA assay (Illumina, Inc.) according to the manufacturer’s protocol. Then paired-end sequenced (101 + 101 cycles, plus indices) on an Illumina HiSeq2500 instrument.
Project description:Comparison of pancreatic islet transcriptome at 4hr intervals around the clock helps to identify genes that are cycling under the control of the circadian clock. We used Agilent microarrays to analyze pancreatic islet transcriptome at different time points across the circadian cycle. Overall design: Pancreatic islets were isloated at ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20 (ZT0 corresponds to lights on - 06:00hr and ZT12 corresponds to lights off - 18:00hr in a 12hr light, 12hr dark cycle). Pooled RNA samples, 3-5 mice per group, were used for the array.
Project description:Altered daily patterns of hormone action are suspected to contribute to metabolic disease. It is poorly understood how the adrenal glucocorticoid hormones contribute to the coordination of daily global patterns of transcription and metabolism. Here, we examined diurnal metabolite and transcriptome patterns in a zebrafish glucocorticoid deficiency model by RNA-Seq, NMR spectroscopy and liquid chromatography-based methods. We observed dysregulation of metabolic pathways including glutaminolysis, the citrate and urea cycles and glyoxylate detoxification. Constant, non-rhythmic glucocorticoid treatment rescued many of these changes, with some notable exceptions among the amino acid related pathways. Surprisingly, the non-rhythmic glucocorticoid treatment rescued almost half of the entire dysregulated diurnal transcriptome patterns. A combination of E-box and glucocorticoid response elements is enriched in the rescued genes. This simple enhancer element combination is sufficient to drive rhythmic circadian reporter gene expression under non-rhythmic glucocorticoid exposure, revealing a permissive function for the hormones in glucocorticoid-dependent circadian transcription. Our work highlights metabolic pathways potentially contributing to morbidity in patients with glucocorticoid deficiency, even under glucocorticoid replacement therapy. Moreover, we provide mechanistic insight into the interaction between the circadian clock and glucocorticoids in the transcriptional regulation of metabolism. Overall design: RNA-Seq from total RNA of zebrafish larvae during (5 dpf) the diurnal cycle. Time-series mRNA profiles of untreated wild type (WT), rx3t25327/t25327 [rx3 strong] and rx3t25181/t25181 [rx3 weak] mutant larvae as well as dexamethasone treated WT and rx strong larvae were generated by deep sequencing.
Project description:Genome-wide expression analysis of two circadian oscillatory mechanisms in the mouse liver; To identify the genes of which the circadian expression is regulated by endogenous glucocorticoids, we performed DNA microarray analysis using hepatic RNA from adrenalectomized (ADX) and sham-operated mice. Mice were housed in a 12:12 h light-dark cycle (LD12:12; lights on at zeitgeber time (ZT) 0) for at least two weeks before the day of the experiment. Liver samples were dissected, quickly frozen, and stored in liquid nitrogen. Total RNA was purified from pools of 3 animal tissues collected at each time-point using ISOGEN (Nippon Gene Co., Ltd., Japan). Hybridization to Affymetrix GeneChip (MG-U74Av2) arrays proceeded as described (Oishi K et al., J Biol Chem, 278, 41519-41527, 2003). The average difference (AD) value for each gene was provided by GeneChip software. To identify putative glucocorticoid-regulated circadian genes, we compared AD values between two time points (ZT2 and ZT14) in sham operated and in ADX mice. We applied three criteria to the selection of putative glucocorticoid-regulated circadian genes: (i) the AD value is marked as “present” by the GeneChip software in at least one of two time points, (ii) the AD value exhibits a 2-fold or greater change in sham-operated mice and (iii) the fold change is below 2-fold in ADX mice. We identified 169 genes that fluctuated between day and night in the livers of sham-operated mice. Among these, 100 lost circadian rhythmicity in ADX mice. On the other hand, the circadian expression of clock or clock-related genes such as mPer2 and DBP remained almost totally intact in the liver of ADX mice. The present study showed that the circadian expression of one type of liver genes in the mouse is governed by core components of the circadian clock such as CLOCK and BMAL1, and the other depends on endogenous glucocorticoids.
Project description:This array set was used to determine clock regulated genes in the adrenal gland that are not necessarily rhythmic but still controlled by the circadian TTL. Keywords: comparative genomic hybridization / time series Overall design: LD entrained animals were released into DD an whole adrenals dissected at the specified time points after "lights off"
Project description:The circadian clock is comprised of proteins that form negative feedback loops, which regulate the timing of global gene expression in a coordinated 24 hour cycle. As a result, the plant circadian clock is responsible for regulating numerous physiological processes central to growth and survival. To date, most plant circadian clock studies have relied on diurnal transcriptome changes to elucidate molecular connections between the circadian clock and observable phenotypes in wild-type plants. Here, we have combined high-throughput RNA-sequencing and mass spectrometry to comparatively characterize the lhycca1, prr7prr9, gi and toc1 circadian clock mutant rosette transcriptome and proteome at the end-of-day and end-of-night.
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.
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 abdominal white adipose tissue 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 white adipose tissue, and to identify the role of circadian regulation in coordinating the functioning of this dynamic tissue. 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 abdominal adipose tissue for gene arrays. Two samples were not used due to RNA degradation: Adipose .25-1 and Adipose 11-2. Time point designations reflect time after lights on in hours.
Project description:This array set was used to determine clock regulated genes in the adrenal gland that are not necessarily rhythmic but still controlled by the circadian TTL. Experiment Overall Design: LD entrained animals were released into DD and whole adrenal glands were dissected at the 46/54 h specified time points after lights off.
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:Cell-autonomous circadian oscillations strongly influence tissue physiology and pathophysiology of peripheral organs. Recent in vivo findings in the heart demonstrate that the circadian clock controls oscillatory gene expression programs in the adult myocardium. However, whether in vitro human embryonic stem (ES) cell-derived cardiomyocytes can establish circadian rhythmicity is unknown. Here we report that while undifferentiated human ES cells do not possess a functional clock, oscillatory expression of known core clock genes emerges during directed cardiac differentiation, with robust rhythms in day 30 cardiomyocytes. Our data reveal a stress related oscillatory network of genes that underlies a time-dependent response to doxorubicin, a frequently used anti-cancer drug with cardiotoxic side effects. These results provide a set of oscillatory genes that is relevant to functional cardiac studies and that can be deployed to uncover the potential contribution of the clock to other processes such as cardiac regeneration. Overall design: Human embryonic stem cells (ES cells) were differentiated via a directed differentiation protocol in vitro towards cardiomyocytes for a period of 30 days. Cardiomyocytes were synchronized with dexamethasone and triplicate samples for RNA extraction and sequencing were taken every 4 hours for 48 hours in total. RNA was then extracted using TRIzol, barcoded and amplified following the CEL-Seq protocol.