Transcriptional analysis of breast circadian clock
ABSTRACT: Circadian clocks drive ~24 hr rhythms in tissue physiology. They rely on transcriptional/translational feedback loops driven by interacting networks of clock complexes.To gain insights into the role of the mammary clock, circadian time-series microarrays were performed to identify rhythmic genes in vivo. Breast tissues were isolated at 4 hr intervals for two circadian (24 hourly) cycles, from mice kept under constant darkness to avoid any light- or dark-driven genes.
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:Odor discrimination behavior displays circadian fluctuations in mice indicating that mammalian olfactory function is under control of the circadian system. This is further supported by the facts that odor discrimination rhythms depend on the presence of clock genes and that olfactory tissues contain autonomous circadian clocks. However, the molecular link between circadian function and olfactory processing is still unknown. In order to elucidate the molecular mechanisms underlying this link, we focused on the olfactory epithelium (OE), the primary target of odors and the site of the initial events in olfactory processing. We asked whether olfactory sensory neurons (OSNs) within the OE possess an autonomous circadian clock and whether olfactory pathways are under circadian control. Employing clock gene-driven bioluminescence reporter assays, immunohistochemistry and a time-dependent microarray-based transcriptome analysis on OE samples, we found robust circadian rhythms of core clock genes and their proteins in OSNs, suggesting that the OE indeed contains an autonomous circadian clock. Furthermore, we identified several OSN-specific components of the olfactory pathway that are under circadian control, including several candidates with putative roles in circadian olfactory processing, such as KIRREL2 -- an established factor involved in short-term OSN activation. The spatiotemporal expression patterns of our candidate proteins suggest that they are involved in short-term anabolic processes to rhythmically prepare the cell for peak performances and to promote circadian function of OSNs. We performed a genome-wide expression study with RNA from OE tissue extracted at 4-h intervals in constant darkness from previously entrained mice. Total RNA of four mice per sampling time was pooled in equal amounts.
Project description:To determine whether immortalized cells derived from the rat SCN (SCN2.2) retain intrinsic rhythm-generating properties characteristic of the SCN, oscillatory properties of the SCN2.2 transcriptome were analyzed and compared to those found in the rat SCN in vivo using rat U34A Affymetrix GeneChips. This SuperSeries is composed of the following subset Series:; GSE1654: Circadian Profiling of the Transcriptome in Immortalized Rat SCN Cells (3 biological replicates); GSE1673: Circadian Profiling of the Transcriptome in Immortalized Rat SCN Cells: Comparison to Long-Evans Rat SCN Experiment Overall Design: Refer to individual Series
Project description:Recent evidence suggest that the circadian timing system plays an important role in the control of renal function and maintaining blood pressure. Here, we analyzed circadian rhythms of urinary excretion of sodium and potassium in wild-type mice and mice lacking circadian transcriptional activator clock. Analysis of urines collected at hourly intervals over a 24-hour period revealed dramatic changes in rhythms of sodium and potassium excretion in clock(-/-) mice. In parallel, significant differences in circadian pattern of plasma aldosterone levels, but not in the 24-hour mean aldosterone levels, were observed. Microarray-based profiling of renal transcriptomes demonstrated that clock(-/-) mice exhibit dysregulation in multiple mechanisms involved in maintaining sodium and potassium balance by the kidney. The most significant changes were detected in the expression levels of several key enzymes (Cyp4a14, Cyp4a12a and Cyp4a12b) required for the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a powerful regulator of renal sodium and potassium excretion, renal vascular tone and blood pressure. The 20-HETE levels measured in kidney microsomes of wild-type mice followed a circadian-like temporal pattern. In clock(-/-) mice, the acrophase of this rhythm was shifted by 8 hours and the 24-hour mean levels of 20-HETE were significantly decreased. These results demonstrate that circadian rhythms of urine electrolyte excretion are largely dependent on the circadian clock activity and indicate that circadian oscillations in renal 20-HETE content could be an important mechanism of blood pressure regulation. We examined the temporal profiles of gene expression in mouse whole kidney. Animals were sacrificed for microdissection every 4 hours, i.e. at ZT0, ZT4, ZT8, ZT12, ZT16 and ZT20 (ZT – 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 pools of RNA composed of equivalent amounts of RNA prepared from teo or three animals at each ZT time-point.
Project description:To gain insights into the mechanisms of TOC1 function in the Arabidopsis circadian clock we performed transcriptional profiling of Wild-Type (WT) and and TOC1 mutant plants (toc1-2) under constant light conditions for two days. Comparisons of WT and toc1-2. Two biological replicates each per array. Two Arabidopsis Oligonucleotide Microarrays (two-color Cy3 and Cy5). synchronized under 12-hour light:12-hour dark (LD) cycles for 10 days followed by two days under constant light conditions. Samples were collected at circadian time 16 (CT16).
Project description:This SuperSeries is composed of the following subset Series:; GSE4238: rhythmic transcriptome in the murine wt adrenal; GSE4239: Transcriptome regulation in the adrenal gland of circadian clock deficient Per2Cry1 double mutant mice Experiment Overall Design: Refer to individual Series
Project description:In the immune system various parameters and immune functions are controlled by the circadian system. To investigate molecular mechanisms that link the circadian clock and the immune system we analyzed the transcriptom of peritoneal macrophages from mice collected in a time course for two consecutive days. We found that more than 8% of expressed genes are under circadian control including many important regulators in pathogen recognition, signal transduction and cytokine secretion. Peritoneal macrophages from mice kept in constant conditions were isolated in 4 h intervalls over a period of 48 h. RNA of CD11b sorted cells was extracted, pooled from 3 mice per time point and used for microarray analysis.
Project description:The hippocampus plays an important role in spatial memory and the conversion of short-term to long-term memory. Several studies have reported the presence of a peripheral oscillator in the hippocampus, and have highlighted the importance of circadian regulation in memory formation. In this study we performed global quantitative phosphoproteome analyses of the murine hippocampus across the circadian cycle, applying spiked-in labeled reference and high accuracy mass spectrometry.
Project description:The mammalian circadian clock involves a transcriptional feedback loop in which CLOCK and BMAL1 activate the Period and Cryptochrome genes, which then feedback and repress their own transcription. We have interrogated the transcriptional architecture of the circadian transcriptional regulatory loop on a genome scale in mouse liver and find a stereotyped, time-dependent pattern of transcription factor binding, RNA polymerase II (RNAPII) recruitment, RNA expression and chromatin states. We find that the circadian transcriptional cycle of the clock consists of three distinct phases — a poised state, a coordinated de novo transcriptional activation state, and a repressed state. Interestingly only 22% of mRNA cycling genes are driven by de novo transcription, suggesting that both transcriptional and post-transcriptional mechanisms underlie the mammalian circadian clock. We also find that circadian modulation of RNAPII recruitment and chromatin remodeling occurs on a genome-wide scale far greater than that seen previously by gene expression profiling. Examination of whole transcriptome every 4hr during the circadian cycle in mouse liver