Extensive dynamic changes in the human transcriptome and its circadian organization during prolonged bed rest.

Physiological and molecular processes including the transcriptome change across the 24-h day, driven by molecular circadian clocks and behavioral and systemic factors. It is not known how the temporal organization of the human transcriptome responds to a long-lasting challenge. This may, however, provide insights into adaptation, disease, and recovery. We investigated the human 24-h time series transcriptome in 20 individuals during a 90-day constant bed rest protocol. We show that the protocol affected 91% of the transcriptome with 76% of the transcriptome still affected after 10 days of recovery. Dimensionality-reduction approaches revealed that many affected transcripts were associated with mRNA translation and immune function. The number, amplitude, and phase of rhythmic transcripts, including clock genes, varied significantly across the challenge. These findings of long-lasting changes in the temporal organization of the transcriptome have implications for understanding the mechanisms underlying health consequences of conditions such as microgravity and bed rest.

Mistimed sleep and waking activity in humans disrupts glucocorticoid signalling transcripts and SP1, but not plasma cortisol rhythms.

Cortisol is a robust circadian signal that synchronises peripheral circadian clocks with the central clock in the suprachiasmatic nucleus via glucocorticoid receptors that regulate peripheral gene expression. Misalignment of the cortisol rhythm with the sleep-wake cycle, as occurs in shift work, is associated with negative health outcomes, but underlying molecular mechanisms remain largely unknown. We experimentally induced misalignment between the sleep-wake cycle and melatonin and cortisol rhythms in humans and measured time series blood transcriptomics while participants slept in-phase and out-of-phase with the central clock. The cortisol rhythm remained unchanged, but many glucocorticoid signalling transcripts were disrupted by mistimed sleep. To investigate which factors drive this dissociation between cortisol and its signalling pathways, we conducted bioinformatic and temporal coherence analyses. We found that glucocorticoid signalling transcripts affected by mistimed sleep were enriched for binding sites for the transcription factor SP1. Furthermore, changes in the timing of the rhythms of SP1 transcripts, a major regulator of transcription, and changes in the timing of rhythms in transcripts of the glucocorticoid signalling pathways were closely associated. Associations between the rhythmic changes in factors that affect SP1 expression and its activity, such as STAT3, EP300, HSP90AA1, and MAPK1, were also observed. We conclude that plasma cortisol rhythms incompletely reflect the impact of mistimed sleep on glucocorticoid signalling pathways and that sleep-wake driven changes in SP1 may mediate disruption of these pathways. These results aid understanding of mechanisms by which mistimed sleep affects health.

Diurnal and circadian rhythmicity of the human blood transcriptome overlaps with organ- and tissue-specific expression of a non-human primate.

BACKGROUND: Twenty-four-hour rhythmicity in mammalian tissues and organs is driven by local circadian oscillators, systemic factors, the central circadian pacemaker and light-dark cycles. At the physiological level, the neural and endocrine systems synchronise gene expression in peripheral tissues and organs to the 24-h-day cycle, and disruption of such regulation has been shown to lead to pathological conditions. Thus, monitoring rhythmicity in tissues/organs holds promise for circadian medicine; however, most tissues and organs are not easily accessible in humans and alternative approaches to quantify circadian rhythmicity are needed. We investigated the overlap between rhythmic transcripts in human blood and transcripts shown to be rhythmic in 64 tissues/organs of the baboon, how these rhythms are aligned with light-dark cycles and each other, and whether timing of tissue-specific rhythmicity can be predicted from a blood sample. RESULTS: We compared rhythmicity in transcriptomic time series collected from humans and baboons using set logic, circular cross-correlation, circular clustering, functional enrichment analyses, and least squares regression. Of the 759 orthologous genes that were rhythmic in human blood, 652 (86%) were also rhythmic in at least one baboon tissue and most of these genes were associated with basic processes such as transcription and protein homeostasis. In total, 109 (17%) of the 652 overlapping rhythmic genes were reported as rhythmic in only one baboon tissue or organ and several of these genes have tissue/organ-specific functions. The timing of human and baboon rhythmic transcripts displayed prominent 'night' and 'day' clusters, with genes in the dark cluster associated with translation. Alignment between baboon rhythmic transcriptomes and the overlapping human blood transcriptome was significantly closer when light onset, rather than midpoint of light, or end of light period, was used as phase reference point. The timing of overlapping human and baboon rhythmic transcriptomes was significantly correlated in 25 tissue/organs with an average earlier timing of 3.21 h (SD 2.47 h) in human blood. CONCLUSIONS: The human blood transcriptome contains sets of rhythmic genes that overlap with rhythmic genes of tissues/organs in baboon. The rhythmic sets vary across tissues/organs, but the timing of most rhythmic genes is similar in human blood and baboon tissues/organs. These results have implications for development of blood transcriptome-based biomarkers for circadian rhythmicity in tissues and organs.

Oxidative stress induces coordinated remodeling of RNA-enzyme interactions.

RNA-binding proteins (RBPs) are key post-transcriptional regulators that play a substantial role during stress adaptation. Recent proteome-wide surveys have uncovered a large number of new and "unconventional" RBPs such as metabolic enzymes, yet little is known about the reconfiguration of the RNA-binding proteome (RBPome) and RNA-enzyme interactions in response to cellular stress. Here, we applied RNA-interactome capture to monitor the dynamics of the mRBPome upon mild oxidative stress in the yeast Saccharomyces cerevisiae. Among the 257 proteins that significantly changed RNA associations, we observed the coordinated remodeling of RNA-binding enzymes - particularly of the central carbon metabolism - that complemented known metabolic responses. Furthermore, we recognized the propensity for paralogous specific alterations of enzyme-RNA interactions. Our results suggest coordinated cross talk between RNA-enzyme interactions and intermediary metabolism to maintain the physiological and molecular balance upon oxidative stress, perhaps through specialization of paralogous during evolution.

A Topological Cluster of Differentially Regulated Genes in Mice Lacking PER3.

Polymorphisms in the human circadian clock gene PERIOD3 (PER3) are associated with a wide variety of phenotypes such as diurnal preference, delayed sleep phase disorder, sleep homeostasis, cognitive performance, bipolar disorder, type 2 diabetes, cardiac regulation, cancer, light sensitivity, hormone and cytokine secretion, and addiction. However, the molecular mechanisms underlying these phenotypic associations remain unknown. Per3 knockout mice (Per3-/- ) have phenotypes related to activity, sleep homeostasis, anhedonia, metabolism, and behavioral responses to light. Using a protocol that induces behavioral differences in response to light in wild type and Per3-/- mice, we compared genome-wide expression in the eye and hypothalamus in the two genotypes. Differentially expressed transcripts were related to inflammation, taste, olfactory and melatonin receptors, lipid metabolism, cell cycle, ubiquitination, and hormones, as well as receptors and channels related to sleep regulation. Differentially expressed transcripts in both tissues co-localized with Per3 on an ∼8Mbp region of distal chromosome 4. The most down-regulated transcript is Prdm16, which is involved in adipocyte differentiation and may mediate altered body mass accumulation in Per3-/- mice. eQTL analysis with BXD mouse strains showed that the expression of some of these transcripts and also others co-localized at distal chromosome 4, is correlated with brain tissue expression levels of Per3 with a highly significant linkage to genetic variation in that region. These data identify a cluster of transcripts on mouse distal chromosome 4 that are co-regulated with Per3 and whose expression levels correlate with those of Per3. This locus lies within a topologically associating domain island that contains many genes with functional links to several of the diverse non-circadian phenotypes associated with polymorphisms in human PER3.

Tandem RNA isolation reveals functional rearrangement of RNA-binding proteins on CDKN1B/p27Kip1 3'UTRs in cisplatin treated cells.

Post-transcriptional control of gene expression is mediated via RNA-binding proteins (RBPs) that interact with mRNAs in a combinatorial fashion. While recent global RNA interactome capture experiments expanded the repertoire of cellular RBPs quiet dramatically, little is known about the assembly of RBPs on particular mRNAs; and how these associations change and control the fate of the mRNA in drug-treatment conditions. Here we introduce a novel biochemical approach, termed tobramycin-based tandem RNA isolation procedure (tobTRIP), to quantify proteins associated with the 3'UTRs of cyclin-dependent kinase inhibitor 1B (CDKN1B/p27Kip1) mRNAs in vivo. P27Kip1 plays an important role in mediating a cell's response to cisplatin (CP), a widely used chemotherapeutic cancer drug that induces DNA damage and cell cycle arrest. We found that p27Kip1 mRNA is stabilized upon CP treatment of HEK293 cells through elements in its 3'UTR. Applying tobTRIP, we further compared the associated proteins in CP and non-treated cells, and identified more than 50 interacting RBPs, many functionally related and evoking a coordinated response. Knock-downs of several of the identified RBPs in HEK293 cells confirmed their involvement in CP-induced p27 mRNA regulation; while knock-down of the KH-type splicing regulatory protein (KHSRP) further enhanced the sensitivity of MCF7 adenocarcinoma cancer cells to CP treatment. Our results highlight the benefit of specific in vivo mRNA-protein interactome capture to reveal post-transcriptional regulatory networks implicated in cellular drug response and adaptation.

Genome-wide analysis of the role of the antibiotic biosynthesis regulator AbsA2 in Streptomyces coelicolor A3(2).

The AbsA1-AbsA2 two component signalling system of Streptomyces coelicolor has long been known to exert a powerful negative influence on the production of the antibiotics actinorhodin, undecylprodiginine and the Calcium-Dependent Antibiotic (CDA). Here we report the analysis of a ΔabsA2 deletion strain, which exhibits the classic precocious antibiotic hyper-production phenotype, and its complementation by an N-terminal triple-FLAG-tagged version of AbsA2. The complemented and non-complemented ΔabsA2 mutant strains were used in large-scale microarray-based time-course experiments to investigate the effect of deleting absA2 on gene expression and to identify the in vivo AbsA2 DNA-binding target sites using ChIP-on chip. We show that in addition to binding to the promoter regions of redZ and actII-orfIV AbsA2 binds to several previously unidentified sites within the cda biosynthetic gene cluster within and/or upstream of SCO3215-SCO3216, SCO3217, SCO3229-SCO3230, and SCO3226, and we relate the pattern of AbsA2 binding to the results of the transcriptomic study and antibiotic phenotypic assays. Interestingly, dual 'biphasic' ChIP peaks were observed with AbsA2 binding across the regulatory genes actII-orfIV and redZ and the absA2 gene itself, while more conventional single promoter-proximal peaks were seen at the CDA biosynthetic genes suggesting a different mechanism of regulation of the former loci. Taken together the results shed light on the complex mechanism of regulation of antibiotic biosynthesis in Streptomyces coelicolor and the important role of AbsA2 in controlling the expression of three antibiotic biosynthetic gene clusters.

Circadian regulation in human white adipose tissue revealed by transcriptome and metabolic network analysis.

Studying circadian rhythms in most human tissues is hampered by difficulty in collecting serial samples. Here we reveal circadian rhythms in the transcriptome and metabolic pathways of human white adipose tissue. Subcutaneous adipose tissue was taken from seven healthy males under highly controlled 'constant routine' conditions. Five biopsies per participant were taken at six-hourly intervals for microarray analysis and in silico integrative metabolic modelling. We identified 837 transcripts exhibiting circadian expression profiles (2% of 41619 transcript targeting probes on the array), with clear separation of transcripts peaking in the morning (258 probes) and evening (579 probes). There was only partial overlap of our rhythmic transcripts with published animal adipose and human blood transcriptome data. Morning-peaking transcripts associated with regulation of gene expression, nitrogen compound metabolism, and nucleic acid biology; evening-peaking transcripts associated with organic acid metabolism, cofactor metabolism and redox activity. In silico pathway analysis further indicated circadian regulation of lipid and nucleic acid metabolism; it also predicted circadian variation in key metabolic pathways such as the citric acid cycle and branched chain amino acid degradation. In summary, in vivo circadian rhythms exist in multiple adipose metabolic pathways, including those involved in lipid metabolism, and core aspects of cellular biochemistry.

REM sleep's unique associations with corticosterone regulation, apoptotic pathways, and behavior in chronic stress in mice.

One of sleep's putative functions is mediation of adaptation to waking experiences. Chronic stress is a common waking experience; however, which specific aspect of sleep is most responsive, and how sleep changes relate to behavioral disturbances and molecular correlates remain unknown. We quantified sleep, physical, endocrine, and behavioral variables, as well as the brain and blood transcriptome in mice exposed to 9 weeks of unpredictable chronic mild stress (UCMS). Comparing 46 phenotypic variables revealed that rapid-eye-movement sleep (REMS), corticosterone regulation, and coat state were most responsive to UCMS. REMS theta oscillations were enhanced, whereas delta oscillations in non-REMS were unaffected. Transcripts affected by UCMS in the prefrontal cortex, hippocampus, hypothalamus, and blood were associated with inflammatory and immune responses. A machine-learning approach controlling for unspecific UCMS effects identified transcriptomic predictor sets for REMS parameters that were enriched in 193 pathways, including some involved in stem cells, immune response, and apoptosis and survival. Only three pathways were enriched in predictor sets for non-REMS. Transcriptomic predictor sets for variation in REMS continuity and theta activity shared many pathways with corticosterone regulation, in particular pathways implicated in apoptosis and survival, including mitochondrial apoptotic machinery. Predictor sets for REMS and anhedonia shared pathways involved in oxidative stress, cell proliferation, and apoptosis. These data identify REMS as a core and early element of the response to chronic stress, and identify apoptosis and survival pathways as a putative mechanism by which REMS may mediate the response to stressful waking experiences.

Identifying and validating blood mRNA biomarkers for acute and chronic insufficient sleep in humans: a machine learning approach.

Acute and chronic insufficient sleep are associated with adverse health outcomes and risk of accidents. There is therefore a need for biomarkers to monitor sleep debt status. None are currently available. We applied elastic net and ridge regression to transcriptome samples collected in 36 healthy young adults during acute total sleep deprivation and following 1 week of either chronic insufficient (<6 hr) or sufficient sleep (~8.6 hr) to identify panels of mRNA biomarkers of sleep debt status. The size of identified panels ranged from 9 to 74 biomarkers. Panel performance, assessed by leave-one-subject-out cross-validation and independent validation, varied between sleep debt conditions. Using between-subject assessments based on one blood sample, the accuracy of classifying "acute sleep loss" was 92%, but only 57% for classifying "chronic sleep insufficiency." A reasonable accuracy for classifying "chronic sleep insufficiency" could only be achieved by a within-subject comparison of blood samples. Biomarkers for sleep debt status showed little overlap with previously identified biomarkers for circadian phase. Biomarkers for acute and chronic sleep loss also showed little overlap but were associated with common functions related to the cellular stress response, such as heat shock protein activity, the unfolded protein response, protein ubiquitination and endoplasmic reticulum-associated protein degradation, and apoptosis. This characteristic response of whole blood to sleep loss can further aid our understanding of how sleep insufficiencies negatively affect health. Further development of these novel biomarkers for research and clinical practice requires validation in other protocols and age groups.

Translational control plays an important role in the adaptive heat-shock response of Streptomyces coelicolor.

Stress-induced adaptations require multiple levels of regulation in all organisms to repair cellular damage. In the present study we evaluated the genome-wide transcriptional and translational changes following heat stress exposure in the soil-dwelling model actinomycete bacterium, Streptomyces coelicolor. The combined analysis revealed an unprecedented level of translational control of gene expression, deduced through polysome profiling, in addition to transcriptional changes. Our data show little correlation between the transcriptome and 'translatome'; while an obvious downward trend in genome wide transcription was observed, polysome associated transcripts following heat-shock showed an opposite upward trend. A handful of key protein players, including the major molecular chaperones and proteases were highly induced at both the transcriptional and translational level following heat-shock, a phenomenon known as 'potentiation'. Many other transcripts encoding cold-shock proteins, ABC-transporter systems, multiple transcription factors were more highly polysome-associated following heat stress; interestingly, these protein families were not induced at the transcriptional level and therefore were not previously identified as part of the stress response. Thus, stress coping mechanisms at the level of gene expression in this bacterium go well beyond the induction of a relatively small number of molecular chaperones and proteases in order to ensure cellular survival at non-physiological temperatures.

Blood transcriptome based biomarkers for human circadian phase.

Diagnosis and treatment of circadian rhythm sleep-wake disorders both require assessment of circadian phase of the brain's circadian pacemaker. The gold-standard univariate method is based on collection of a 24-hr time series of plasma melatonin, a suprachiasmatic nucleus-driven pineal hormone. We developed and validated a multivariate whole-blood mRNA-based predictor of melatonin phase which requires few samples. Transcriptome data were collected under normal, sleep-deprivation and abnormal sleep-timing conditions to assess robustness of the predictor. Partial least square regression (PLSR), applied to the transcriptome, identified a set of 100 biomarkers primarily related to glucocorticoid signaling and immune function. Validation showed that PLSR-based predictors outperform published blood-derived circadian phase predictors. When given one sample as input, the R2 of predicted vs observed phase was 0.74, whereas for two samples taken 12 hr apart, R2 was 0.90. This blood transcriptome-based model enables assessment of circadian phase from a few samples.

Conserved mRNA-binding proteomes in eukaryotic organisms.

RNA-binding proteins (RBPs) are essential for post-transcriptional regulation of gene expression. Recent high-throughput screens have dramatically increased the number of experimentally identified RBPs; however, comprehensive identification of RBPs within living organisms is elusive. Here we describe the repertoire of 765 and 594 proteins that reproducibly interact with polyadenylated mRNAs in Saccharomyces cerevisiae and Caenorhabditis elegans, respectively. Furthermore, we report the differential association of mRNA-binding proteins (mRPBs) upon induction of apoptosis in C. elegans L4-stage larvae. Strikingly, most proteins composing mRBPomes, including components of early metabolic pathways and the proteasome, are evolutionarily conserved between yeast and C. elegans. We speculate, on the basis of our evidence that glycolytic enzymes bind distinct glycolytic mRNAs, that enzyme-mRNA interactions relate to an ancient mechanism for post-transcriptional coordination of metabolic pathways that perhaps was established during the transition from the early 'RNA world' to the 'protein world'.

Exploiting human and mouse transcriptomic data: Identification of circadian genes and pathways influencing health.

The power of the application of bioinformatics across multiple publicly available transcriptomic data sets was explored. Using 19 human and mouse circadian transcriptomic data sets, we found that NR1D1 and NR1D2 which encode heme-responsive nuclear receptors are the most rhythmic transcripts across sleep conditions and tissues suggesting that they are at the core of circadian rhythm generation. Analyzes of human transcriptomic data show that a core set of transcripts related to processes including immune function, glucocorticoid signalling, and lipid metabolism is rhythmically expressed independently of the sleep-wake cycle. We also identify key transcripts associated with transcription and translation that are disrupted by sleep manipulations, and through network analysis identify putative mechanisms underlying the adverse health outcomes associated with sleep disruption, such as diabetes and cancer. Comparative bioinformatics applied to existing and future data sets will be a powerful tool for the identification of core circadian- and sleep-dependent molecules.

A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing.

Streptomyces coelicolor is a model for studying bacteria renowned as the foremost source of natural products used clinically. Post-genomic studies have revealed complex patterns of gene expression and links to growth, morphological development and individual genes. However, the underlying regulation remains largely obscure, but undoubtedly involves steps after transcription initiation. Here we identify sites involved in RNA processing and degradation as well as transcription within a nucleotide-resolution map of the transcriptional landscape. This was achieved by combining RNA-sequencing approaches suited to the analysis of GC-rich organisms. Escherichia coli was analysed in parallel to validate the methodology and allow comparison. Previously, sites of RNA processing and degradation had not been mapped on a transcriptome-wide scale for E. coli. Through examples, we show the value of our approach and data sets. This includes the identification of new layers of transcriptional complexity associated with several key regulators of secondary metabolism and morphological development in S. coelicolor and the identification of host-encoded leaderless mRNA and rRNA processing associated with the generation of specialized ribosomes in E. coli. New regulatory small RNAs were identified for both organisms. Overall the results illustrate the diversity in mechanisms used by different bacterial groups to facilitate and regulate gene expression.

A human sleep homeostasis phenotype in mice expressing a primate-specific PER3 variable-number tandem-repeat coding-region polymorphism.

In humans, a primate-specific variable-number tandem-repeat (VNTR) polymorphism (4 or 5 repeats 54 nt in length) in the circadian gene PER3 is associated with differences in sleep timing and homeostatic responses to sleep loss. We investigated the effects of this polymorphism on circadian rhythmicity and sleep homeostasis by introducing the polymorphism into mice and assessing circadian and sleep parameters at baseline and during and after 12 h of sleep deprivation (SD). Microarray analysis was used to measure hypothalamic and cortical gene expression. Circadian behavior and sleep were normal at baseline. The response to SD of 2 electrophysiological markers of sleep homeostasis, electroencephalography (EEG) θ power during wakefulness and δ power during sleep, were greater in the Per3(5/5) mice. During recovery, the Per3(5/5) mice fully compensated for the SD-induced deficit in δ power, but the Per3(4/4) and wild-type mice did not. Sleep homeostasis-related transcripts (e.g., Homer1, Ptgs2, and Kcna2) were differentially expressed between the humanized mice, but circadian clock genes were not. These data are in accordance with the hypothesis derived from human data that the PER3 VNTR polymorphism modifies the sleep homeostatic response without significantly influencing circadian parameters.

Mistimed sleep disrupts circadian regulation of the human transcriptome.

Circadian organization of the mammalian transcriptome is achieved by rhythmic recruitment of key modifiers of chromatin structure and transcriptional and translational processes. These rhythmic processes, together with posttranslational modification, constitute circadian oscillators in the brain and peripheral tissues, which drive rhythms in physiology and behavior, including the sleep-wake cycle. In humans, sleep is normally timed to occur during the biological night, when body temperature is low and melatonin is synthesized. Desynchrony of sleep-wake timing and other circadian rhythms, such as occurs in shift work and jet lag, is associated with disruption of rhythmicity in physiology and endocrinology. However, to what extent mistimed sleep affects the molecular regulators of circadian rhythmicity remains to be established. Here, we show that mistimed sleep leads to a reduction of rhythmic transcripts in the human blood transcriptome from 6.4% at baseline to 1.0% during forced desynchrony of sleep and centrally driven circadian rhythms. Transcripts affected are key regulators of gene expression, including those associated with chromatin modification (methylases and acetylases), transcription (RNA polymerase II), translation (ribosomal proteins, initiation, and elongation factors), temperature-regulated transcription (cold inducible RNA-binding proteins), and core clock genes including CLOCK and ARNTL (BMAL1). We also estimated the separate contribution of sleep and circadian rhythmicity and found that the sleep-wake cycle coordinates the timing of transcription and translation in particular. The data show that mistimed sleep affects molecular processes at the core of circadian rhythm generation and imply that appropriate timing of sleep contributes significantly to the overall temporal organization of the human transcriptome.