Gene Expression In Multiple Tissues Research Articles

GEPREP: A comprehensive data atlas of RNA-seq-based gene expression profiles of exercise responses

BackgroundPhysical activity can regulate and affect gene expression in multiple tissues and cells. Recently, with the development of next-generation sequencing, a large number of RNA-sequencing (RNA-seq)-based gene expression profiles about physical activity have been shared in public resources; however, they are poorly curated and underutilized. To tackle this problem, we developed a data atlas of such data through comprehensive data collection, curation, and organization. MethodsThe data atlas, termed gene expression profiles of RNA-seq-based exercise responses (GEPREP), was built on a comprehensive collection of high-quality RNA-seq data on exercise responses. The metadata of each sample were manually curated. Data were uniformly processed and batch effects corrected. All the information was well organized in an easy-to-use website for free search, visualization, and download. ResultsGEPREP now includes 69 RNA-seq datasets of pre- and post-exercise, comprising 26 human datasets (1120 samples) and 43 mouse datasets (1006 samples). Specifically, there were 977 (87.2%) human samples of skeletal muscle and 143 (12.8%) human samples of blood. There were also samples across 9 mice tissues with skeletal muscle (359, 35.7%) and brain (280, 27.8%) accounting for the main fractions. Metadata—including subject, exercise interventions, sampling sites, and post-processing methods—are also included. The metadata and gene expression profiles are freely accessible at http://www.geprep.org.cn/. ConclusionGEPREP is a comprehensive data atlas of RNA-seq-based gene expression profiles responding to exercise. With its reliable annotations and user-friendly interfaces, it has the potential to deepen our understanding of exercise physiology.

The forgotten variable? Does the euthanasia method and sample storage condition influence an organisms transcriptome – a gene expression analysis on multiple tissues in pigs

BackgroundTranscriptomic studies often require collection of fresh tissues post euthanasia. The chosen euthanasia method might have the potential to induce variations in gene expressions that are unlinked with the experimental design. The present study compared the suitability of ‘nitrogen gas in foam’ (ANOXIA) in comparison to a non-barbiturate anaesthetic, T-61® (T61), for euthanizing piglets used in transcriptome research. Further, the effect of common tissue storage conditions, RNAlater™ (RL) and snap freezing in liquid nitrogen (LN2), on gene expression profiles were also analysed.ResultsOn comparison of the 3’mRNA-Seq data generated from pituitary, hypothalamus, liver and lung tissues, no significant differential expression in the protein coding genes were detected between the euthanasia methods. This implies that the nitrogen anoxia method could be a suitable alternative for euthanasia of piglets used in transcriptomic research. However, small nuclear RNAs (snRNAs) that constitute the eukaryotic spliceosomal machinery were found to be significantly higher (log2fold change ≥ 2.0, and adjusted p value ≤ 0.1) in pituitary samples collected using ANOXIA. Non-protein coding genes like snRNAs that play an important role in pre-mRNA splicing can subsequently modify gene expression. Storage in RL was found to be superior in preserving RNA compared to LN2 storage, as evidenced by the significantly higher RIN values in representative samples. However, storage in RL as opposed to LN2, also influenced differential gene expression in multiple tissues, perhaps as a result of its inability to inhibit biological activity during storage. Hence such external sources of variations should be carefully considered before arriving at research conclusions.ConclusionsSource of biological variations like euthanasia method and storage condition can confound research findings. Even if we are unable to prevent the effect of these external factors, it will be useful to identify the impact of these variables on the parameter under observation and thereby prevent misinterpretation of our results.

Abstract 102: Targeted Disruption Of Ctcf Binding Near The Ren Gene Alters Local Gene Expression In Response To Dietary Salt

Blood pressure regulation and hypertension risk may be impacted by changes in chromatin conformation (CC) caused by common genetic variations. CCCTCF-binding factor (CTCF) is a DNA architecture protein that regulates CC and thereby gene expression. We hypothesized that disruption of local CC surrounding the renin gene can alter local gene expression and influence blood pressure changes to dietary salt. Three mutant rat models were created using CRISPR-Cas9 in the Dahl salt-sensitive rat with mutations in one or more CTCF motifs surrounding the renin gene. Disruption of CTCF binding was confirmed at specific mutated sites in all three models. Plasma renin activity (PRA) was measured and RNA-seq analysis was performed in multiple tissues of wild-type (WT) and mutant rats fed a 0.1%, 0.4% or 4.0% NaCl diet for four days. While PRA in WT males responded as expected (17.52±0.81, 12.87±1.00, and 7.13±0.19 ng/mL; p<0.05 for 0.1% and 4.0% vs. 0.4%) on these diets respectively, males from two of three mutant strains failed to increase their PRA on 0.1% NaCl diet. Females were unaffected. At the transcriptional level in kidney cortex, one mutant strain failed to increase renin mRNA expression on 0.1% NaCl diet, while another did not decrease expression on 4.0% NaCl diet. Other local genes Kiss1 and Snrpe were also significantly disrupted in kidney cortex. Renin expression was also disrupted in liver and adrenal gland in two of three mutant strains. Blood pressure and urinary protein excretion were measured in CTCF mutant and WT littermates over the course of 28 days at baseline (3 days, 0.4% NaCl diet), followed by seven days on 0.1% NaCl, then 21 days 4.0% NaCl diet. While no significant effects were observed between WT and mutant males or females on 0.4% or 0.1% NaCl diet, mean arterial pressure response to 4.0% NaCl diet was significantly attenuated in females of one of three mutant strains on days 16, 17 and 20 (169.6±7.09 vs. 144.2±2.69 mmHg at Day 20; p<0.005). No changes in urinary albumin or protein excretion in mutants of either sex were detected. Collectively, these data suggest that disruption of CTCF binding around the renin gene alters PRA and local gene expression in multiple tissues in response to dietary salt.

Feeding during hibernation shifts gene expression toward active season levels in brown bears (Ursus arctos).

Hibernation in bears involves a suite of metabolical and physiological changes, including the onset of insulin resistance, that are driven in part by sweeping changes in gene expression in multiple tissues. Feeding bears glucose during hibernation partially restores active season physiological phenotypes, including partial resensitization to insulin, but the molecular mechanisms underlying this transition remain poorly understood. Here, we analyze tissue-level gene expression in adipose, liver, and muscle to identify genes that respond to midhibernation glucose feeding and thus potentially drive postfeeding metabolical and physiological shifts. We show that midhibernation feeding stimulates differential expression in all analyzed tissues of hibernating bears and that a subset of these genes responds specifically by shifting expression toward levels typical of the active season. Inferences of upstream regulatory molecules potentially driving these postfeeding responses implicate peroxisome proliferator-activated receptor gamma (PPARG) and other known regulators of insulin sensitivity, providing new insight into high-level regulatory mechanisms involved in shifting metabolic phenotypes between hibernation and active states.

Blood-derived mitochondrial DNA copy number is associated with gene expression across multiple tissues and is predictive for incident neurodegenerative disease.

Mitochondrial DNA copy number (mtDNA-CN) is a proxy for mitochondrial function and is associated with aging-related diseases. However, it is unclear how mtDNA-CN measured in blood can reflect diseases that primarily manifest in other tissues. Using the Genotype-Tissue Expression Project, we interrogated relationships between mtDNA-CN measured in whole blood and gene expression from whole blood and 47 additional tissues in 419 individuals. mtDNA-CN was significantly associated with expression of 700 genes in whole blood, including nuclear genes required for mtDNA replication. Significant enrichment was observed for splicing and ubiquitin-mediated proteolysis pathways, as well as target genes for the mitochondrial transcription factor NRF1. In nonblood tissues, there were more significantly associated genes than expected in 30 tissues, suggesting that global gene expression in those tissues is correlated with blood-derived mtDNA-CN. Neurodegenerative disease pathways were significantly associated in multiple tissues, and in an independent data set, the UK Biobank, we observed that higher mtDNA-CN was significantly associated with lower rates of both prevalent (OR = 0.89, CI = 0.83; 0.96) and incident neurodegenerative disease (HR = 0.95, 95% CI = 0.91;0.98). The observation that mtDNA-CN measured in blood is associated with gene expression in other tissues suggests that blood-derived mtDNA-CN can reflect metabolic health across multiple tissues. Identification of key pathways including splicing, RNA binding, and catalysis reinforces the importance of mitochondria in maintaining cellular homeostasis. Finally, validation of the role of mtDNA CN in neurodegenerative disease in a large independent cohort study solidifies the link between blood-derived mtDNA-CN, altered gene expression in multiple tissues, and aging-related disease.

Numerical method for the generalized nonnegative tensor factorization problem

In this paper, we consider the generalized nonnegative tensor factorization (GNTF) problem, which arises in multiple-tissue gene expression and multi-target tracking. Based on the Karhsh-Kuhn-Tucker conditions, the necessary condition of the local solution for the GNTF problem is given. The proximal alternating nonnegative least squares method is designed to solve it, and its convergence theorem is also derived. Numerical examples show that the new method is feasible and effective.

Isolation and identification of endogenous RFamide-related peptides 1 and 3 in the mouse hypothalamus.

Although the RFamide-related peptide (RFRP) preproprotein sequence is known in mice, until now, the molecular structure of the mature, functional peptides processed from the target precursor molecule has not been determined. In the present study, we purified endogenous RFRP1 and RFRP3 peptides from mouse hypothalamic tissue extracts using an immunoaffinity column conjugated with specific antibodies against the mouse C-terminus of RFRP-1 and RFRP-3. Employing liquid chromatography coupled with mass spectrometry, we demonstrated that RFRP1 consists of 15 amino acid residues and RFRP3 consists of 10 amino acid residues (ANKVPHSAANLPLRF-NH2 and SHFPSLPQRF-NH2, respectively). To investigate the distribution of RFRPs in the mouse central nervous system, we performed immunohistochemical staining of the brain sections collected from wild-type and Rfrp knockout animals. These data, together with gene expression in multiple tissues, provide strong confidence that RFRP-immunoreactive neuronal cells are localised in the dorsomedial hypothalamic nucleus (DMH) and between the DMH and the ventromedial hypothalamic nuclei. The identification of RFRP1 and RFRP3 peptides and immunohistochemical visualisation of targeting RFRPs neurones in the mice brain provide the basis for further investigations of the functional biology of RFRPs.

Comparing allele specific expression and local expression quantitative trait loci and the influence of gene expression on complex trait variation in cattle

BackgroundThe mutations changing the expression level of a gene, or expression quantitative trait loci (eQTL), can be identified by testing the association between genetic variants and gene expression in multiple individuals (eQTL mapping), or by comparing the expression of the alleles in a heterozygous individual (allele specific expression or ASE analysis). The aims of the study were to find and compare ASE and local eQTL in 4 bovine RNA-sequencing (RNA-Seq) datasets, validate them in an independent ASE study and investigate if they are associated with complex trait variation.ResultsWe present a novel method for distinguishing between ASE driven by polymorphisms in cis and parent of origin effects. We found that single nucleotide polymorphisms (SNPs) driving ASE are also often local eQTL and therefore presumably cis eQTL. These SNPs often, but not always, affect gene expression in multiple tissues and, when they do, the allele increasing expression is usually the same. However, there were systematic differences between ASE and local eQTL and between tissues and breeds. We also found that SNPs significantly associated with gene expression (p < 0.001) were likely to influence some complex traits (p < 0.001), which means that some mutations influence variation in complex traits by changing the expression level of genes.ConclusionWe conclude that ASE detects phenomenon that overlap with local eQTL, but there are also systematic differences between the SNPs discovered by the two methods. Some mutations influencing complex traits are actually eQTL and can be discovered using RNA-Seq including eQTL in the genes CAST, CAPN1, LCORL and LEPROTL1.

The effects of sex and age on the metabolic response to methionine deprivation, a novel intervention for the treatment of obesity and diabetes

Obesity has become an increasing health problem in the United States and worldwide; effective interventions that promote weight loss are urgently needed. Dietary restriction of methionine promotes leanness and improves metabolic health in mice and humans. However, poor long‐term adherence to this diet limits its translational potential. To address this problem, we have developed short‐term methionine deprivation as a rapid and effective strategy to reduce adiposity and promote metabolic health.We examined the effects of a short‐term MD regimen on the metabolic health of C57BL/6J mice of both sexes, including 1) young mice raised on a chow diet; 2) young mice preconditioned with a Western high‐fat, high‐sucrose diet for 12 weeks; and 3) aged mice on a chow diet. We examined weight, body composition, glucose and insulin tolerance, food intake, activity and energy expenditure. We further examined hepatic steatosis and gene expression in multiple tissues. Using metabolomic and proteomic approaches, we also determined methionine metabolite levels and evaluated global histone post‐translational modification profiles in the liver. Finally, as dietary methionine is an agonist of the protein kinase mTORC1 (mechanistic Target of Rapamycin Complex 1), which is proposed to play a key role in the metabolic response to amino acid‐restricted diets, we examined the role of hepatic mTORC1 in the metabolic response to MD using a mouse model of constitutive hepatic mTORC1 activity.We find that a short‐term MD regimen preferentially reduces fat mass, restoring normal body weight and glycemic control to diet‐induced obese mice of both sexes. Additionally, we have examined the persistence of these metabolic changes in young and aged mice. We find that the benefits of MD do not accrue from calorie restriction, but instead result from increased energy expenditure. Intriguingly, MD promotes increased energy expenditure in both sexes, but induces the FGF21‐UCP1 axis only in males. We also observed sex‐specific effects of MD on lipid metabolism. The metabolic analysis revealed that MD significantly decreases levels of methionine as well as the methionine metabolite SAM (S‐adenosyl methionine) in the liver, and that accordingly, histone methylation in the liver is dramatically downregulated compared to control. Furthermore, using liver‐specific TSC1 knockout mice, which have constitutively active hepatic mTORC1, we determined that the metabolic effects of MD do not depend upon reduced hepatic mTORC1 signaling.Our study sheds new light on the mechanisms by which dietary methionine regulates metabolic health. In particular, our results suggest that sex‐dependent mechanisms may mediate the metabolic response to decreased dietary methionine, and that the FGF21‐UCP1 axis may be dispensable for the metabolic benefits of MD in females. Our results also demonstrate that the metabolic benefits of MD are not mediated by suppression of hepatic mTORC1 signaling. Our study clearly demonstrates the translational potential of MD or MD‐mimetics for the treatment of obesity and type 2 diabetes, diseases which are highly prevalent in the aged.Support or Funding InformationThis research was supported in part by grants from the NIH AG041765 (D.W.L.), AG050135 (D.W.L.), AG051974 (D.W.L.), GM059789‐15/P250VA (J.M.D), a New Investigator Program Award (D.W.L.) and a Collaborative Health Sciences Program Award (V.L.C.) from the Wisconsin Partnership Program, the V. Foundation for Cancer Research (V.L.C.), and a Glenn Foundation Award for Research in the Biological Mechanisms of Aging (D.W.L.), as well as startup funds from the UW‐Madison School of Medicine and Public Health and the UW‐Madison Department of Medicine (V.L.C. and D.W.L.). This research was conducted while D.W.L. was an AFAR Research Grant recipient from the American Federation for Aging Research. D.Y. is supported in part by a fellowship from the American Heart Association (17PRE33410983). S.A.H. is supported by a training grant from the NIH/NIDDK (T32DK007665). This work was supported using facilities and resources from the William S. Middleton Memorial Veterans Hospital. This work does not represent the views of the Department of Veterans Affairs or the United States Government.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bayesian Variable Selection Regression of Multivariate Responses for Group Data

We propose two multivariate extensions of the Bayesian group lasso for variable selection and estimation for data with high dimensional predictors and multi-dimensional response variables. The methods utilize spike and slab priors to yield solutions which are sparse at either a group level or both a group and individual feature level. The incorporation of group structure in a predictor matrix is a key factor in obtaining better estimators and identifying associations between multiple responses and predictors. The approach is suited to many biological studies where the response is multivariate and each predictor is embedded in some biological grouping structure such as gene pathways. Our Bayesian models are connected with penalized regression, and we prove both oracle and asymptotic distribution properties under an orthogonal design. We derive efficient Gibbs sampling algorithms for our models and provide the implementation in a comprehensive R package called MBSGS available on the Comprehensive R Archive Network (CRAN). The performance of the proposed approaches is compared to state-of-the-art variable selection strategies on simulated data sets. The proposed methodology is illustrated on a genetic dataset in order to identify markers grouping across chromosomes that explain the joint variability of gene expression in multiple tissues.

Incorporating covariates into integrated factor analysis of multi-view data.

In modern biomedical research, it is ubiquitous to have multiple data sets measured on the same set of samples from different views (i.e., multi-view data). For example, in genetic studies, multiple genomic data sets at different molecular levels or from different cell types are measured for a common set of individuals to investigate genetic regulation. Integration and reduction of multi-view data have the potential to leverage information in different data sets, and to reduce the magnitude and complexity of data for further statistical analysis and interpretation. In this article, we develop a novel statistical model, called supervised integrated factor analysis (SIFA), for integrative dimension reduction of multi-view data while incorporating auxiliary covariates. The model decomposes data into joint and individual factors, capturing the joint variation across multiple data sets and the individual variation specific to each set, respectively. Moreover, both joint and individual factors are partially informed by auxiliary covariates via nonparametric models. We devise a computationally efficient Expectation-Maximization (EM) algorithm to fit the model under some identifiability conditions. We apply the method to the Genotype-Tissue Expression (GTEx) data, and provide new insights into the variation decomposition of gene expression in multiple tissues. Extensive simulation studies and an additional application to a pediatric growth study demonstrate the advantage of the proposed method over competing methods.

In vivo imaging of clock gene expression in multiple tissues of freely moving mice.

Clock genes are expressed throughout the body, although how they oscillate in unrestrained animals is not known. Here, we show an in vivo imaging technique that enables long-term simultaneous imaging of multiple tissues. We use dual-focal 3D tracking and signal-intensity calibration to follow gene expression in a target area. We measure circadian rhythms of clock genes in the olfactory bulb, right and left ears and cortices, and the skin. In addition, the kinetic relationship between gene expression and physiological responses to experimental cues is monitored. Under stable conditions gene expression is in phase in all tissues. In response to a long-duration light pulse, the olfactory bulb shifts faster than other tissues. In Cry1−/− Cry2−/− arrhythmic mice circadian oscillation is absent in all tissues. Thus, our system successfully tracks circadian rhythms in clock genes in multiple tissues in unrestrained mice.

Functional characterization of insulin-like growth factors in an ancestral fish species, the Shovelnose sturgeon Scaphirhynchus platorhynchus

Observations from the present study provide the first characterization of the GH-IGF axis in Shovelnose sturgeon Scaphirhynchus platorhynchus, an ancestral fish species. An initial characterization of steady-state IGF-I and IGF-II gene expression in multiple tissues was conducted using real-time RT-qPCR. Overall, the tissues had significantly different profiles of IGF-I gene expression, with the highest IGF-I expression observed in the liver. The highest IGF-II gene expression was also observed in the liver, with minimal or no detection in muscle. A comparison between IGF-I and IGF-II expression within individual tissues revealed higher levels of IGF-II than IGF-I mRNA in the spleen, stomach and trunk kidney, and higher levels of relative IGF-I mRNA expression in the intestine and muscle. The GH-IGF axis was further elucidated by observing the effects of exogenous GH on IGF-I and IGF-II expression in liver and muscle tissue. The results revealed a significant dose-dependent response of both hepatic IGF-I and IGF-II, and muscle IGF-I mRNA expression following rbGH administration. At the highest rbGH concentration (240μg/g BW), IGF-I mRNA levels in liver and muscle peaked significantly at 48h, indicating both hepatic and muscle IGF-I expression to be stimulated by GH. Hepatic IGF-II expression was also stimulated 48h following rbGH administration. Expression of IGF-II mRNA was not inducible in the muscle. Few studies have evaluated the effects of exogenous GH on IGF expression in ancestral vertebrate species, and as such, this research provides valuable insight into the evolution of the somatotropic axis in vertebrates.

Functional analysis and transcriptional output of the Göttingen minipig genome.

BackgroundIn the past decade the Göttingen minipig has gained increasing recognition as animal model in pharmaceutical and safety research because it recapitulates many aspects of human physiology and metabolism. Genome-based comparison of drug targets together with quantitative tissue expression analysis allows rational prediction of pharmacology and cross-reactivity of human drugs in animal models thereby improving drug attrition which is an important challenge in the process of drug development.ResultsHere we present a new chromosome level based version of the Göttingen minipig genome together with a comparative transcriptional analysis of tissues with pharmaceutical relevance as basis for translational research. We relied on mapping and assembly of WGS (whole-genome-shotgun sequencing) derived reads to the reference genome of the Duroc pig and predict 19,228 human orthologous protein-coding genes. Genome-based prediction of the sequence of human drug targets enables the prediction of drug cross-reactivity based on conservation of binding sites. We further support the finding that the genome of Sus scrofa contains about ten-times less pseudogenized genes compared to other vertebrates. Among the functional human orthologs of these minipig pseudogenes we found HEPN1, a putative tumor suppressor gene. The genomes of Sus scrofa, the Tibetan boar, the African Bushpig, and the Warthog show sequence conservation of all inactivating HEPN1 mutations suggesting disruption before the evolutionary split of these pig species. We identify 133 Sus scrofa specific, conserved long non-coding RNAs (lncRNAs) in the minipig genome and show that these transcripts are highly conserved in the African pigs and the Tibetan boar suggesting functional significance. Using a new minipig specific microarray we show high conservation of gene expression signatures in 13 tissues with biomedical relevance between humans and adult minipigs. We underline this relationship for minipig and human liver where we could demonstrate similar expression levels for most phase I drug-metabolizing enzymes. Higher expression levels and metabolic activities were found for FMO1, AKR/CRs and for phase II drug metabolizing enzymes in minipig as compared to human. The variability of gene expression in equivalent human and minipig tissues is considerably higher in minipig organs, which is important for study design in case a human target belongs to this variable category in the minipig. The first analysis of gene expression in multiple tissues during development from young to adult shows that the majority of transcriptional programs are concluded four weeks after birth. This finding is in line with the advanced state of human postnatal organ development at comparative age categories and further supports the minipig as model for pediatric drug safety studies.ConclusionsGenome based assessment of sequence conservation combined with gene expression data in several tissues improves the translational value of the minipig for human drug development. The genome and gene expression data presented here are important resources for researchers using the minipig as model for biomedical research or commercial breeding. Potential impact of our data for comparative genomics, translational research, and experimental medicine are discussed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2119-7) contains supplementary material, which is available to authorized users.

Synchronized age-related gene expression changes across multiple tissues in human and the link to complex diseases.

Aging is one of the most important biological processes and is a known risk factor for many age-related diseases in human. Studying age-related transcriptomic changes in tissues across the whole body can provide valuable information for a holistic understanding of this fundamental process. In this work, we catalogue age-related gene expression changes in nine tissues from nearly two hundred individuals collected by the Genotype-Tissue Expression (GTEx) project. In general, we find the aging gene expression signatures are very tissue specific. However, enrichment for some well-known aging components such as mitochondria biology is observed in many tissues. Different levels of cross-tissue synchronization of age-related gene expression changes are observed, and some essential tissues (e.g., heart and lung) show much stronger “co-aging” than other tissues based on a principal component analysis. The aging gene signatures and complex disease genes show a complex overlapping pattern and only in some cases, we see that they are significantly overlapped in the tissues affected by the corresponding diseases. In summary, our analyses provide novel insights to the co-regulation of age-related gene expression in multiple tissues; it also presents a tissue-specific view of the link between aging and age-related diseases.

Dissecting the Rev-erbα Cistrome and the Mechanisms Controlling Circadian Transcription in Liver.

Circadian clocks maintain whole-body metabolic homeostasis by coordinating rhythmic gene expression in multiple tissues. Core clock regulators sustain their own oscillation and confer expression rhythmicity on clock-controlled genes (CCGs). Our unbiased examination of enhancer RNA (eRNA) transcription around the clock in mouse liver identified functional enhancers of circadian genes driven by phase-specific transcription factors (TFs). Rev-erbα emerged as a primary driver of circadian enhancers, leading to oscillating gene expression in opposite phases through direct and indirect regulation. Among Rev-erbα target genes were core clock components and metabolic CCGs. Oscillation of clock genes was enforced by direct competition between Rev-erbα and RORα for binding to cognate motifs in the genome, whereas metabolic CCGs were governed by recruitment of the NCoR/HDAC3 complex to enhancers where Rev-erbα is tethered by tissue-specific TFs. The DNA sequence-mediated competition between Rev-erbα and RORα ensures consistent clock control across all tissues. In contrast, the tethered binding mechanism is tissue-specific and thus allows Rev-erbα to dictate an epigenomic rhythm tailored to the specific need of that tissue. Therefore, discrete modes of recruitment allow Rev-erbα to link the clock to cell-specific functions, including metabolism.

Circadian clocks and pregnancy.

The recognition that 24-h rhythmic processes (“circadian”) underlie many endocrine functions has added a fascinating new temporal dimension to our appreciation of their complexity. Research from various laboratories has revealed circadian gene expression in multiple tissues in the reproductive system of non-pregnant and pregnant mammals. However, many questions regarding role of circadian clocks in the female reproductive system remain unresolved. This Research Topic on Circadian Clocks and Pregnancy brings together a broad and innovative group of researchers to discuss, evaluate, and summarize the key aspects of circadian function as it relates to all aspects of pregnancy from ovulation and implantation to fetal development and parturition. In the initial review, Ditisheim et al. (1) address preeclampsia, a pregnancy-related disease that involves an imbalance of angiogenic factors, which leads to abnormal placentation, marked systemic inflammatory responses, and hypertension. One particularly relevant feature of this disease is the loss of the nocturnal decrease in arterial blood pressure. The authors assess the question of whether disruption of circadian function may represent an occupational risk factor for preeclampsia and make the case that while this condition should be considered a major public health concern, the evidence for shift work increasing the risk of preeclampsia is not yet persuasive enough to draw any solid conclusions. Using a DNA microarray approach, Tasaki et al. (2) analyzed gene expression in uterine endometrial stromal cells during the implantation stage in the rat. In particular, they focused on the evaluation of genes compromising the circadian clockwork as well as downstream clock-controlled genes, since mutations in the former are known to associate with implantation failure and other reproductive defects. Among their findings was evidence for the clock gene Bmal1 controlling inhibin-β, IGF-1, and other endometrial genes. They also developed the hypothesis that alterations in the circadian system during decidualization are important for regulating the expression of genes vital for the formation of the placenta. In his review, Sellix (3) highlights our current understanding of circadian clock functions in the female reproductive system as it relates to the timing of gonadotropin secretion at ovulation and the timing of parturition. He proposes that the neuroendocrine-reproductive axis should be considered a partnership of synchronized and cooperative oscillators whose disruption can help to explain some forms of fertility disorders. In their contribution, Gamble et al. (4) examine the importance of circadian clocks for successful pregnancy. It is well known that shift work increases the risk of miscarriages, preterm birth, low birth weights, etc. These authors identify key gaps in our knowledge of circadian clock function in reproductive physiology and offer useful strategies for better understanding how shift work-induced circadian misalignment leads to reproductive dysfunctions. The complex relationship between cholesterol metabolism, estrogens, and female reproductive cycles is reviewed by Urlep and Rozman (5). For example, various genes in the cholesterol and steroidogenic pathway are rhythmic and are regulated by clock genes. Conversely, the central circadian clock in the suprachiasmatic nuclei (SCN) expresses the estrogen receptor, ERβ, which is also under clock gene control. Thus, perturbations in clock gene expression can have profound effects at multiple levels of the neuroendocrine-reproductive axis. Watanabe et al. (6) address the growing recognition that circadian rhythms during pregnancy have important consequences on fetal development. In their interesting clinical trials, they have determined that preterm babies (week 31 of pregnancy) already have pupillary reflexes and that exposure of these infants to a regular light/dark cycle (as opposed to the more common constant light environment of neonatal units) promotes more daytime activity and body growth. In other words, the direct use of natural circadian cycles of light and dark would appear to offer the preterm infant a surrogate rhythmic environment more conducive to optimal postnatal development, underscoring the importance of the circadian system during pregnancy. While it has long been recognized that female reproductive cycles are under the control of the central circadian clock in the SCN, only in the last few years have the specific details of this hypothalamic regulation been clarified. Miller and Takahashi (7) provide a lucid overview of the contributions of circadian vasopressin signals to the kisspeptin neurons that regulate hypothalamic GnRH secretion as well as the VIP regulation of pituitary prolactin rhythms. They also make a compelling case for the effects of clock gene mutations on pregnancy having important similarities to the impact of senescence on reproductive abnormalities. As documented by this Research Topic, the study of cellular circadian clocks on reproductive systems is still fertile territory for endocrinologists, cell biologists, and clinicians alike. It has great potential for providing testable hypotheses and unified explanations for disparate processes, such as blood pressure and gene expression rhythms, effects of shift work and artificial lighting on fetal development, hormonal rhythms and fertility, etc. An exciting era awaits!

Serine 249 phosphorylation by ATM protein kinase regulates hepatocyte nuclear factor-1α transactivation

Hepatocyte nuclear factor-1 alpha (HNF1α) exerts important effects on gene expression in multiple tissues. Several studies have directly or indirectly supported the role of phosphorylation processes in the activity of HNF1α. However, the molecular mechanism of this phosphorylation remains largely unknown. Using microcapillary liquid chromatography MS/MS and biochemical assays, we identified a novel phosphorylation site in HNF1α at Ser249. We also found that the ATM protein kinase phosphorylated HNF1α at Ser249 in vitro in an ATM-dependent manner and that ATM inhibitor KU55933 treatment inhibited phosphorylation of HNF1α at Ser249 in vivo. Coimmunoprecipitation assays confirmed the association between HNF1α and ATM. Moreover, ATM enhanced HNF1α transcriptional activity in a dose-dependent manner, whereas the ATM kinase-inactive mutant did not. The use of KU55933 confirmed our observation. Compared with wild-type HNF1α, a mutation in Ser249 resulted in a pronounced decrease in HNF1α transactivation, whereas no dominant-negative effect was observed. The HNF1αSer249 mutant also exhibited normal nuclear localization but decreased DNA-binding activity. Accordingly, the functional studies of HNF1αSer249 mutant revealed a defect in glucose metabolism. Our results suggested that ATM regulates the activity of HNF1α by phosphorylation of serine 249, particularly in glucose metabolism, which provides valuable insights into the undiscovered mechanisms of ATM in the regulation of glucose homeostasis.

Cell-specific dysregulation of microRNA expression in obese white adipose tissue.

Obesity is characterized by the excessive accumulation of dysfunctional white adipose tissue (WAT), leading to a strong perturbation of metabolic regulations. However, the molecular events underlying this process are not fully understood. MicroRNAs (miRNAs) are small noncoding RNAs acting as posttranscriptional regulators of gene expression in multiple tissues and organs. However, their expression and roles in WAT cell subtypes, which include not only adipocytes but also immune, endothelial, and mesenchymal stem cells as well as preadipocytes, have not been characterized. Design/Results: By applying differential miRNome analysis, we demonstrate that the expression of several miRNAs is dysregulated in epididymal WAT from ob/ob and high-fat diet-fed mice. Adipose tissue-specific down-regulation of miR-200a and miR-200b and the up-regulation of miR-342-3p, miR-335-5p, and miR-335-3p were observed. Importantly, a similarly altered expression of miR-200a and miR-200b was observed in obese diabetic patients. Furthermore, cell fractionation of mouse adipose tissue revealed that miRNAs are differentially expressed in adipocytes and in subpopulations from the stromal vascular fraction. Finally, integration of transcriptomic data showed that bioinformatically predicted miRNA target genes rarely showed anticorrelated expression with that of targeting miRNA, in contrast to experimentally validated target genes. Taken together, our data indicate that the dysregulated expression of miRNAs occurs in distinct cell types and is likely to affect cell-specific function(s) of obese WAT.

Tissue-specific gene expression and regulation in liver and muscle following chronic corticosteroid administration.

Although corticosteroids (CSs) affect gene expression in multiple tissues, the array of genes that are regulated by these catabolic steroids is diverse, highly tissue specific, and depends on their functions in the tissue. Liver has many important functions in performing and regulating diverse metabolic processes. Muscle, in addition to its mechanical role, is critical in maintaining systemic energy homeostasis and accounts for about 80% of insulin-directed glucose disposal. Consequently, a better understanding of CS pharmacogenomic effects in these tissues would provide valuable information regarding the tissue-specificity of transcriptional dynamics, and would provide insights into the underlying molecular mechanisms of action for both beneficial and detrimental effects.We performed an integrated analysis of transcriptional data from liver and muscle in response to methylprednisolone (MPL) infusion, which included clustering and functional annotation of clustered gene groups, promoter extraction and putative transcription factor (TF) identification, and finally, regulatory closeness (RC) identification.This analysis allowed the identification of critical transcriptional responses and CS-responsive functions in liver and muscle during chronic MPL administration, the prediction of putative transcriptional regulators relevant to transcriptional responses of CS-affected genes which are also potential secondary bio-signals altering expression levels of target-genes, and the exploration of the tissue-specificity and biological significance of gene expression patterns, CS-responsive functions, and transcriptional regulation.The analysis provided an integrated description of the genomic and functional effects of chronic MPL infusion in liver and muscle.