mRNA Transcription Rate Research Articles

Direct Molecular Action of Taurine on Hepatic Gene Expression Associated with the Amelioration of Hypercholesterolemia in Rats.

Taurine can ameliorate hypercholesterolemia by facilitating cholesterol efflux and increasing cytochrome P450 7A1 (CYP7A1) without clear underlying molecular mechanisms. This study aims to elucidate the molecular action of taurine in diet-induced hypercholesterolemia. Male Wistar rats were fed a high cholesterol diet containing 5% taurine for 14 days. Three-dimensional primary hepatocytes from rats were exposed to 10 mM taurine for 24 h. Transcriptome analyses of both the liver and hepatocytes were performed using DNA microarray. Taurine significantly decreased serum cholesterol levels and increased hepatic CYP7A1 mRNA levels and transcription rates in rats. Taurine altered the expression of seventy-seven genes in the liver, involving lipid, drug, amino acid metabolism, and gluconeogenesis pathways. The small heterodimer partner (SHP), a transcription factor regulated by taurine, was suppressed. "Network analysis" revealed a negative correlation between the SHP and induction of CYP7A1 and cytochrome P450 8B1 (CYP8B1). However, CYP7A1 and CYP8B1 levels were not altered by taurine in 3D-primary hepatocytes. Venn diagram analyses of the transcriptomes in both hepatocytes and the liver indicated a consistent upregulation of organic anion transporting polypeptide 2 (OATP2) and betaine homocysteine methyltransferase (BHMT). Taurine ameliorated hypercholesterolemia in rats fed a high cholesterol diet by directly enhancing the hepatic expression of BHMT and OATP2, which modulated the SHP and induced CYP7A1 and CYP8B1, thereby promoting cholesterol catabolism and lowering blood cholesterol levels.

Cell-type-specific mRNA transcription and degradation kinetics in zebrafish embryogenesis from metabolically labeled single-cell RNA-seq

During embryonic development, pluripotent cells assume specialized identities by adopting particular gene expression profiles. However, systematically dissecting the relative contributions of mRNA transcription and degradation to shaping those profiles remains challenging, especially within embryos with diverse cellular identities. Here, we combine single-cell RNA-Seq and metabolic labeling to capture temporal cellular transcriptomes of zebrafish embryos where newly-transcribed (zygotic) and pre-existing (maternal) mRNA can be distinguished. We introduce kinetic models to quantify mRNA transcription and degradation rates within individual cell types during their specification. These models reveal highly varied regulatory rates across thousands of genes, coordinated transcription and destruction rates for many transcripts, and link differences in degradation to specific sequence elements. They also identify cell-type-specific differences in degradation, namely selective retention of maternal transcripts within primordial germ cells and enveloping layer cells, two of the earliest specified cell types. Our study provides a quantitative approach to study mRNA regulation during a dynamic spatio-temporal response.

An in silico FSHD muscle fiber for modeling DUX4 dynamics and predicting the impact of therapy.

Facioscapulohumeral muscular dystrophy (FSHD) is an incurable myopathy linked to the over-expression of the myotoxic transcription factor DUX4. Targeting DUX4 is the leading therapeutic approach, however, it is only detectable in 0.1-3.8% of FSHD myonuclei. How rare DUX4 drives FSHD and the optimal anti-DUX4 strategy are unclear. We combine stochastic gene expression with compartment models of cell states, building a simulation of DUX4 expression and consequences in FSHD muscle fibers. Investigating iDUX4 myoblasts, scRNAseq, and snRNAseq of FSHD muscle we estimate parameters including DUX4 mRNA degradation, transcription and translation rates, and DUX4 target gene activation rates. Our model accurately recreates the distribution of DUX4 and targets gene-positive cells seen in scRNAseq of FSHD myocytes. Importantly, we show DUX4 drives significant cell death despite expression in only 0.8% of live cells. Comparing scRNAseq of unfused FSHD myocytes to snRNAseq of fused FSHD myonuclei, we find evidence of DUX4 protein syncytial diffusion and estimate its rate via genetic algorithms. We package our model into freely available tools, to rapidly investigate the consequences of anti-DUX4 therapy.

Dynamically regulated transcription factors are encoded by highly unstable mRNAs in the Drosophila larval brain.

The level of each RNA species depends on the balance between its rates of production and decay. Although previous studies have measured RNA decay across the genome in tissue culture and single-celled organisms, few experiments have been performed in intact complex tissues and organs. It is therefore unclear whether the determinants of RNA decay found in cultured cells are preserved in an intact tissue, and whether they differ between neighboring cell types and are regulated during development. To address these questions, we measured RNA synthesis and decay rates genome wide via metabolic labeling of whole cultured Drosophila larval brains using 4-thiouridine. Our analysis revealed that decay rates span a range of more than 100-fold, and that RNA stability is linked to gene function, with mRNAs encoding transcription factors being much less stable than mRNAs involved in core metabolic functions. Surprisingly, among transcription factor mRNAs there was a clear demarcation between more widely used transcription factors and those that are expressed only transiently during development. mRNAs encoding transient transcription factors are among the least stable in the brain. These mRNAs are characterized by epigenetic silencing in most cell types, as shown by their enrichment with the histone modification H3K27me3. Our data suggest the presence of an mRNA destabilizing mechanism targeted to these transiently expressed transcription factors to allow their levels to be regulated rapidly with high precision. Our study also demonstrates a general method for measuring mRNA transcription and decay rates in intact organs or tissues, offering insights into the role of mRNA stability in the regulation of complex developmental programs.

Hepatic CCAAT/Enhancer Binding Protein (C/EBP-α and C/EBP-β) Expression Changes with Riboflavin Deficiency, Diet Restriction and Starvation in Rats , ,

To study the role of nuclear regulatory proteins in mediating dietary effects, hepatic CCAAT/enhancer binding protein (C/EBP), mRNA and transcription rate were measured for C/EBP-alpha and C/EBP-beta in nutritional states that profoundly alter energy metabolism and growth. Weanling male Sprague-Dawley rats were fed riboflavin-sufficient (R+) or deficient (R-) diets for 4 wk. A diet-restricted, pair-fed (RP) group was maintained concurrently, because riboflavin-deficient rats voluntarily decrease food consumption by approximately 50% compared with controls. Half of each group was deprived of food for 48 h. The 4-wk treatment altered hepatic levels of both proteins (P < 0.05). C/EBP-alpha protein levels were increased -twofold by diet restriction. C/ EBP-beta protein levels were increased nearly threefold by riboflavin deficiency. Starvation had no significant effect on the expression of either protein. We investigated the mechanism responsible for increased protein by measuring steady-state mRNA levels and transcription rates for C/EBP-alpha and C/EBP-beta. In both isoforms, increases in mRNA were parallel to increases in transcription rates. The nutrient-induced changes in protein, mRNA and transcription rates could not be attributed only to alterations in serum glucagon or insulin concentrations. We conclude that 1) C/EBP-alpha and C/EBP-beta expression responds to diet but may involve different dietary signals for diet restriction vs. riboflavin deficiency; 2) the dietary regulation of C/EBP-alpha and C/EBP-beta expression seems to be controlled in part at the level of gene transcription; and 3) C/EBP-alpha and C/EBP-beta nuclear proteins, by virtue of their increased quantities, may participate in regulating altered energy metabolism and growth by influencing hepatic transcription of key metabolic enzymes.

A newly emerging alphasatellite affects banana bunchy top virus replication, transcription, siRNA production and transmission by aphids.

Banana bunchy top virus (BBTV) is a six-component ssDNA virus (genus Babuvirus, family Nanoviridae) transmitted by aphids, infecting monocots (mainly species in the family Musaceae) and likely originating from South-East Asia where it is frequently associated with self-replicating alphasatellites. Illumina sequencing analysis of banana aphids and leaf samples from Africa revealed an alphasatellite that should be classified in a new genus, phylogenetically related to alphasatellites of nanoviruses infecting dicots. Alphasatellite DNA was encapsidated by BBTV coat protein and accumulated at high levels in plants and aphids, thereby reducing helper virus loads, altering relative abundance (formula) of viral genome components and interfering with virus transmission by aphids. BBTV and alphasatellite clones infected dicot Nicotiana benthamiana, followed by recovery and symptomless persistence of alphasatellite, and BBTV replication protein (Rep), but not alphasatellite Rep, induced leaf chlorosis. Transcriptome sequencing revealed 21, 22 and 24 nucleotide small interfering (si)RNAs covering both strands of the entire viral genome, monodirectional Pol II transcription units of viral mRNAs and pervasive transcription of each component and alphasatellite in both directions, likely generating double-stranded precursors of viral siRNAs. Consistent with the latter hypothesis, viral DNA formulas with and without alphasatellite resembled viral siRNA formulas but not mRNA formulas. Alphasatellite decreased transcription efficiency of DNA-N encoding a putative aphid transmission factor and increased relative siRNA production rates from Rep- and movement protein-encoding components. Alphasatellite itself spawned the most abundant siRNAs and had the lowest mRNA transcription rate. Collectively, following African invasion, BBTV got associated with an alphasatellite likely originating from a dicot plant and interfering with BBTV replication and transmission. Molecular analysis of virus-infected banana plants revealed new features of viral DNA transcription and siRNA biogenesis, both affected by alphasatellite. Costs and benefits of alphasatellite association with helper viruses are discussed.

Computational modeling of RNase, antisense ORF0 RNA, and intracellular compartmentation and their impact on the life cycle of the line retrotransposon

Nearly half of the human genome is occupied by repetitive sequences of ancient virus-like genetic elements. The largest class, comprising 17% of the genome, belong to the type 1 Long INterspersed Elements (LINE-1) and are the only class capable of autonomous propagation in the genome. When epigenetic silencing mechanisms of LINE-1 fail, the proteins encoded by LINE-1 engage in reverse transcription to make new copies of their own or other DNAs that are pasted back into the genome. To elucidate how LINE-1 is dysregulated as a result of carcinogen exposure, we developed a computational model of key elements in the LINE-1 lifecycle, namely, the role of cytosolic ribonuclease (RNase), RNA interference (RNAi) by the antisense ORF0 RNA, and sequestration of LINE-1 products into stress granules and multivesicular structures.The model showed that when carcinogen exposure is represented as either a sudden increase in LINE-1 mRNA count, or as an increase in mRNA transcription rate, the retrotransposon copy number exhibits a distinct threshold behavior above which LINE-1 enters a positive feedback loop that allows the cDNA copy number to grow exponentially. We also found that most of the LINE-1 RNA was degraded via the RNAase pathway and that neither ORF0 RNAi, nor the sequestration of LINE-1 products into granules and multivesicular structures, played a significant role in regulating the retrotransposon’s life cycle. Several aspects of the prediction agree with experimental results and indicate that the model has significant potential to inform future experiments related to LINE-1 activation.

Crystal Structure of a Variant PAM2 Motif of LARP4B Bound to the MLLE Domain of PABPC1.

Eukaryotic cells determine the protein output of their genetic program by regulating mRNA transcription, localization, translation and turnover rates. This regulation is accomplished by an ensemble of RNA-binding proteins (RBPs) that bind to any given mRNA, thus forming mRNPs. Poly(A) binding proteins (PABPs) are prominent members of virtually all mRNPs that possess poly(A) tails. They serve as multifunctional scaffolds, allowing the recruitment of diverse factors containing a poly(A)-interacting motif (PAM) into mRNPs. We present the crystal structure of the variant PAM motif (termed PAM2w) in the N-terminal part of the positive translation factor LARP4B, which binds to the MLLE domain of the poly(A) binding protein C1 cytoplasmic 1 (PABPC1). The structural analysis, along with mutational studies in vitro and in vivo, uncovered a new mode of interaction between PAM2 motifs and MLLE domains.

Size-Dependent Increase in RNA Polymerase II Initiation Rates Mediates Gene Expression Scaling with Cell Size.

Cell size varies during the cell cycle and in response to external stimuli. This requires the tight coordination, or "scaling," of mRNA and protein quantities with the cell volume in order to maintain biomolecule concentrations and cell density. Evidence in cell populations and single cells indicates that scaling relies on the coordination of mRNA transcription rates with cell size. Here, we use a combination of single-molecule fluorescence in situ hybridization (smFISH), time-lapse microscopy, and mathematical modeling in single fission yeast cells to uncover the precise molecular mechanisms that control transcription rates scaling with cell size. Linear scaling of mRNA quantities is apparent in single fission yeast cells during a normal cell cycle. Transcription of both constitutive and periodic genes is a Poisson process with transcription rates scaling with cell size and without evidence for transcriptional off states. Modeling and experimental data indicate that scaling relies on the coordination of RNA polymerase II (RNAPII) transcription initiation rates with cell size and that RNAPII is a limiting factor. We show using real-time quantitative imaging that size increase is accompanied by a rapid concentration-independent recruitment of RNAPII onto chromatin. Finally, we find that, in multinucleated cells, scaling is set at the level of single nuclei and not the entire cell, making the nucleus a determinant of scaling. Integrating our observations in a mechanistic model of RNAPII-mediated transcription, we propose that scaling of gene expression with cell size is the consequence of competition between genes for limiting RNAPII.

Thiouridine-to-Cytidine Conversion Sequencing (TUC-Seq) to Measure mRNA Transcription and Degradation Rates.

The study of RNA dynamics, specifically RNA transcription and decay rates, has gained increasing attention in recent years because various mechanisms have been discovered that affect mRNA half-life, thereby ultimately controlling protein output. Therefore, there is a need for methods enabling minimally invasive, simple and high-throughput determination of RNA stability that can be applied to determine RNA transcription and decay rates in cells and organisms. We have recently developed a protocol which we named TUC-seq to directly distinguish newly synthesized transcripts from the preexisting pool of transcripts by metabolic labeling of nascent RNAs with 4-thiouridine (4sU) followed by osmium tetroxide-mediated conversion of 4sU to cytidine (C) and direct sequencing. In contrast to other related methods (SLAM-seq, TimeLapse-seq), TUC-seq converts 4sU to a native C instead of an alkylated or otherwise modified nucleoside derivative. TUC-seq can be applied to any cell type that is amenable to 4sU labeling. By employing different labeling strategies (pulse or pulse-chase labeling), it is suitable for a broad field of applications and provides a fast and highly efficient means to determine mRNA transcription and decay rates.

Mitochondrial Activity Identifies a Transcriptionally and Functionally Distinct Subset of Aged HSCs with Lineage-Balanced Output

During ageing, the haematopoietic stem cell (HSC) pool expands numerically, but declines functionally. This functional decline is characterised by myeloid skewing and decreased long-term reconstitution potential and clinically manifests as anaemia, immune compromise and increased risk of clonal malignancy. We postulate that HSCs do not synchronously functionally decline with age, but instead represent a spectrum in which physiologically aged HSCs become dominant. In this study, we aimed to reveal properties that might identify physiologically young HSCs during chronological ageing and to exploit these in an attempt to rescue haematopoietic ageing. Young adult ((y), 2-3 months) and aged (old (o), &gt;18 months) mouse HSCs were profiled and we observed a significant decrease (30-50%) in mitochondrial membrane potential (MMP) in oHSCs. Interestingly, a small fraction (15%) of oHSCs maintained a similar MMP to the bulk (70%) of yHSCs. We explored, initially at the transcriptional level, whether these MMPhigh HSCs are distinct from MMPlow HSCs. Strikingly, RNA sequencing of MMPhigh and MMPlow young and aged HSCs revealed that samples cluster by MMP over age. MMPhigh young and aged HSCs were characterised by upregulation of lymphoid and erythroid lineage markers, as well as RNA processing, MYC and E2F pathways. MMPlow young and aged HSCs were transcriptionally associated with ageing, inflammation and myeloid bias. Based on these results, we hypothesised that enhancing MMP in oHSCs might restore lineage-balanced peripheral blood (PB) output, used as a measure of functional improvement. To achieve this, we chemically enhanced MMP in oHSCs in vivo, using the mitochondrially-targeted drug mitoquinol (MQ). Interestingly, our initial experiments show that a 5-day treatment with MQ significantly shifted the B-cell/myeloid ratio in PB from 0.6 (aged) to 1.5 (MQ), in the direction of the ratio observed in young mice (2.9). This ratio change was not due to numerical depletion of myeloid cells, but due to restoration of B-cell numbers, including IgM+ B-cells commonly reduced with age. To assess whether the effect of MQ was due to HSC-intrinsic changes and could be sustained over time, HSCs were isolated from MQ-treated or untreated aged mice and transplanted into lethally irradiated recipients. Our experiments to date show that HSCs isolated from MQ-treated aged mice show superior engraftment and faster and greater B-cell reconstitution than HSCs from age-matched untreated animals, and that these improvements are stable over the 16-week assay. Based on our sequencing results indicating that RNA processing, MYC and E2F pathways are associated with MMPhigh HSCs, and previous work reporting a relationship between MMP and rate of mRNA transcription (das Neves et al., PLoS Biol. 2010; Johnston et al., PLoS Comput. Biol. 2012), we explored the possibility that MMP might orchestrate the observed changes by altering transcriptional rate of HSCs. We tested transcription rate in HSCs in vivo and found that yHSCs display a particularly high rate of mRNA transcription. Rate of transcription was significantly reduced in oHSCs compared to yHSCs, equivalent to their quantitative reduction in MMP. We could demonstrate a direct correlation between MMP and transcription rate in HSCs, by showing that MMPhigh sorted HSCs were transcribing nearly twice as fast as MMPlow sorted HSCs. Furthermore, in vivo injection with mitochondrial uncoupler CCCP caused a similar reduction in transcription rate of HSCs (&gt;50%) as did conventional RNA Pol-II inhibitors (DRB, Flavopiridol), hereby demonstrating that transcription rate directly depends on MMP. This work indicates that mitochondrial state can separate HSCs with distinct transcriptional profiles linked to different cell fates. We speculate that changes in transcriptional profile arise from MMP-driven regulation of transcriptional rate in HSCs. This would open up the possibility that pharmacological manipulation of mitochondrial activity can alter transcriptional programs of HSCs with consequences for functionality. Disclosures No relevant conflicts of interest to declare.

Obtaining insights from high-dimensional data: sparse principal covariates regression

BackgroundData analysis methods are usually subdivided in two distinct classes: There are methods for prediction and there are methods for exploration. In practice, however, there often is a need to learn from the data in both ways. For example, when predicting the antibody titers a few weeks after vaccination on the basis of genomewide mRNA transcription rates, also mechanistic insights about the effect of vaccinations on the immune system are sought. Principal covariates regression (PCovR) is a method that combines both purposes. Yet, it misses insightful representations of the data as these include all the variables.ResultsHere, we propose a sparse extension of principal covariates regression such that the resulting solutions are based on an automatically selected subset of the variables. Our method is shown to outperform competing methods like sparse principal components regression and sparse partial least squares in a simulation study. Furthermore good performance of the method is illustrated on publicly available data including antibody titers and genomewide transcription rates for subjects vaccinated against the flu: the selected genes by sparse PCovR are higly enriched for immune related terms and the method predicts the titers for an independent test sample well. In comparison, no significantly enriched terms were found for the genes selected by sparse partial least squares and out-of-sample prediction was worse.ConclusionsSparse principal covariates regression is a promising and competitive tool for obtaining insights from high-dimensional data.AvailabilityThe source code implementing our proposed method is available from GitHub, together with all scripts used to extract, pre-process, analyze, and post-process the data: https://github.com/katrijnvandeun/SPCovR.

Inferring transcriptional logic from multiple dynamic experiments.

MotivationThe availability of more data of dynamic gene expression under multiple experimental conditions provides new information that makes the key goal of identifying not only the transcriptional regulators of a gene but also the underlying logical structure attainable.ResultsWe propose a novel method for inferring transcriptional regulation using a simple, yet biologically interpretable, model to find the logic by which a set of candidate genes and their associated transcription factors (TFs) regulate the transcriptional process of a gene of interest. Our dynamic model links the mRNA transcription rate of the target gene to the activation states of the TFs assuming that these interactions are consistent across multiple experiments and over time. A trans-dimensional Markov Chain Monte Carlo (MCMC) algorithm is used to efficiently sample the regulatory logic under different combinations of parents and rank the estimated models by their posterior probabilities. We demonstrate and compare our methodology with other methods using simulation examples and apply it to a study of transcriptional regulation of selected target genes of Arabidopsis Thaliana from microarray time series data obtained under multiple biotic stresses. We show that our method is able to detect complex regulatory interactions that are consistent under multiple experimental conditions.Availability and implementationPrograms are written in MATLAB and Statistics Toolbox Release 2016b, The MathWorks, Inc., Natick, Massachusetts, United States and are available on GitHub https://github.com/giorgosminas/TRS and at http://www2.warwick.ac.uk/fac/sci/systemsbiology/research/software.Supplementary information Supplementary data are available at Bioinformatics online.

Chronic exposure to cannabinoids during adolescence causes long-lasting behavioral deficits in adult mice.

Regular use of marijuana during adolescence enhances the risk of long‐lasting neurobiological changes in adulthood. The present study was aimed at assessing the effect of long‐term administration of the synthetic cannabinoid WIN55212.2 during adolescence in young adult mice. Adolescent mice aged 5 weeks were subjected daily to the pharmacological action of WIN55212.2 for 3 weeks and were then left undisturbed in their home cage for a 5‐week period and finally evaluated by behavioral testing. Mice that received the drug during adolescence showed memory impairment in the Morris water maze, as well as a dose‐dependent memory impairment in fear conditioning. In addition, the administration of 3 mg/kg WIN55212.2 in adolescence increased adult hippocampal AEA levels and promoted DNA hypermethylation at the intragenic region of the intracellular signaling modulator Rgs7, which was accompanied by a lower rate of mRNA transcription of this gene, suggesting a potential causal relation. Although the concrete mechanisms underlying the behavioral observations remain to be elucidated, we demonstrate that long‐term administration of 3 mg/kg of WIN during adolescence leads to increased endocannabinoid levels and altered Rgs7 expression in adulthood and establish a potential link to epigenetic changes.

Dynamic regulation of mRNA decay during neural development.

BackgroundGene expression patterns are determined by rates of mRNA transcription and decay. While transcription is known to regulate many developmental processes, the role of mRNA decay is less extensively defined. A critical step toward defining the role of mRNA decay in neural development is to measure genome-wide mRNA decay rates in neural tissue. Such information should reveal the degree to which mRNA decay contributes to differential gene expression and provide a foundation for identifying regulatory mechanisms that affect neural mRNA decay.ResultsWe developed a technique that allows genome-wide mRNA decay measurements in intact Drosophila embryos, across all tissues and specifically in the nervous system. Our approach revealed neural-specific decay kinetics, including stabilization of transcripts encoding regulators of axonogenesis and destabilization of transcripts encoding ribosomal proteins and histones. We also identified correlations between mRNA stability and physiologic properties of mRNAs; mRNAs that are predicted to be translated within axon growth cones or dendrites have long half-lives while mRNAs encoding transcription factors that regulate neurogenesis have short half-lives. A search for candidate cis-regulatory elements identified enrichment of the Pumilio recognition element (PRE) in mRNAs encoding regulators of neurogenesis. We found that decreased expression of the RNA-binding protein Pumilio stabilized predicted neural mRNA targets and that a PRE is necessary to trigger reporter-transcript decay in the nervous system.ConclusionsWe found that differential mRNA decay contributes to the relative abundance of transcripts involved in cell-fate decisions, axonogenesis, and other critical events during Drosophila neural development. Neural-specific decay kinetics and the functional specificity of mRNA decay suggest the existence of a dynamic neurodevelopmental mRNA decay network. We found that Pumilio is one component of this network, revealing a novel function for this RNA-binding protein.Electronic supplementary materialThe online version of this article (doi:10.1186/s13064-015-0038-6) contains supplementary material, which is available to authorized users.

Cidan inhibits liver cancer cell growth by reducing COX-2 and VEGF expression and cell cycle arrest.

Cidan is a traditional Chinese medicine formula that has been used for >10 years as an antitumor drug. In the present study, the antitumor effect of cidan on hepatocellular carcinoma (HCC) and the underlying molecular mechanisms were investigated. A total of 372 patients with primary HCC, as confirmed by pathological examination in the Eastern Hepatobiliary Surgery Hospital and Beijing Oncology Hospital of Weida TCM, were prospectively enrolled in the study. In total, 92 patients were treated with cidan capsules for three months postoperatively, while 280 patients served as controls. The efficacy of cidan was analyzed by monitoring associated symptoms and liver function tests, including measuring the levels of α-1-fetoprotein, α-L-fucosidase, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase and γ-glutamyl transferase. In addition, in vivo analysis was performed using mice Hepa1-6 xenograft models, while in vitro studies were performed with SMMC-7721 and CSQT-1 cells; this included cidan-dependent cell viability and migration assays, cell cycle analyses and the evaluation of cidan effects on cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) mRNA transcription rates using quantitative polymerase chain reaction. The postoperative two-year overall survival (77 and 58% for the cidan and control groups, respectively; P<0.01) and disease-free survival (36 and 24% for the cidan and control groups, respectively; P<0.01) rates were superior in the cidan-treated group when compared with the control. In addition, the size and weight of the tumor xenografts in the C57BL/6 mice were significantly reduced in a time- and dose-dependent manner following cidan treatment (P<0.01). Cidan significantly reduced the cell viability of SMMC-7721 and CSQT-1 cells after four and five days when compared with the control (P<0.01). Furthermore, COX-2 and VEGF mRNA expression levels decreased following cidan treatment (P<0.01), and cidan treatment resulted in enhanced G1 and G2/M cell cycle arrest of CSQT-1 cells. Therefore, cidan effectively inhibited cell proliferation, reduced cell viability and downregulated COX-2 and VEGF expression levels in hepatoma cells.

Transient Changes in Intercellular Protein Variability Identify Sources of Noise in Gene Expression

Protein levels differ considerably between otherwise identical cells, and these differences significantly affect biological function and phenotype. Previous work implicated various noise mechanisms that drive variability in protein copy numbers across an isogenic cell population. For example, transcriptional bursting of mRNAs has been shown to be a major source of noise in the expression of many genes. Additional expression variability, referred to as extrinsic noise, arises from intercellular variations in mRNA transcription and protein translation rates attributed to cell-to-cell differences in cell size, abundance of ribosomes, etc. We propose a method to determine the magnitude of different noise sources in a given gene of interest. The method relies on blocking transcription and measuring changes in protein copy number variability over time. Our results show that this signal has sufficient information to quantify both the extent of extrinsic noise and transcription bursting in gene expression. Moreover, if the mean mRNA count is known, then the relative contributions of transcription versus translation rate fluctuations to extrinsic noise can also be determined. In summary, our study provides an easy-to-implement method for characterizing noisy protein expression that complements existing techniques for studying stochastic dynamics of genetic circuits.

Heritable variation of mRNA decay rates in yeast.

Gene expression levels are determined by the balance between rates of mRNA transcription and decay, and genetic variation in either of these processes can result in heritable differences in transcript abundance. Although the genetics of gene expression has been a subject of intense interest, the contribution of heritable variation in mRNA decay rates to gene expression variation has received far less attention. To this end, we developed a novel statistical framework and measured allele-specific differences in mRNA decay rates in a diploid yeast hybrid created by mating two genetically diverse parental strains. We estimate that 31% of genes exhibit allelic differences in mRNA decay rates, of which 350 can be identified at a false discovery rate of 10%. Genes with significant allele-specific differences in mRNA decay rates have higher levels of polymorphism compared to other genes, with all gene regions contributing to allelic differences in mRNA decay rates. Strikingly, we find widespread evidence for compensatory evolution, such that variants influencing transcriptional initiation and decay have opposite effects, suggesting that steady-state gene expression levels are subject to pervasive stabilizing selection. Our results demonstrate that heritable differences in mRNA decay rates are widespread and are an important target for natural selection to maintain or fine-tune steady-state gene expression levels.

Targeting Estrogen-Related Receptor Alpha Inhibits Epithelial-to-Mesenchymal Transition and Stem Cell Properties of Ovarian Cancer Cells

Epithelial-mesenchymal transition represents a key event in cancer progression and has emerged as a promising anticancer target. Estrogen-related receptor alpha (ERRα) is frequently elevated in advanced-stage ovarian cancer, but its potential role in tumor progression is not known. Here we show that ERRα functions in epithelial-mesenchymal transition and in subsequent stem cell traits responsible for the acquisition of high degree of aggressiveness and potential for metastasis that are characteristic of ovarian cancer. Importantly, targeted inhibition of ERRα also inhibited the expression of Snail, a repressor of E-cadherin and an inducer of epithelial-mesenchymal transition. Interestingly, induction of Snail resulted from not only changes in mRNA transcription rate but also mRNA stability. We thus identified the miR-200 family as a new player in the ERRα-mediated posttranscriptional regulation of Snail, and antagonism of miR-200a/b could revert the decreased expression of Snail and reversal of epithelial-mesenchymal transition and stem cell characteristics due to ERRα depletion. Finally, we showed that RNA interference-mediated inhibition of ERRα significantly reduced tumor burden, ascites formation, and metastatic peritoneal nodules in vivo in an orthotopic model of ovarian cancer. These results suggest ERRα activation as a mechanism of tumor aggressiveness and imply that targeting ERRα may be a promising approach in ovarian cancer treatment.

Statistical inference of regulatory networks for circadian regulation.

We assess the accuracy of various state-of-the-art statistics and machine learning methods for reconstructing gene and protein regulatory networks in the context of circadian regulation. Our study draws on the increasing availability of gene expression and protein concentration time series for key circadian clock components in Arabidopsis thaliana. In addition, gene expression and protein concentration time series are simulated from a recently published regulatory network of the circadian clock in A. thaliana, in which protein and gene interactions are described by a Markov jump process based on Michaelis-Menten kinetics. We closely follow recent experimental protocols, including the entrainment of seedlings to different light-dark cycles and the knock-out of various key regulatory genes. Our study provides relative network reconstruction accuracy scores for a critical comparative performance evaluation, and sheds light on a series of highly relevant questions: it quantifies the influence of systematically missing values related to unknown protein concentrations and mRNA transcription rates, it investigates the dependence of the performance on the network topology and the degree of recurrency, it provides deeper insight into when and why non-linear methods fail to outperform linear ones, it offers improved guidelines on parameter settings in different inference procedures, and it suggests new hypotheses about the structure of the central circadian gene regulatory network in A. thaliana.