Changes In Rate Of Transcription Research Articles

Rapid-Kinetics Degron Benchmarking Reveals Off-Target Activities and Mixed Agonism-Antagonism of MYB Inhibitors

The transcription factor (TF) MYB is a critical transcriptional dependency of acute myeloid leukemia (AML) where it has been a long-term focus of drug development efforts. However, development of transcriptional inhibitors is hampered by the lack of a generally accepted functional cellular readout to characterize their target specificity and on-target activity. We reasoned that the specificity and on-target activity of MYB inhibitors would be best evaluated in a rapid kinetics system where their immediate transcriptional effects are benchmarked against targeted MYB degradation. We began by engineering a chemical degron model and established the direct gene-regulatory functions of MYB in a nascent transcriptomics assay. We fused the C-terminus of MYB with an FKBP12 F36V (dTAG) domain and a fluorescent tag by a homozygous knock-in of the FKBP12 F36V-coding DNA sequence into the endogenous MYB locus in MV411 cells. The resulting fusion protein was nearly completely degraded after a 1-hour treatment with dTAG V-1, a highly specific VHL-engaging PROTAC. As expected, degradation of MYB resulted in a profound loss of cell viability, consistent with the effects of a genetic MYB knockout in AML cells. To establish MYB's direct gene-regulatory functions, we measured genome-wide rates of nascent mRNA synthesis by thiol (SH)-linked alkylation metabolic sequencing of RNA (SLAM-seq) after a 1-hour MYB degradation. Defining direct targets as those genes which displayed significant changes in transcription rates (FDR<0.0), we detected 450 genes directly regulated by MYB. Of these, 319 genes were downregulated and 131 genes were upregulated, indicating that MYB acts as a transcriptional activator and repressor of these genes, respectively. In parallel, we performed SLAM-seq in AML cells treated with six agents reported as MYB inhibitors: MYBMIM, celastrol, naphthol AS-E phosphate, mebendazole, plumbagin and all-trans retinoic acid (ATRA). For comparison, we treated MV411 cells with the BET bromodomain inhibitor JQ1, which has been reported to indirectly inhibit MYB by interfering with its expression and function. The MYB inhibitors displayed a dramatic variability in the number of dysregulated genes, varying from 19 (naphthol) to 1123 (MYBMIM). In contrast, JQ1 caused widespread and bimodal effects, altering the transcription rates of >2000 genes in both directions. On pairwise overlap, the MYB inhibitors captured a relatively minor portion of the direct MYB program (between 5-155, or 1-34%, of the 450 direct MYB targets), compared with 43% of the MYB program captured by JQ1. Nonetheless, the MYB inhibitors displayed a stronger specificity for MYB target genes compared to JQ1, because they elicited much narrower responses. Thus, while MYB inhibitors displayed strong enrichments for primary MYB targets, they did not attenuate the entire MYB transcriptional program, and significant portions of their activities appeared to be off-target. In addition, the inhibitors displayed bimodal effects on the transcription of MYB-regulated genes, further activating subsets of genes that were repressed by MYB degradation, and vice versa. These observations uncovered unexpected activities of MYB inhibitors as context-dependent mixed agonists-antagonists. We conclude that MYB has a narrow direct transcriptional program in AML cells, which is only partially captured by the existing MYB inhibitors, which display partial specificity for MYB targets and act as mixed agonists-antagonists. Our work will serve as a proof-of-principle demonstration of the use of rapid kinetic resolution of TF degron models for a more precise characterization of the target specificity and efficacy of TF-directed inhibitors. Benchmarking of TF modulators against degron models in nascent transcriptomics assays should be considered as an important criterion in their functional characterization.

Buffering of transcription rate by mRNA half-life is a conserved feature of Rett syndrome models

Transcriptional changes in Rett syndrome (RTT) are assumed to directly correlate with steady-state mRNA levels, but limited evidence in mice suggests that changes in transcription can be compensated by post-transcriptional regulation. We measure transcription rate and mRNA half-life changes in RTT patient neurons using RATEseq, and re-interpret nuclear and whole-cell RNAseq from Mecp2 mice. Genes are dysregulated by changing transcription rate or half-life and are buffered when both change. We utilized classifier models to predict the direction of transcription rate changes and find that combined frequencies of three dinucleotides are better predictors than CA and CG. MicroRNA and RNA-binding Protein (RBP) motifs are enriched in 3ʹUTRs of genes with half-life changes. Nuclear RBP motifs are enriched on buffered genes with increased transcription rate. We identify post-transcriptional mechanisms in humans and mice that alter half-life or buffer transcription rate changes when a transcriptional modulator gene is mutated in a neurodevelopmental disorder.

Regulation of perineuronal net components in the synaptic bouton vicinity on lumbar α-motoneurons in the rat after spinalization and locomotor training: New insights from spatio-temporal changes in gene, protein expression and WFA labeling

Chondroitin sulfate proteoglycans (CSPGs) consist of core proteins and glycosaminoglycan side chains. Tenascins, and hyaluronan and proteoglycan link protein 1 (HAPLN), link CSPGs with a hyaluronan backbone to constitute perineuronal nets (PNNs), which ensheath preferentially highly active neurons to maintain architecture and stabilize synapses, but restrict repair plasticity. Spinal cord injury increases CSPG core protein levels in the lesion proximity, limiting permissiveness of the extracellular milieu for fiber regrowth, however regulation of PNNs structure in the vicinity of distant α-motoneurons (MNs) in the course of degeneration and reorganization of their inputs requires research. Here, we examined early and late changes in CSPGs, HAPLN1, tenascin-R, and glial activation along the spinal cord in male rats with complete spinal cord transection (Th10), and their impact on PNNs ensheathing lumbar MNs innervating ankle extensor and flexor muscles, which are in different loading states in paraplegic rats. We show that (1) distance from the lesion site and time after injury (2–5 weeks) differentiate degree of changes in transcription rates (measured with RT-qPCR) of PNNs proteins with increased CSPGs and decreased HAPLN1 transcripts, suggesting long-term PNN destabilization in majority of spinal segments, (2) in lumbar segments PNN composition is not MN-class (extensor vs flexor) specific, both showing early decrease and late upregulation of Wisteria floribunda agglutinin (WFA) labeling in vicinity of synaptic boutons on MNs, (3) long-term locomotor training tends to reduce WFA(+) PNNs, but not their protein components (immunofluorescence measurements) around MNs. Our results suggest that training-induced regulation may target glycan structures of CSPGs.

Metabolic labeling of RNA uncovers the contribution of transcription and decay rates on hypoxia-induced changes in RNA levels.

Cells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. However, most transcriptomic studies do not distinguish the relative contribution of transcription, RNA processing, and RNA degradation processes to cellular homeostasis. Here we used metabolic labeling followed by massive parallel sequencing of newly transcribed and preexisting RNA fractions to simultaneously analyze RNA synthesis and decay in primary endothelial cells exposed to low oxygen tension. We found that changes in transcription rates induced by hypoxia are the major determinant of changes in RNA levels. However, degradation rates also had a significant contribution, accounting for 24% of the observed variability in total mRNA. In addition, our results indicated that hypoxia led to a reduction of the overall mRNA stability from a median half-life in normoxia of 8.7 h, to 5.7 h in hypoxia. Analysis of RNA content per cell confirmed a decrease of both mRNA and total RNA in hypoxic samples and that this effect is dependent on the EGLN/HIF/TSC2 axis. This effect could potentially contribute to fundamental global responses such as inhibition of translation in hypoxia. In summary, our study provides a quantitative analysis of the contribution of RNA synthesis and stability to the transcriptional response to hypoxia and uncovers an unexpected effect on the latter.

The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription.

The signal recognition particle (SRP), responsible for co-translational protein targeting and delivery to cellular membranes, depends on the native long-hairpin fold of its RNA to confer functionality. Since RNA initiates folding during its synthesis, we used high-resolution optical tweezers to follow in real time the co-transcriptional folding of SRP RNA. Surprisingly, SRP RNA folding is robust to transcription rate changes and the presence or absence of its 5'-precursor sequence. The folding pathway also reveals the obligatory attainment of a non-native hairpin intermediate (H1) that eventually rearranges into the native fold. Furthermore, H1 provides a structural platform alternative to the native fold for RNase P to bind and mature SRP RNA co-transcriptionally. Delays in attaining the final native fold are detrimental to the cell, altogether showing that a co-transcriptional folding pathway underpins the proper biogenesis of function-essential SRP RNA.

In vitro study on the regulation of cellular mRNA levels by changes in transcription rate and transcript stability in fish red blood cells.

The interplay of transcriptional and post-transcriptional processes in the regulation of gene expression has been extensively studied in mammals but little is known in other vertebrates so far. Most non-mammalian vertebrates are faced with environmental cues and stressors distinct from those experienced by mammals and thus it is likely that the gene expression strategies differ from those of mammals. Here we performed experiments to study in vitro the various levels of gene expression regulation in nucleated fish red blood cells. Three critical environmental cues frequently experienced by fish were chosen: exposure to hypoxia (2.5%), ambient water temperature increase by 10°C (from 10°C to 20°C), and exposure to stress hormones (represented by the β-adrenergic agonist isoproterenol). We found that β-adrenergic stimulation increases the stability of the β3b-adrenergic receptor (β3b-ar) mRNA, suggesting that mRNA stability can play a role in the regulation of hormonal stress responses in fish. The β3b-ar gene encodes a unique β-adrenergic receptor subtype in fish red blood cells which controls the β-Na+/H+ exchanger activity - an important component of responses to oxygen limitations. Our results furthermore show a yet undescribed link between the Hif1a signaling pathway and the β-adrenergic receptor response. After β-adrenergic stimulation, the transcription of hif1a was activated significantly after 4 hours of exposure. So far, such a response has only been described from mammalian species. This indicates that the β-AR is fundamental to the molecular and physiological responses to hypoxia and that Hif1a might have additional functions than those already known.

Abstract 2214: Dynamic transcriptome analysis of DNA damage pathways under hypoxia

Abstract During the outgrowth of tumor, gradients of hypoxic regions develop due to reduced oxygen diffusion. Intratumoral hypoxia is a prognostic factor associated with decreased disease-free survival and increased resistance to radiotherapy and chemotherapy in different solid cancers. We have previously shown that chronic hypoxia suppresses DNA damage repair pathways partly through decreased protein translation efficiency. The RNA levels of repair pathway genes have also been shown to be downregulated in hypoxia; however, most of the RNA studies do not distinguish between RNA synthesis and degradation. Here, we use novel metabolic labeling of newly synthesized RNA to investigate the dynamic changes in transcription rates of DNA damage repair pathways under hypoxia. 4-thiouridine (4sU), a uridine analogue, was added to the media of DU145 cells (prostate cancer cell line) that have been cultured in 21% or 0.2% oxygen concentrations for 72 hours. Total RNA was extracted and 4sU-tagged RNA was biotinylated and separated from untagged pre-existing RNA using streptavidin coated magnetic beads. Real-time RT-PCR was used to quantify the expression of repair pathways in three RNA subsets (total, pre-existing, and newly synthesized). Parallel experiments were performed in RNA harvested from the cytoplasmic and nuclear factions of cells. We found that with 30-minute 4sU incubation, most 4sU-tagged RNA is still in the nucleus, and is subjected to little degradation, thus reflecting the average transcription rate. The percentage of newly synthesized RNA is lower in hypoxic than in normoxic cells. Importantly, the transcript levels of homologous recombination (HR) repair pathway regulators, RAD51, BRCA1 and BRCA2, are decreased in both total and newly-synthesized RNA of hypoxic cells. Our results demonstrate that in addition to translational regulation, hypoxia may also downregulate repair pathways through suppression of transcription. This 4sU assay provides opportunities for further analysis of differential RNA synthesis and decay of DNA damage repair pathways between hypoxia and normoxia. Citation Format: Winnie W. Lo, Gaetano Zafarana, Robert G. Bristow. Dynamic transcriptome analysis of DNA damage pathways under hypoxia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2214. doi:10.1158/1538-7445.AM2015-2214

Direct measurement of transcription rates reveals multiple mechanisms for configuration of the Arabidopsis ambient temperature response

BackgroundSensing and responding to ambient temperature is important for controlling growth and development of many organisms, in part by regulating mRNA levels. mRNA abundance can change with temperature, but it is unclear whether this results from changes in transcription or decay rates, and whether passive or active temperature regulation is involved.ResultsUsing a base analog labelling method, we directly measured the temperature coefficient, Q10, of mRNA synthesis and degradation rates of the Arabidopsis transcriptome. We show that for most genes, transcript levels are buffered against passive increases in transcription rates by balancing passive increases in the rate of decay. Strikingly, for temperature-responsive transcripts, increasing temperature raises transcript abundance primarily by promoting faster transcription relative to decay and not vice versa, suggesting a global transcriptional process exists that controls mRNA abundance by temperature. This is partly accounted for by gene body H2A.Z which is associated with low transcription rate Q10, but is also influenced by other marks and transcription factor activities.ConclusionsOur data show that less frequent chromatin states can produce temperature responses simply by virtue of their rarity and the difference between their thermal properties and those of the most common states, and underline the advantages of directly measuring transcription rate changes in dynamic systems, rather than inferring rates from changes in mRNA abundance.

Extracting rate changes in transcriptional regulation from MEDLINE abstracts

BackgroundTime delays are important factors that are often neglected in gene regulatory network (GRN) inference models. Validating time delays from knowledge bases is a challenge since the vast majority of biological databases do not record temporal information of gene regulations. Biological knowledge and facts on gene regulations are typically extracted from bio-literature with specialized methods that depend on the regulation task. In this paper, we mine evidences for time delays related to the transcriptional regulation of yeast from the PubMed abstracts.ResultsSince the vast majority of abstracts lack quantitative time information, we can only collect qualitative evidences of time delays. Specifically, the speed-up or delay in transcriptional regulation rate can provide evidences for time delays (shorter or longer) in GRN. Thus, we focus on deriving events related to rate changes in transcriptional regulation. A corpus of yeast regulation related abstracts was manually labeled with such events. In order to capture these events automatically, we create an ontology of sub-processes that are likely to result in transcription rate changes by combining textual patterns and biological knowledge. We also propose effective feature extraction methods based on the created ontology to identify the direct evidences with specific details of these events. Our ontologies outperform existing state-of-the-art gene regulation ontologies in the automatic rule learning method applied to our corpus. The proposed deterministic ontology rule-based method can achieve comparable performance to the automatic rule learning method based on decision trees. This demonstrates the effectiveness of our ontology in identifying rate-changing events. We also tested the effectiveness of the proposed feature mining methods on detecting direct evidence of events. Experimental results show that the machine learning method on these features achieves an F1-score of 71.43%.ConclusionsThe manually labeled corpus of events relating to rate changes in transcriptional regulation for yeast is available in https://sites.google.com/site/wentingntu/data. The created ontologies summarized both biological causes of rate changes in transcriptional regulation and corresponding positive and negative textual patterns from the corpus. They are demonstrated to be effective in identifying rate-changing events, which shows the benefits of combining textual patterns and biological knowledge on extracting complex biological events.

Compromised paraspeckle formation as a pathogenic factor in FUSopathies

Paraspeckles are nuclear bodies formed by a set of specialized proteins assembled on the long non-coding RNA NEAT1; they have a role in nuclear retention of hyperedited transcripts and are associated with response to cellular stress. Fused in sarcoma (FUS) protein, linked to a number of neurodegenerative disorders, is an essential paraspeckle component. We have shown that its recruitment to these nuclear structures is mediated by the N-terminal region and requires prion-like activity. FUS interacts with p54nrb/NONO, a major constituent of paraspeckles, in an RNA-dependent manner and responds in the same way as other paraspeckle proteins to alterations in cellular homeostasis such as changes in transcription rates or levels of protein methylation. FUS also regulates NEAT1 levels and paraspeckle formation in cultured cells, and FUS deficiency leads to loss of paraspeckles. Pathological gain-of-function FUS mutations might be expected to affect paraspeckle function in human diseases because mislocalized amyotrophic lateral sclerosis (ALS)-linked FUS variants sequester other paraspeckle proteins into aggregates formed in cultured cells and into neuronal inclusions in a transgenic mouse model of FUSopathy. Furthermore, we detected abundant p54nrb/NONO-positive inclusions in motor neurons of patients with familial forms of ALS caused by FUS mutations, but not in other ALS cases. Our results suggest that both loss and gain of FUS function can trigger disruption of paraspeckle assembly, which may impair protective responses in neurons and thereby contribute to the pathogenesis of FUSopathies.

Abstract 2316: Mutations in the p53 gene family reveal conservation of structure-function in the L1 and L3 loops and a response element code for transcriptional specificity.

Abstract The p53, p63 and p73 family of sequence-specific transcription factors share similar biochemical properties and structural features resulting in the recognition of DNA with identical amino acids and similar binding affinity. However, loss of individual members results in very different phenotypes in mice as well as humans. We have addressed functional relationships between p53 family proteins using a combination of mutants, target response elements (REs) and sequence-specific transactivation assays based in yeast and human cells. p53 alleles exhibiting enhanced transcriptional activation and altered promoter selectivity were previously identified in the L1 and L3 loop of the DNA binding domain of the protein. Included was S121F that results in defective p21 induction and confers a strong apoptotic phenotype in mammalian cells. Guided by result with p53-S121F, -T123A and -S240N mutants, the corresponding p63 and p73 alleles were investigated for ability to drive transactivation at over fifty p53 REs in isogenic yeast reporter strains. The phenotypes observed with p53 alleles were reproduced with the p63- and p73- alleles. Focusing on p73-S139F (corresponding to p53-S121F) transactivation at many RE sequences, we identify and experimentally confirm an RE target sequence code that predicts enhanced or reduced transactivation potentials. The correlation is particularly strong for induction at p53 targets within promoters of genes associated with apoptosis. Furthermore, p73 S139F showed an enhanced DNA-binding cooperativity in vitro, and loop L1 shows conformational changes in the crystal structure of the protein-DNA complex. The altered function phenotype was confirmed not only in gene reporter assays but also for endogenous gene expression in the p53 null HCT116 human cell line, suggesting that target recognition can predict relative changes in transcription rates at different loci. Based on the correlation between RE code and associated gene functions, we propose the selection during evolution of an RE sequence code within target promoters that can affect intrinsic conformational flexibility / DNA binding affinity of p53 proteins thereby modulating in vivo selectivity, specifically favoring the activation of apoptotic target genes Citation Format: Yari Ciribilli, Paola Monti, Alessandra Bisio, H. T. Nguyen, A S. Ethayathulla, Micheal A. Resnick, Hector Viadiu, Gilberto Fronza, Alberto Inga. Mutations in the p53 gene family reveal conservation of structure-function in the L1 and L3 loops and a response element code for transcriptional specificity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2316. doi:10.1158/1538-7445.AM2013-2316

A Design Principle for a Posttranslational Biochemical Oscillator

Multisite phosphorylation plays an important role in biological oscillators such as the circadian clock. Its general role, however, has been elusive. In this theoretical study, we show that a simple substrate with two modification sites acted upon by two opposing enzymes (e.g., a kinase and a phosphatase) can show oscillations in its modification state. An unbiased computational analysis of this oscillator reveals two common characteristics: a unidirectional modification cycle and sequestering of an enzyme by a specific modification state. These two motifs cause a substrate to act as a coupled system in whicha unidirectional cycle generates single-molecule oscillators, whereas sequestration synchronizes the population by limiting the available enzyme under conditions in which substrate is in excess. We also demonstrate the conditions under which the oscillation period is temperature compensated, an important feature of the circadian clock. This theoretical model will provide a framework for analyzing and synthesizing posttranslational oscillators.

A mechanism for robust circadian timekeeping via stoichiometric balance

Circadian (∼24 h) timekeeping is essential for the lives of many organisms. To understand the biochemical mechanisms of this timekeeping, we have developed a detailed mathematical model of the mammalian circadian clock. Our model can accurately predict diverse experimental data including the phenotypes of mutations or knockdown of clock genes as well as the time courses and relative expression of clock transcripts and proteins. Using this model, we show how a universal motif of circadian timekeeping, where repressors tightly bind activators rather than directly binding to DNA, can generate oscillations when activators and repressors are in stoichiometric balance. Furthermore, we find that an additional slow negative feedback loop preserves this stoichiometric balance and maintains timekeeping with a fixed period. The role of this mechanism in generating robust rhythms is validated by analysis of a simple and general model and a previous model of the Drosophila circadian clock. We propose a double-negative feedback loop design for biological clocks whose period needs to be tightly regulated even with large changes in gene dosage.

Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells

Regulation of RNA levels is determined through the interplay between RNA production, processing and degradation. However, since most global studies of RNA regulation do not distinguish the separate contributions of these processes, relatively little is known about how they are temporally integrated to determine changes in RNA levels. In particular, while some studies emphasize the role of changes in the rate of transcription, others suggest a prominent involvement of time-varying degradation rates. Here, we combine metabolic labeling of RNA at high temporal resolution with advanced RNA quantification assays and computational modeling to estimate RNA transcription and degradation rates during the model response of immune dendritic cells (DCs) to pathogens. We find that changes in transcription rates determine the majority of temporal changes in RNA levels, but that changes in degradation rate are important for shaping sharp ‘peaked’ responses. Furthermore, transcription rate changes precede corresponding changes in RNA level by a small lag (15-30 min), which is shorter for induced than for repressed genes. We used massively parallel sequencing of the newly-transcribed RNA population – including non-polyadenylated transcripts – to estimate constant RNA degradation and processing rates. We find that temporally constant degradation rates vary significantly between genes and contribute substantially to the observed differences in the dynamic response, and that specific groups of transcripts, mostly cytokines and transcription factors, are undergoing faster mRNA maturation. Our study provides a new quantitative approach to study key steps in the integrative process of RNA regulation.

The origins and consequences of heterogeneous neurons in a molecular oscillator model of the mammalian circadian clock

The mammalian suprachiasmatic nuclei (SCN) contain 20,000 neurons capable of generating near 24‐h rhythms. Recent data from functionally isolated SCN neurons show that single cells can exhibit a range of oscillatory phenotypes when removed from their network. The implications of variability in the quality of oscillations between circadian cells remain unknown. We exploited known bifurcations in a set of parameters from an existing deterministic model to reveal their contributions to different behaviors in SCN neurons. By simulating single cells with a range of parameter values, the authors (1) produced a range of oscillatory phenotypes similar to biological cells, (2) found that phenotype was more sensitive to changes in rates of transcription and degradation of Period than to changes in rates of translation and phosphorylation, and (3) determined phenotype‐dependent properties using velocity response curves. The authors next looked for potential biological consequences of building networks from these heterogeneous oscillators and (4) observed that the rate of resynchronization in the network depends on the number and location of damped cells, such that when coupling is reinstated, network topologies in which damped oscillators are highly connected restore rhythmicity and synchrony faster. These results indicate that heterogeneity in oscillatory ability amongst neurons is beneficial and their position within the network can influence the behavior of the system overall. Thus, a range of oscillation types is crucial for a resynchronizing network and damped cells in particular aid the speed of synchronization.

Heat Shock Response in Yeast Involves Changes in Both Transcription Rates and mRNA Stabilities

We have analyzed the heat stress response in the yeast Saccharomyces cerevisiae by determining mRNA levels and transcription rates for the whole transcriptome after a shift from 25°C to 37°C. Using an established mathematical algorithm, theoretical mRNA decay rates have also been calculated from the experimental data. We have verified the mathematical predictions for selected genes by determining their mRNA decay rates at different times during heat stress response using the regulatable tetO promoter. This study indicates that the yeast response to heat shock is not only due to changes in transcription rates, but also to changes in the mRNA stabilities. mRNA stability is affected in 62% of the yeast genes and it is particularly important in shaping the mRNA profile of the genes belonging to the environmental stress response. In most cases, changes in transcription rates and mRNA stabilities are homodirectional for both parameters, although some interesting cases of antagonist behavior are found. The statistical analysis of gene targets and sequence motifs within the clusters of genes with similar behaviors shows that both transcriptional and post-transcriptional regulons apparently contribute to the general heat stress response by means of transcriptional factors and RNA binding proteins.

Transcription‐dependence of histone H3 lysine 27 trimethylation at the Arabidopsis polycomb target gene FLC

The FLC gene encodes a MADS box repressor of flowering that is the main cause of the late-flowering phenotype of many Arabidopsis ecotypes. Expression of FLC is repressed by vernalization; maintenance of this repression is associated with the deposition of histone 3 K27 trimethylation (H3K27me3) at the FLC locus. However, whether this increased H3K27me3 is a consequence of reduced FLC transcription or the cause of transcriptional repression is not well defined. In this study we investigate the effect of changes in transcription rate on the abundance of H3K27me3 in the FLC gene body, a chromatin region that includes sequences required to maintain FLC repression following vernalization. We show that H3K27me3 is inversely correlated with transcription across the FLC gene body in a range of ecotypes and mutants with different flowering times. We demonstrate that the FLC gene body becomes marked with H3K27me3 in the absence of transcription. When transcription of the gene body is directed by an inducible promoter, H3K27me3 is removed following activation of transcription and H3K27me3 is added after transcription is decreased. The rate of addition of H3K27me3 to the FLC transgene following inactivation of transcription is similar to that observed in the FLC gene body following vernalization. Our data suggest that reduction of FLC transcription during vernalization leads to an increase of H3K27me3 levels in the FLC gene body that in turn maintains FLC repression.

Human Food Chain and Microorganisms: A Case of Co-Evolution

Human Food Chain and Microorganisms: A Case of Co-Evolution

Angiopoietin-like 4 (ANGPTL4, Fasting-induced Adipose Factor) Is a Direct Glucocorticoid Receptor Target and Participates in Glucocorticoid-regulated Triglyceride Metabolism

Glucocorticoids are important regulators of lipid homeostasis, and chronically elevated glucocorticoid levels induce hypertriglyceridemia, hepatic steatosis, and visceral obesity. The occupied glucocorticoid receptor (GR) is a transcription factor. However, those genes regulating lipid metabolism under GR control are not fully known. Angiopoietin-like 4 (ANGPTL4, fasting-induced adipose factor), a protein inhibitor of lipoprotein lipase, is synthesized and secreted during fasting, when circulating glucocorticoid levels are physiologically increased. We therefore tested whether the ANGPTL4 gene (Angptl4) is transcriptionally controlled by GR. We show that treatment with the synthetic glucocorticoid dexamethasone increased Angptl4 mRNA levels in primary hepatocytes and adipocytes (2-3-fold) and in the livers and white adipose tissue of mice (approximately 4-fold). We tested the mechanism of this increase in H4IIE hepatoma cells and found that dexamethasone treatment increased the transcriptional rate of Angptl4. Using bioinformatics and chromatin immunoprecipitation, we identified a GR binding site within the rat Angptl4 sequence. A reporter plasmid containing this site was markedly activated by dexamethasone, indicative of a functional glucocorticoid response element. Dexamethasone treatment also increased histone H4 acetylation and DNase I accessibility in genomic regions near this site, further supporting that it is a glucocorticoid response element. Glucocorticoids promote the flux of triglycerides from white adipose tissue to liver. We found that mice lacking ANGPTL4 (Angptl4(-/-)) had reductions in dexamethasone-induced hypertriglyceridemia and hepatic steatosis, suggesting that ANGPTL4 is required for this flux. Overall, we establish that ANGPTL4 is a direct GR target that participates in glucocorticoid-regulated triglyceride metabolism.

Specific and global regulation of mRNA stability during osmotic stress in Saccharomyces cerevisiae

Hyperosmotic stress yields reprogramming of gene expression in Saccharomyces cerevisiae cells. Most of this response is orchestrated by Hog1, a stress-activated, mitogen-activated protein kinase (MAPK) homologous to human p38. We investigated, on a genomic scale, the contribution of changes in transcription rates and mRNA stabilities to the modulation of mRNA amounts during the response to osmotic stress in wild-type and hog1 mutant cells. Mild osmotic shock induces a broad mRNA destabilization; however, osmo-mRNAs are up-regulated by increasing both transcription rates and mRNA half-lives. In contrast, mild or severe osmotic stress in hog1 mutants, or severe osmotic stress in wild-type cells, yields global mRNA stabilization and sequestration of mRNAs into P-bodies. After adaptation, the absence of Hog1 affects the kinetics of P-bodies disassembly and the return of mRNAs to translation. Our results indicate that regulation of mRNA turnover contributes to coordinate gene expression upon osmotic stress, and that there are both specific and global controls of mRNA stability depending on the strength of the osmotic stress.