Gene transcription in Daphnia magna: effects of acute exposure to a carbamate insecticide and an acetanilide herbicide.

Daphnia magna is a key invertebrate in the freshwater environment and is used widely as a model in ecotoxicological measurements and risk assessment. Understanding the genomic responses of D. magna to chemical challenges will be of value to regulatory authorities worldwide. Here we exposed D. magna to the insecticide methomyl and the herbicide propanil to compare phenotypic effects with changes in mRNA expression levels. Both pesticides are found in drainage ditches and surface water bodies standing adjacent to crops. Methomyl, a carbamate insecticide widely used in agriculture, inhibits acetylcholinesterase, a key enzyme in nerve transmission. Propanil, an acetanilide herbicide, is used to control grass and broad-leaf weeds. The phenotypic effects of single doses of each chemical were evaluated using a standard immobilisation assay. Immobilisation was linked to global mRNA expression levels using the previously estimated 48h-EC(1)s, followed by hybridization to a cDNA microarray with more than 13,000 redundant cDNA clones representing >5000 unique genes. Following exposure to methomyl and propanil, differential expression was found for 624 and 551 cDNAs, respectively (one-way ANOVA with Bonferroni correction, P

Transcription factor NF-kappaB is transported to the nucleus via cytoplasmic dynein/dynactin motor complex in hippocampal neurons

Background Long-term changes in synaptic plasticity require gene transcription, indicating that signals generated at the synapse must be transported to the nucleus. Synaptic activation of hippocampal neurons is known to trigger retrograde transport of transcription factor NF-κB. Transcription factors of the NF-κB family are widely expressed in the nervous system and regulate expression of several genes involved in neuroplasticity, cell survival, learning and memory. Principal Findings In this study, we examine the role of the dynein/dynactin motor complex in the cellular mechanism targeting and transporting activated NF-κB to the nucleus in response to synaptic stimulation. We demonstrate that overexpression of dynamitin, which is known to dissociate dynein from microtubules, and treatment with microtubule-disrupting drugs inhibits nuclear accumulation of NF-κB p65 and reduces NF-κB-dependent transcription activity. In this line, we show that p65 is associated with components of the dynein/dynactin complex in vivo and in vitro and that the nuclear localization sequence (NLS) within NF-κB p65 is essential for this binding. Conclusion This study shows the molecular mechanism for the retrograde transport of activated NF-κB from distant synaptic sites towards the nucleus.

Ascl1 coordinately regulates gene expression and the chromatin landscape during neurogenesis

The proneural transcription factor Ascl1 coordinates gene expression in both proliferating and differentiating progenitors along the neuronal lineage. Here, we used a cellular model of neurogenesis to investigate how Ascl1 interacts with the chromatin landscape to regulate gene expression when promoting neuronal differentiation. We find that Ascl1 binding occurs mostly at distal enhancers and is associated with activation of gene transcription. Surprisingly, the accessibility of Ascl1 to its binding sites in neural stem/progenitor cells remains largely unchanged throughout their differentiation, as Ascl1 targets regions of both readily accessible and closed chromatin in proliferating cells. Moreover, binding of Ascl1 often precedes an increase in chromatin accessibility and the appearance of new regions of open chromatin, associated with de novo gene expression during differentiation. Our results reveal a function of Ascl1 in promoting chromatin accessibility during neurogenesis, linking the chromatin landscape at Ascl1 target regions with the temporal progression of its transcriptional program.

Estrogen receptor-mediated transcription involves the activation of multiple kinase pathways in neuroblastoma cells

While many physiological effects of estrogens (E) are due to regulation of gene transcription by liganded estrogen receptors (ERs), several effects are also mediated, at least in part, by rapid non-genomic actions of E. Though the relative importance of rapid versus genomic effects in the central nervous system is controversial, we showed previously that membrane-limited effects of E, initiated by an estradiol bovine serum albumin conjugate (E2-BSA), could potentiate transcriptional effects of 17beta-estradiol from an estrogen response element (ERE)-reporter in neuroblastoma cells. Here, using specific inhibitors and activators in a pharmacological approach, we show that activation of phosphatidylinositol-3-phosphate kinase (PI3K) and mitogen activated protein kinase (MAPK) pathways, dependent on a Galphaq coupled receptor signaling are important in this transcriptional potentiation. We further demonstrate, using ERalpha phospho-deficient mutants, that E2-BSA mediated phosphorylation of ERalpha is one mechanism to potentiate transcription from an ERE reporter construct. This study provides a possible mechanism by which signaling from the membrane is coupled to transcription in the nucleus, providing an integrated view of hormone signaling in the brain.

Influence of mutations affecting gonadotropin production or responsiveness on expression of inhibin subunit mRNA and protein in the mouse ovary

Measurement of inhibins A and B in the serum of normal cyclic rodents has implicated FSH in the regulation of these peptides within the ovary. To extend these observations we have used a panel of mutant mice carrying mutations which affect either the production of, or the ability to respond to, FSH and LH. As a consequence, the females are infertile and show different degrees of follicular development. The aim of this study was to measure inhibin gene transcription in the ovaries of these mutant females together with inhibin protein levels in ovaries and serum and to relate these to follicular development within the ovary. Comparison was made with a pool of normal/heterozygous females. In hpg females where lack of GnRH production results in the absence of gonadotropin synthesis, in FSHbeta knockout (FSHbetaKO) females where disruption of the gene encoding FSHbeta results in the absence of FSH production, and in FSH receptor knockout (FSHRKO) females which are unable to respond to circulating FSH, follicular development remains at the pre-antral stage in these three mutants. Only in the hpg females were common inhibin alpha subunit mRNA levels significantly lower than normal. In these three mutants, however, mRNA levels for both the betaA and betaB subunits were extremely low compared with normal mice. At the protein level, neither inhibin A nor B was detected in the serum of these three mutants; however inhibin B, albeit at very low levels, was detectable within the ovaries. These observations confirm a major role for FSH in the control of transcription of the RA and betaB genes but suggest that the constitutive transcription of the alpha subunit is less dependent on FSH. In contrast, in LH receptor knockout (LuRKO) female mice inhibin betaA subunit mRNA levels were similar to those measured in normal/heterozygous females but levels of inhibin alpha and betaB subunit mRNAs were significantly higher than in the normal group. This was reflected in significantly higher inhibin B protein levels in ovaries and serum. An inability to respond to LH combined with high circulating levels of FSH leads to a high proportion of antral follicles in LuRKO females, with granulosa cells constituting the major cell type within the ovary. The high percentage of antral granulosa cells is likely to account for the significantly higher levels of inhibin B production in these ovaries.

Isoflavones and endothelial function

Dietary isoflavones are thought to be cardioprotective due to their structural similarity to oestrogen. Oestrogen is believed to have beneficial effects on endothelial function and may be one of the mechanisms by which premenopausal women are protected against CVD. Decreased NO production and endothelial NO synthase activity, and increased endothelin-1 concentrations, impaired lipoprotein metabolism and increased circulating inflammatory factors result from oestrogen deficiency. Oestrogen acts by binding to oestrogen receptors alpha and beta. Isoflavones have been shown to bind with greater affinity to the latter. Oestrogen replacement therapy is no longer thought to be a safe treatment for prevention of CVD; isoflavones are a possible alternative. Limited evidence from human intervention studies suggests that isoflavones may improve endothelial function, but the available data are not conclusive. Animal studies provide stronger support for a role of isoflavones in the vasculature, with increased vasodilation and endothelial NO synthase activity demonstrated. Cellular mechanisms underlying the effects of isoflavones on endothelial cell function are not yet clear. Possible oestrogen receptor-mediated pathways include modulation of gene transcription, and also non-genomic oestrogen receptor-mediated signalling pathways. Putative non-oestrogenic pathways include inhibition of reactive oxygen species production and up regulation of the protein kinase A pathway (increasing NO bioavailability). Further research is needed to unravel effects of isoflavones on intracellular regulation of the endothelial function. Moreover, there is an urgent need for adequately powered, robustly designed human intervention studies in order to clarify the present equivocal findings.

Cellular mechanisms of cardiac hypertrophy.

Hypertrophy of myocytes in the heart ventricles is an important adaptation that in vivo occurs in response to a requirement for increased contractile power. It involves changes at the level of gene transcription, stimulation of the rate of protein synthesis (translation), and increased assembly of myofibrils. There is mounting evidence of the involvement of reversible protein phosphorylation and dephosphorylation in most of these processes. Protein kinase C, mitogen-activated protein kinases, and transcription factors have been implicated in the modulation of the transcriptional changes. Activation of translation may also be mediated through protein phosphorylation/dephosphorylation, although this has not been clearly established in the heart. Here we provide a critical overview of the signalling pathways involved in the hypertrophic response and provide a scheme to account for many of its features.

Endothelin signalling in the cardiac myocyte and its pathophysiological relevance

Endothelin A (ET(A)) transmembrane receptors predominate in rat cardiac myocytes. These are G protein-coupled receptors whose actions are mediated by the G(q) heterotrimeric G proteins. Through these, ET-1 binding to ET(A)-receptors stimulates the hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to diacylglycerol and inositol 1,4,5-trisphosphate. Diacylglycerol remains in the membrane whereas inositol 1,4,5-trisphosphate is soluble (though its importance in the cardiac myocyte is still debated). Isoforms of the phospholipid-dependent protein kinase, protein kinase C (PKC), are intracellular receptors for diacylglycerol. Cytoplasmic nPKCdelta and nPKCepsilon detect increases in membrane diacylglycerols and translocate to the membrane. This brings about PKC activation, though modifications additional to binding to phospholipids and diacylglycerol are involved. The next event (probably associated with PKC activation) is the activation of the membrane-bound small G protein Ras by exchange of GTP for GDP. Ras.GTP loading translocates Raf family mitogen-activated protein kinase (MAPK) kinase kinases to the membrane, initiates the activation of Raf, and thus activates the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade. Over longer times, two analogous protein kinase cascades, the c-Jun N-terminal kinase and p38-mitogen-activated protein kinase cascades, become activated. As the signals originating from the ET(A) receptor are transmitted through these protein kinase pathways, other signalling molecules become phosphorylated, thus changing their biological activities. For example, ET-1 increases the expression of the c-jun transcription factor gene, and increases abundance and phosphorylation of c-Jun protein. These changes in c-Jun expression and phosphorylation are likely to be important in the regulation of gene transcription.

Genesis and expansion of metazoan transcription factor gene classes

We know little about the genomic events that led to the advent of a multicellular grade of organization in animals, one of the most dramatic transitions in evolution. Metazoan multicellularity is correlated with the evolution of embryogenesis, which presumably was underpinned by a gene regulatory network reliant on the differential activation of signaling pathways and transcription factors. Many transcription factor genes that play critical roles in bilaterian development largely appear to have evolved before the divergence of cnidarian and bilaterian lineages. In contrast, sponges seem to have a more limited suite of transcription factors, suggesting that the developmental regulatory gene repertoire changed markedly during early metazoan evolution. Using whole- genome information from the sponge Amphimedon queenslandica, a range of eumetazoans, and the choanoflagellate Monosiga brevicollis, we investigate the genesis and expansion of homeobox, Sox, T- box, and Fox transcription factor genes. Comparative analyses reveal that novel transcription factor domains ( such as Paired, POU, and T- box) arose very early in metazoan evolution, prior to the separation of extant metazoan phyla but after the divergence of choanoflagellate and metazoan lineages. Phylogenetic analyses indicate that transcription factor classes then gradually expanded at the base of Metazoa before the bilaterian radiation, with each class following a different evolutionary trajectory. Based on the limited number of transcription factors in the Amphimedon genome, we infer that the genome of the metazoan last common ancestor included fewer gene members in each class than are present in extant eumetazoans. Transcription factor orthologues present in sponge, cnidarian, and bilaterian genomes may represent part of the core metazoan regulatory network underlying the origin of animal development and multicellularity.

Modelling negative feedback networks for activating transcription factor 3 predicts a dominant role for miRNAs in immediate early gene regulation

Activating transcription factor 3 (Atf3) is rapidly and transiently upregulated in numerous systems, and is associated with various disease states. Atf3 is required for negative feedback regulation of other genes, but is itself subject to negative feedback regulation possibly by autorepression. In cardiomyocytes, Atf3 and Egr1 mRNAs are upregulated via ERK1/2 signalling and Atf3 suppresses Egr1 expression. We previously developed a mathematical model for the Atf3-Egr1 system. Here, we adjusted and extended the model to explore mechanisms of Atf3 feedback regulation. Introduction of an autorepressive loop for Atf3 tuned down its expression and inhibition of Egr1 was lost, demonstrating that negative feedback regulation of Atf3 by Atf3 itself is implausible in this context. Experimentally, signals downstream from ERK1/2 suppress Atf3 expression. Mathematical modelling indicated that this cannot occur by phosphorylation of pre-existing inhibitory transcriptional regulators because the time delay is too short. De novo synthesis of an inhibitory transcription factor (ITF) with a high affinity for the Atf3 promoter could suppress Atf3 expression, but (as with the Atf3 autorepression loop) inhibition of Egr1 was lost. Developing the model to include newly-synthesised miRNAs very efficiently terminated Atf3 protein expression and, with a 4-fold increase in the rate of degradation of mRNA from the mRNA/miRNA complex, profiles for Atf3 mRNA, Atf3 protein and Egr1 mRNA approximated to the experimental data. Combining the ITF model with that of the miRNA did not improve the profiles suggesting that miRNAs are likely to play a dominant role in switching off Atf3 expression post-induction.

Triacylglycerol-rich lipoprotein-gene interactions in endothelial cells

Lipoproteins such as LDL (low-density lipoprotein) and oxidized LDL have potentially adverse effects on endothelial cells due to their ability to activate pro-inflammatory pathways regulated via the transcription factor NF-kappaB (nuclear factor kappaB). Triacylglycerol-rich lipoproteins (the chylomicrons, very-low-density lipoprotein and their respective remnant particles) have also been implicated in the induction of a pro-inflammatory phenotype and up-regulation of adhesion molecule expression. Although early studies supported the proposal that LPL (lipoprotein lipase)-mediated hydrolysis of TRLs (triglyceride-rich lipoproteins) at the endothelium could activate the NFkappaB pathway, more recent studies provide evidence of pro-and anti-inflammatory responses when cells are exposed to fatty acids of TRL particles. A large number of genes are up- and down-regulated when cells are exposed to TRL, with the net effect reflecting receptor- and nonreceptor-mediated pathways that are activated or inhibited depending on fatty acid type, the lipid and apolipoprotein composition of the TRL and the presence or absence of LPL. Early concepts of TRL particles as essentially pro-inflammatory stimuli to the endothelium provide an overly simplistic view of their impact on the vascular compartment.

Analysis of wheat SAGE tags reveals evidence for widespread antisense transcription

BACKGROUND: Serial Analysis of Gene Expression (SAGE) is a powerful tool for genome-wide transcription studies. Unlike microarrays, it has the ability to detect novel forms of RNA such as alternatively spliced and antisense transcripts, without the need for prior knowledge of their existence. One limitation of using SAGE on an organism with a complex genome and lacking detailed sequence information, such as the hexaploid bread wheat Triticum aestivum, is accurate annotation of the tags generated. Without accurate annotation it is impossible to fully understand the dynamic processes involved in such complex polyploid organisms. Hence we have developed and utilised novel procedures to characterise, in detail, SAGE tags generated from the whole grain transcriptome of hexaploid wheat. RESULTS: Examination of 71,930 Long SAGE tags generated from six libraries derived from two wheat genotypes grown under two different conditions suggested that SAGE is a reliable and reproducible technique for use in studying the hexaploid wheat transcriptome. However, our results also showed that in poorly annotated and/or poorly sequenced genomes, such as hexaploid wheat, considerably more information can be extracted from SAGE data by carrying out a systematic analysis of both perfect and "fuzzy" (partially matched) tags. This detailed analysis of the SAGE data shows first that while there is evidence of alternative polyadenylation this appears to occur exclusively within the 3' untranslated regions. Secondly, we found no strong evidence for widespread alternative splicing in the developing wheat grain transcriptome. However, analysis of our SAGE data shows that antisense transcripts are probably widespread within the transcriptome and appear to be derived from numerous locations within the genome. Examination of antisense transcripts showing sequence similarity to the Puroindoline a and Puroindoline b genes suggests that such antisense transcripts might have a role in the regulation of gene expression. CONCLUSION: Our results indicate that the detailed analysis of transcriptome data, such as SAGE tags, is essential to understand fully the factors that regulate gene expression and that such analysis of the wheat grain transcriptome reveals that antisense transcripts maybe widespread and hence probably play a significant role in the regulation of gene expression during grain development.

Pre-B-cell transcription factor 1 and steroidogenic factor 1 synergistically regulate adrenocortical growth and steroidogenesis

variety of transcription factors including Wilms tumor gene (Wt-1), steroidogenic factor 1 (Sf-1), dosage-sensitive sex reversal, adrenal hypoplasia congenita on the X-chromosome, Gene 1 (Dax-1), and pre-B-cell transcription factor 1 (Pbx1) have been defined as necessary for regular adrenocortical development. However, the role of Pbx1 for adrenal growth and function in the adult organism together with the molecular relationship between Pbx1 and these other transcription factors have not been characterized. We demonstrate that Pbx haploinsufficiency (Pbx1(+/-)) in mice is accompanied by a significant lower adrenal weight in adult animals compared with wild-type controls. Accordingly, baseline proliferating cell nuclear antigen levels are lower in Pbx1(+/-) mice, and unilateral adrenalectomy results in impaired contralateral compensatory adrenal growth, indicating a lower proliferative potential in the context of Pbx1 haploinsufficiency. In accordance with the key role of IGFs in adrenocortical proliferation and development, real-time RT-PCR demonstrates significant lower expression levels of the IGF-I receptor, and up-regulation of IGF binding protein-2. Functionally, Pbx1(+/-) mice display a blunted corticosterone response after ACTH stimulation coincident with lower adrenal expression of the ACTH receptor (melanocortin 2 receptor, Mc2-r). Mechanistically, in vitro studies reveal that Pbx1 and Sf-1 synergistically stimulates Mc2-r promoter activity. Moreover, Sf-1 directly activates the Pbx1 promoter activity in vitro and in vivo. Taken together, these studies provide evidence for a role of Pbx1 in the maintenance of a functional adrenal cortex mediated by synergistic actions of Pbx1 and Sf-1 in the transcriptional regulation of the critical effector of adrenocortical differentiation, the ACTH receptor.

Restricted expression of NADPH oxidase/peroxidase gene (Duox) in zone VII of the ascidian endostyle

The ascidian Ciona intestinalis, a marine invertebrate chordate, is an emerging model system for developmental and evolutionary studies. The endostyle, one of the characteristic organs of ascidians, is a pharyngeal structure with iodine-concentrating and peroxidase activities and is therefore considered to be homologous to the follicular thyroid of higher vertebrates. We have previously reported that a limited part of the endostyle (zone VII) is marked by the expression of orthologs of the thyroid peroxidase (TPO) and thyroid transcription factor-2 (TTF-2/FoxE) genes. In this study, we have identified the Ciona homolog of NADPH oxidase/peroxidase (Duox), which provides hydrogen peroxide (H2O2) for iodine metabolism by TPO in the vertebrate thyroid. Expression patterns assessed by in situ hybridization have revealed that Ciona Duox (Ci-Duox) is predominantly expressed in the dorsal part of zone VII of the endostyle. Furthermore, two-color fluorescent in situ hybridization with Ci-Duox and Ciona TPO (CiTPO) has revealed that the ventral boundary of the Ci-Duox domain of expression is more dorsal than that of CiTPO. We have also characterized several genes, such as Ci-Fgf8/17/18, 5HT7, and Ci-NK4, which are predominantly expressed in the ventral part of zone VII, in a region complementary to the Ci-Duox expression domain. These observations suggest that, at the molecular level, zone VII has a complex organization that might have some impact on the specification of cell types and functions in this thyroid-equivalent element of the ascidian endostyle.

Comparative expression analysis of transcription factor genes in the endostyle of invertebrate chordates

The endostyle of invertebrate chordates is a pharyngeal organ that is thought to be homologous with the follicular thyroid of vertebrates. Although thyroid-like features such as iodine-concentrating and peroxidase activities are located in the dorsolateral part of both ascidian and amphioxus endostyles, the structural organization and numbers of functional units are different. To estimate phylogenetic relationships of each functional zone with special reference to the evolution of the thyroid, we have investigated, in ascidian and amphioxus, the expression patterns of thyroid-related transcription factors such as TTF-2/MoxE4 and Pax2/5/8, as well as the forkhead transcription factors FoxQ1 and FoxA. Comparative gene expression analyses depicted an overall similarity between ascidians and amphioxus endostyles, while differences in expression patterns of these genes might be specifically related to the addition or elimination of a pair of glandular zones. Expressions of Ci-FoxE and BbFoxE4 suggest that the ancestral FoxE class might have been recruited for the formation of thyroid-like region in a possible common ancestor of chordates. Furthermore, coexpression of FoxE4, Pax2/5/8, and TPO in the dorsolateral part of both ascidian and amphioxus endostyles suggests that genetic basis of the thyroid function was already in place before the vertebrate lineage. (c) 2005 Wiley-Liss, Inc.