The discovery of interchromosomal interactions in higher eukaryotes points to a functional interplay between genome architecture and gene expression, challenging the view of transcription as a one-dimensional process. However, the extent of interchromosomal interactions and the underlying mechanisms are unknown. Here we present the first genome-wide analysis of transcriptional interactions using the mouse globin genes in erythroid tissues. Our results show that the active globin genes associate with hundreds of other transcribed genes, revealing extensive and preferential intra- and interchromosomal transcription interactomes. We show that the transcription factor Klf1 mediates preferential co-associations of Klf1-regulated genes at a limited number of specialized transcription factories. Our results establish a new gene expression paradigm, implying that active co-regulated genes and their regulatory factors cooperate to create specialized nuclear hot spots optimized for efficient and coordinated transcriptional control.

Expression of the nuclear orphan receptor gene Nur77 in neuronal cells is induced by activity-dependent increases in intracellular Ca2+ ions. Ca2+ responsiveness of the Nur77 gene has been attributed to two distinct DNA regulatory regions that recruit the transcription factors cAMP response element binding protein (CREB) and myocyte enhancer factor-2 (MEF2). Here we used dominant interfering and constitutively active mutants of CREB and MEF2 proteins to assess their relative contribution to depolarization-induced Nur77 expression in undifferentiated PC12 cells and hippocampal neurons. We show that while CREB is necessary for Ca2+-activated Nur77 expression MEF2 functions to modulate CREB-dependent Nur77 expression by acting as a repressor in quiescent cells.

Cohesin is a chromatin-associated protein complex that mediates sister chromatid cohesion by connecting replicated DNA molecules. Cohesin also has important roles in gene regulation, but the mechanistic basis of this function is poorly understood. In mammalian genomes, cohesin co-localizes with CCCTC binding factor (CTCF), a zinc finger protein implicated in multiple gene regulatory events. At the imprinted IGF2-H19 locus, CTCF plays an important role in organizing allele-specific higher-order chromatin conformation and functions as an enhancer blocking transcriptional insulator. Here we have used chromosome conformation capture (3C) assays and RNAi-mediated depletion of cohesin to address whether cohesin affects higher order chromatin conformation at the IGF2-H19 locus in human cells. Our data show that cohesin has a critical role in maintaining CTCF-mediated chromatin conformation at the locus and that disruption of this conformation coincides with changes in IGF2 expression. We show that the cohesin-dependent, higher-order chromatin conformation of the locus exists in both G1 and G2 phases of the cell cycle and is therefore independent of cohesin's function in sister chromatid cohesion. We propose that cohesin can mediate interactions between DNA molecules in cis to insulate genes through the formation of chromatin loops, analogous to the cohesin mediated interaction with sister chromatids in trans to establish cohesion.

Chromatin regulates many key processes in the nucleus by controlling access to the underlying DNA. SNF2-like factors are ATP-driven enzymes that play key roles in the dynamics of chromatin by remodelling nucleosomes and other nucleoprotein complexes. Even simple eukaryotes such as yeast contain members of several subfamilies of SNF2-like factors. The FUN30/ETL1 subfamily of SNF2 remodellers is conserved from yeasts to humans, but is poorly characterized. We show that the deletion of FUN30 leads to sensitivity to the topoisomerase I poison camptothecin and to severe cell cycle progression defects when the Orc5 subunit is mutated. We demonstrate a role of FUN30 in promoting silencing in the heterochromatin-like mating type locus HMR, telomeres and the rDNA repeats. Chromatin immunoprecipitation experiments demonstrate that Fun30 binds at the boundary element of the silent HMR and within the silent HMR. Mapping of nucleosomes in vivo using micrococcal nuclease demonstrates that deletion of FUN30 leads to changes of the chromatin structure at the boundary element. A point mutation in the ATP-binding site abrogates the silencing function of Fun30 as well as its toxicity upon overexpression, indicating that the ATPase activity is essential for these roles of Fun30. We identify by amino acid sequence analysis a putative CUE motif as a feature of FUN30/ETL1 factors and show that this motif assists Fun30 activity. Our work suggests that Fun30 is directly involved in silencing by regulating the chromatin structure within or around silent loci.

Embryonic stem cell (ESC) pluripotency is dependent on an intrinsic gene regulatory network centered on Oct4. Propagation of the pluripotent state is stimulated by the cytokine leukemia inhibitory factor (LIF) acting through the transcriptional regulator Stat3. Here, we show that this extrinsic stimulus converges with the intrinsic circuitry in Krüppel-factor activation. Oct4 primarily induces Klf2 while LIF/Stat3 selectively enhances Klf4 expression. Overexpression of either factor reduces LIF dependence, but with quantitative and qualitative differences. Unlike Klf4, Klf2 increases ESC clonogenicity, maintains undifferentiated ESCs in the genetic absence of Stat3, and confers resistance to BMP-induced differentiation. ESCs expanded with Klf2 remain capable of contributing to adult chimeras. Postimplantation-embryo-derived EpiSCs lack both Klf2 and Klf4 and expression of either can reinstate naive pluripotency. These findings indicate that Oct4 and Stat3 intersect in directing expression of Klf transcriptional regulators with overlapping properties that additively reinforce ground-state ESC pluripotency, identity, and self-renewal.

Exchanging and sharing scientific results are essential for researchers in the field of computational modelling. BioModels.net defines agreed-upon standards for model curation. A fundamental one, MIRIAM (Minimum Information Requested in the Annotation of Models), standardises the annotation and curation process of quantitative models in biology. To support this standard, MIRIAM Resources maintains a set of standard data types for annotating models, and provides services for manipulating these annotations. Furthermore, BioModels.net creates controlled vocabularies, such as SBO (Systems Biology Ontology) which strictly indexes, defines and links terms used in Systems Biology. Finally, BioModels Database provides a free, centralised, publicly accessible database for storing, searching and retrieving curated and annotated computational models. Each resource provides a web interface to submit, search, retrieve and display its data. In addition, the BioModels.net team provides a set of Web Services which allows the community to programmatically access the resources. A user is then able to perform remote queries, such as retrieving a model and resolving all its MIRIAM Annotations, as well as getting the details about the associated SBO terms. These web services use established standards. Communications rely on SOAP (Simple Object Access Protocol) messages and the available queries are described in a WSDL (Web Services Description Language) file. Several libraries are provided in order to simplify the development of client software. BioModels.net Web Services make one step further for the researchers to simulate and understand the entirety of a biological system, by allowing them to retrieve biological models in their own tool, combine queries in workflows and efficiently analyse models.

Two main approaches in exploring causal relationships in biological systems using time-series data are the application of Dynamic Causal model (DCM) and Granger Causal model (GCM). These have been extensively applied to brain imaging data and are also readily applicable to a wide range of temporal changes involving genes, proteins or metabolic pathways. However, these two approaches have always been considered to be radically different from each other and therefore used independently. Here we present a novel approach which is an extension of Granger Causal model and also shares the features of the bilinear approximation of Dynamic Causal model. We have first tested the efficacy of the extended GCM by applying it extensively in toy models in both time and frequency domains and then applied it to local field potential recording data collected from in vivo multi-electrode array experiments. We demonstrate face discrimination learning-induced changes in inter- and intra-hemispheric connectivity and in the hemispheric predominance of theta and gamma frequency oscillations in sheep inferotemporal cortex. The results provide the first evidence for connectivity changes between and within left and right inferotemporal cortexes as a result of face recognition learning.

Cardiac hypertrophy is associated with profound remodeling of Ca(2+) signaling pathways. During the early, compensated stages of hypertrophy, Ca(2+) fluxes may be enhanced to facilitate greater contraction, whereas as the hypertrophic heart decompensates, Ca(2+) homeostatic mechanisms are dysregulated leading to decreased contractility, arrhythmia and death. Although ryanodine receptor Ca(2+) release channels (RyR) on the sarcoplasmic reticulum (SR) intracellular Ca(2+) store are primarily responsible for the Ca(2+) flux that induces myocyte contraction, a role for Ca(2+) release via the inositol 1,4,5-trisphosphate receptor (InsP(3)R) in cardiac physiology has also emerged. Specifically, InsP(3)-induced Ca(2+) signals generated following myocyte stimulation with an InsP(3)-generating agonist (e.g., endothelin, ET-1), lead to modulation of Ca(2+) signals associated with excitation-contraction coupling (ECC) and the induction of spontaneous Ca(2+) release events that cause cellular arrhythmia. Using myocytes from spontaneously hypertensive rats (SHR), we recently reported that expression of the type 2 InsP(3)R (InsP(3)R2) is significantly increased during hypertrophy. Notably, this increased expression was restricted to the junctional SR in close proximity to RyRs. There, enhanced Ca(2+) release via InsP(3)Rs serves to sensitize neighboring RyRs causing an augmentation of Ca(2+) fluxes during ECC as well as an increase in non-triggered Ca(2+) release events. Although the sensitization of RyRs may be a beneficial consequence of elevated InsP(3)R expression during hypertrophy, the spontaneous Ca(2+) release events are potentially of pathological significance giving rise to cardiac arrhythmia. InsP(3)R2 expression was also increased in hypertrophic hearts from patients with ischemic dilated cardiomyopathy and aortically-banded mice demonstrating that increased InsP(3)R expression may be a general phenomenon that underlies Ca(2+) changes during hypertrophy.

Ino80 is an ATP-dependent nucleosome-remodeling enzyme involved in transcription, replication, and the DNA damage response. Here, we characterize the fission yeast Ino80 and find that it is essential for cell viability. We show that the Ino80 complex from fission yeast mediates ATP-dependent nucleosome remodeling in vitro. The purification of the Ino80-associated complex identified a highly conserved complex and the presence of a novel zinc finger protein with similarities to the mammalian transcriptional regulator Yin Yang 1 (YY1) and other members of the GLI-Krüppel family of proteins. Deletion of this Iec1 protein or the Ino80 complex subunit arp8, ies6, or ies2 causes defects in DNA damage repair, the response to replication stress, and nucleotide metabolism. We show that Iec1 is important for the correct expression of genes involved in nucleotide metabolism, including the ribonucleotide reductase subunit cdc22 and phosphate- and adenine-responsive genes. We find that Ino80 is recruited to a large number of promoter regions on phosphate starvation, including those of phosphate- and adenine-responsive genes that depend on Iec1 for correct expression. Iec1 is required for the binding of Ino80 to target genes and subsequent histone loss at the promoter and throughout the body of these genes on phosphate starvation. This suggests that the Iec1-Ino80 complex promotes transcription through nucleosome eviction.

Ribosomal protein S6 (rpS6) is a key component of the translational machinery in eukaryotic cells and is essential for ribosome biogenesis. rpS6 is phosphorylated on evolutionarily conserved serine residues, and data indicate that rpS6 phosphorylation might regulate cell growth and protein synthesis. Studies in cell lines have shown an important role for the serine kinase mammalian target of rapamycin (mTOR) in rpS6 phosphorylation, further linking rpS6 to control of cellular metabolism. rpS6 is essential in T cells because its deletion in mouse double-positive thymocyte cells results in a complete block in T cell development; however, the signaling pathway leading to rpS6 phosphorylation downstream of TCR stimulation has yet to be fully characterized. We show that maximal TCR-induced rpS6 phosphorylation in CD8 T cells requires both Lck and Fyn activity and downstream activation of PI3K, mTOR, and MEK/ERK MAPK pathways. We demonstrate that there is cross-talk between the PI3K and MAPK pathways as well as PI3K-independent mTOR activity, which result in differential phosphorylation of specific rpS6 serine residues. These results place rpS6 phosphorylation as a point of convergence for multiple crucial signaling pathways downstream of TCR triggering.

Mice can learn a food preference from odor cues transmitted on the breath of a conspecific, even if the "demonstrator" is anesthetized. To our knowledge there are no studies examining the effect of anesthetizing the "observer" on development of memory for socially transmitted food preferences (STFP). In Experiment 1 we found that 2-4 month-old F2 C57Bl/6x129sv male and female mice demonstrated a STFP after a 5min exposure to an anesthetized demonstrator mouse when tested 24h later. In Experiment 2, observer mice anesthetized with Sagatal (60 mg/kg) prior to the "social interaction" preferentially avoided the cued food when tested 24h later. This aversion was not due to any overt aversive effects of this dose of Sagatal because mice that ate the food and were then anesthetized, or could only smell the food for 5 min while anesthetized, showed no preference or aversion. In a third experiment we found that the Sagatal-induced aversion was not a general property of anesthesia because there were varied results produced by observer mice treated with anesthetic drugs with different mechanisms of action. Vetalar (200mg/kg) and Rompun (10 mg/kg) treated animals ate similar amounts of cued and non-cued food at test, indicating an absence of learning. Hypnorm (0.5 ml/kg) treated animals showed a preference for the cued food whereas those treated with Hypnovel (2.5 ml/kg) showed an aversion to the cued food. These results show that the food aversion observed with Sagatal is not a general property of anesthetic agents, but appears to be restricted to those acting primarily on the GABAergic system. Thus, we have shown that under certain conditions it is possible for an anesthetized observer mouse to learn a preference or aversion of a socially-linked olfactory cue.

Fibroblast growth factors (FGFs) acting through their cognate receptors (FGFRs) play vital roles in development and de-regulation of FGF/FGFR signalling is associated with many developmental syndromes. In addition there is much interest in inhibiting FGF/FGFR signalling as a therapeutic approach to cancer. FGF/FGFR signalling is certainly important in tumour angiogenesis but studies in the last few years have uncovered increasing evidence that FGFRs are driving oncogenes in certain cancers and act in a cell autonomous fashion to maintain the malignant properties of tumour cells. These observations make FGFRs increasingly attractive as targets for therapeutic intervention in cancer. In this article, we review FGFR signalling and describe recent advances in cancer genomics and cancer cell biology that demonstrate that specific tumour types are dependent upon or addicted to de-regulated FGFR. We also describe the range of therapeutic strategies currently employed or in development to antagonise de-regulated FGFRs including antibodies and small molecule tyrosine kinase inhibitors.

Nitric oxide (NO) controls numerous physiological processes by activation of its receptor, guanylyl cyclase (sGC), leading to the accumulation of 3'-5' cyclic guanosine monophosphate (cGMP). Ca(2+)-calmodulin (CaM) regulates both NO synthesis by NO synthase and cGMP hydrolysis by phosphodiesterase-1. We report that, unexpectedly, the CaM antagonists, calmidazolium, phenoxybenzamine and trifluoperazine, also inhibited cGMP accumulation in cerebellar cells evoked by an exogenous NO donor, with IC(50) values of 11, 80 and 180 microM respectively. Here we sought to elucidate the underlying mechanism(s).

Lymphocyte migration is crucial for immunological surveillance. A better understanding of the mechanisms that regulate lymphocyte migration will help appreciate how lymphocytes deliver specific functions to appropriate anatomical sites. Phosphoinositide-3 kinases (PI3Ks) are lipid kinases that regulate numerous cellular responses, including cell motility and chemotaxis. Here we discuss how PI3K isoforms differentially regulate lymphocyte migration and trafficking, with an emphasis on natural killer cells.

Sphingosine-1-phosphate (S1P), formed by sphingosine kinases (SphKs), regulates cellular proliferation and migration by acting as an agonist at specific receptors or intracellularly. Since S1P's effects are probably dependent on subcellular localization of its formation and degradation, we have studied the influence of G protein-coupled receptors on the localization of SphK1. Activation of Gq-coupled receptors induced a profound, rapid (half-life 3-5 s) and long-lasting (> 2 h) translocation of SphK1 to the plasma membrane. This was mimicked by expression of constitutively active G protein alpha-subunits specifically of the Gq family. Classical Gq signalling pathways, or phosphorylation at Ser225, phospholipase D and Ca2+/calmodulin were not involved in M3 receptor-induced SphK1 translocation in HEK-293 cells. Translocation was associated with S1P receptor internalization, which was dependent on catalytic activity of SphK1 and S1P receptor binding and thus resulted from S1P receptor cross-activation. It is concluded that SphK1 is an important effector of Gq-coupled receptors, linking them via cross-activation of S1P receptors to G(i) and G12/13 signalling pathways.

Russell bodies (RBs) are intracellular inclusions filled with protein aggregates. In diverse lymphoid disorders these occur as immunoglobulin (Ig) deposits, accumulating in abnormal plasma or Mott cells. In heavy-chain deposition disease truncated antibody heavy-chains (HCs) are found, which bear a resemblance to diverse polypeptides produced in Ig light-chain (LC)-deficient (L(-/-)) mice. In L(-/-) animals, the known functions of LC, providing part of the antigen-binding site of an antibody and securing progression of B-cell development, may not be required. Here, we show a novel function of LC in preventing antibody aggregation. L(-/-) mice produce truncated HC naturally, constant region (C)gamma and Calpha lack C(H)1, and Cmicro is without C(H)1 or C(H)1 and C(H)2. Most plasma cells found in these mice are CD138(+) Mott cells, filled with RBs, formed by aggregation of HCs of different isotypes. The importance of LC in preventing HC aggregation is evident in knock-in mice, expressing Cmicro without C(H)1 and C(H)2, which only develop an abundance of RBs when LC is absent. These results reveal that preventing antibody aggregation is a major function of LC, important for understanding the physiology of heavy-chain deposition disease, and in general recognizing the mechanisms, which initiate protein conformational diseases.

Caloric restriction (CR) protects against aging and disease, but the mechanisms by which this affects mammalian life span are unclear. We show in mice that deletion of ribosomal S6 protein kinase 1 (S6K1), a component of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway, led to increased life span and resistance to age-related pathologies, such as bone, immune, and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian life-span and suggest that therapeutic manipulation of S6K1 and AMPK might mimic CR and could provide broad protection against diseases of aging.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder caused by an increase in amyloid metabolism. The calcium hypothesis of AD explores how activation of the amyloidogenic pathway may function to remodel the neuronal Ca(2+) signaling pathways responsible for cognition. Hydrolysis of the beta-amyloid precursor protein (APP) yields two products that can influence Ca(2+) signaling. Firstly, the amyloids released to the outside form oligomers that enhance the entry of Ca(2+) that is pumped into the endoplasmic reticulum (ER). An increase in the luminal level of Ca(2+) within the ER enhances the sensitivity of the ryanodine receptors (RYRs) to increase the amount of Ca(2+) being released from the internal stores. Secondly, the APP intracellular domain may alter the expression of key signaling components such as the RYR. It is proposed that this remodeling of Ca(2+) signaling will result in the learning and memory deficits that occur early during the onset of AD. In particular, the Ca(2+) signaling remodeling may erase newly acquired memories by enhancing the mechanism of long-term depression that depends on activation of the Ca(2+)-dependent protein phosphatase calcineurin. The alteration in Ca(2+) signaling will also contribute to the neurodegeneration that characterizes the later stages of dementia.

We have hypothesized that variation in imprinted growth-promoting fetal genes may affect maternal glucose concentrations in pregnancy. To test this hypothesis we evaluated the effects of fetal disruption of murine H19(Delta13) on maternal glucose concentrations in pregnancy.

The study of small molecules in body fluids has become an important tool to monitor the state of biological organisms. Applications range from model studies using cell lines to applications where human body fluids are used to monitor disease states or drug responses. NMR spectroscopy has been an important tool for metabolomics although severe overlap of signals has limited the number of compounds, which can be unambiguously identified and quantified. Therefore, deconvolution of NMR spectra is one of the greatest challenges for NMR-based metabolomics. This has commonly been achieved by using multidimensional spectra that have the disadvantage of requiring significantly longer acquisition times. Recently, a number of methods have been described to record NMR spectra much faster. Here, we explore the use of Hadamard-encoded TOCSY spectra to simultaneously select multiple lines from crowded NMR spectra of blood serum samples to acquire pseudo-two-dimensional spectra in minutes which would otherwise require many hours. The potential of this approach is demonstrated for the detection of a signature for colorectal cancer from human blood samples.

The BCL-2 homology domain 3 (BH3)-only protein, B-cell lymphoma 2 interacting mediator of cell death (BIM) is a potent pro-apoptotic protein belonging to the B-cell lymphoma 2 protein family. In recent years, advances in basic biology have provided a clearer picture of how BIM kills cells and how BIM expression and activity are repressed by growth factor signalling pathways, especially the extracellular signal-regulated kinase 1/2 and protein kinase B pathways. In tumour cells these oncogene-regulated pathways are used to counter the effects of BIM, thereby promoting tumour cell survival. In parallel, a new generation of targeted therapeutics has been developed, which show remarkable specificity and efficacy in tumour cells that are addicted to particular oncogenes. It is now apparent that the expression and activation of BIM is a common response to these new therapeutics. Indeed, BIM has emerged from this marriage of basic and applied biology as an important mediator of tumour cell death in response to such drugs. The induction of BIM alone may not be sufficient for significant tumour cell death, as BIM is more likely to act in concert with other BH3-only proteins, or other death pathways, when new targeted therapeutics are used in combination with traditional chemotherapy agents. Here we discuss recent advances in understanding BIM regulation and review the role of BIM as a mediator of tumour cell death in response to novel oncogene-targeted therapeutics.

Pleckstrin homology (PH) domains are modules characterised by a conserved three-dimensional protein fold. Several PH domains bind phosphoinositides with high affinity and specificity whilst most others do not. ARAP3 is a dual GTPase activating protein for Arf6 and RhoA which was identified in a screen for phosphatidylinositol-(3,4,5)-trisphophate (PtdIns(3,4,5)P(3)) binding proteins. It is a regulator of cell shape and adhesion, and is itself regulated by PtdIns(3,4,5)P(3,) which acts to recruit ARAP3 to the plasma membrane and to catalytically activate it. We show here that ARAP3 binds to PtdIns(3,4,5)P(3) in an unusual, PH domain-dependent manner. None of the five PH domains are sufficient to bind PtdIns(3,4,5)P(3) in isolation. Instead, the minimal PtdIns(3,4,5)P(3) binding fragment comprises ARAP3's N-terminal tandem PH domains, and an N-terminal linker region. For substantial binding, the N-terminal sterile alpha motif (SAM) domain is also required. Site-directed mutagenesis of either of the two N-terminal PH domains within the fragment greatly reduces binding to PtdIns(3,4,5)P(3), however, in the context of the full-length protein, point mutations in the second PH domain have a lesser effect on binding, whilst deletion of any one of the five PH domains abolishes PtdIns(3,4,5)P(3) binding. We propose a mechanism by which basic residues from the N-terminal tandem PH domains, and from elsewhere in the protein synergise to mediate strong, specific PtdIns(3,4,5)P(3) binding.