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The Babraham Institute Publications database contains details of all publications resulting from our research groups and scientific facilities. Pre-prints by Institute authors can be viewed on the Institute's bioRxiv channel. We believe that free and open access to the outputs of publicly‐funded research offers significant social and economic benefits, as well as aiding the development of new research. We are working to provide Open Access to as many publications as possible and these can be identified below by the padlock icon. Where this hasn't been possible, subscriptions may be required to view the full text.
 

Petry CJ, Ong KK, Barratt BJ, Wingate D, Cordell HJ, Ring SM, Pembrey ME, Reik W, Todd JA, Dunger DB, Epigenetics

Common genetic variation at genes that are imprinted and exclusively maternally expressed could explain the apparent maternal-specific inheritance of low birthweight reported in large family pedigrees. We identified ten single nucleotide polymorphisms (SNPs) in H19, and we genotyped three of these SNPs in families from the contemporary ALSPAC UK birth cohort (1,696 children, 822 mothers and 661 fathers) in order to explore associations with size at birth and cord blood IGF-II levels.

+view abstract BMC genetics, PMID: 15885138 2005

Chakalova L, Carter D, Debrand E, Goyenechea B, Horton A, Miles J, Osborne C, Fraser P

The beta-globin genes have become a classical model for studying regulation of gene expression. Wide-ranging studies have revealed multiple levels of epigenetic regulation that coordinately ensure a highly specialised, tissue- and stage-specific gene transcription pattern. Key players include cis-acting elements involved in establishing and maintaining specific chromatin conformations and histone modification patterns, elements engaged in the transcription process through long-range regulatory interactions, transacting general and tissue-specific factors. On a larger scale, molecular events occurring at the locus level take place in the context of a highly dynamic nucleus as part of the cellular epigenetic programme.

+view abstract Progress in molecular and subcellular biology, PMID: 15881896 2005

AM Condliffe, K Davidson, KE Anderson, CD Ellson, T Crabbe, K Okkenhaug, B Vanhaesebroeck, M Turner, L Webb, MP Wymann, E Hirsch, T Ruckle, M Camps, C Rommel, SP Jackson, ER Chilvers, LR Stephens, PT Hawkins Immunology

It is well established that preexposure of human neutrophils to proinflammatory cytokines markedly augments the production of reactive oxygen species (ROS) to subsequent stimuli. This priming event is thought to be critical for localizing ROS to the vicinity of the inflammation, maximizing their role in the resolution of the inflammation, and minimizing the damage to surrounding tissue. We have used a new generation of isoform-selective phosphoinositide 3-kinase (PI3K) inhibitors to show that ROS production under these circumstances is regulated by temporal control of class I PI3K activity. Stimulation of tumor necrosis factor-alpha (TNF-alpha)-primed human neutrophils with N-formyl-methionyl-leucyl-phenylalanine (fMLP) results in biphasic activation of PI3K; the first phase is largely dependent on PI3Kgamma, and the second phase is largely dependent on PI3Kdelta. The second phase of PI3K activation requires the first phase; it is this second phase that is augmented by TNF-alpha priming and that regulates parallel activation of ROS production. Surprisingly, although TNF-alpha-primed mouse bone marrow-derived neutrophils exhibit superficially similar patterns of PI3K activation and ROS production in response to fMLP, these responses are substantially lower and largely dependent on PI3Kgamma alone. These results start to define which PI3K isoforms are responsible for modulating neutrophil responsiveness to infection and inflammation.

+view abstract Blood, PMID: 15878979 2005

Sinnecker D, Voigt P, Hellwig N, Schaefer M Epigenetics

Color variants of green fluorescent protein (GFP) are increasingly used for multicolor imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP). Here we show that experimental settings commonly used in these imaging experiments may induce an as yet uncharacterized reversible photobleaching of fluorescent proteins, which is more pronounced at acidic pH. Whereas the reversible photobleaching spectrum of eCFP corresponds to its absorption spectrum, reversible photobleaching spectra of yellow variants resemble absorption spectra of their protonated states. Fluorescence intensities recover spontaneously with time constants of 25-58 s. The recovery of eCFP can be further accelerated by illumination. The resulting steady-state fluorescence reflects a variable equilibrium between reversible photobleaching, spontaneous recovery, and light-induced recovery. These processes can cause significant artifacts in commonly applied imaging techniques, photobleach-based FRET determinations, and FRAP assays.

+view abstract Biochemistry, PMID: 15865453

PJ Rugg-Gunn, AC Ferguson-Smith, RA Pedersen Epigenetics

We examined the allele-specific expression of six imprinted genes and the methylation profiles of three imprinting control regions to assess the epigenetic status of human embryonic stem cells. We identified generally monoallelic gene expression and normal methylation patterns. During prolonged passage, one cell line became biallelic with respect to H19, but without loss of the gametic methylation imprint. These data argue for a substantial degree of epigenetic stability in human embryonic stem cells.

+view abstract Nature genetics, PMID: 15864307 2005

David DC, Hauptmann S, Scherping I, Schuessel K, Keil U, Rizzu P, Ravid R, Dröse S, Brandt U, Müller WE, Eckert A, Götz J Signalling

Transgenic mice overexpressing the P301L mutant human tau protein exhibit an accumulation of hyperphosphorylated tau and develop neurofibrillary tangles. The consequences of tau pathology were investigated here by proteomics followed by functional analysis. Mainly metabolism-related proteins including mitochondrial respiratory chain complex components, antioxidant enzymes, and synaptic proteins were identified as modified in the proteome pattern of P301L tau mice. Significantly, the reduction in mitochondrial complex V levels in the P301L tau mice revealed using proteomics was also confirmed as decreased in human P301L FTDP-17 (frontotemporal dementia with parkinsonism linked to chromosome 17) brains. Functional analysis demonstrated a mitochondrial dysfunction in P301L tau mice together with reduced NADH-ubiquinone oxidoreductase activity and, with age, impaired mitochondrial respiration and ATP synthesis. Mitochondrial dys-function was associated with higher levels of reactive oxygen species in aged transgenic mice. Increased tau pathology as in aged homozygous P301L tau mice revealed modified lipid peroxidation levels and the up-regulation of antioxidant enzymes in response to oxidative stress. Furthermore, P301L tau mitochondria displayed increased vulnerability toward beta-amyloid (Abeta) peptide insult, suggesting a synergistic action of tau and Abeta pathology on the mitochondria. Taken together, we conclude that tau pathology involves a mitochondrial and oxidative stress disorder possibly distinct from that caused by Abeta.

+view abstract The Journal of biological chemistry, PMID: 15831501

Reik W, Lewis A Epigenetics

Recent studies have revealed mechanistic parallels between imprinted X-chromosome inactivation and autosomal imprinting. We suggest that neither mechanism was present in ancestral egg-laying mammals, and that both arose when the evolution of the placenta exerted selective pressure to imprint growth-related genes. We also propose that non-coding RNAs and histone modifications were adopted for the imprinting of growth suppressors on the X chromosome and on autosomes. This provides a unified hypothesis for the evolution of X-chromosome inactivation and imprinting.

+view abstract Nature reviews. Genetics, PMID: 15818385 2005

Mead KI, Zheng Y, Manzotti CN, Perry LC, Liu MK, Burke F, Powner DJ, Wakelam MJ, Sansom DM Signalling

CTLA-4 is an essential protein in the regulation of T cell responses that interacts with two ligands found on the surface of APCs (CD80 and CD86). CTLA-4 is itself poorly expressed on the T cell surface and is predominantly localized to intracellular compartments. We have studied the mechanisms involved in the delivery of CTLA-4 to the cell surface using a model Chinese hamster ovary cell system and compared this with activated and regulatory human T cells. We have shown that expression of CTLA-4 at the plasma membrane (PM) is controlled by exocytosis of CTLA-4-containing vesicles and followed by rapid endocytosis. Using selective inhibitors and dominant negative mutants, we have shown that exocytosis of CTLA-4 is dependent on the activity of the GTPase ADP ribosylation factor-1 and on phospholipase D activity. CTLA-4 was identified in a perinuclear compartment overlapping with the cis-Golgi marker GM-130 but did not colocalize strongly with lysosomal markers such as CD63 and lysosome-associated membrane protein. In regulatory T cells, activation of phospholipase D was sufficient to trigger release of CTLA-4 to the PM but did not inhibit endocytosis. Taken together, these data suggest that CTLA-4 may be stored in a specialized compartment in regulatory T cells that can be triggered rapidly for deployment to the PM in a phospholipase D- and ADP ribosylation factor-1-dependent manner.

+view abstract Journal of immunology (Baltimore, Md. : 1950), PMID: 15814706 2005

Morgan HD, Santos F, Green K, Dean W, Reik W Epigenetics

Epigenetic marking systems confer stability of gene expression during mammalian development. Genome-wide epigenetic reprogramming occurs at stages when developmental potency of cells changes. At fertilization, the paternal genome exchanges protamines for histones, undergoes DNA demethylation, and acquires histone modifications, whereas the maternal genome appears epigenetically more static. During preimplantation development, there is passive DNA demethylation and further reorganization of histone modifications. In blastocysts, embryonic and extraembryonic lineages first show different epigenetic marks. This epigenetic reprogramming is likely to be needed for totipotency, correct initiation of embryonic gene expression, and early lineage development in the embryo. Comparative work demonstrates reprogramming in all mammalian species analysed, but the extent and timing varies, consistent with notable differences between species during preimplantation development. Parental imprinting marks originate in sperm and oocytes and are generally protected from this genome-wide reprogramming. Early primordial germ cells possess imprinting marks similar to those of somatic cells. However, rapid DNA demethylation after midgestation erases these parental imprints, in preparation for sex-specific de novo methylation during gametogenesis. Aberrant reprogramming of somatic epigenetic marks after somatic cell nuclear transfer leads to epigenetic defects in cloned embryos and stem cells. Links between epigenetic marking systems appear to be developmentally regulated contributing to plasticity. A number of activities that confer epigenetic marks are firmly established, while for those that remove marks, particularly methylation, some interesting candidates have emerged recently which need thorough testing in vivo. A mechanistic understanding of reprogramming will be crucial for medical applications of stem cell technology.

+view abstract Human molecular genetics, PMID: 15809273 2005

West AG, Fraser P

In this review, we look at the most recent studies of DNA elements that function over long genomic distances to regulate gene transcription and will discuss the mechanisms genes employ to overcome the positive and negative influences of their genomic neighbourhood in order to achieve accurate programmes of expression. Enhancer elements activate high levels of transcription of linked genes from distal locations. Recent technological advances have demonstrated chromatin loop interactions between enhancers and their target promoters. Moreover, there is increasing evidence that these dynamic interactions regulate the repositioning of genes to foci of active transcription within the nucleus. Enhancers have the potential to activate a number of neighbouring genes over a large chromosomal region, hence, their action must be restricted in order to prevent activation of non-target genes. This is achieved by specialized DNA sequences, termed enhancer blockers (or insulators), that interfere with an enhancer's ability to communicate with a target promoter when positioned between the two. Here, we summarize current models of enhancer blocking activity and discuss recent findings of how it can be dynamically regulated. It has become clear that enhancer blocking elements should not be considered only as structural elements on the periphery of gene loci, but as regulatory elements that are crucial to the outcome of gene expression. The transcription potential of a gene can also be susceptible to heterochromatic silencing originating from its chromatin environment. Insulator elements can act as barriers to the spread of heterochromatin. We discuss recent evidence supporting a number of non-exclusive mechanisms of barrier action, which mostly describe the modulation of chromatin structure or modification.

+view abstract Human molecular genetics, PMID: 15809261 2005

Taly A, Delarue M, Grutter T, Nilges M, Le Novère N, Corringer PJ, Changeux JP Signalling

We present a three-dimensional model of the homopentameric alpha7 nicotinic acetylcholine receptor (nAChR), that includes the extracellular and membrane domains, developed by comparative modeling on the basis of: 1), the x-ray crystal structure of the snail acetylcholine binding protein, an homolog of the extracellular domain of nAChRs; and 2), cryo-electron microscopy data of the membrane domain collected on Torpedo marmorata nAChRs. We performed normal mode analysis on the complete three-dimensional model to explore protein flexibility. Among the first 10 lowest frequency modes, only the first mode produces a structural reorganization compatible with channel gating: a wide opening of the channel pore caused by a concerted symmetrical quaternary twist motion of the protein with opposing rotations of the upper (extracellular) and lower (transmembrane) domains. Still, significant reorganizations are observed within each subunit, that involve their bending at the domain interface, an increase of angle between the two beta-sheets composing the extracellular domain, the internal beta-sheet being significantly correlated to the movement of the M2 alpha-helical segment. This global symmetrical twist motion of the pentameric protein complex, which resembles the opening transition of other multimeric ion channels, reasonably accounts for the available experimental data and thus likely describes the nAChR gating process.

+view abstract Biophysical journal, PMID: 15805177 2005

Plagge A, Isles AR, Gordon E, Humby T, Dean W, Gritsch S, Fischer-Colbrie R, Wilkinson LS, Kelsey G Epigenetics

Genomic imprinting results in parent-of-origin-dependent monoallelic expression of selected genes. Although their importance in development and physiology is recognized, few imprinted genes have been investigated for their effects on brain function. Gnas is a complex imprinted locus whose gene products are involved in early postnatal adaptations and neuroendocrine functions. Gnas encodes the stimulatory G-protein subunit Gsalpha and two other imprinted protein-coding transcripts. Of these, the Nesp transcript, expressed exclusively from the maternal allele, codes for neuroendocrine secretory protein 55 (Nesp55), a chromogranin-like polypeptide associated with the constitutive secretory pathway but with an unknown function. Nesp is expressed in restricted brain nuclei, suggesting an involvement in specific behaviors. We have generated a knockout of Nesp55 in mice. Nesp55-deficient mice develop normally, excluding a role of this protein in the severe postnatal effects associated with imprinting of the Gnas cluster. Behavioral analysis of adult Nesp55 mutants revealed, in three separate tasks, abnormal reactivity to novel environments independent of general locomotor activity and anxiety. This phenotype may be related to prominent Nesp55 expression in the noradrenergic locus coeruleus. These results indicate a role of maternally expressed Nesp55 in controlling exploratory behavior and are the first demonstration that imprinted genes affect such a fundamental behavior.

+view abstract Molecular and cellular biology, PMID: 15798190 2005

S Suire, J Coadwell, GJ Ferguson, K Davidson, P Hawkins, L Stephens Signalling

A variety of genetic and inhibitor studies have shown that phosphoinositide 3-kinase gamma (PI3Kgamma) plays an essential role in a number of physiological responses, including neutrophil chemotaxis, mast cell degranulation, and cardiac function []. PI3Kgamma is currently thought to be composed of a p110gamma catalytic subunit and a single regulatory subunit, p101. The binding of p110gamma to p101 dramatically increases the activation of the complex by Gbetagamma subunits and, hence, is thought to be critical for the coupling of PI3Kgamma to G protein coupled receptors []. Here, we characterize a new regulatory subunit for PI3Kgamma. p84 is present in human, mouse, chicken, frog, and fugu genomes and is located beside the p101 locus. It is broadly expressed in cells of the murine immune system. Both recombinant and endogenous p84 bind p110gamma specifically and with high affinity. Binding of p84 to p110gamma substantially increases the ability of Gbetagamma to stimulate phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) production both in vitro and in vivo. However, the p84/p110gamma heterodimer is approximately 4-fold less sensitive to Gbetagammas than p101/p110gamma. Endogenous murine p84 expression is substantially reduced in the absence of p110gamma expression. We conclude that p110gamma has two potential regulatory subunits in vivo, p84 and p101.

+view abstract Current biology : CB, PMID: 15797027 2005

Liston A, Lesage S, Gray DH, Boyd RL, Goodnow CC Immunology

The cause of common organ-specific autoimmune diseases is poorly understood because of genetic and cellular complexity in humans and animals. Recent advances in the understanding of the mechanisms of the defects underlying autoimmune disease in autoimmune polyendocrinopathy syndrome type 1 and non-obese diabetic mice suggest that failures in central tolerance play a key role in predisposition towards organ-specific autoimmunity. The lessons from such rare monogenic autoimmune disorders and well-characterized polygenic traits demonstrate how subtle quantitative trait loci can result in large changes in the susceptibility to autoimmunity. These data allow us to propose a model relating efficiency of thymic deletion to T-cell tolerance and susceptibility to autoimmunity.

+view abstract Immunological reviews, PMID: 15790352 2005

Puri KD, Doggett TA, Huang CY, Douangpanya J, Hayflick JS, Turner M, Penninger J, Diacovo TG

Phosphoinositide 3-kinase gamma (PI3Kgamma) in neutrophils plays a critical role in the directed migration of these cells into inflamed tissues. In this study, we demonstrate the importance of the endothelial component of PI3Kgamma activity relative to its leukocyte counterpart in supporting neutrophil interactions with the inflamed vessel wall. Despite the reconstitution of class-Ib PI3K function in neutrophils of p110gamma-/- mice, we observed a 45% reduction in accumulation of these cells in an acute lung injury model. Mechanistically, this appears to result from a perturbation in selectin-mediated adhesion as manifested by a 70% reduction in wild-type (WT) neutrophil attachment to and 17-fold increase in rolling velocities on p110gamma-/- microvessels in vivo in response to tumor necrosis factor alpha (TNFalpha). This alteration in adhesion was further augmented by a deficiency in p110delta, suggesting that the activity of both catalytic subunits is required for efficient capture of neutrophils by cytokine-stimulated endothelium. Interestingly, E-selectin-mediated adhesion in p110gamma-/-) mice was impaired by more than 95%, but no defect in nuclear factor kappa B (NF-kappaB)-induced gene expression was observed. These findings suggest a previously unrecognized partnership between class-I PI3Ks expressed in leukocytes and endothelium, the combination of which is required for the efficient trafficking of immunocompetent cells to sites of inflammation.

+view abstract Blood, PMID: 15769890 2005

Zouwail S, Pettitt TR, Dove SK, Chibalina MV, Powner DJ, Haynes L, Wakelam MJ, Insall RH Signalling

PLD (phospholipase D) activity catalyses the generation of the lipid messenger phosphatidic acid, which has been implicated in a number of cellular processes, particularly the regulation of membrane traffic. In the present study, we report that disruption of PLD signalling causes unexpectedly profound effects on the actin-based motility of Dictyostelium. Cells in which PLD activity is inhibited by butan-1-ol show a complete loss of actin-based structures, accompanied by relocalization of F-actin into small clusters, and eventually the nucleus, without a visible fall in levels of F-actin. Addition of exogenous phosphatidic acid reverses the effects of butan-1-ol, confirming that these effects are caused by inhibition of PLD. Loss of motility correlates with complete inhibition of endocytosis and a reduction in phagocytosis. Inhibition of PLD caused a major decrease in the synthesis of PtdIns(4,5)P2, which could again be reversed by exogenously applied phosphatidic acid. Thus the essential role of PLD signalling in both motility and endocytosis appears to be mediated directly via regulation of PtdIns(4)P kinase activity. This implies that localized PLD-regulated synthesis of PtdIns(4,5)P2 is essential for Dictyostelium actin function.

+view abstract The Biochemical journal, PMID: 15769249 2005

Santos F, Peters AH, Otte AP, Reik W, Dean W Epigenetics

On fertilisation, gametes undergo epigenetic reorganisation and re-establish totipotency. Here, we investigate links between chromatin remodelling and asymmetric maintenance of DNA methylation in the early mouse embryo. Using antibodies for lysine specific H3 methylation reveals that the male pronucleus is negative for di- and trimethyl H3-K9 yet the female is positive for these residues. However, the male is positive for monomethyl H3-K9 and H3-K27 and these signals increase during pronuclear maturation. Non-histone chromatin proteins of the Polycomb group are found in the paternal compartment as early as sperm decondensation. However, trimethyl H3-K27 is not observed in the male until the completion of DNA replication. Heterochromatin protein 1 beta (HP1beta) is abundant in the male pronucleus, despite the absence of di- and trimethyl H3-K9, and co-localises with monomethyl H3-K9. Recent evidence identifies monomethyl H3-K9 as the preferred substrate of Suvar39h, the histone methyl transferase (HMT) responsible for heterochromatic H3-K9 trimethylation. The association of HP1beta with monomethyl H3-K9 may assist in preventing further modification of H3-K9. Association of dimethylation but not trimethylation of H3-K9 with DNA methylation, in the female pronucleus, suggests a mechanistically significant link. These differences begin to provide a chromatin based explanation for paternal-specific active DNA demethylation and maternal specific protection in the mouse.

+view abstract Developmental biology, PMID: 15766761 2005

AE Corcoran

Lymphocytes are characterised by monoclonal expression of antigen receptors. This is achieved by silencing of one of two homologous antigen receptor alleles, a process known as allelic exclusion. This process is regulated both before and after V(D)J recombination, by a variety of mechanisms. These include nuclear localisation, changes in chromatin structure and histone modifications, non-coding sense and antisense RNA transcription, epigenetic alterations at the DNA level, feedback signalling from expressed alleles, locus contraction and decontraction, recruitment to heterochromatin. This review will focus on recent advances in the immunoglobulin heavy and kappa light chain loci. The current picture is of a complex, temporally ordered sequence of events, in which these loci share many contributory mechanisms, but clear and intriguing differences are emerging.

+view abstract Seminars in immunology, PMID: 15737575 2005

R Ley, K Hadfield, E Howes, SJ Cook Signalling

The BH3-only protein, Bim, exists as three splice variants (Bim(S), Bim(L), and Bim(EL)) of differing pro-apoptotic potency. Bim(EL), the least effective killer, is degraded by the proteasome in response to phosphorylation by extracellular signal-regulated kinases 1 and 2 (ERK1/2). ERK1/2-dependent phosphorylation correlates with the presence of a domain unique to the Bim(EL) splice variant that includes the major ERK1/2 phosphorylation site Ser(65). However, efficient phosphorylation by ERK1/2, c-Jun N-terminal kinase, or p38 requires the presence in the substrate of a discrete kinase-docking domain as well as the phosphoacceptor site. Here we show that the region unique to Bim(EL) (amino acids 41-97) harbors two potential DEF-type ERK1/2 kinase-docking domains, DEF1 and DEF2. Peptide competition assays revealed that the DEF2 peptide could act autonomously to bind active ERK1/2, whereas the DEF1 peptide did not. Truncation analysis identified a minimal region, residues 80-97, containing the DEF2 motif as sufficient for ERK1/2 binding. Mutation of key residues in the DEF2 motif abolished the interaction of ERK1/2 and Bim(EL) and also abolished ERK1/2-dependent phosphorylation of Bim(EL) in vivo, thereby stabilizing the protein and enhancing cytotoxicity. Our results identify a new physiologically relevant functional motif in Bim(EL) that may account for the distinct biological properties of this splice variant.

+view abstract The Journal of biological chemistry, PMID: 15728578 2005

H Rooks, J Bergounioux, L Game, JP Close, C Osborne, S Best, T Senior, S Height, R Thompson, N Hadzic, P Fraser, P Bolton-Maggs, SL Thein

We have characterized three novel epsilon gamma delta beta-thalassaemia deletions in three English families. Two of the deletions, 114 and 439 kb, removed the entire beta-globin gene complex, including a variable number of flanking olfactory receptor (HOR) genes. The 98-kb deletion extended 90-kb upstream of the epsilon gene to 8 kb upstream of the G gamma-gene, leaving the gamma,delta and beta-genes intact. The 439 kb deletion is the largest deletion reported so far to cause epsilon gamma delta beta-thalassaemia; heterozygotes for this deletion were variably affected by neonatal haemolytic anaemia. Two of the deletions were de novo. Breakpoints of all three deletions occurred within regions of L1 or Alu repeats and contained short regions of direct homology between the flanking sequences, a feature that is likely to have contributed to the illegitimate recombinations.

+view abstract British journal of haematology, PMID: 15725095 2005

Houseley JM,Wang Z,Brock GJ,Soloway J,Artero R,Perez-Alonso M,O'Dell KM,Monckton DG Epigenetics

Myotonic dystrophy type 1 is an autosomal dominant disorder associated with the expansion of a CTG repeat in the 3' untranslated region (UTR) of the DMPK gene. Recent data suggest that pathogenesis is predominantly mediated by a gain of function of the mutant transcript. In patients, these expanded CUG repeat-containing transcripts are sequestered into ribonuclear foci that also contain the muscleblind-like proteins. To provide further insights into muscleblind function and the pathogenesis of myotonic dystrophy, we generated Drosophila incorporating CTG repeats in the 3'-UTR of a reporter gene. As in patients, expanded CUG repeats form discrete ribonuclear foci in Drosophila muscle cells that co-localize with muscleblind. Unexpectedly, however, foci are not observed in all cell types and muscleblind is neither necessary nor sufficient for their formation. The foci are dynamic transient structures with short half-lifes that do not co-localize with the proteasome, suggesting they are unlikely to contain mis-folded proteins. However, they do co-localize with non-A, the human orthologs of which are implicated in both RNA splicing and attachment of dsRNA to the nuclear matrix. Muscleblind is also revealed as having a previously unrecognized role in stabilizing CUG transcripts. Most interestingly, Drosophila expressing (CUG)162 repeats has no detectable pathological phenotype suggesting that in contrast to expanded polyglutamine-containing proteins, neither the expanded CUG repeat RNA nor the ribonuclear foci are directly toxic.

+view abstract Human molecular genetics, PMID: 15703191 2005

Beirowski B, Adalbert R, Wagner D, Grumme DS, Addicks K, Ribchester RR, Coleman MP Signalling

The progressive nature of Wallerian degeneration has long been controversial. Conflicting reports that distal stumps of injured axons degenerate anterogradely, retrogradely, or simultaneously are based on statistical observations at discontinuous locations within the nerve, without observing any single axon at two distant points. As axon degeneration is asynchronous, there are clear advantages to longitudinal studies of individual degenerating axons. We recently validated the study of Wallerian degeneration using yellow fluorescent protein (YFP) in a small, representative population of axons, which greatly improves longitudinal imaging. Here, we apply this method to study the progressive nature of Wallerian degeneration in both wild-type and slow Wallerian degeneration (WldS) mutant mice.

+view abstract BMC neuroscience, PMID: 15686598 2005

J Wu, F Iwata, JA Grass, CS Osborne, L Elnitski, P Fraser, O Ohneda, M Yamamoto, EH Bresnick

The process whereby the primitive vascular network develops into the mature vasculature, known as angiogenic vascular remodeling, is controlled by the Notch signaling pathway. Of the two mammalian Notch receptors expressed in vascular endothelium, Notch1 is broadly expressed in diverse cell types, whereas Notch4 is preferentially expressed in endothelial cells. As mechanisms that confer Notch4 expression were unknown, we investigated how NOTCH4 transcription is regulated in human endothelial cells and in transgenic mice. The NOTCH4 promoter and the 5' portion of NOTCH4 assembled into an endothelial cell-specific histone modification pattern. Analysis of NOTCH4 primary transcripts in human umbilical vein endothelial cells by RNA fluorescence in situ hybridization revealed that 36% of the cells transcribed one or both NOTCH4 alleles. The NOTCH4 promoter was sufficient to confer endothelial cell-specific transcription in transfection assays, but intron 1 or upstream sequences were required for expression in the vasculature of transgenic mouse embryos. Cell-type-specific activator protein 1 (AP-1) complexes occupied NOTCH4 chromatin and conferred endothelial cell-specific transcription. Vascular angiogenic factors activated AP-1 and reprogrammed the endogenous NOTCH4 gene in HeLa cells from a repressed to a transcriptionally active state. These results reveal an AP-1-Notch4 pathway, which we propose to be crucial for transducing angiogenic signals and to be deregulated upon aberrant signal transduction in cancer.

+view abstract Molecular and cellular biology, PMID: 15684396 2005

T Barthlott, H Moncrieffe, M Veldhoen, CJ Atkins, J Christensen, A O'Garra, B Stockinger Immunology

Maintenance of homeostasis in the immune system involves competition for resources between T lymphocytes, which avoids the development of immune pathology seen in lymphopenic mice. CD25+ CD4+ T cells are important for homeostasis, but there is as yet no consensus on their mechanisms of action. Although CD25+ CD4+ T cells cause substantial down-regulation of IL-2 mRNA in responder T cells in an in vitro co-culture system, the presence of IL-protein can be demonstrated by intracellular staining. As a consequence of competition for IL-2, CD25+ CD4+ T cells further up-regulate the IL-2R alpha chain (CD25), a process that is strictly dependent on IL-2, whereas responder T cells fail to up-regulate CD25. Similarly, adoptive transfer into lymphopenic mice showed that CD25+ CD4+ T cells interfere with CD25 up-regulation on co-transferred naive T cells, while increasing their own CD25 levels. IL-2 sequestration by CD25+ CD4+ T cells is not a passive phenomenon but instead initiates--in conjunction with signals through the TCR--their differentiation to IL-10 production. Although IL-10 is not required for in vitro suppression, it is vital for the in vivo function of regulatory T cells. Our data provide a link explaining the apparent difference in regulatory mechanisms in vitro and in vivo.

+view abstract International immunology, PMID: 15684039 2005