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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.
 

M Gehring, W Reik, S Henikoff Epigenetics

Active DNA demethylation underlies key facets of reproduction in flowering plants and mammals and serves a general genome housekeeping function in plants. A family of 5-methylcytosine DNA glycosylases catalyzes plant demethylation via the well-known DNA base-excision-repair process. Although the existence of active demethylation has been known for a longer time in mammals, the means of achieving it remain murky and mammals lack counterparts to the plant demethylases. Several intriguing experiments have indicated, but not conclusively proven, that DNA repair is also a plausible mechanism for animal demethylation. Here, we examine what is known from flowering plants about the pathways and function of enzymatic demethylation and discuss possible mechanisms whereby DNA repair might also underlie global demethylation in mammals.

+view abstract Trends in genetics : TIG, PMID: 19144439 2009

M Chotalia, SA Smallwood, N Ruf, C Dawson, D Lucifero, M Frontera, K James, W Dean, G Kelsey Epigenetics

Genomic imprinting requires the differential marking by DNA methylation of genes in male and female gametes. In the female germline, acquisition of methylation imprint marks depends upon the de novo methyltransferase Dnmt3a and its cofactor Dnmt3L, but the reasons why specific sequences are targets for Dnmt3a and Dnmt3L are still poorly understood. Here, we investigate the role of transcription in establishing maternal germline methylation marks. We show that at the Gnas locus, truncating transcripts from the furthest upstream Nesp promoter disrupts oocyte-derived methylation of the differentially methylated regions (DMRs). Transcription through DMRs in oocytes is not restricted to this locus but occurs across the prospective DMRs at many other maternally marked imprinted domains, suggesting a common requirement for transcription events. The transcripts implicated here in gametic methylation are protein-coding, in contrast to the noncoding antisense transcripts involved in the monoallelic silencing of imprinted genes in somatic tissues, although they often initiate from alternative promoters in oocytes. We propose that transcription is a third essential component of the de novo methylation system, which includes optimal CpG spacing and histone modifications, and may be required to create or maintain open chromatin domains to allow the methylation complex access to its preferred targets.

+view abstract Genes & development, PMID: 19136628 2009

DC Berwick, M Calissano, JD Corness, SJ Cook, DS Latchman Signalling

The POU family transcription factor Brn-3a is required for the differentiation and survival of sensory neurones, and is phosphorylated in neuroblastoma cells following treatment with all-trans retinoic acid (RA). Mutation of serines-121 and -122 of Brn-3a to alanine blocks its phosphorylation and impairs RA-mediated neurite outgrowth. Here we show that this deficit in differentiation is mimicked by a single mutation at serine-122, and demonstrate a similar requirement for a second residue, threonine-39. Like Brn-3a, the neuropeptide Galanin has been implicated in the development of sensory neurones. We show that Brn-3a over-expression acts synergistically with RA treatment to up-regulate Galanin promoter activity; that the activity of the N-terminal transcriptional activation domain of Brn-3a is increased following RA treatment; and that both these effects require threonine-39 and serine-122. In addition, we demonstrate that the RA-mediated activation of Galanin promoter activity and Brn-3a N-terminal transcriptional activity are both blocked by pan-MEK inhibitors, and show that the expression of a constitutively-active mutant of MEK1, but not MEK5, is sufficient to increase Brn-3a activity. These results reveal an important role for the ERK1/2 pathway in Brn-3a regulation during RA-mediated neuronal differentiation and define the neuropeptide Galanin as a novel target of this transcription factor.

+view abstract Brain research, PMID: 19135033 2009

Kaden D, Voigt P, Munter LM, Bobowski KD, Schaefer M, Multhaup G Epigenetics

The molecular association between APP and its mammalian homologs has hardly been explored. In systematically addressing this issue, we show by live cell imaging that APLP1 mainly localizes to the cell surface, whereas APP and APLP2 are mostly found in intracellular compartments. Homo- and heterotypic cis interactions of APP family members could be detected by FRET and co-immunoprecipitation analysis and occur in a modular mode. Only APLP1 formed trans interactions, supporting the argument for a putative specific role of APLP1 in cell adhesion. Deletion mutants of APP family members revealed two highly conserved regions as important for the protein crosstalk. In particular, the N-terminal half of the ectodomain was crucial for APP and APLP2 interactions. By contrast, multimerization of APLP1 was only partially dependent on this domain but strongly on the C-terminal half of the ectodomain. We further observed that coexpression of APP with APLP1 or APLP2 leads to diminished generation of Abeta42. The current data suggest that this is due to the formation of heteromeric complexes, opening the way for novel therapeutic strategies targeting these complexes.

+view abstract Journal of cell science, PMID: 19126676

Ribeiro de Almeida C, Heath H, Krpic S, Dingjan GM, van Hamburg JP, Bergen I, van de Nobelen S, Sleutels F, Grosveld F, Galjart N, Hendriks RW Immunology

Differentiation of naive CD4+ cells into Th2 cells is accompanied by chromatin remodeling at the Th2 cytokine locus allowing the expression of the IL-4, IL-5, and IL-13 genes. In this report, we investigated the role in Th2 differentiation of the transcription regulator CCCTC-binding factor (CTCF). Chromatin immunoprecipitation analysis revealed multiple CTCF binding sites in the Th2 cytokine locus. Conditional deletion of the Ctcf gene in double-positive thymocytes allowed development of peripheral T cells, but their activation and proliferation upon anti-CD3/anti-CD28 stimulation in vitro was severely impaired. Nevertheless, when TCR signaling was circumvented with phorbol ester and ionomycin, we observed proliferation of CTCF-deficient T cells, enabling the analysis of Th2 differentiation in vitro. We found that in CTCF-deficient Th2 polarization cultures, transcription of IL-4, IL-5, and IL-13 was strongly reduced. By contrast, CTCF deficiency had a moderate effect on IFN-gamma production in Th1 cultures and IL-17 production in Th17 cultures was unaffected. Consistent with a Th2 cytokine defect, CTCF-deficient mice had very low levels of IgG1 and IgE in their serum, but IgG2c was close to normal. In CTCF-deficient Th2 cultures, cells were polarized toward the Th2 lineage, as substantiated by induction of the key transcriptional regulators GATA3 and special AT-rich binding protein 1 (SATB1) and down-regulation of T-bet. Also, STAT4 expression was low, indicating that in the absence of CTCF, GATA3 still operated as a negative regulator of STAT4. Taken together, these findings show that CTCF is essential for GATA3- and SATB1-dependent regulation of Th2 cytokine gene expression.

+view abstract Journal of immunology (Baltimore, Md. : 1950), PMID: 19124743 2009

M Veldhoen, K Hirota, J Christensen, A O'Garra, B Stockinger Immunology

Th17 cell differentiation is dependent on interleukin (IL)-6 and transforming growth factor (TGF)-beta, and it is modulated by activation of the aryl hydrocarbon receptor (AhR). In this study, we show that differentiation of Th17 cells, but not Th1 or induced regulatory T (iT reg) cells, is increased by endogenous AhR agonists present in culture medium. Th17 development from wild-type mice is suboptimal in the presence of the AhR antagonist CH-223191, similar to the situation in AhR-deficient mice, which show attenuated IL-17 production and no IL-22 production. The presence of natural AhR agonists in culture medium is also revealed by the induction of CYP1A1, a downstream target of AhR activation. However, the most commonly used medium, RPMI, supports very low levels of Th17 polarization, whereas Iscove's modified Dulbecco's medium, a medium richer in aromatic amino acids, which give rise to AhR agonists, consistently results in higher Th17 expansion in both mouse and human cells. The relative paucity of AhR agonists in RPMI medium, coupled with the presence of factors conducive to IL-2 activation and enhanced Stat5 phosphorylation, conspire against optimal Th17 differentiation. Our data emphasize that AhR activation plays an essential part in the development of Th17 cells and provide a rational explanation for the poor in vitro polarization of Th17 cells that is reported in the majority of publications for both mouse and human cells.

+view abstract The Journal of experimental medicine, PMID: 19114668 2009

R Zhao, GA Follows, PA Beer, LM Scott, BJ Huntly, AR Green, DR Alexander

The myeloproliferative disorders are clonal disorders with frequent somatic gain-of-function alterations affecting tyrosine kinases. In these diseases, there is an increase in DNA damage and a risk of progression to acute leukemia. The molecular mechanisms in myeloproliferative disorders that prevent apoptosis induced by damaged DNA are obscure.

+view abstract The New England journal of medicine, PMID: 19109573 2008

E Fahy, S Subramaniam, RC Murphy, M Nishijima, CR Raetz, T Shimizu, F Spener, G van Meer, MJ Wakelam, EA Dennis Signalling,Lipidomics

In 2005, the International Lipid Classification and Nomenclature Committee under the sponsorship of the LIPID MAPS Consortium developed and established a "Comprehensive Classification System for Lipids" based on well-defined chemical and biochemical principles and using an ontology that is extensible, flexible, and scalable. This classification system, which is compatible with contemporary databasing and informatics needs, has now been accepted internationally and widely adopted. In response to considerable attention and requests from lipid researchers from around the globe and in a variety of fields, the comprehensive classification system has undergone significant revisions over the last few years to more fully represent lipid structures from a wider variety of sources and to provide additional levels of detail as necessary. The details of this classification system are reviewed and updated and are presented here, along with revisions to its suggested nomenclature and structure-drawing recommendations for lipids.

+view abstract Journal of lipid research, PMID: 19098281 2009

C Dumont, A Corsoni-Tadrzak, S Ruf, J de Boer, A Williams, M Turner, D Kioussis, VL Tybulewicz Immunology

The Rac1 and Rac2 GTPases play important roles in many processes including cytoskeletal reorganization, proliferation, and survival, and are required for B-cell development. Previous studies had shown that deficiency in Rac2 did not affect T-cell development, whereas the function of Rac1 in this process has not been investigated. We now show that simultaneous absence of both GTPases resulted in a very strong developmental block at the pre-TCR checkpoint and in defective positive selection. Unexpectedly, deficiency of Rac1 and Rac2 also resulted in the aberrant survival of thymocytes lacking expression of TCR beta, showing hallmarks of hyperactive Notch signaling. Furthermore, we found a similar novel phenotype in the absence of Vav1, Vav2, and Vav3, which function as guanine nucleotide exchange factors for Rac1 and Rac2. These results show that a pathway containing Vav and Rac proteins may negatively regulate Notch signaling during early thymic development.

+view abstract Blood, PMID: 19088377 2009

U Singh, F Rizvi, Y Yu, W Shi, A Orth, M Karimi, TJ Ekström, A Plagge, G Kelsey, R Fundele Epigenetics

Interspecific hybridization in mammals causes hybrid dysgenesis effects, such as sterility and abnormal placentation. Here, we describe a novel obesity syndrome caused by interspecific hybridization in the genus Mus and show that this obesity, appearing sporadically in F1 littermates derived from inbred strains, has an epigenetic basis. Mus hybrids from various strains of M. musculus and M. spretus were generated and the sporadic obese phenotype was confirmed through assessment of physiological and biochemical parameters in littermates. To understand the underlying mechanisms, large-scale and candidate gene expression assays, global DNA methylation assays and allelic expression analysis were performed. Studies showed that obese hybrids are similar to other known models of obesity. While increased axial growth indicated a defect in POMC pathway, comparison of global gene expression patterns in brain of obese F1 and obese Pomc mutant mice showed little similarity. In F1 obese mice many genes involved in the maintenance of epigenetic states, as well as several imprinted genes, were differentially expressed. Global DNA methylation analysis in brain showed that increased methylation levels were associated with obesity. The imprinted gene Gnasxl, known to be important in lipid homeostasis, was found over expressed in the obese hybrids. Allelic expression and methylation analysis of Gnasxl showed that alterations of epigenetic marks underlying F1 obesity are probably many and multi-factorial.

+view abstract Archives of physiology and biochemistry, PMID: 19085233 2008

Y Zhan, S Guo, KM Kendrick, J Feng

Novel approaches to effectively reduce noise in data recorded from multi-trial physiology experiments have been investigated using two-dimensional filtering methods, adaptive Wiener filtering and reduced update Kalman filtering. Test data based on signal and noise model consisting of different conditions of signal components mixed with noise have been considered with filtering effects evaluated using analysis of frequency coherence and of time-dependent coherence. Various situations that may affect the filtering results have been explored and reveal that Wiener and Kalman filtering can considerably improve the coherence values between two channels of multi-trial data and suppress uncorrelated components. We have extended our approach to experimental data: multi-electrode array (MEA) local field potential (LFPs) recordings from the inferotemporal cortex of sheep and LFP vs. electromyogram (LFP-EMG) recording data during resting tremor in Parkinson's disease patients. Finally general procedures for implementation of these filtering techniques are described.

+view abstract Bio Systems, PMID: 19084574 2009

D Lim, SC Bowdin, L Tee, GA Kirby, E Blair, A Fryer, W Lam, C Oley, T Cole, LA Brueton, W Reik, F Macdonald, ER Maher Epigenetics

Beckwith-Wiedemann syndrome (BWS) is a model imprinting disorder resulting from mutations or epigenetic events affecting imprinted genes at 11p15.5. Most BWS cases are sporadic and result from imprinting errors (epimutations) involving either of the two 11p15.5 imprinting control regions (IC1 and IC2). Previously, we and other reported an association between sporadic BWS and assisted reproductive technologies (ARTs).

+view abstract Human reproduction (Oxford, England), PMID: 19073614 2009

DF Robbiani, A Bothmer, E Callen, B Reina-San-Martin, Y Dorsett, S Difilippantonio, DJ Bolland, HT Chen, AE Corcoran, A Nussenzweig, MC Nussenzweig

Chromosomal translocation requires formation of paired double-strand DNA breaks (DSBs) on heterologous chromosomes. One of the most well characterized oncogenic translocations juxtaposes c-myc and the immunoglobulin heavy-chain locus (IgH) and is found in Burkitt's lymphomas in humans and plasmacytomas in mice. DNA breaks in IgH leading to c-myc/IgH translocations are created by activation-induced cytidine deaminase (AID) during antibody class switch recombination or somatic hypermutation. However, the source of DNA breaks at c-myc is not known. Here, we provide evidence for the c-myc promoter region being required in targeting AID-mediated DNA damage to produce DSBs in c-myc that lead to c-myc/IgH translocations in primary B lymphocytes. Thus, in addition to producing somatic mutations and DNA breaks in antibody genes, AID is also responsible for the DNA lesions in oncogenes that are required for their translocation.

+view abstract Cell, PMID: 19070574 2008

J Houseley, L Rubbi, M Grunstein, D Tollervey, M Vogelauer Epigenetics

The extensively studied yeast GAL1-10 gene cluster is tightly regulated by environmental sugar availability. Unexpectedly, under repressive conditions the 3' region of the GAL10 coding sequence is trimethylated by Set1 on histone H3 K4, normally characteristic of 5' regions of actively transcribed genes. This reflects transcription of a long noncoding RNA (GAL10-ncRNA) that is reciprocal to GAL1 and GAL10 mRNAs and driven by the DNA-binding protein Reb1. Point mutations in predicted Reb1-binding sites abolished Reb1 binding and ncRNA synthesis. The GAL10-ncRNA is transcribed approximately once every 50 min and targeted for degradation by the TRAMP and exosome complexes, resulting in low steady-state levels (approximately one molecule per 14 cells). GAL10-ncRNA transcription recruits the methyltransferase Set2 and histone deacetylation activities in cis, leading to stable changes in chromatin structure. These chromatin modifications act principally through the Rpd3S complex to aid glucose repression of GAL1-10 at physiologically relevant sugar concentrations.

+view abstract Molecular cell, PMID: 19061643 2008

R Adalbert, A Nogradi, E Babetto, L Janeckova, SA Walker, M Kerschensteiner, T Misgeld, MP Coleman Signalling

Synapse loss precedes cell death in Alzheimer's disease, but the timing of axon degeneration relative to these events, and the causal relationships remain unclear. Axons become so severely dystrophic near amyloid plaques that their interruption, causing permanent loss of function, extensive synapse loss, and potentially cell death appears imminent. However, it remains unclear whether axons are truly interrupted at plaques and whether cell bodies fail to support their axons and dendrites. We traced TgCRND8 mouse axons longitudinally through, distal to, and proximal from dystrophic regions. The corresponding neurons not only survived but remained morphologically unaltered, indicating absence of axonal damage signalling or a failure to respond to it. Axons, no matter how dystrophic, remained continuous and initially morphologically normal outside the plaque region, reflecting support by metabolically active cell bodies and continued axonal transport. Immunochemical and ultrastructural studies showed dystrophic axons were tightly associated with disruption of presynaptic transmission machinery, suggesting local functional impairment. Thus, we rule out long-range degeneration axons or dendrites as major contributors to early synapse loss in this model, raising the prospect of a therapeutic window for functional rescue of individual neurons lasting months or even years after their axons become highly dystrophic. We propose that multi-focal pathology has an important role in the human disease in bringing about the switch from local, and potentially recoverable, synapse loss into permanent loss of neuronal processes and eventually their cell bodies.

+view abstract Brain : a journal of neurology, PMID: 19059977 2009

RA Hill, DJ Flint, JM Pell Signalling

Physiologists have routinely used understanding of the immune system to generate antibodies against regulatory molecules, growth factors, plasma membrane receptors, and other mammalian molecules in the development of analytical tools and assays. In taking this notion further, antibodies have been used in vivo to modulate physiological systems and to improve our understanding of their molecular interactions. To develop antibodies with physiological activity (efficacy), physiologists have worked with immunologists in developing interdisciplinary insights, requiring basic knowledge of immune system function in designing strategies to generate antibodies that interact with endogenous molecules of physiological interest, in vivo. Antibodies in different physiological systems have been shown to enhance or inhibit endogenous molecular functions. Two approaches have been used: passive and active immunization. Antibodies in these contexts have provided tools to develop further insights into molecular physiological mechanisms. Perhaps surprisingly, enhancing antibodies have been developed against a diverse set of target molecules including several members of the growth hormone/insulin-like growth factor-I axes and those of the beta(2)-adrenoceptor axis. Antibodies that inhibit the actions of somatostatin have also been developed. A further novel approach has been the development of antibodies that interact with adipose cells in vivo. These have the potential to be used in therapeutic antiobesity approaches. Antibodies with efficacy in vivo have provided new insights into molecular physiological mechanisms, enhancing our understanding of these complex processes.

+view abstract Advances in physiology education, PMID: 19047502 2008

A Ademokun, M Turner Immunology

Post-transcriptional control of gene expression is an important mechanism for maintaining cellular homoeostasis and regulating the immune response to infection. It allows control of mRNA abundance, translation and localization. Mechanisms for post-transcriptional control involve RNA-binding proteins and miRNAs (microRNAs). The TTP(tristetraprolin) family of proteins recognize and bind AU-rich elements. Deletion of TTP led to a systemic autoimmune syndrome with excess circulating TNFalpha (tumour necrosis factor alpha) and GM-CSF (granulocyte/macrophage colony-stimulating factor) due to aberrantly stabilized mRNA. The family may also have a role in control of lymphocyte development and function. miRNAs regulate gene expression by promoting decay or inhibiting translation of transcripts with base pair complementarity. The importance of miRNAs in lymphocytes is highlighted by the T-cell-specific deletion of Dicer, an enzyme required for miRNA-mediated processing and from the phenotype of bic (B-cell integration cluster)/miR-155 (miRNA 155)-deficient mice.

+view abstract Biochemical Society transactions, PMID: 19021522 2008

T Henley, D Kovesdi, M Turner Immunology

B-cell activating factor of the TNF family (BAFF) is critical for the survival and maturation of B cells. The molecular mechanisms by which BAFF regulates the survival of developing B cells are becoming better understood. Recent evidence has begun to emerge demonstrating a role for the PI3K/Akt signalling pathway in response to BAFF. However, the importance of the PI3K family for BAFF-signalling and the effects of loss of PI3K function on BAFF responses are still unknown. We therefore investigated the BAFF-mediated responses of B cells deficient for the PI3K catalytic subunit P110delta. We find that the loss of P110delta impairs the BAFF-mediated survival of cultured B cells demonstrating a direct role for this member of the PI3K family in regulating the survival of B cells in response to BAFF. P110delta was required for the growth of B cells in response to BAFF and was critical for the upregulation of the receptor for BAFF following BCR crosslinking. Our findings reveal an important role for p110delta in regulating B-cell responses to BAFF.

+view abstract European journal of immunology, PMID: 19016531 2008

TT Zhang, K Okkenhaug, BF Nashed, KD Puri, ZA Knight, KM Shokat, B Vanhaesebroeck, AJ Marshall Immunology

Recent studies indicate that pharmaceutical blockade of phosphoinositide 3-kinase (PI3K) signaling enzymes might be effective in reducing allergic airway inflammation. Signals generated by the p110delta PI3K isoform play critical roles in signaling through antigen and cytokine receptors and were shown to be required for induction of type 2, but not type 1, cytokine responses.

+view abstract The Journal of allergy and clinical immunology, PMID: 19014771 2008

MJ Berridge

Studies on control of fluid secretion by an insect salivary gland led to the discovery of inositol trisphosphate (IP3) and its role in calcium signalling. Many cell stimuli act on receptors that are coupled to phospholipase C that hydrolyses phosphatidylinosol 4,5-bisphosphate (PIP2) to release IP3 to the cytosol. IP3 receptors located on the endoplasmic reticulum respond to this elevation of IP3 by releasing Ca2+, which is often organized into characteristic spatial (elementary events and waves) and temporal (Ca2+ oscillations) patterns. This IP3/Ca2+ pathway is a remarkably versatile signalling system that has been adapted to control processes as diverse as fertilization, proliferation, contraction, cell metabolism, vesicle and fluid secretion and information processing in neuronal cells.

+view abstract Biochimica et biophysica acta, PMID: 19010359 2009

T Nagano, JA Mitchell, LA Sanz, FM Pauler, AC Ferguson-Smith, R Feil, P Fraser

A number of large noncoding RNAs (ncRNAs) epigenetically silence genes through unknown mechanisms. The Air ncRNA is imprinted--monoallelically expressed from the paternal allele. Air is required for allele-specific silencing of the cis-linked Slc22a3, Slc22a2, and Igf2r genes in mouse placenta. We show that Air interacts with the Slc22a3 promoter chromatin and the H3K9 histone methyltransferase G9a in placenta. Air accumulates at the Slc22a3 promoter in correlation with localized H3K9 methylation and transcriptional repression. Genetic ablation of G9a results in nonimprinted, biallelic transcription of Slc22a3. Truncated Air fails to accumulate at the Slc22a3 promoter, which results in reduced G9a recruitment and biallelic transcription. Our results suggest that Air, and potentially other large ncRNAs, target repressive histone-modifying activities through molecular interaction with specific chromatin domains to epigenetically silence transcription.

+view abstract Science (New York, N.Y.), PMID: 18988810 2008

K Eder, H Guan, HY Sung, J Ward, A Angyal, M Janas, G Sarmay, E Duda, M Turner, SK Dower, SE Francis, DC Crossman, E Kiss-Toth Immunology

Inflammatory activation of monocytes is an essential part of both innate immune responses and the pathogenesis of conditions such as atherosclerosis. However, the mechanisms which modulate the response of monocytes to inflammatory stimuli are still poorly understood. Here, we report that tribbles-2 (trb-2) is a novel regulator of inflammatory activation of monocytes. Down-regulation of trb-2 levels potentiates LPS-induced IL-8 production via enhanced activation of the extracellular signal-regulated kinase and jun kinase mitogen-activated protein kinase (MAPK) pathways. In keeping with this, the endogenous level of trb-2 expression in human primary monocytes is inversely correlated to the cell's ability to produce IL-8. We show that trb-2 is a binding partner and a negative regulator of selected MAPKs. The potential in vivo relevance of these findings is highlighted by the observation that modified low-density lipoprotein profoundly down-regulates trb-2 expression, which may, in turn, significantly contribute to the inflammatory processes in the development of vascular disease. Taken together, our results define trb-2 as a potent novel regulator of monocyte biology, controlling the activation of these cells.

+view abstract International immunology, PMID: 18952906 2008

RR Pandey, T Mondal, F Mohammad, S Enroth, L Redrup, J Komorowski, T Nagano, D Mancini-Dinardo, C Kanduri

Recent investigations have implicated long antisense noncoding RNAs in the epigenetic regulation of chromosomal domains. Here we show that Kcnq1ot1 is an RNA polymerase II-encoded, 91 kb-long, moderately stable nuclear transcript and that its stability is important for bidirectional silencing of genes in the Kcnq1 domain. Kcnq1ot1 interacts with chromatin and with the H3K9- and H3K27-specific histone methyltransferases G9a and the PRC2 complex in a lineage-specific manner. This interaction correlates with the presence of extended regions of chromatin enriched with H3K9me3 and H3K27me3 in the Kcnq1 domain in placenta, whereas fetal liver lacks both chromatin interactions and heterochromatin structures. In addition, the Kcnq1 domain is more often found in contact with the nucleolar compartment in placenta than in liver. Taken together, our data describe a mechanism whereby Kcnq1ot1 establishes lineage-specific transcriptional silencing patterns through recruitment of chromatin remodeling complexes and maintenance of these patterns through subsequent cell divisions occurs via targeting the associated regions to the perinucleolar compartment.

+view abstract Molecular cell, PMID: 18951091 2008

P Cuddon, MD Bootman, GR Richards, AJ Smith, PB Simpson, HL Roderick

Neurons are a diverse cell type exhibiting hugely different morphologies and neurotransmitter specifications. Their distinctive phenotypes are established during differentiation from pluripotent precursor cells. The signalling pathways that specify the lineage down which neuronal precursor cells differentiate remain to be fully elucidated. Among the many signals that impinge on the differentiation of neuronal cells, cytosolic calcium (Ca2+) has an important role. However, little is known about the nature of the Ca2+ signals involved in fate choice in neuronal precursor cells, or their sources. In this study, we show that activation of either muscarinic or platelet-derived growth factor (PDGF) receptors induces a biphasic increase in cytosolic Ca2+ that consists of release from intracellular stores followed by sustained entry across the plasma membrane. For both agonists, the prolonged Ca2+ entry occurred via a store-operated pathway that was pharmacologically indistinguishable from Ca2+ entry initiated by thapsigargin. However, muscarinic receptor-activated Ca2+ entry was inhibited by siRNA-mediated knockdown of TRPC6, whereas Ca2+ entry evoked by PDGF was not. These data provide evidence for agonist-specific activation of molecularly distinct store-operated Ca2+ entry pathways, and raise the possibility of privileged communication between these Ca2+ entry pathways and downstream processes.

+view abstract Biological research, PMID: 18949136 2008