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EMBO World Program EMBO Workshop on Genomic Imprinting 21 – 24 September 2008 Temasek LifeSciences Laboratory, Singapore Organizers: Marisa Bartolomei, Robert Feil and Fred Berger 1 TIMETABLE Sunday 21 September Arrival, Registration at the hotels from 6 pm till 9 pm, Free evening, Monday 22 September 8:00 – 9:00 Registration and Poster display 9:00-9:45 Azim Surani – Gurdon Institute, Cambridge, U.K. – Keynote Lecture sponsored by Development: “Genetic and epigenetic regulators of the germ line.” Session 1 Comparative studies of imprinting and dosage compensation Chair: Hugh Dickinson 9:45-10:15 Neil Brockdorff -University of Oxford, Oxford, U.K. “X chromosome inactivation – a model for epigenetic regulation of the genome.” 10:15-10:45 Christine Disteche – University of Washington, Washington, U.S.A. “Dosage regulation of the mammalian X chromosome.” 10:45-11:45 Coffee break and Posters 11:45-12:15 Sunil Jayaramaiah Raja – EMBL, Heidelberg, Germany “Dosage compensation in Drosophila.” 12:15-12:45 Clara Goday – CSIC, Madrid, Spain “Imprinting and chromosome elimination in sciarid flies.” 12:45-14:45 Lunch and Poster Session 14:00-14:30 Samuel Kho – Carl Zeiss, Singapore - “Isolating pure DNA, RNA, proteins and living cells by non-contact laser capture micro-dissection” 14:45-15:15 William G. Kelly – Emory University, Atlanta, U.S.A. “Epigenetic Regulation of the X Chromosome in C. elegans.” 15:15-15:30 Arp Schnittger -IBMP, CNRS, Strasbourg, France – “Dissection of cell proliferation and chromatin regulation during seed development.” 15:30-16:00 Frédéric Berger – Temasek LifeScience Laboratory, Singapore “Regulation of DNA methylation controlling imprinting in plants.” 2 16:00-16:45 Coffee break Session 2 Gametogenesis and Imprinting Chair: Denise Barlow 16:45-17:15 Benjamin Loppin – IGM, CNRS, Lyon, France – “Replacement of chromosomal proteins during Drosophila spermiogenesis, fertilization and early development”. 17:15-17:45 Hugh Dickinson – Oxford University, Oxford, U.K. “Imprinting in the maize endosperm; dissecting the control elements.” 17:45-18:15 Tetsu Kinoshita – Nara, Japan – “Establishment of genomic imprinting by ALARM CLOCK genes in Arabidopsis.” 18:15-18:30 Déborah Bourc’his – Institut Jacques Monod, Paris, France “Epigenetic cooperation in DNA methylation in the mammalian germ line “ 18:30-19:15 Robert Feil – CNRS Montpellier France – “Role and regulation of histone methylation at imprinted mouse loci.” Dinner and Poster Session Tuesday 23 September 9:00-9:45 Steve Henikoff - Fred Hutchinson Cancer Research Institute, Seattle, U.S.A. –Keynote Lecture: “Genome-wide profiling of histone variants, active chromatin and DNA methylation”. Session 2 Gametogenesis and Imprinting (continued) 9:45-10:15 Wolf Reik – The Babraham Institute, Cambridge, U.K. – “Regulation of imprinting and epigenetic reprogramming in mammalian development.” 10:15-10:45 Hiroyuki Sasaki – National Institute of Genetics, Mishima, Japan “Gametogenesis and imprinting in mice.” 3 10:45-11:15 Coffee break and Posters Session 3 Physiological regulation of imprinting in plants and mammals Chair: Bill Kelly 11:15-11:45 Bob Fischer – University of California, Berkeley, U.S.A. – “Regulation of gene imprinting in the Arabidopsis endosperm.” 11:45-12:00 José Gutierrez-Marcos– University of Warwick – “Nutrient allocation in the plant endosperm is regulated by a group of imprinted genes.” 12:00-12:30 Claudia Köhler – ETH, Zurich, Switzerland "Imprinting control at the PHERES1 locus in Arabidopsis.” 12:30-12.45 Amanda Fortier – MacGill University, Montreal, Canada ”The effect of superovulation on expression of imprinted genes and fetal weights at mid- to late-gestation in the mouse.” 12:45-13:15 Denise Barlow - Research Center for Molecular Medicine of the Austrian Academy of Science, Vienna, Austria “Control of imprinted expression by the Air macro ncRNA.” 13:15-16:00 Lunch and Poster Session 15:00 – 15h30 Lee Chee How – Riche Diagnostic Asia Pacific Singapore – “Epigenetics: An Insights into the Science of Change with Roche NimbleGen Microarrays” 16:00-16:30 Ruth Shemer – The Hebrew University, Jerusalem, Israel – “Mechanism of imprinting at the Prader Willi/Angelman syndromes domain.” 16:300-17:00 Jo Peters - MRC, Harwell, U.K. “Control of imprinting at the Gnas locus”. 17h00 –17:15 Hitomi Matsuzaki – University of Tsukuba, Tsukuga, Japan “Acquisition of parent-of-origin-specific methylation in the transgenic mouse carrying 2.9-kbp H19 ICR DNA fragment.” 17:15-17:45 Marisa Bartolomei - University of Philadelphia, Philadelphia, U.S.A. “Epigenetic regulation of the H19/IGF2 loci.” Poster session and Conference Dinner 4 _______________________________________________________________ Wednesday 24 September 7:00-9:30 Breakfast in the Jungle Session 4 Biological functions of imprinted genes. Chair: Hiroyuki Sasaki 10:00-10:30 Sébastien Smallwood – The Babraham Institute, Cambridge, U.K. “Imprinted genes and the regulation of metabolism in mice.” 10:30-11:00 Lawrence Wilkinson – Cardiff University, Cardiff, U.K. “Imprints on the brain; neurodevelopment and function.” 11:00-11:15 Michael Cowley – University of Bath, Bath U.K. ”Milking Grb10 for novel roles.” 11:15-11:30 Tomas Babak – Rosetta Inpharmatics, Seatlle, U.S.A. “Using whole transcriptome sequencing to map imprinted transcription from the mouse embryo.” 11:30-12:00 Coffee break 12:00-12:30 Anne Ferguson-Smith – University of Cambridge, Cambridge, U.K. – “Establishment and early developmental maintenance of genomic imprints in mouse.” 12:30-13:00 Fumitoshi Ishino -Tokyo Medical and Dental University, Tokyo, Japan – “Roles of Peg11/Rtl1 in neo- and postnatal growth.” 13:00-13:30 Gudrun Moore –Institute of Child Health, London, U.K. “Conservation of imprinting in the human placenta.” 13:30-14:30 Lunch Session 5 Evolution of imprinting mechanisms Chair: Anne Ferguson-Smith 14:30-15:00 Paul Vrana -University of California, Irvine, U.S.A. – “A system for assessing the effects of natural genetic variation on genomic imprinting.” 5 15:00-15:30 Luca Comai – University of California, Davis, U.S.A. – “Role of imprinting in speciation in Arabidopsis relatives.” 15:30-15:45 Radhika Das – Duke University, Durham, U.S.A. – “Phylogenetic analysis of genomic imprinting in the marsupial Monodelphis domestica reveals incomplete conservation of Eutherian imprint regulatory features. “ 15:45-16:00 Tasman Daish – University of Adelaide, Adelaide, Australia ”Meiotic sex chromosome inactivation in monotremes.“ 16:00-16:30 Coffee break 16:30-17:00 Rebecca Oakey – King’s College, London, U.K. “Transposons and the evolution of imprinting.” 17:00-17:30 Phil Avner – Pasteur Institute, Paris, France – “Pluriopotency factors couple directly X-inactivation programming to developmental processes.” 17:30-18:00 Marylin Renfree - University of Melbourne, Melbourne, Australia – “The evolution of imprinting in Marsupials.” General Discussion Departure 6 SPEAKERS ABSTRACTS Azim Surani [email protected] Genetic and epigenetic regulators of the germ line Specification of primordial germ cells (PGCs) in mammals occurs according to the 'stem cell model' in which pluripotent stem cells give rise to both the PGCs and somatic cells in response to signaling molecules. Expression of a transcriptional repressor, Blimp1/Prdm1, in pluripotent cells initiates the germ cell program and induces repression of the somatic program in nascent PGCs. This is followed by dynamic epigenetic changes in nascent PGCs leading to the establishment of the germ cell-specific chromatin signature. Thereafter, the presence of Blimp1/Prmt5 complex in the nucleus of germ cells prevents their reversion to a pluripotent state and maintains the monopotent PGCs as they migrate into the gonads. However, PGCs in vitro can undergo dedifferentiation into pluripotent embryonic germ cells (EG) in response to FGF2 or Trichostatin A (an inhibitor of histone deacetylases). At this time, Blimp1 is rapidly downregulated resulting in the re-expression of the direct targets of Blimp1/Prmt5 complex, which include Dhx38, cMyc and Klf4. However, Prmt5, an arginine methylase, is retained in EG cells but it translocates from the nucleus to the cytoplasm. Indeed, Prmt5 is present in the cytoplasm of all types of pluripotent stem cells. Evidence shows that Prmt5 has independent roles since it is also essential for the maintenance of pluripotency in embryonic stem cells, and apparently also during the initiation of PGC specification. It is interesting to note that the homologue of Prmt5 has a critical role in germ cell specification in flies as shown by others. These studies provide insights into the mechanism underlying the reversible phenotypic changes between monopotent PGCs and pluripotent stem cells. Lecture sponsored by Development 7 Neil Brockdorff [email protected] X chromosome inactivation – a model for epigenetic regulation of the genome. Work in my lab is geared towards understanding epigenetic regulation of the genome during differentiation and development. Much of this centres on studying X chromosome inactivation, a classical epigenetic model system. X inactivation is the mechanism used in mammals to achieve sex chromosome dosage compensation. In early XX female embryos both X chromosomes are active. Chromosome silencing of one X chromosome, normally chosen at random, then occurs coincident with cellular differentiation. Once chromosome silencing has been established it is stably maintained through all subsequent cell divisions. We are trying to understand developmental regulation of X inactivation, how X chromosome silencing is established and maintained, and how specific pluripotent lineages can reverse stable silencing of the inactive X. To achieve these goals we are analysing different epigenetic mechanisms known to contribute to the establishment of heritable silencing in X inactivation. These include the cis-acting non-coding RNA, Xist, that acts as the primary trigger for chromosome wide silencing, histone tail modifications, for example global deacetylation, methylation of histone H3 lysine 27 and mono-ubiquitylation of histone H2A, incorporation/exclusion of specific histone variants, for example macroH2A, DNA methylation of CpG islands on the inactive X, and mechanisms influencing nuclear organisation/folding/compaction of the chromosome. 8 Christine Disteche, Xinxian Deng, Fan Yang, Joel Berletch [email protected] Dosage regulation of the mammalian X chromosome Differentiation of the sex chromosomes in mammals has led to mechanisms of dosage compensation, including X inactivation and X up-regulation. We have previously shown that the mammalian X chromosome is up-regulated two-fold to balance expression between X-linked genes and autosomal genes. Pseudoautosomal genes are not up-regulated, as expected for genes with equivalent copies on the sex chromosomes. In contrast, genes that escape X inactivation are up-regulated. Escape from X inactivation results in moderate but consistent differences in gene expression between males and females. Analyses of triploid cell lines confirm a basic doubling of gene expression from the X chromosome with additional adjustment likely mediated by chromatin modifications of the genome. Analyses of the chromatin structure of the active X chromosome and of genes that escape X inactivation are ongoing. In particular, we have found that CTCF, a chromatin insulator, plays a role in X chromosome location and in structuring the chromatin around genes that escape. 9 Sunil Jayaramaiah Raja [email protected] Dosage compensation in Drosophila Dosage compensation, mediated by the MSL complex, regulates X-chromosomal geneexpression in Drosophila. Here we report that the histone H4 lysine 16 (H4K16) specific histone acetyltransferase MOF displays differential binding behavior depending on whether the target gene is located on the X chromosome versus the autosomes. More specifically, on the male X chromosome, where MSL1 and MSL3 are preferentially associated with the 3' end of dosage compensated genes, MOF displays a bimodal distribution binding to promoters and the 3' ends of genes. In contrast, on MSL1/MSL3 independent X-linked genes and autosomal genes in males and females, MOF binds primarily to promoters. Binding of MOF to autosomes is functional, as H4K16 acetylation and the transcription levels of a number of genes are affected upon MOF depletion. Therefore, MOF is not only involved in the onset of dosage compensation, but also acts as a regulator of gene expression in the Drosophila genome. 10 Clara Goday [email protected] Imprinting and chromosome elimination in sciarid flies A most outstanding example of chromosome elimination and genomic imprinting is found in sciarid flies, where whole chromosomes of exclusive parental origin are selectively discarded from the genome during development. Three types of tissue-specific chromosome elimination events occur in sciarids. During early cleavages, one or two paternal chromosomes is/are discarded from somatic cells of embryos, depending on the sex of the embryo. In early germ cells, a single paternal X chromosome is eliminated in embryos of both sexes and in male meiotic cells the whole paternal complement is discarded from spermatocytes. As a consequence, only maternally derived chromosomes are included in the sperm nucleus. We are currently investigating chromatin differences between parental chromosomes in Sciara in germline cells by analyzing histone acetylation/methylation modifications. In early germ nuclei, maternal chromosomes show high levels of di- and trimethylated histone H3 at Lys4 whereas this histone modification is not detected in paternal chromosomes that show high levels of acetylated histones H4 (Lys8 and Lys12) and H3 (Lys9 and Lys14). In male meiosis, by contrast, maternal chromosomes are highly acetylated for histones H4 and H3 while paternal chromosomes exhibit high levels of di- and trimethylated histone H3 at Lys4. Our findings are consistent with a model that integrates covalent histone modifications, intranuclear parental chromosome distribution and chromosome elimination in Sciara germ cells. We concluded that specific covalent modifications are involved in the imprinted behaviour of germline chromosomes in Sciara. 11 Samuel Ko (Carl Zeiss Singapore) [email protected] Isolating pure DNA, RNA, proteins and living cells by non-contact laser capture microdissection Understanding cellular mechanisms and intercellular communication requires pure samples to study key biomolecules such as DNA, RNA and proteins. The extraction of homogeneous specimens derived from a morphologically defined origin became one of the most challenging tasks in biological sciences. With the technology of laser capture microdissection that made non-contact sampling at microscopic level, clear defined specimen without contamination and with precision at the micrometer level opens up new perspectives in both plant (Angeles et al., Planta, 224:228; Fominaya et al., Cytogenet Genome Res, 109:8) and human biology (Bazan et al., J Cell Physiol, 202:503; Chaudhary et al., Toxicol Sci, 90:149). With the use of LCM Robosoftware, isolation of DNA, RNA, proteins or living cells can be done easily in different fields of applications such as botany, cell biology, molecular pathology, cytogenetics, stem cell research and forensic science. 12 Jackelyn Arico, Bill Kelly [email protected] Epigenetic regulation of the X chromosome in C. elegans The X chromosome has no partner during XO male meiosis in C. elegans, and as such is targeted for repression by meiotic silencing mechanisms recognizing unsynapsed/unpaired chromatin. The near-complete paucity of germlineexpressed genes on the X chromosome is presumably an evolved consequence of meiotic silencing. The paucity of X-linked genes expressed during meiosis has resulted in a whole chromosome that, during male meiosis, is largely transcriptionally inert. In contrast, during early XX meiosis the X chromosomes are also largely quiescent but then activate at later stages of meiosis and oogenesis, as predicted by the normal representation of oocyte-specific genes on the X. In the embryo, the different transcriptional histories of the Xp and Xm correlate with gamete of origin differences in transcription-independent, postfertilization chromatin remodeling events that occur in the early zygote. In contrast to that of the Xm and all autosomes, the Xp chromatin uniquely and dramatically fails to accumulate histone H3 lysine 4 dimethylation (H3K4me2) in early stages. In later stages, however, H3K4me2 gradually appears in Xp chromatin in a manner consistent with replication-coupled dilution of an unknown imprinted chromatin “mark”. The chromatin imprint on the Xp is solely dependent on its passage in spermatocytes, which can be derived from either XO male or XX hermaphrodite spermatogenesis. The correlation of the absence of transcription during male meiosis with the establishment of imprinted chromatin assembly in the embryo suggests a causative link. Using transgenes with differential expression in germ cells, we observe that transcriptional history during meiosis can faithfully recapitulate imprinted chromatin assembly in the embryo. Furthermore, chromatin remodeling mechanisms that are associated with transcriptionally active meiotic chromatin seem to play a role in the imprint establishment. These results imply that germline sex-specific expression of imprinted loci and/or their regulators can contribute to imprint establishment mechanisms in the parent that affect epigenetic regulation in the offspring. 13 Moritz Nowack, Alexander Ungru, Schnittger [email protected] Reza Shirzadi, Paul Grini, Arp Dissection of cell proliferation and chromatin regulation Focus of our research is the control of the plant cell cycle. In particular, we are interested in cell growth and proliferation patterns during seed development and how the different parts that comprise a seed coordinate their development. Research from many groups has shown that epigenetic regulation plays an import role during seed development. Among other epigenetic mechanisms it has been observed that similar to mammals certain genes in seeds are expressed depending on their parental origin, i.e. are imprinted. Recently, we have identified a mutant in the major cell cycle regulator CDKA;1, the Arabidopsis homolog of the yeast Cdc2/Cdc28 kinase. The mutant has enabled us to dissect seed development and to show that bypassing of imprinting allows seed development in certain genetic situations. Here we show how the cdka;1 mutant can be further used to analyze cell proliferation and chromatin regulation during seed growth. By a pilot mutagenesis screen we have identified one suppressor of the cdka;1 mutant. This suppressor shows features of seed development without fertilization, a phenotype characteristic for mutants in FIS class genes, which encode components of the Arabidopsis PRC2 complex. In a second line of research we show here that the cdka;1 mutant can be used to tunnel genes that are important for chromatin regulation during seed development and as a proof of concept we have generated homozygous mutants in the FIE gene, that is a key component in presumably all PRC2 complexes in Arabidopsis. 14 Pauline Jullien, Jonathan FitzGerald and Frederic Berger [email protected] Regulation of DNA methylation controlling imprinting in plants Parental genomic imprinting causes preferential expression of one of the two parental alleles. Parental genomic imprinting in plants relies in part on DNA methylation by the methyltransferase MET1. In contrast to mammals, plant imprints are created by differential removal of silencing marks during gametogenesis. In Arabidopsis, DNA demethylation is mediated by the DNA glycosylase DEMETER (DME) causing activation of imprinted genes at the end of female gametogenesis. We show that in addition to DME, the plant homologues of the human Retinoblastoma (Rb) and its binding partner RbAp48 are required for the activation of the imprinted genes FIS2 and FWA. This Rbdependent activation is mediated by direct transcriptional repression of MET1 during female gametogenesis. We have thus identified a new mechanism required for imprinting establishment, outlining a new role for the Retinoblastoma pathway, which may be conserved in mammals. 15 Guillermo A. Orsi, Couble Pierre, Benjamin Loppin [email protected] Replacement of chromosomal proteins during Drosophila spermiogenesis, fertilization and early development. In many animal species, the sperm chromatin is characterized by a nonnucleosomal organization whereby histones are replaced with protamines, during the course of spermatid differentiation. At fertilization, the reverse process takes place and allows the paternal nucleus to decondense and participate in the formation of the diploid zygote. We are studying this process in Drosophila with the analysis of HIRA, a chromatin assembly factor that is essential for the remodeling of the male nucleus at fertilization. Our work could participate in determining the chromatin environment in which imprinted marks are established in other model organisms. 16 Liliana Costa, Pepe Gutierrez-Marcos, and Hugh Dickinson [email protected] Imprinting in the maize endosperm; dissecting the control elements. A combination of gene dosage and genomic imprinting in the endosperm results in a strong maternal control over early seed development in maize. The recently duplicated Fie1 and Fie2 sequences, which encode Polycomb Group transcription factors are both expressed solely from the maternal alleles early in development, although the duration of this imprinting controlled monoallelic expression differs between the two genes. Using ‘imprinted’ reporter constructs (1) we have shown that monoallelic expression is likely to be regulated by differentially methylated regions (DMRs). Surprisingly, while the DMRs of Fie1 receive their asymmetric methylation in the gametes, differential methylation of these regions in Fie2 takes place in the primary endosperm cells, after fertilisation (2). Through functional analysis of these control regions, both in mutant lines of maize and in Arabidopsis, we are endeavouring to unravel the different mechanisms by which one DMR becomes methylated during gametogenesis, and the other after nuclear fusion. Why such recently duplicated and subfunctionalised genes are imprinted by two different processes is also unclear, but may be related to the differing expression pattern of these two sequences throughout the plant life history. 1.Gutierrez-Marcos, J, Costa, L.M, Biderre-Petit, C., O’Sullivan, D., Perez, P. and Dickinson, H.G. (2004) maternally expressed gene 1 is a novel maize endosperm transfer cell-specific gene with a maternal parent of origin pattern of expression. Plant Cell 16, 1288-1301. 2. Gutiérrez-Marcos, J.F., Costa, L.M., Dal Prà,, Scholten, S., Perez, P. and Dickinson, H.G. (2006) Epigenetic asymmetry of imprinted genes in plant gametes. Nature Genetics 38, 876-878. 17 Tetsu Kinoshita [email protected] Establishment of genomic imprinting by ALARM CLOCK genes in Arabidopsis Genomic imprinting in flowering plants is restricted to the endosperm, which supports embryonic growth. Imprinting of the maternally expressed genes MEDEA, FIS2 and FWA is established in the maternal central cell, which is the progenitor cell of endosperm before fertilization, by the activity of DEMETER, a DNA demethylase gene, while the paternl allele is held silent. This one-way activation mechanism of genomic imprinting is unique to flowering plants. A process of imprinting likely begins with the recognition of imprinted genes, followed by DNA demethylation and remodeling of silent chromatin to the active state. We have carried out genetic screening to gain insight into this process using Arabidopsis plant. We have isolated five alac (alarm clock for FWA imprinting) mutants that are defective in FWA-GFP expression, and found that ALAC1 encodes a conserved chromatin-related protein that controls all maternally expressed imprinted genes. 18 Schaefer Christopher, Aravin Alexei, Hannon Gregory, Déborah Bourc'his [email protected] Epigenetic cooperation in DNA methylation in the mammalian germ line Retrotransposon activity is controlled by several layers of repressive mechanisms in the mammalian germ line. Transcriptional silencing though DNA methylation is established in prenatal male germ cells under the control of the DNA-methyltransferase co-factor Dnmt3L. Piwi-interacting small RNAs (piRNAs) provide a level of post-transcriptional silencing through RNA degradation, and potentially cooperate in DNA methylation targeting to retrotransposons. Dnmt3L deficiency results in the reactivation of several classes of retrotransposons and leads to impaired spermatogenesis in mutant mice. A similar phenotype is observed in piRNA-deficient males that are mutant for Miwi2 or Mili, two members of the Piwi family. We analyzed by genetic means the cumulative effects of Dnmt3L and Piwi proteins on retrotransposon methylation and mobilization in Dnmt3L/Miwi2 double-mutants. Consequences of retrotranscript accumulation on the production of retrotransposon-associated piRNAs were moreover examined in Dnmt3L mutant germ cells. This study provides insight into the cooperative role of DNA-methyltransferases and the piRNA pathway in de novo DNA methylation and retrotransposon control in the mammalian germ line. 19 Alexandre Wagschal, Maëlle Pannetier, Lionel Sanz, Amandine Henckel, Szabolcs Toth, Herry Herman, Philippe Arnaud and Robert Feil [email protected] Role and regulation of histone methylation at imprinted mouse loci We are interested in the role and regulation of histone methylation at imprinting control regions (ICRs), the sequence elements that control the allelic gene expression at imprinted domains in mammals. Previously, we reported that the DNA-methylated allele of ICRs is consistently associated with trimethylation at H3 lysine-9 (H3K9me3) and H4 lysine-20 (H4K20me3), similarly as pericentric heterochromatin. We find that the histone methyltransferases (HMTs) Suv4-20h1/h2 are recruited to the ICRs’ DNA-methylated allele and are essential for the allelic H4K20me3. Indirectly, their expression influences levels of H3K9me3 and H4K20me1 as well. H4K20me1, however, is present on both the parental alleles and is mediated by Pr-Set7. It remains unclear which HMT(s) regulates the allelic H3K9me3 at ICRs, but, unlike at pericentric heterochromatin, Suv39h1/h1 is not involved. In a related theme, we explore the role of histone methylation in the control of tissues-specific imprinting. One of our models is the Grb10 gene on proximal chromosome 11 which displays paternal allele-specific expression in the brain. The relevant promoter region corresponds to the putative ICR (with maternal DNA methylation) and is marked by ‘bivalent chromatin’ (enriched in both H3K4me2 and H3K27me3) on the paternal allele from early embryonic stages onwards. Our data support a model in which bivalent chromatin controls the paternal expression of Grb10 in association with neuronal factors. As concerns imprinted gene expression in the placenta, our recent collaborative work reveals an important role for the H3K9-specific HMT G9a at different imprinted gene clusters, including the Kcnq1 domain on distal chromosome 7 and the Igf2r domain on chromosome 17. 20 Steven Henikoff, Mary Gehring and Daniel Zilberman [email protected] Genome-wide profiling of histone variants, active chromatin and DNA methylation We have applied genome-wide profiling to histone variants, nucleosome density, active chromatin and DNA methylation to gain a better understanding of chromatin dynamics in plants and animals. Nucleosomes are assembled on newly replicated DNA, but over the course of the cell cycle their component histones are dynamically replaced by replication-independent assembly pathways at active genes and regulatory sites. Our high-resolution mapping of sites of histone replacement using replication-independent histone variants in Drosophila has revealed a correspondence to sites responsible for epigenetic regulation and to promoter regions genome-wide. We have also applied a salt fractionation procedure to isolate active chromatin and to profile nucleosome stability. Unstable nucleosomes appear to represent intermediates in replicationindependent nucleosome assembly and are especially conspicuous at regions generally thought to be nucleosome-free. In Arabidopsis, our exploration of the relationship between histone variants and DNA methylation has revealed an unexpected mutual exclusivity between these different features of the epigenomic landscape. Although DNA methylation is a stable feature of the Arabidopsis genome, only two genes are known to show differential methylation during development. To expand this list, we have profiled genome-wide methylation patterns in Arabidopsis torpedo-stage seeds by dissecting out the embryo and endosperm. These data offer the opportunity to 1) determine how dynamic DNA methylation patterns are across sister tissues 2) potentially discover new imprinted genes by identifying genes that are less methylated in the endosperm compared to the embryo and 3) determine what types of sequences are subject to methylation changes. 21 G Smits, A Mungall, R Ng, C Farthing, G Ficz, F Santos, C Popp, W Dean, M Hemberger, I Dunham, M Renfree, W Reik [email protected] Regulation of imprinting and epigenetic reprogramming in mammalian development We are interested in the regulation and evolution of epigenetic systems in mammals. Comparisons between eutherians and marsupials suggest limited conservation of the molecular mechanisms that control genomic imprinting in mammals. We have studied the evolution of the imprinted IGF2-H19 locus in therians. The H19 non coding RNA orthologs show miR-675 and exon structure conservation, suggesting functional selection on both features. As in eutherians, marsupial H19 is maternally expressed; paternal methylation upstream of the gene originates in the male germline, encompasses a CTCF insulator, and spreads somatically into the H19 gene. The conservation in all therians of the mechanism controlling imprinting of the IGF2-H19 locus suggests a sequential model of imprinting evolution. We propose that the evolution of the IGF2-H19 system in the therian ancestor more than 150 million years ago preceded the emergence in eutherians of complex imprinting clusters regulated by long noncoding RNAs, such as XIST, Air, or Kcnq1ot1. Epigenetic reprogramming and lineage specific acquisition of epigenetic marking occurs in mammalian preimplantation embryos. Active and passive demethylation is followed by de novo methylation which preferentially occurs in inner cell mass cells in the blastocyst. We have begun to carry out genome wide profiling studies of various pluripotent, multipotent, and terminally committed celltypes. We find that at a global level methylation of gene promoters is remarkably similar in pluripotent cells such as ES and EG cells and in sperm. This suggests that while sperm is a highly differentiated cell, its genome is epigenetically already largely reprogrammed to a pluripotent state. Nevertheless, a small number of key regulators of pluripotency including Nanog, Oct4, and Lefty1 were found to be highly methylated in sperm. Presumably these need to be tightly inactivated during a period of germ cell differentiation in which pluripotency is lost. The Nanog promoter was rapidly demethylated after fertilisation and devoid of methylation at the morula stage when expression of Nanog commences. We have also begun to screen for differences in promoter methylation between ES and TS cells, in order to potentially identify genes whose epigenetic regulation is critical for commitment to the inner cell mass and trophectoderm lineage, respectively. Finally, we are studying the mechanisms of demethylation in the germline and the early embryo, which are critical for the transmission of pluripotency. 22 Hiroyuki Sasaki [email protected] Gametogenesis and imprinting in mice Imprinted genes are associated with, and controlled by, differentially methylated regions (DMRs) that are methylated differently between the paternal and the maternal chromosome. The differential methylation of at least fifteen DMRs is established during gametogenesis in mice. A number of studies have shown that a de novo DNA methyltransferase Dnmt3a and its regulator Dnmt3L are responsible for the imprint establishment in both male and female germline. However, how the Dnmt3a/Dnmt3L complex is recruited to different sets of genes in the two germlines is unknown. Also, whether other epigenetic mechanisms are involved in the imprint establishment is yet unclear. I will discuss some of our recent data in light of these issues. 23 Robert Fischer, Jin Hoe Huh, Mary Gehring, Tzung-Fu Hsieh, Yeonhee Choi [email protected] Regulation of gene imprinting in the Arabidopsis endosperm Alleles of imprinted genes are expressed differently depending on whether they are inherited from the male or female parent. Imprinting regulates a number of genes essential for normal development in mammals and angiosperms. In mammals, imprinted genes contribute to the control of fetal growth and placental development. Diseases are linked to mutations in imprinted genes or aberrant regulation of their expression. The endosperm, one of the products of angiosperm double fertilization, is an important site of imprinting in plants. The endosperm has functions analogous to the placenta and supports seed growth and development. Failure to imprint certain genes results in embryo abortion and seed death. Using the model plant Arabidopsis, the researchers discovered how gene imprinting is epigenetically regulated. Epigenetic information resides in chromatin, DNA and its associated proteins, which exists in an open conformation with the genes accessible to transcription factors or in a compacted conformation that silences genes. Two interdependent processes regulate chromatin conformation: the chemical modification (methylation and acetylation) of histone proteins around which the DNA is wrapped, and the methylation of cytosine (5-methylcytosine). Both mechanisms contribute to the regulation of gene imprinting. A DNA glycosylase, called DEMETER (DME), excises 5methylcytosine and creates an abasic site. Other enzymes (AP endonuclease, DNA polymerase, ligase) nick the DNA, insert cytosine, and seal the DNA. This pathway, reminiscent of the base-excision DNA repair pathway, demethylates DNA by replacing 5-methylcytosine with cytosine. DME specifically demethylates and activates expression of the maternal MEDEA (MEA) allele in the central cell of the female gametophyte. DME is not present in sperm cells and does not activate expression of the paternal MEA allele. The endosperm, generated from the fertilized central cell, inherits an active maternal MEA allele and a silenced paternal MEA allele. The maternal MEA allele encodes a SET-domain Polycomb group protein that, in turn, methylates histones at the paternal MEA allele, causing the chromatin to be condensed, and ensuring that the paternal MEA allele is silenced. Seed, the end product of plant reproduction, represents the primary nutrient source for humans and domesticated animals. Understanding the epigenetic mechanism of gene imprinting provides scientists with information to improve seed growth, development and viability. 24 Liliana Costa, Hugh Dickinson, Jose Gutierrez-Marcos [email protected] Nutrient allocation in the plant endosperm is regulated by a group of imprinted genes A key biological question in sexual reproduction is how the transmission of signals and metabolites between the maternal tissue and offspring is regulated. In flowering plants, this process takes place at the maternal/filial interface, in the region located between the mother plant and the developing embryo and neighbouring endosperm. This region comprises a group of specialized cells, termed transfer cells. We have identified a novel group of transfer cell specific genes that show exclusive maternal expression during the early phases of endosperm development. We show that these imprinted genes encode small, secreted peptides that are necessary for correct seed development. Mutations that affect the expression of these transcripts also affect metabolic transport, and thus have a major impact on seed size. Our data support the view that in plants, imprinted maternal factors are important for the allocation of nutrients between maternal tissues and the developing embryo- thus representing yet another example of convergent evolution between flowering plants and mammals. 25 Claudia Köhler [email protected] Imprinting control at the PHERES1 locus in Arabidopsis Imprinting of the plant gene PHERES1 requires the function of the FERTILIZATION INDEPENDENT SEED (FIS) Polycomb group (PcG) complex for repression of the maternal PHERES1 allele. We addressed the question whether the FIS complex is sufficient to establish imprinting of PHERES1. We find that PcG silencing is necessary, but not sufficient for imprinting establishment of PHERES1. We provide evidence that silencing of the maternal PHERES1 allele depends on a distantly located region downstream of the PHERES1 locus. Similar to the regulation of several imprinted genes in mammals, this region needs to be methylated to ensure PHERES1 expression, but it must be demethylated for PHERES1 repression. We propose a model that predicts how the FIS targeted promoter and the distantly located region act together to establish PHERES1 imprinting. 26 Amanda Fortier, Flavia Lopes, Jacquetta Trasler [email protected] The effect of superovulation on expression of imprinted genes and fetal weights at mid- to late-gestation in the mouse The stimulation of follicular growth and ovulation via the administration of exogenous gonadotropins is commonly used in human assisted reproduction as well as animal research. The concern has been raised that this procedure may either force oocytes to go through the final growth and maturation process too rapidly or result in poor quality oocytes, possibly affecting the establishment of methylation imprints. We have previously shown that superovulation followed by embryo transfer at E3.5 results in the abnormal biallelic expression of H19 in the mouse placenta by E9.5. The goal of this study was to examine the effects of superovulation on fetal outcome and genomic imprinting in embryonic and extraembryonic tissues at later stages of development. Naturally cycling or superovulated CD1 females were mated with CAST-7 males such that parental alleles were identifiable, followed by embryo transfer at E3.5. Embryo, placenta and yolk sac samples were collected at E14.5 and E18.5, weights and measures were taken, and allele-specific expression of H19, Snrpn and Kcnq1ot1 were examined using fluorescent hybridization probes. At E14.5, there were no differences in embryo or placenta weight or size, or in the proportion of samples exhibiting biallelic expression of either Snrpn or Kcnq1ot1 between embryos from naturally-cycling control donor females or superovulated donors. However, there was a significant increase in the proportion of embryo, placenta and yolk sac samples exhibiting biallelic expression of H19 following superovulation. At E18.5, there was a significant decrease in embryo weight and size following superovulation, while placental weight and size remained unaffected. Superovulation did not increase the proportion of samples exhibiting biallelic expression of any of the genes examined at E18.5. In conclusion, superovulation affects the maintenance of monoallelic expression of the paternally imprinted gene H19 in embryonic and extra-embryonic tissues at midgestation. While this effect is no longer apparent in late gestation, early alterations in placental imprinted gene expression may affect placental function resulting in lower fetal weights at term. (Supported by the Canadian Institutes of Health Research) 27 Denise Barlow, Paulina Latos, Stefan Huang, Irena Vlatkovic [email protected] Stricker, Florian Pauler, Ru Control of imprinted expression by the Air macro ncRNA Macro non-coding RNAs with gene regulatory functions have the possibility of becoming a common feature of mammalian gene regulation, following the discovery that the majority of the transcriptome comprises ncRNAs. The prototype has long been the Xist ncRNA that induces X-chromosome inactivation in female cells. However, a new paradigm is emerging - the silencing of imprinted gene clusters by macro ncRNAs. The Igf2r imprinted cluster is an epigenetic silencing model in which expression of a macro ncRNA silences multiple genes in cis (Seidl et al., 2006 EMBO J.25:3565). We have recently used genome tiling arrays to map parental-specific epigenetic modifications in a 250kb region in mouse embryo fibroblast cells, to show that repressive histone modifications on the silent Igf2r gene are localized to discrete regions (Regha et al., 2007 Mol.Cell 27:353). This demonstrates that the Air ncRNA does not spread 'silence' in a manner similar to that proposed for the Xist ncRNA during X chromosome inactivation. Thus, the Air ncRNA may directly act to silence genes in the Igf2r imprinted gene cluster in a manner dependent only on its transcription and not on its ncRNA product (reviewed in Pauler et al., 2007 TIG 23:284). We have now examined the kinetics of Air ncRNA mediated silencing in a new ES cell in vitro differentiation model that recapitulates the onset of Igf2r silencing seen in the early preimplantation embryo. The role of the Air ncRNA in directly silencing Igf2r has been investigated in this ES model system using targeted alleles carrying different lengths of the Air ncRNA and the results provide further support for a transcription-based silencing model. 28 Yotam Kaufman, Shiri Solsky-Rabinovitz, Jonathan Perk, Maya Heled, Aharon Razin and Ruth Shemer [email protected] Mechanism of imprinting at the Prader-Willi /Angelman syndromes domain Imprinting of the 2 Mb domain on human chromosome 15q11-13 is under the control of a 4.3 kb imprinting center (PWS-IC) that functions on the paternal allele as a bidirectional activator of the domain. The apparent inactivation of PWS-IC on the maternal allele requires the 880 bp AS-IC. AS-IC serves as the primary imprint since it is methylated in sperm and unmethylated in oocytes, while the PWS-IC is unmethylated in both types of gametes, as judged by bisulfite analysis of an imprinted transgene. We asume that the AS-IC confers silencing of the maternal PWS-IC already in the oocyte, since AS-IC becomes methylated at the pre-implantation stage while the methylation of the maternal PWS-IC is established post implantation. Seven cis elements in the AS-IC sequence have been identified and shown to bind nuclear proteins that act probably in concert to preform the AS-IC repressory function. Mutating each of these elements in a transgene revealed that these elements are essential to the establishment of the unmethylated primary imprint of AS-IC on the maternal allele and in faciliting repression of PWS-IC. The activation and the imprinted status of the genes across the domain on the paternal allele depends on the imprinted status of PWS-IC, as concluded from the results of transgenic experiments. The NDN gene was methylated and silenced upon both parental transmissions when present alone in the transgene, active on the two alleles when ligated to PWS-IC and imprinted in a construct that contains both PWS-IC and AS-IC. We also found that activation of genes by PWS-IC is not dependent on their sequence since a non-imprinted human APOAI reporter gene was imprinted in a transgenic line when ligated to PWS-IC and AS-IC. 29 Jo Peters, S.Ball, C. Beechey, S.Mehta, C.Ottway, C.Williamson [email protected] Control of imprinting at the Gnas locus Imprinted genes occur in clusters and gene silencing throughout the cluster is controlled by an imprinting centre (IC). We are interested in the mechanisms whereby an IC controls the expression of imprinted genes. We use the Gnas cluster as a model system. It has two developmentally important protein coding transcripts, Gnas (maternally expressed in some tissues), and Gnasxl (paternally expressed). Imprinted expression of these transcripts is under the overall control of the IC. This is a germline differentially methylated region (DMR) that is unmethylated and active on the paternal chromosome. The IC contains the promoter for Nespas, a paternally expressed noncoding transcript that runs antisense to a maternally expressed transcript, Nesp. Nesp has the furthest upstream start site in the Gnas cluster and is transcribed through the entire cluster. The Nesp promoter lies in a DMR that acquires methylation post fertilisation. To control the imprinted expression of Gnas the IC must interact with a second germline DMR, the Exon 1A DMR. We have shown that paternal transmission of a deletion of the IC, or paternal transmission of an insertion that leads to truncation of the Nespas transcript resulted in loss of methylation of the Nesp promoter and full expression of Nesp. In both cases the Exon 1A DMR gained methylation by midgestation and remained methylated at later stages. We propose that a primary role of the paternally active IC at the Nespas DMR is to silence Nesp, so that the Exon 1A DMR remains unmethylated and Gnas is silenced. When the IC is inactive, Nesp can be transcribed and is required for methylation and inactivation of the Exon 1A DMR and expression of Gnas. Thus the interaction between the IC and the Exon 1A DMR occurs via a transcript, Nesp. 30 Hitomi Matsuzaki, Keiji Tanimoto [email protected] Acquisition of parent-of-origin-specific methylation in the transgenic mouse carrying 2.9-kb H19 ICR DNA fragment The mono-allelic expression in the mouse Igf2/H19 imprinted locus requires parental-specific methylation of the imprinting control region (ICR) that is located 5' to the H19 gene. Although it is reported that methylation imprinting of the H19 ICR is established during gametogenesis and maintained after fertilization, underlying molecular mechanism in this process is not fully understood. Here, we explored the mechanism of methylation imprinting by using transgenic mouse (TgM) methodology. To test if H19 ICR activity is autonomous, the 2.9-kbp ICR DNA sequence alone was used to generate a transgene construct, in which the ICR fragment was flanked by loxP sequences and the ~0.5-kbp human beta-globin sequences on both sides, in order to distinguish transgenic from endogenous ICR. We established six TgM lines, each of which carried from one to five copies of the transgenes inserted randomly at non-imprinted genomic loci. In addition, TgM carrying five copies of transgenes was crossed with TgM expressing the Cre recombinase to derive low-copy number ICR TgM in vivo, which ended up with generation of TgM lines retaining one or four copies of transgenic ICR at a same chromosomal position. Methylation status of the transgenic ICR was then analyzed by Southern blotting and bisulfite sequencing. In all but one TgM lines, paternally inherited transgenic ICR was more heavily methylated than the maternal one in DNA from nucleated erythrocytes and tail tips. These results suggest that the transgenic ICR fragment could autonomously mark its parental origin independent of its copy number and its genomic integration site. 31 Le-Ben Wan, Shu Lin, Marisa Bartolomei [email protected] Epigenetic regulation of the H19/Igf2 loci The opposite imprinting of H19 and Igf2 is mediated through shared 3’ enhancers and a 2 kb differentially methylated domain (DMD, or imprinting control region, ICR). The DMD is hypermethylated on the repressed paternal H19 allele and hypersensitive to nucleases on the active maternal allele and is required for H19 and Igf2 imprinted expression on both chromosomes. It is postulated that the DMD acts as a methylation-sensitive insulator and hypermethylated mediator of H19 repression on the maternal and paternal alleles, respectively. The insulator activity is mediated, at least in part, by the multi-functional 11-zinc finger protein CTCF. The goal of our experiments is to determine the cis-acting sequences and epigenetic modifications and the trans-acting factors that are establishing and maintaining imprinting at the H19 locus. One approach that we have taken is to ablate candidate epigenetic regulators in the oocyte and preimplantation embryo using RNAi technology. For example, by using transgenes expressing inverted repeats in oocytes and dsRNA injections and infections, we have ascertained essential roles for CTCF and MBD3 in imprinted regulation of the H19 gene. Finally, we and others have recently shown that cohesin subunits colocalize at CTCF binding sites, including those in the H19 DMD: when the CTCF binding sites are deleted at the H19 locus, the cohesin subunits no longer bind to the DMD. The function of this relationship at H19 and other imprinted loci that interact with CTCF is unclear and is currently being pursued. 32 Sebastien Smallwood, Mita Chotalia, Claire Dawson, Nico Ruf, Marga Frontera, Diana Lucifero, Wendy Dean, Gavin Kelsey [email protected] The role of transcriptional events in establishment of DNA methylation imprint marks in the female germline. Genomic imprinting in mammals arises because certain genes are marked differently in the male and female germlines at differentially methylated regions (DMRs). An essential component of these marks is DNA methylation. In the female germline, acquisition of methylation at DMRs depends upon the de novo methyltransferase Dnmt3a and its co-factor Dnmt3L. The reasons why specific sequences are selected as targets for Dnmt3a and 3L, and thus the fundamental choice about whether to imprint a gene, are poorly understood. Amongst factors that have been implicated are the association of tandem repeats with CpG islands, periodicity in the spacing of CpGs and the methylation status of lysine 4 of histone H3. Various lines of evidence indicate that additional factors are required. These include the preferential intronic location of maternal germline DMRs and the existence of discrete, remote sequence elements necessary for methylation at some DMRs that have been identified from microdeletions in imprinted gene syndromes. These observations encouraged us to investigate the role of transcription in imprint establishment. We show that at the mouse Gnas locus, disruption of the upstream Nesp transcript results in the loss of maternal germline-derived methylation marks. Furthermore, we show that transcription occurs in oocytes across DMRs at a number of other imprinted loci, and that these transcripts often have oocyte-specific alternative promoters, suggesting that transcriptional events are generally required for imprint establishment. We propose a model to explain the dependence of de novo methylation on transcription events. Our findings have important implications for the nature of events causing imprinting errors and for how imprinting evolves. 33 Lawrence Wilkinson [email protected] Imprints on the brain - neurodevelopment and function. As evidence for the existence of brain-expressed imprinted genes accumulates, we need to address what they are doing in this tissue, especially in terms of organizational themes and the challenges posed by reconciling imprinted gene action in brain with current evolutionary theories attempting to explain the origin and maintenance of genomic imprinting. We are at the beginning of this endeavour and much work remains to be done but already it is clear that imprinted genes have the potential to influence diverse behavioural processes via multiple brain mechanisms. There are also strong grounds to believe that imprinting may contribute to risk of mental and neurological disease (www.bgg.cardiff.ac.uk). 34 Michael Cowley, Alastair Garfield, Joanne Stewart-Cox, Kim Moorwood, Andrew Ward [email protected] Milking Grb10 for novel roles We have shown that, uniquely, Grb10 demonstrates mono-allelic expression from each of the parental alleles in a tissue-specific manner, and that maternallyand paternally-expressed Grb10 influence distinct physiological processes (growth/metabolism and behaviour, respectively). Recently, our work has revealed that the transcription factor Stat5 is crucial for Grb10 expression in three specific tissues: brain, placental labyrinth and mammary epithelium. This result is suggestive of another, previously uncharacterised, role of Grb10. Our current model proposes that Grb10, through Stat5, is implicated in prolactin signalling and feedback in these three tissues. Further, Grb10 is paternally-expressed in the brain, maternally-expressed in the mammary epithelium and biallelic in the placental labyrinth, thus demonstrating that this role is independent of the imprinting status of Grb10, unlike the previously characterised roles specific to the maternally- and paternally-derived alleles. 35 Tomas Babak, Brian DeVeale, Chris Armour, Chris Raymond, Michele Cleary, Derek van der Kooy, Jason Johnson, Lee Lim [email protected] Using whole transcriptome sequencing to map imprinted transcription from the mouse embryo We report the analysis of sequenced whole-transcriptomes from mouse embryos derived from a reciprocal cross of CAST/EiJ and C57Bl/6J mice. Sequencing of total RNA allows simultaneous identification and quantification of allele-biased expression by discrimination of inter-strain polymorphisms. Isolating sex-specific from strain-specific parental differences enabled construction of a fine-scale, genome-wide map of imprinted transcription. We identified 18 genomic imprinted regions, including 14 (of 17) known loci and experimentally confirmed novel imprinting of at least 11 independent transcripts selected from 199 novel imprinted polymorphic regions. More than half of all imprinted SNPs detected do not overlap previously annotated imprinted transcripts, suggesting that characterization of imprinted regions is still incomplete: most of this uncharacterized imprinting occurs as new noncoding transcription within known loci. The ability to map allelic expression will provide a resource for exploration of the extent and mechanisms of imprinting. We are currently identifying tissuespecific imprints in mice and generating genome-wide imprinting maps in other mammals. 36 Xiajun Li,Mitsuteru Ito, Neil Youngson, Fen Shou, Dionne Gray, Philip Leder, Anne C Ferguson-Smith. [email protected] Establishment and early developmental maintenance of genomic imprints in mouse. The monoallelic expression/repression at imprinted domains is regulated by regional cis-acting imprinting control regions (ICRs) such that imprinting is lost upon targeted deletion of an ICR. The two functionally distinct parental chromosomes acquire differential DNA methylation at ICRs in the male and female germlines and this is essential for imprinting. After fertilisation during normal preimplantation development genome-wide demethylation occurs presumably contributing to the initiation of the developmental programme in stem cells. Remarkably, germline methylation imprints are maintained during this process allowing the somatic heritability of the germline methylation marks. The mechanisms associated with maintaining methylation imprints during the preimplantaion period are not understood. During a screen to identify genes down-regulated upon differentiation, a maternal-zygotic factor has been identified that plays a role in the establishment and early developmental maintenance of germline methylation imprints suggesting specific targeting of methylation to ICRs during reprogramming. These findings uncover a new mechanism of epigenetic control in early development. 37 Yoichi Sekita, Masayo Kagami, Daisuke Endo, Ryuichi Ono, Takashi Kohda, Tsutomu Ogata, Tomoko Kaneko-Ishino, Fumitoshi Ishino [email protected] Roles of Peg11/Rtl1 in neo- and postnatal growth Peg11/Rtl1 (paternally expressed 11/retrotransposon-like 1) that locates in the large imprinted region on mouse distal chromosome 12, plays an essential role in late placenta function via maintenance of fetal capillaries where nutrient and gas exchange between fetal and maternal blood occurs. A half of Peg11/Rtl1 knockout mice exhibit late fetal lethality, and the remaining half exhibit late fetal growth retardation associated with neonatal death. However, in some genetic background, such as B6/DBA2 and B6/JF1, Peg11/Rtl1 KO neonates survive and grow to adulthood despite of their sever pre- and postnatal growth retardation like human maternal UPD14 patients. Overexpression of Peg11/Rtl1 due to lack of maternally expressed antiPeg11/Rtl1 that degrade Peg11/Rtl1 mRNA by RNAi mechanism causes late fetal or neonatal lethality associated with overgrowth of placenta where the fetal capillaries anomaly expanded. In this case, rare survivors also show postnatal growth retardation. Such placental overgrowth, neonatal lethality and survivor’s postnatal growth retardation are commonly observed in human UPD14 patients, however, thorax malformation (bell-shaped thorax) observed in humans is severe than that in mice. These results indicate that mouse Peg11/Rtl1 and human PEG11/RTL1 have also important roles in neonatal growth as well as in placental function. Peg11/Rtl1 mRNA expresses in many neonatal tissues and organs, however, Peg11/Rtl1 protein is rarely detected in most cases. We would like to discuss the target tissues and organs of mouse Peg11/Rtl1 and human PEG11/RTL1 associated with several neonatal phenotypes in these two species. References: 1. Sekita Y et al. Nat Genet 40, 243-248 (2008). 2. Kagami M and Sekita Y et al. Nat Genet 40, 237-242 (2008) 38 Gudrun Moore, Philip Stanier, Sayeda Abu-Amero, Sophia Apostolidou, David Monk, Jenny Frost, Caroline Daelemans, Jiehan Chan, Miho Ishida [email protected] Conservation of Imprinting in the Human Placenta Fetal growth is a dynamic process influenced by many factors including genes and the environment. Human fetal growth in utero is most marked from the second trimester and is reliant on the placenta as an interface for nutrient transfer from the mother to the fetus. In the third trimester, the baby’s weight can increase by up to 200 grams/week, ideally resulting in a birth weight of approximately 3.2 kg. Genomic imprinting is an epigenetic phenomenon that results in monoallelic expression of a subset of genes in a parent-of-origin dependent manner. It is found almost exclusively in eutherian mammals, and has been suggested to be one of the most important regulatory pathways involved in the development and function of the placenta. Approximately 90 imprinted genes have been described in the mouse with about 50 of these also displaying imprinting in the human. It has been shown that this small group of genes can have profound effects on fetal and placenta growth, as well as postnatal growth and behaviour (http://www.mgu.har.mrc.ac.uk/research/imprinted; http://www.otago.ac.nz/IGC). Work on mouse models has shown that several of these imprinted genes can influence growth through the function of specific placental cell types. Paternally expressed genes such as Igf2, Mest and Peg3 are found in the labyrinthine trophoblast and spongiotrophoblast, with Igf2 found additionally in glycogen cells and Mest in fetal blood vessels. Paternally inherited null alleles for these three transcripts result in smaller sized placentae. Genes that are maternally expressed in the placenta can also affect its growth and development. H19, Igf2r, Grb10 and Cdkn1c are widely expressed throughout the placenta, whereas expression of Phlda2 and Mash2 is limited to the labyrinthine and spongiotrophoblast respectively. Deletions of Igf2r, Grb10 and Cdkn1c all result in hyperplasia of all placental layers. Most data regarding specific functions of imprinted genes has come from studies of the mouse. In human, the role of genomic imprinting in fetal growth restriction is still poorly understood. We have used a large cohort of normal human trios (comprising of placental DNA and RNA, and parental DNAs) to look at the conservation of imprinted genes from mouse to human (Monk et al. 2006). We found that imprinting was not conserved in the human for the mouse placental-specific imprinted genes. Also for many of the imprinted candidate genes studied, there was no correlation between expression levels in the placenta with any growth parameters measured at term. However, the expression of PHLDA2 is significantly, negatively associated with birth weight in our cohort (Apostolidou et al. 2007). The data on a selection of imprinted genes and further analysis of PHLDA2 with respect to human fetal growth will be discussed. Monk D, et al. (2006) Limited evolutionary conservation of imprinting in the human placenta. Proc Natl Acad Sci USA 103(7): 6623-6628. Apostolidou S, et al. 2007 Elevated placental expression of the imprinted PHLDA2 gene is associated with low birth weight. J Mol Med 85:379-387. 39 Paul Vrana [email protected] A system for assessing the effects of natural genetic variation on genomic imprinting The native North American rodents of the genus Peromyscus (aka deer mice) to are used to study the effects of natural genetic variation on imprinted loci and imprinting control. This group is ~30 million years diverged from house mice and rats. The P. maniculatus species complex is particularly widespread and varied, consisting of a series of partially reproductively isolated populations. The best characterized example of hybrid dysgenesis occurs between the prairie deer mouse (P. maniculatus bairdii; stock = BW), and populations of the Oldfield mouse (P. polionotus; stock = PO). While the two are the same size at birth, BW females mated to PO males produce growth-retarded offspring (bw x po). The bw x po hybrid animals are otherwise healthy and fertile. Imprinting perturbations in the bw x po hybrids appear minimal and confined to extraembryonic tissues. In contrast, PO females mated to BW males (PO x BW) produce overgrown and severely dysmorphic conceptuses. Roughly half of all PO x BW breedings end in complete death of the litter by mid-gestation . PO x BW hybrids which survive to late gestation display numerous developmental defects, many reminiscent of human syndromes. The placenta is particularly affected in both crosses. Approximately 10-15% of PO x BW conceptuses lack embryonic structures, analogous to human molar pregnancies. The more severe PO x BW phenotypes correlate with 1. a genetic interaction between an X-linked locus and an autosomal locus linked to the Peg3 gene, and 2. perturbations of imprinted gene expression. The imprinting disruptions are mediated by a PO maternal effect locus whose product cannot sustain the epigenetic processes in the presence of BW chromatin. Here I will give an overview of the genome wide patterns of hybrid imprinting and DNA methylation status at imprint control regions (ICRs). No obvious pattern is discernable. For example, while many paternal loci (e.g. Peg3, Peg10, Lit1) lose imprinting (LOI) in the PO x BW hybrids, Igf2 does not. In contrast, the linked maternally-expressed H19 locus displays LOI. The Dlk1/Gtl2 cluster displays some similarities to the Igf2/H19 cluster. However, the maternally expressed Gtl2 maintains imprinting in the PO x BW hybrids. Additionally, I will report on the imprinting of the dopamine receptor 4 (Drd4) gene in the CNS of both hybrid types. The Drd4 gene is thought to confer susceptibility to schizophrenia and attention-deficit disorder. Finally, a survey of the Peg3 and Peg10 ICRs in multiple Peromyscus species and populations within the P.m. species complex reveals the rapid evolution 40 Radhika Das, Nathan Anderson, MaryEllen Koran, Jennifer Weidman, Tarjei Mikkelsen, Michael Kamal, Kerstin Linblad-Toh, John Greally, Randy Jirtle [email protected] Phylogenetic analysis of genomic imprinting in the marsupial Monodelphis domestica reveals incomplete conservation of Eutherian imprint regulatory features Genomic imprinting is a non-Mendelian inherited epigenetic form of gene regulation that results in parent-of-origin dependent monoallelic expression. The conflict theory predicts that imprinted genes evolved in placental mammals in order to modulate intra-uterine resource allocation amongst multiple progeny. However, it is unclear whether the phenomenon arose independently in each of the placental lineages, or if it was maintained through evolution subsequent to its establishment in a common ancestor. To address this question, we investigated imprint control mechanisms operating at Metatherian orthologues of Eutherian imprinted loci. The recently completed sequence of the Monodelphis domestica (gray short-tailed opossum) genome facilitated our search for such conserved loci. On analyzing seven orthologues of loci known to be imprinted in Eutherian mammals, we found that L3MBTL, DIO3 and HTR2A were monoallelically expressed. Moreover, PEG1/MEST showed one imprinted and one non-imprinted transcript, as has previously been reported in Eutherians and the tammar wallaby. IMPACT, COPG2 and PLAGL1, however, showed non-imprinted expression in M. domestica. Genomic imprinting therefore appears to be conserved at a substantial proportion of loci between Eutherians and Metatherians, but not at all loci. In order to elucidate the regulatory mechanisms governing monoallelic expression, we investigated both differential methylation and histone modifications surrounding loci found to be imprinted in the opossum. Up until recently, there was no evidence that imprinted genes in Metatherians had the parent of origin-specific cytosine methylation characteristic of imprintregulatory regions in Eutherians. In our analyses, we were unable to locate Differentially Methylated Regions (DMRs) around any of the novel imprinted loci, but surprisingly, found a DMR in the imprinted gene IGF2R, which has previously been reported to exhibit no differential methylation. Interestingly, this DMR was located at a region not conserved with any other Eutherian species, as is the case for the PEG10 and IGF2 DMRs investigated elsewhere. This suggests the independent evolution of these differentially methylated domains from the ones reported in Eutherians. Our investigation also revealed that the promoter regions of all the imprinted loci were enriched for the activating histone modification H3 Lysine 4 dimethylation. This indicates that chromatin modifications may serve as the “primordial imprint mark” in Metatherians, while differential methylation may have evolved later to reinforce the imprint status. In Eutherians, both mechanisms of allele-specific marking are well-developed and are possibly equally important in conferring the parent-of-origin specific expression of such important growthregulatory genes. This indicates that genomic imprinting may have evolved independently in these two lineages, and that its establishment probably involved utilization of regulatory mechanisms which had already originated in the ancestors of each of these mammals. 41 Tasman Daish, Frank Grutzner, Enkhjargal Tsend-Ayush [email protected] Meiotic sex chromosome inactivation in monotremes In mammals, pairing of sex chromosomes at male meiosis is restricted with the unpaired regions being subject to silencing during prophase 1. This silencing is achieved by the accumulation of specific histone variants and histone modifications leading to the formation of the heterochromatic sex body. Monotremes have a unique and complex sex chromosome system involving the formation of a meiotic sex chromosome chain however it remains unknown whether any or all of the sex chromosomes undergo meiotic silencing. Hallmarks of meiotic silencing include accumulation of the histone variant mH2A and phosphorylation of H2AX. Platypus male meiotic cells immunostain for mH2A adjacent the nucleolus organising region. Using sex chromosomespecific BAC clones for FISH probes, we observed some sex chromosomes in the chain to be excluded from the mH2A enriched regions suggesting differential silencing of sex chromosomes may occur. Phosphorylation of H2AX (gH2AX) is a rapid and highly conserved response to double strand break (DSB) formation and also accompanies meiotic sex chromosome silencing however we could not detect this modification in platypus testis histone extracts. However, we could induce gH2AX formation by irradiating platypus fibroblasts but this response was significantly reduced and delayed compared to that observed in mouse fibroblasts. We conclude there to be differential sex chromosome silencing occurring in platypus and that monotremes may not have a conserved DSB response mechanism as part of the meiotic sex chromosome silencing pathway. 42 Andrew Wood, Ruth McCole, Reiner Schulz, Rebecca Oakey [email protected] Transposons and the evolution of imprinting Parent-of-origin effects arise due to differential epigenetic reprogramming in the male and female germ-line. Methylation at CpG dinucleotides is known to play a role, and germ-line differentially methylated regions are located near most imprinted genes. In addition to this role, DNA methylation also suppresses the activity of retrotransposon promoters. This connection suggests that the two processes may be mechanistically linked, this is further supported by four imprinted genes with retrotransposon-like properties. These genes are associated with a CpG island, located in the intron of a host gene and are derived from a parent gene on the X-Chromosome. A computational screen identified one of these loci with at least two protein-coding genes: H13 and Mcts2. Mcts2 is a duplicate of the X-linked Mcts1 gene that originated by retrotransposition from the X-linked locus into the fourth intron of H13. Unlike most retrotransposed gene copies, Mcts2 has maintained the capacity to encode a protein, and shares 94% amino acid identity with the X-linked paralogue over 65 million years after the gene duplication event. There is evidence at this locus that epigenetic modifications can influence alternative polyadenylation. 43 Pablo Navarro, Ian Chambers, Corinne Chureau, Céline Morey, Claire Rougeulle, Philip Avner [email protected] Pluripotency factors couple directly X-inactivation programming to developmental processes X-inactivation is a highly regulated process ensuring dosage compensation in females. In eutherian mammals, the initiation of X-inactivation involves the decoration of the presumptive inactive X by the non-coding Xist RNA followed by the recruitment of a series of repressive proteins and modifications to histone constituents making up the X chromosome. Xist upregulation is widely accepted to be one of the earliest, if not the earliest step in X-inactivation onset, suggesting the importance of understanding the mechanisms controlling Xist regulation for our understanding of the wider process. Previous findings from our laboratory which have underlined the importance of transcriptional control mechanisms for Xist regulation and demonstrated a role for the Tsix antisense in this process have also suggested that other control mechanisms allowing integration of the onset of the Xinactivation with wider developmental processes are likely to exist. We will present results demonstrating the direct coupling of the dynamics of Xinactivation programming and reprogramming to development through pluripotency factors which include Nanog,Oct3/4 and Sox2. 44 Marilyn Renfree, Geoff Shaw, Andrew Pask [email protected] The evolution of genomic imprinting in marsupials Eutherians and marsupials diverged at least 125 million years ago, but share a viviparous mode of reproduction that relies on placentation for embryo survival. The allantois and yolk sac can both form a placenta. The yolk sac, or choriovitelline, placenta of the tammar wallaby provides a unique opportunity to assess the importance genomic imprinting in the broader context of mammalian placentation. Parent-of-origin specific gene expression, genomic imprinting, is widespread amongst eutherian mammals. Initial descriptions for two marsupials, the North American opossum Didelphis virginiana and the South American grey short-tailed opossum Monodelphis domestica, demonstrated that marsupials had genomic imprinting but functional studies were not conducted nor was any placental tissues examined. We have now characterized several genes in the developing fetus and placenta of an Australian marsupial, the tammar wallaby Macropus eugenii that are found in separate imprinted clusters in eutherian mammals. The receptor for insulin-like growth factor 2, IGF2R, insulin-like growth factor 2, IGF2, insulin, INS, and paternally expressed 1/mesoderm specific transcript, PEG1/MEST are imprinted in the fetus and placenta, but p57KIP2, a gene that is usually imprinted in eutherian mammals, is not imprinted in the tammar. Similarly imprinting is absent in DIO3, DLK and SNRPN. Although no differentially methylated regions (DMRs) known to be essential for imprinting in eutherians have been found in the IGF2R, IGF2 and (PEG1/MEST) imprinted genes, there is at least one so far identified, namely paternally-expressed placental gene (PEG10) that has a DMR. Since this gene is not imprinted in the platypus, we can assume that this retrotransposon-derived gene was inserted into the therian mammal genome at least 130 million years ago. However its function in the tammar placenta is not yet known. Other DMR regulated genes are under investigation in the tammar. These results show that the same mechanisms have evolved in marsupials and eutherians. As yet there is no evidence for genomic imprinting in the egg laying monotreme mammals, but genomic imprinting appears to be of functional importance in marsupials as in eutherians and confirms an intimate association between imprinting and the evolution of mammalian placentation. 45 POSTER ABSTRACTS 1. Sayeda Abu-Amero, John Whittaker, Mike Preece, Philip Stanier, Gudrun Moore [email protected] Analysis of the methylation status at the ICR1 and ICR2 on human chromosome 11p15.5 in a SRS, IUGR and normal cohort Silver-Russell Syndrome (SRS) is characterised by intra-uterine growth restriction (IUGR), poor post-natal growth, body asymmetry, typical triangular face and clinodactyly. Ten percent of SRS patients have maternal uniparental disomy for chromosome 7 (mUPD 7). More recently the telomeric imprinting control region (ICR1) regulating the imprinted genes H19 and IGF2 on chromosome 11p15.5 has been implicated in 35-65% of SRS patients. Hypomethylation of ICR1 should cause relaxation of imprinting of H19 resulting in biallelic expression of H19 and consequently down regulation of IGF2, potentially causing growth restriction. We have examined the H19 promoter (somatic differentially methylated region (DMR)) and the H19 DMD (germline DMR) in the ICR1 in our cohort of SRS patients (n=70), a normal birth weight white European cohort (n=180) and a cohort of severely growth restricted babies (n=36) to look for methylation defects. We also looked at methylation of the ICR2 KvDMR1 region located more centromerically on chromosome 11p15.5. Hypomethylation was seen for both the H19 promoter and H19 DMD region in the SRS patients. No hypomethylation was observed for the KvDMR1 region in the SRS cohort. We found a strong correlation in SRS patients for hypomethylation of the H19 DMD and severity of symptoms (p = 0.017) after correcting for confounding factors such as sex, gestational age etc. There was also a specific correlation for H19 DMD (p = 0.047) and clinodactyly after correction for confounding factors. We found no correlation between methylation ratio in the normal cohort or the severely growth restricted cohort for the H19 promoter, H19 DMD or KvDMR1 and birth weight, placental weight or head circumference before and after correcting for confounding factors such as sex and gestational age. Hypomethylation of 11p15.5 therefore seems to be an epigenetic abnormality found exclusively in > 50% of SRS patients. The higher the absence of methylation at the H19 DMD the more severe the clinical spectrum of the syndrome, and the more likely for those individuals to exhibit body asymmetry and clinodactyly. 46 2. Corina Belle Villar, Grigory Makarevich, Raphael Troesch, Claudia Köhler [email protected] Mechanisms of PcG-mediated silencing of PHERES1 The type I MADS-box gene PHERES1 is expressed dependent on the parent-oforigin, a phenomenon known as genomic imprinting. The paternal allele of PHERES1 is expressed, whereas the maternal allele is silenced. PHERES1 is repressed by the FIS Polycomb-group complex that includes MEDEA (MEA), FERTILIZATION INDEPENDENT ENDOSPERM (FIE), FERTILIZATION INDEPENDENT SEED 2 (FIS2),and MULTICOPY SUPPRESSOR of ira1 (MSI1). PHERES1 repression by the FIS complex is mediated through chromatin modification, i.e., histone methylation. However, our research revealed that FIS Polycomb group-mediated repression of PHERES1 is not sufficient to establish PHERES1 imprinting. I am investigating the nature of the imprint and how this imprint is established and maintained. 47 3. Desmond C Brabazon, Sarah Furlong, Fiona M O'Sullivan, John J Callanan, Catherine M Nolan [email protected] Imprinting of IGF2, H19 and IGF2R in a novel mammalian order, Carnivora Most studies of genomic imprinting have involved rodents (chiefly mice) and primates (chiefly humans), both of which belong to the mammalian superordinal clade, Euarchontoglires. The domestic dog Canis familiaris, is a carnivore, and thus belongs to the Laurasiatheria, a sister group of the Euarchontoglires. This phylogenetic distance of dogs from mice and humans means that comparative studies that involve the canine should help to distinguish ancestral and derived features of imprinted genes. In addition the dog represents a potentially useful animal in which to probe the contribution of imprinted genes to disease, and to the evolution of behavioural and morphological diversity. With this in mind, we have initiated a study of genomic imprinting in the canine. We initially focused on canine orthologs of well-characterized imprinted genes, and have shown that canine IGF2R, IGF2 and H19 are imprinted, with monoallelic expression patterns that are similar to those seen in other mammals (O’Sullivan et al, 2007; Brabazon et al, unpublished data). We also demonstrated differential methylation of CpG islands associated with these genes. Using sperm derived genomic DNA, we demonstrate paternal allele-associated methylation of a putative intergenic imprint control element for IGF2-H19. We also show that the intron 2 CpG island of canine IGF2R is not methylated in sperm, suggesting that the differential methylation of this region that we observed in somatic tissues is oocyte-derived. Thus many features of canine imprinted genes resemble those of mice and humans. However, a surprise finding was that canine IGF2R is imprinted in the absence of promoter methylation or of an antisense transcript originating in the intron 2DMR (canine AIR). In this respect this gene is more similar to the imprinted opossum IGF2R than to the mouse Igf2r. This may indicate that murine Air is a derived feature of imprinting at the mammalian IGF2R locus, a somewhat controversial suggestion. To investigate this possibility further, we are examining the canine MAS1-IGF2R intergenic region for evidence of transcription that could be involved in silencing the paternally-derived IGF2R. O’Sullivan, FM, Murphy, SK, Simel, LR, McCann, A, Callanan, JJ and Nolan CM (2007) Imprinted expression of the canine IGF2R, in the absence of an antisense transcript or promoter methylation. Evolution and Development 9, 579589. 48 4. Katie Burton, Antonius Plagge [email protected] Comparative characterisation of Gnasxl (XLαs) expression in postnatal and adult mouse brain. Our work so far has indicated that the paternally expressed Gnasxl transcript (XLαs protein) of the imprinted Gnas locus is required for postnatal feeding, growth and the establishment of adipose reserves. Although a high neonatal mortality rate is associated with the knockout mutation, some mutants survive to weaning age and into adulthood displaying a milder phenotype at this stage, which is characterised by hypermetabolism, continued leanness (despite increased food intake), glucose tolerance and insulin sensitivity. Amelioration of the phenotype is correlated with downregulation of Gnasxl towards adulthood in some peripheral tissues, e. g. adipose tissue. Central nervous system pathways regulating energy homeostasis and sympathetic tone are hypothesised to play an important role in these phenotypes, since Gnasxl is expressed in defined areas of the brainstem, midbrain and hypothalamus at postnatal stages. We are currently analysing changes in the Gnasxl expression pattern in adult versus postnatal brain. Initial data indicate a downregulation in specific midbrain nuclei, while expression appears more widespread in regions of the adult brainstem. Colocalisation studies with neural markers are currently being undertaken, to further characterise XLαs positive CNS pathways. Developmental changes in the brain expression pattern of Gnasxl are in line with the observed phenotypical changes of knockout mice towards adulthood. 49 4. Michael Chao, Lahiru Handunnetthi, Katie Morrison, Helen Lockstone, Jennifer Taylor, George Ebers [email protected] Differential DNA methylation in MZ twins discordant for MS The evidence that multiple sclerosis (MS) is a complex genetic disease is overwhelming with the major loci for susceptibility located within the HLA class II region of the MHC. Genetic-epidemiological studies have found a parent-oforigin effect and gender bias for MHC loci, suggesting the involvement of epigenetic mechanisms in this region in MS susceptibility. AimThe purpose of this study is to determine whether DNA methylation is involved in MS risk. The potential role of epigenetic modifications in MS susceptibility was investigated by examining the MHC region for variations in methylation levels in monozygotic (MZ) female-female twin pairs discordant for MS (N=15). Controls included 15 pairs of female-female concordant MZ pairs and 15 pairs of discordant unlike-sexed pairs. We used MeDIP (methylated DNA immunoprecipitation) coupled with DNA tiling microarrays that allow rapid identification of methylated regions in a high-throughput manner throughout chromosome 6, including the MHC. ResultsWe found that DNA methylation was highly variable within discordant twin pairs. Following stratification by affection status (affected versus unaffected), a distinct pattern of DNA methylation was shared among affected twins as well as among pooled unaffected twins. The general trend is that the affected twins have significantly less number of methylation sites as compared with the unaffected twins. Conclusion: Differential methylation within MZ twins discordant for MS suggests that MHC-related MS risk is a manifestation of a general pattern of hypomethylation affecting distant parts of the genome uninvolved in risk. 50 5. Marika Charalambous, Simao da Rocha, Anne Ferguson-Smith [email protected] The mouse chromosome 12 imprinted domain controls prenatal growth and directs postnatal energy expenditure A 1Mb cluster of imprinted genes on mouse chromosome 12 contains three protein-encoding genes expressed from the paternally-inherited chromosome (Dlk1, Rtl1 and Dio3), and multiple non-coding RNAs (including Gtl2, Rtl1as and Mirg) expressed from the maternally-inherited chromosome. Opposite parental inheritance of a regulatory mutation (the Gtl2LacZ insertion) at this locus results in non-lethal phenotypes that reveal novel roles for chromosome 12 genes in postnatal life. Moderate maternalisation of the paternally-inherited chromosome ( /Gtl2LacZ) leads to pre- and postnatal growth retardation, as previously described. Paternalisation of the maternally-inherited chromosome (Gtl2LacZ/ ) results in normal embryonic growth with a marked postnatal failure to thrive. In Gtl2LacZ/ adults, glucose homeostasis is disrupted and fat deposition is increased, a Metabolic Syndrome-like phenotype. Late prenatal/early postnatal failure to thrive in humans is associated with increased incidence of the Metabolic Syndrome, and often progresses to Type II Diabetes and cardiovascular disease. We propose that chromosome 12 imprinted gene dosage is crucial for establishing and maintaining set points of energy balance in the adult organism. Furthermore, epigenetic alterations at this locus may mechanistically explain how an insult in early life can have significant consequences for adult health. 51 6. Hatsune Chiba, Ryutaro Hirasawa, Masahiro Kaneda, Takashi Sado, En Li, Hiroyuki Sasaki [email protected] De novo DNA methyltransferases are dispensable for imprinting X chromosomes in oocytes In female preimplantation embryos and extraembryonic tissues, the paternal X chromosome (Xp) is selectively inactivated, whereas the maternal X chromosome (Xm) remains active. There is evidence that the Xm is imprinted so that it can maintain its active status, however, the nature of the imprint is not yet identified. DNA methylation is one candidate, since it serves as germline imprints in autosomal imprinting. In this study, we examined whether disruption of the two de novo DNA methyltransferases in oocytes affects imprinted X-chromosome inactivation (XCI) in resulting embryos. We analyzed the expression of X-linked genes and accumulation of histone H3 lysine-27 trimethylation on X chromosomes in mutant-derived embryos and found that the imprinted XCI occurred appropriately. The results argue that de novo DNA methylation is dispensable for imprinting X chromosomes in oocytes and underscore the difference between the X-chromosomal and autosomal imprinting. 52 7. Tasman Daish, Frank Grutzner, Enkhjargal Tsend-Ayush [email protected] Meiotic sex chromosome inactivation in monotremes In mammals, pairing of sex chromosomes at male meiosis is restricted with the unpaired regions being subject to silencing during prophase 1. This silencing is achieved by the accumulation of specific histone variants and histone modifications leading to the formation of the heterochromatic sex body. Monotremes have a unique and complex sex chromosome system involving the formation of a meiotic sex chromosome chain however it remains unknown whether any or all of the sex chromosomes undergo meiotic silencing. Hallmarks of meiotic silencing include accumulation of the histone variant mH2A and phosphorylation of H2AX. Platypus male meiotic cells immunostain for mH2A adjacent the nucleolus organising region. Using sex chromosomespecific BAC clones for FISH probes, we observed some sex chromosomes in the chain to be excluded from the mH2A enriched regions suggesting differential silencing of sex chromosomes may occur. Phosphorylation of H2AX (gH2AX) is a rapid and highly conserved response to double strand break (DSB) formation and also accompanies meiotic sex chromosome silencing however we could not detect this modification in platypus testis histone extracts. However, we could induce gH2AX formation by irradiating platypus fibroblasts but this response was significantly reduced and delayed compared to that observed in mouse fibroblasts. We conclude there to be differential sex chromosome silencing occurring in platypus and that monotremes may not have a conserved DSB response mechanism as part of the meiotic sex chromosome silencing pathway. 53 8. Brian DeVeale, Tomas Babak, Christopher Armour, Christopher Raymond, Michele Cleary, Derek van der Kooy, Jason Johnson, Lee Lim [email protected] Confirmation and functional assessment of novel mouse imprinted transcripts detected using whole transcriptome sequencing We decided to test the extent to which previous experimental approaches were successful in identifying imprinted genes. Using transcriptome sequencing on embryos obtained from a reciprocal cross of CAST/EiJ and C57Bl/6J mice, we constructed a fine-scale, strand-specific, genome-wide map of imprinted transcription. The cutoffs we used for analysis led to confirmation of 14 of 17 known loci. Here we report confirmation of 16 novel transcribed regions identified by screening for expression of parentally-biased SNPs. These include extensions of known mouse imprinted transcripts, novel imprinted loci associated with known clusters and novel imprinted coding and noncoding transcripts distant to any known imprinted locus. While the majority of imprinted transcripts that registered in our screen were known, we noted numerous confirmations that were not associated with previously established imprinted loci where they might be discounted –from an evolutionary perspective- as bystanders of conserved imprinted genes. To predict function, of which there was little annotation for many existing imprinted genes and the novel imprinted genes discovered herein, we used a mouse expression atlas of 41,422 transcripts across 59 tissues and cell lines to predict membership of imprinted genes to all available Gene Ontology categories. The majority of the novel functional inferences pertained to metabolic regulation (e.g. “homoiothermy and energy reserve metabolism” for Gnasxl and “coenzyme/cofactor metabolic process” for TSSC5), consistent with prevailing theories. Here we present our list of confirmed novel imprinted genes and discuss their predicted function along with those of other poorly characterized imprinted genes in the context of the evolution of imprinting. 54 9. Christine Doe, Alastair Garfield, Lawrence Wilkinson, Anthony Isles [email protected] Trevor Humby, Dinko Relkovic, 5-HT2CR pre-RNA editing, alternate splicing and function in a mouse model of Prader-Willi Syndrome Prader-Willi Syndrome (PWS) is a complex genetic disorder caused by the loss of paternal gene expression from chromosome 15q11-q13. In addition to a number of coding genes, there are several imprinted small nucleolar (sno)RNAs present in the PWS cluster. Recent research has demonstrated that one of these, HBII-52, has a regulatory function in that it reduces alternate splicing of the RNA-editing region of the serotonin 2C receptor (5-HT2CR) pre-RNA, and that loss of expression of this snoRNA may lead to abnormal molecular processing of 5-HT2CR in the PWS brain. In turn, these molecular modifications of 5-HT2CR pre-RNA lead to a much less functional receptor. Using a mouse model of PWS (PWS-IC /del) we are investigating the consequences of the loss of mbii-52 (mouse homologue of HBII-52) for 5-HT2CR functioning at a molecular, neurochemical and behavioural level. To achieve this we have examined the expression levels and extent of RNA-editing and alternate splicing of the 5-HT2CR pre-RNA in the brains of PWS-IC /del mice. Behavioural studies have focussed on those known to be influenced by serotonin. These include spontaneous behaviours, such as marble burying and locomotor activity; and complex cognitive processes, including attention and impulsivity, measured on the 5-choice serial reaction time test (5-csrtt). The specificity of any abnormalities in PWS-IC /del mice was tested by examining the effects of pharmacological manipulations with 5-HT2CR specific drugs. Our findings suggest that abnormalities in 5-HT2CR functioning may underlie aspects of the behavioural phenotype seen in PWS, and that the imprinted snoRNA mbii-52 plays an important role in sculpting brain and behaviour. 55 10. Yucel Erbilgin, Muge Sayitoglu, Ozden Hatırnaz, Ugur Ozbek [email protected] Epigenetic inactivation of WNT5A by promotor methylation in acute leukemia patients WNT5A is a crucial member of soluble-secreted growth factor family (WNT) and required for the proliferation of hematopoietic progenitor cells during embryogenesis and development. WNT5A protein is detectable in a number of normal tissues and its expression increases in a wide variety human tumors. In contrast, here we found evidence that WNT5A may play a role as a tumor suppressor gene and showed the WNT5A gene methylated in acute leukemia patients. WNT5A and its interactive receptor FZ5 gene expressions was revealed by using quantitative real time PCR (QRT-PCR), and WNT5A promoter methylation status were determined with methylation specific PCR (MS-PCR) in 140 acute leukemia patients (n=41 AML and n=98 ALL). We analyzed the mRNA levels of WNT5A and FZ5, and did not detect any significant expression for both genes. As supporting evidence WNT5A proximal promoter methylation was found to be 82% for ALL patients and 84% for AML patients. Hypermethylation of WNT5A promoter is associated with mRNA expression disappearance. Based on the results of our present study, we can propose that, WNT5A act as a tumorsuppressor in acute leukemia patients. There is no prognostic relationship between WNT5A promotor methylation and ALL patients. This study represents the first demonstration of the WNT5A gene hypermethylation in acute leukemia patients. 56 11. Jennifer Frost, Dave Monk, Stephen Minger, Pascale Guillot, Harry Moore, Gudrun Moore [email protected] Epigenetic stability of human stem cells Human stem cells harbour huge potential for the treatment of degenerative disorders and disease. It is very important to characterise both the stem cells themselves and any potential effects they may have on their potential recipient. Epigenetics, defined as heritable and reversible regulation of DNA, is responsible for cellular phenotype changes beyond that dictated by the genome. Through direct modification of DNA or changes to chromatin stucture, control of cellular fate is achieved, whether division, differentiation or apoptosis. The propensity of a stem cell to differentiate into a particular lineage, or to undergo neoplastic transformation, is in part modified by epigenetics. The epigenetic stability of two different types of human stem cell, embryonic and fetal mesenchymal, was characterised using imprinted genes as an index for stability. All of the clusters of imprinted genes in the genome were evaluated both for their level of gene expression, allele specificity, and the integrity of respective controlling differentially methylated regions. Disruption of monoallelic expression was found in both types of stem cell. This disruption is cluster specific, does not reflect changes in methylation and may be linked by a common mechanism, one particularly susceptible to the pressures of in vitro cell culture. Work supported by the Medical Research Council. 57 12. Vivian Fu, Joe Dobosy, Josh Dosetelle, Jon Ewald, Rajini Srinivasan, Mark Berres, John Svaren, Richard Weindruch, David Jarrard [email protected] Aging and cancer-related loss of insulin-like growth factor 2 (Igf2) imprinting in the mouse and human prostate The epigenome is susceptible to modulation by many factors associated with aging, including dietary and oxidative stress. We determined whether the normal imprint (i.e. the epigenetic silencing of one allele) is altered for the Igf2 gene with aging in the prostate. Igf2 functions as a potent cancer promoter in the prostate. Prostate and other tissues from C57/B6 mice (containing a Cast IGF2-H19 allele) were serially harvested (3-24 months) and analyzed for alterations in IGF2 imprinting using a primer extension assay (FLuPE). Quantitative PCR for expression, DNA methylation sequencing, and Chromatin immunoprecipitation (CHiP) analyses were performed on the harvested tissues. Histologically normal human prostate specimens without cancer (40 samples) and containing cancer (25 samples) were also assessed. A significant loss of imprinting (LOI) for Igf2 is demonstrated specifically in the dorsolateral prostate (DLP) beginning at 11 months of age when compared to young sexually mature animals (3mo). No alteration in Igf2 imprinting is seen in the ventral prostate (VP) nor in other non-prostate tissues examined (kidney, liver, muscle). The LOI in the DLP found with aging is associated with a significant increase in Igf2 expression at the RNA and protein level. As a mechanism, CHiP analysis comparing 3 to 24 mo animals reveals a 2-fold decrease in CTCF expression and binding to the H19 imprint control region (ICR). Forced downregulation of CTCF using siRNA induced a LOI in prostate cells. Peripheral prostate samples from humans (17-72yo) reveal a significant association between increasing age and relaxation of IGF2 imprinting(p=0.05). LOI in histologically normal prostate tissues from men with cancer is significantly greater than men without cancer (p=0.02). These results demonstrate that degradation of the epigenome occurs with aging which may lead to a field effect of cancer susceptibility in the prostate. These findings in human tissues raise the possibility for utilizing the relaxation of Igf2 imprinting as a test to identify men who are at risk of having clinically significant prostate cancer. Furthermore, LOI may serve as a model for testing dietary and other genetic factors that modulate the aging-related loss of epigenetic control. 58 13. Tasuku Nomura, Mika Kimura, Takuro Horii, Sumiyo Morita, Hidenobu Soejima, Shinichi Kudo, Izuho Hatada [email protected] MeCP2-dependent repression of an imprinted miR-184 released by depolarization Both Fragile X syndrome and Rett syndrome are commonly associated with autism spectrum disorders (ASD) and involve defects in synaptic plasticity. MicroRNA is implicated in synaptic plasticity because the fragile X mental retardation protein (FMRP) was recently shown to be linked to the microRNA pathway. DNA methylation is also involved in synaptic plasticity since methyl CpG-binding protein 2 (MeCP2) is mutated in patients with Rett syndrome. Here we report that a brain-specific microRNA called miR-184 is an imprinted gene. Its expression is repressed by the binding of MeCP2 to its promoter, and expression is upregulated by the release of MeCP2 after depolarization. The restricted release of MeCP2 from the paternal allele results in the paternal allele-specific expression of miR-184. Our findings provides a clue to the link between the microRNA and DNA methylation pathways. 59 14. Ru Huang, Irena M. Vlatkovic, Florian M. Pauler, Renping Qiao, Federica Santoro, Mathew A. Sloane, Denise P. Barlow [email protected] Mapping and regulation of imprinted macro ncRNAs in the mouse and human genome Genomic imprinting results in parental-specific gene expression and offers one of the best examples of an epigenetic gene silencing mechanism in mammals. The analysis of imprinted gene expression in mouse models has identified two important but unexpected, epigenetic mechanisms. First, that DNA methylation acts to silence macro ncRNAs (long non-protein-coding RNAs with few or no introns). Second, that macro ncRNAs act to silence flanking genes in cis. To date, 85 imprinted genes are found to be clustered in 26 imprinted regions. Amongst these, 10 are imprinted macro ncRNAs that display reciprocal parentalspecific expression, relative to imprinted protein-coding genes in the cluster. This reciprocal expression indicates a functional role for macro ncRNAs in silencing imprinted protein-coding genes. Two typical examples of imprinted macro ncRNAs with a silencing function are already known (Air and Kcnq1ot1). We have developed a screen for the presence of macro ncRNAs of all imprinted gene clusters in the mouse and human genome by using a newly established technique, RNA Expression on Tiling Arrays (RETA). Our goals are to test if macro ncRNA is a common feature of imprinted regions in the mouse and human genome. The preliminary results show we can identify all 6 well-known imprinted macro ncRNAs in different developmental stages of mouse tissues and cells, and from different human cell lines as well. Some novel macro ncRNA candidates have been detected. We are now trying to characterize the transcriptional and post-transcriptional features of the newly identified macro ncRNAs. 60 15. Yoko Ikeda, Yuki Kinoshita, Tetsuji Kakutani, Tetsu Kinoshita [email protected] Analysis of Arabidopsis alac1 mutant defective in genomic imprinting Flowering plants control seed development in part by genomic imprinting in the central cell and endosperm. The establishment of imprinting is known to require DNA demethylation by DNA glycosylase, but little is know about how DNA demethylation is regulated. To better understand the regulatory mechanism, we screened alarm clock for FWA imprinting (alac) mutants that are defective in genomic imprinting using GFP reporter constructs of a maternally expressed gene, FWA. We successfully isolated two alleles of the alac1 mutant that were deficient in the activation of FWA-GFP. The expression of two other maternally imprinted genes, MEDEA and FIS2, was also low in alac1 mutants. Furthermore, DNA methylation levels within the 5’ differentially methylated region of FWA remained higher in the endosperms of mutants than in WT. Role of ALAC1 in the establishment of genomic imprinting and DNA demethylation will be discussed. 61 16. Ryo Ishikawa, Mitsugu Eiguchi, Nori Kurata, Tetsu Kinoshita [email protected] Genomic imprinting as a reproductive barrier in hybrid rice endosperms Interspecific crosses often result in defective endosperm development, including seed abortion, which is thus a post-fertilization reproductive barrier. Several reports have suggested that defective endosperm development is caused by functional differences between parental genomes. One of the possible molecular mechanisms underlying this phenomenon is genomic imprinting, which is an epigenetic mechanism that results in parent-of-origin-dependent mono-allelic gene expression. Interspecific crosses between O. sativa cv. Nipponbare and O. longistaminata, O punctata or O. australiensis produced developmental defects in the hybrid endosperm with precocious or delayed cellularization, depending on parental combinations. This phenotype is similar to the imprinting mutants of Arabidopsis. Possible molecular mechanisms and the involvement of genomic imprinting on the reproductive barrier will be discussed. 62 17. Anthony Isles, Trevor Humby, Antonius Plagge, Alastair Garfield, Andrew Ward, Gavin Kelsey, Lawrence Wilkinson, Jon Wilkins [email protected] Genomic imprinting and risk-taking: brain and behavioural analysis of Nesp55 null animals One scenario thought to have led to the evolution of genomic imprinting is when maternally and paternally derived genomes are subject to differential selective pressures, resulting in intra-genomic conflict. This idea successfully explains the role of many imprinted genes in mother-to-offspring resource allocation. However, why imprinted genes should play a role in adult behaviour is less clear. A new theoretical argument suggests that imprinted genes in the brain will influence risk-taking behaviour. In most mammals, males have a higher variance of reproductive success than females. Paternally derived alleles, which were in a high-variance state in the previous generation, are therefore under selective pressure to reduce their exposure to risk. In contrast, maternally derived alleles, having not been exposed to the same degree of variation in reproductive success, will be less risk-averse. We tested this prediction in mice lacking Nesp55, which is maternally expressed in discrete areas of the brain. Behavioural analysis (exploration of a novel environment; delay-discounting) shows that maternally expressed Nesp55 normally promotes risk-taking. Further studies indicate this may be due to altered neurochemical release in these animals, suggesting a neural basis for the behaviour. Importantly, a number of other expressed imprinted genes, most notably Grb10, show a striking degree of overlap in terms brain expression with Nesp55. This suggests that these genes (both maternally and paternally expressed) may impact on the same behaviour. This work provides a new framework for the evolution of imprinted genes based on intra-genomic conflict, but predicting a novel function for imprinted gene action in the brain. 63 18. Meaghan Jones, Louis Lefebvre [email protected] Epigenetic regulation of a GFP insertional allele by genomic imprinting While introducing a truncation on distal mouse chromosome 7 (MMU7), we obtained an insertion allele carrying a single copy of the Green Fluorescent Protein (GFP) gene 3.3kb distal to the mouse Ins2 gene, between the two imprinted sub-domains on distal 7. We report here that this insertion, called Tel7Knock-In or Tel7KI, shares many features with endogenous maternally expressed imprinted genes. When Tel7KI is maternally transmitted, GFP is expressed in approximately 40% of the cells of the developing embryo, whereas upon paternal transmission, GFP expression is undetectable by fluorescence microscopy, flow cytometry, or RT-PCR. This silencing is reversible such that the GFP reporter is reactivated in the progeny of paternal-heterozygous females. DNA methylation analysis of the promoter driving GFP expression has shown increased methylation on the paternal allele in embryos, but no methylation in sperm or in GFP-expressing cells of maternal transmission embryos. Interestingly we noted that upon paternal transmission of the allele, GFP expression was observed in the placenta of transgenic embryos in a punctuate pattern reminiscent of the localization of trophoblast giant cells. These cells have been hypothesized to show epigenetic instability due to their extremely high ploidy. We are investigating whether the observed loss of imprinting (LOI) of GFP reflects LOI at endogenous loci in these cells. To obtain and examine these giant cells in vitro, we cultured ectoplacental cones of embryos heterozygous for castaneus polymorphisms on distal MMU7. The resulting giant cells will be examined for methylation and expression of the Tel7KI allele as well as other imprinted genes on MMU7. We hope to show that the Tel7KI mouse line is a reporter for LOI in vivo, and a useful non-invasive tool to study the maintenance and loss of imprinting during mouse development and tumorigenesis. 64 19. Pauline Jullien, Mathieu Ingouff, Frédéric Berger [email protected] Retinoblastoma and its binding partner MSI1 control imprinting in Arabidopsis Parental genomic imprinting causes preferential expression of one of the two parental alleles. In mammals the differential sex-dependent deposition of silencing DNA methylation marks during gametogenesis initiates a new cycle of imprinting. Parental genomic imprinting has been detected in plants and relies on DNA methylation by the methyltransferase MET1. However, in contrast to mammals, plant imprints are created by differential removal of silencing marks during gametogenesis. In the female gamete DNA demethylation is mediated by the DNA glycosylase DEMETER (DME). Based on genetic interactions we show that in addition to DME, the plant homologues of the human Retinoblastoma (Rb) and its binding partner RbAp48 are required for the activation of the imprinted genes FIS2 and FWA. This activation by the Rb pathway is mediated by direct transcriptional repression of MET1 demonstrated by Chromatin immunoprecipitation. Our data identifies a new mechanism required for imprinting, relying on a Retinoblastoma dependent pathway. The potential conservation of this pathway suggests that Rb might also control imprinting in mammals. 65 20. Tomoko Kaneko-Ishino, Shunsuke Suzuki, Andrew Pask, Geoffrey Shaw, Takashi Kohda, Jennifer Graves, Marilyn Renfree, Fumitoshi Ishino [email protected] Origins of retrotransposon-derived PEG10 and imprinted regulation by DNA methylation We have demonstrated that two imprinted genes, Peg10 (paternally expressed 10) and Peg11/Rtl1 (paternally expressed 11/retrotransposon-like 1) that are derived from a sushi-ichi related retrotransposon, play essential roles in early placenta formation and in maintenance of fetal capillaries in late placenta, respectively. As these genes do not exist in non-mammal animals, such as birds and fish, it is very interesting to know exactly when these genes were acquired in our ancestors and how they contributed to establishment of mammalian viviparity. From analysis of orthologous chromosomal regions of tammar wallaby (an Australian marsupial species) and platypus (an Australian monotreme species), we have found that PEG10 exists in marsupials in the form similar to eutherian PEG10 but not in monotremes, suggesting its close relationship to the origin of placentation in mammals. We have also discovered a missing link of the imprinting mechanism between eutherians and marsupials, DMR (differentially methylated region) in marsupial PEG10 promoter region because this is the first DMR identified in marsupial imprinted genes. These results demonstrate that insertion of the original PEG10 retrotransposon occurred after diversion of protherian and therian mammals and suggest that retrotransposon silencing by DNA methylation can promote genomic imprinting in this region. References: 1. Ono R et al. Nat Genet 38, 101-106 (2006). 2. Sekita Y et al. Nat Genet 40, 243-248 (2008). 3. Suzuki S et al. PLoS Genet 3, e55 (2007). 66 21. Ana Katusic, E.G Van Donselaar, A.J Verkleij, Maja Vlahovic, Ljiljana Serman, Nino Sincic, Frane Paic, Floriana Bulic-Jakus [email protected] Expression of apoptotic markers in fetal rat testis after exposure to 5-azaC DNA methylation is important mechanism of gene regulation in embryogenesis and gametogenesis. 5-azacytidine (5-azaC) is cytosine based analogue, which causes DNA hypomethylation if incorporated during the process of cell proliferation. Weekly administration of this drug disturbs spermatogenesis of adult rat or mice while its teratogenic effects were also described, mostly caused by a single administration of the pregnant animal. We have studied the effect of single 5-azaC administration on the development of fetal rat testis. Pregnant female Fisher rats have received single intraperitoneal injection of 5-azaC in dose of 5mg/kg on 13th, 14th, 15th or 16th day of pregnancy. Fetal testes were isolated on the day 20 and processed for routine histology and immunohistochemistry. Apoptotic-like cells were found in testes of all experimental groups with the highest incidence of apoptotic cells in tubules of the group treated on day 15. Electron microsopy revealed cells in various stages of apoptosis which are presumed to be gonocytes. Moreover, TUNEL analysis and staining against early apoptotic marker γ-H2AX (Asp175) (Cell Signaling Technology) were done on semi-thin Tokuyasu cryosections. TUNEL positive cells were mostly detected in testes which were exposed to the drug on 15th and 16th day of pregnancy. On the other hand, less γ-H2AX positive cells were found, with the γ-H2AX foci in the nucleus and the morphology of early-to-middle apoptotic stage. Testes of control fetuses were negative for the presence of apoptotic cells or any apoptotic marker. Described results suggest that 5-azaC disturbs cell homeostasis in fetal rat testis by causing apoptosis, with the most effect when applied on 15th day of pregnancy. Moreover, most of apoptotic cells were in late stage with few of them in earlier stage, where γ-H2AX foci were found. 67 22. Neelam Kedia-Mokashi, Shilpa Pathak, Nafisa Balasinor [email protected] Epigenetic paternal development factors affecting early embryo and placental Imprinting is known to have evolved to promote placental development and imprinted genes are known modulators of embryonic growth and development. Studies from our laboratory with paternal administration of tamoxifen, a selective estrogen receptor modulator (SERM), to adult rats at a dose of 0.4 mg/kg/day for 60 days revealed an increase in post-implantation loss at around mid gestation. Expression of Igf2, a paternally expressed imprinted gene and a pivotal growth factor, was down regulated in the resorbed embryos (Kedia et al., 2004). Igf2H19 ICR displayed hypomethylation in the resorbed embryos as well as spermatozoa obtained from treated group. The above studies suggested that paternal drug treatment with tamoxifen could have lead to errors in imprint establishment and/maintenance during spermatogenesis and this could be responsible for embryo loss due to deregulation in the expression of imprinted genes. Igf2 KO mice are 60% the weight of their normal litter mates whereas resorbed embryos obtained after tamoxifen treatment are not viable by day 20 of gestation. To dwell further into the details of the mechanism of embryo loss following paternal drug treatment, microarray studies using Agilent rat whole genome slides were carried out. The differential gene expression profile generated by comparing the normal vs resorbed embryos revealed the disruption of growth factor signaling and cell cycle arrest. Apoptosis has just set-in as inflammatory caspases were up regulated but most pro-apoptotic genes were down regulated. Also up-regulation of Dlk1, a paternally expressed imprinted gene, which is a part of Delta-Notch signaling, was observed. The expression of Dlk1 has been associated with cellular processes characterized by the presence of an inflammatory response. Stochastic expression of genes important for trophoblast formation and differentiation was observed in the resorbed embryos. 3-4 fold down regulation of imprinted genes viz. Mash2, p57, Phlda2, Gpc3, and Grb10 along with upregulation of Peg3 was observed in the resorbed embryos. The current study demonstrates that paternal drug treatment can affect epigenetic reprogramming occurring during placenta formation. Defects in either imprint establishment and/maintenance which are crucial for formation of a functional placenta can disrupt harmonious interactions between the placenta and fetus which regulate supply of nutrients, hormones and growth factors. Any deviations from this interplay can be detrimental to fetal growth thereby causing fetal loss. Kedia N, Gill-Sharma MK, Parte P, Juneja HS, Balasinor N.(2004) Mol Reprod Dev 69:22-30 68 22. Rosemary Oh, Aaron Bogutz, Louis Lefebvre [email protected] Regulation of imprinting and trophoblast-specific expression by the imprint control region KvDMR1 (IC2) The distal end of mouse chromosome 7 (MMU7) arbors a large domain regulated by genomic imprinting. Imprinted expression in this region implicates two cisacting imprinting centres, IC1 (H19 DMR) and IC2 (KvDMR1), defining proximal and distal sub-domains, respectively. To assess the functional independence of IC1 in the context of MMU7, we developed a new recombinase-mediated chromosome truncation strategy in embryonic stem (ES) cells and engineered a terminal deletion with a breakpoint in between the two imprinted sub-domains (DelTel7 allele). We obtained stable germline transmission of this truncated MMU7 and viable paternal heterozygotes. Our results confirm the absence of additional paternally expressed genes within the 2.7 Mb deleted region. Conversely, maternal transmission of DelTel7 causes a mid-gestational lethality, consistent with loss of maternally expressed genes in the distal sub-domain. Expression and DNA methylation analyses on reciprocal DelTel7 heterozygotes confirmed the independent imprinting of IC1 in absence of the entire IC2 sub-domain. Furthermore, we show that the developmental phenotype of maternal heterozygotes is rescued by a paternally inherited deletion of IC2 itself (viable DelTel7/IC2 KO compound heterozygotes), demonstrating that all the essential imprinted genes in the region are under IC2 regulation. Since the entire IC2 domain is deleted in DelTel7, this truncation offers the possibility to perform allele-specific studies in reciprocal hemizygotes for the IC2 region. We conducted a detailed analysis of placental development in the hemizygous DelTel7/IC2 KO “rescued” embryos, since several of the IC2-regulated genes are implicated in this process. Our analysis suggests that some of these genes, notably Cdkn1c and Phlda2, show abnormal expressions from the IC2 KO allele. We present a model in which the methylated maternal IC2 is bi-functional in regulating the long-range monoallelic expression of protein-coding genes in the region(i) it silences the production of the Kcnq1ot1 ncRNA, thereby preventing the establishment of a repressive chromatin in the domain; and (ii) it plays a role in the regulation of tissue-specific expression of neighboring genes, perhaps via a methyl-CpG binding complex. 69 23. Flavia L. Lopes, Daniel R. Arnold, Kirsten Niles, Jacquetta M Trasler [email protected] Serge McGraw, Knockdown of DNMT3L reveals variable sensitivity of imprinted genes to disruption DNA methylation is an epigenetic mechanism involved in a variety of biological and developmental processes, and is catalyzed by a family of DNA methyltransferases (DNMTs). Genomic imprinting involves the formation of epigenetic ‘marks’ in a parent-of-origin-specific manner such that genes are expressed monoallelically. Deletion of Dnmt3L is embryonic lethal and results in biallelic expression of maternally methylated imprinted genes. To investigate how modulation rather than ablation of DNMT3L in oocytes affects acquisition of methylation patterns, we produced transgenic mice harboring an oocyte-specific expression of DNMT3L hairpin dsRNA, resulting in knockdown of Dnmt3L in oocytes. Four lines were investigated, with residual DNMT3L mRNA expression of approximately 40, 30, 10 and <5%. Fertility evaluated during 5 months was unaffected in the lines with 30 and 40% residual expression, whereas significantly reduced average numbers of pups born/female were observed in the lines with 10 and <5% expression (2.5±0.6 and 4.1±0.9 pups/female, respectively; vs 10.5±0.9 in wild type). Similar to the knockout model, the majority of embryos from these two lines die between 9.5 and 10.5 dpc. Defects in neural tube closure and pericardial edema were commonly observed, as were dilated allantois, reduced vascularization of the yolk sac and delayed embryonic development. Quantitative analysis of methylation by Real-Time PCR (qAMP) of embryos at 9.5 dpc of partial DMRs of Snrpn, Lit1, U2as-rf1, Peg1 and Igf2r revealed abnormal patterns of methylation in these genes. Interestingly, within each embryo, certain genes appeared to be more resistant to loss of methylation than others. In conclusion, oocyte reduction of DNMT3L affects development of embryos and extra-embryonic membranes, as well as methylation of imprinted genes, in a gene-specific manner. (Supported by CIHR) 70 24. Marta Madon, Alastair Garfield, Andrew Ward [email protected] Characterisation of the imprinted Grb10 and Dlk1 genes in mouse The majority of mammalian genes are equally expressed from both the maternal and paternal allele, but a small group of genes is known to be regulated by the phenomenon of genomic imprinting, which leads to predominant expression from only one parental allele. Imprinted genes play important roles in the development and growth of embryonic and extra-embryonic lineages. According to the parental conflict theory, genomic imprinting evolved due to the antagonistic interests of maternal and paternal genomes. Paternally expressed genes are predicted to enhance growth and promote the use of maternal resources, while maternally expressed genes act to reduce growth and limit use of maternal resources. Grb10 and Dlk1 are imprinted genes which perfectly fit with this theory, Grb10 being a maternally expressed growth inhibitor and Dlk1 a paternally expressed growth enhancer. Not only are Grb10 and Dlk1 oppositely imprinted, but knocking out the Grb10 and Dlk1 genes results in the opposite phenotypes in mice. Therefore, the hypothesis has been generated that the Grb10 and Dlk1 genes might be acting through a common genetic pathway. We aim to test this hypothesis by characterizing in detail the Grb10/Dlk1 double mutant mice and comparing their phenotype to wild type, Grb10 knockout and Dlk1 knockout animals. If our hypothesis proves to be correct, these findings will contribute to better understanding of mechanisms of imprinting and epigenetic modifications during early developmental processes. 71 25. Kirsten McEwen, Anne Ferguson-Smith [email protected] Epigenetic profiling of all imprinted genes and comparison to the mouse Dlk1-Dio3 domain during neural differentiation The mechanisms acting to control imprinting at the mouse Dlk1-Dio3 domain are currently unknown. DNA methylation is known to play an important role and it is likely that other epigenetic mechanisms also act to establish and/or maintain the imprinted status of genes within the cluster. Here we assess the histone modifications present across the cluster in a mouse model of embryonic stem cell (ESC) neural differentiation. This system enables marks of differentiation to be distinguished from marks of imprinting and provides insight into the genome-wide action of epigenetics. The profile at the Dlk1-Dio3 domain is compared to that of other imprinted clusters and also to all known imprinted genes. This assessment is made through a meta-analysis of low- and high-throughput studies of histone modifications at mouse and human imprinted genes. This analysis has on its own uncovered previously uncharacterised patterns, including distinct profiles at imprinted compared to non-imprinted genes and between maternally and paternally expressed imprinted genes. Interestingly, a very specific profile is observed in mouse ESCs at all imprinting control regions (ICRs). This may prove to be a useful tool for identifying ICRs for newly discovered imprinted genes. 72 26. Serge McGraw, Christopher C Oakes, Josee Martel, Flavia L Lopes, J. Richard Chaillet, Jacquetta M Trasler [email protected] Loss of DNMT1o disrupts DNA methylation in extraembryonic tissues DNA methylation is an epigenetic mark catalyzed by DNA cytosine methyltransferase (DNMT) enzymes and is involved in a variety of developmental processes. In mice, the oocyte-derived DNMT1o protein is restricted to the cytoplasm during early preimplantation embryonic cleavage stages, and is later transiently localized in the nuclei of 8-cell embryos. Embryos born to Dnmt1odeficient mothers (Dnmt1o mat-/-) have disrupted genomic imprinting and display significant anatomical abnormalities. Here, we have found that placentas from 9.5 dpc Dnmt1o mat-/- conceptuses have an elevated frequency of malformations (misshaped and larger size) compared to wild-type controls. Interestingly, placentas from female Dnmt1o mat-/- conceptuses had a much higher incidence of abnormalities compared to males. We assessed the methylation status of CCGG sites using thin layer chromatography (TLC) and uncovered that Dnmt1o-deficiency in 9.5 dpc embryos and placentas does not result in an alteration of embryonic DNA methylation; however, placental methylation is reduced in a sex-specific manner. Dnmt1o mat-/- female placentas displayed reduced levels of global methylation compare to their male counterparts, which were mostly normal. Further analysis revealed that the repetitive DNA elements (IAP repeat and minor satellite) demonstrate selective hypomethylation in XX Dnmt1o mat-/- placentas. By means of a more sensitive method, restriction landmark genomic scanning (RLGS), we investigated the methylation status of approximately 2000 single-copy loci and found that Dnmt1o mat-/- 9.5 dpc embryos and placentas display a range of aberrations (at less than 5 to more than 60 loci) compared to controls. Individual embryos and placentas of both sexes predominantly exhibited hypomethylation; however, hypermethylation events were also observed. Interestingly, almost all sites that were consistently hypomethylated in XX versus XY Dnmt1o mat-/- placentas are X-linked CpG island-containing loci. Together these studies show that a lack of DNMT1o activity in the 8-cell preimplantation embryo stage results in widespread DNA methylation defects in extraembryonic tissues. Methylation defects on the X chromosome may provide a clue to the underlying nature of the sexspecific bias in placental morphology and global methylation. 73 27. John McLaughlin [email protected] Consequences of imprinting status on hematopoiesis in parthenogenetic embryonic stem cell versus parthenogenetic aggregation chimeras Parthenogenetic (PG) aggregation chimeras and those produced by blastocyst injection of PG ES cells exhibit various phenotypic differences, including the absence of growth deficits and restriction of tissue contribution in the latter. This suggests that ES cell derivation and culture change the developmental capacity of PG cells, consistent with observations that paternally expressed imprinted genes can become reactivated in PG ES cells. The extent and consistency of changes in imprinting in PG ES cells compared to those of primary embryonic origin in aggregation chimeras is unknown, and consequences on the formation of particular cell types are poorly understood. Changes in cell phenotype and differentiation capacity that can be associated with epigenetic markers or gene expression patterns, however, can be highly valuable for the evaluation of the therapeutic potential of any cell type maintained in vitro. As an approach towards this, we are investigating the in vivo hematopoietic differentiation capacity and functionality of PG cells, both in PG aggregation and ES cell chimeras, with the outlook to relate these to gene expression changes and epigenetic markers. In fetal stage chimeras produced by aggregation of four-cell stage EGFP transgenic PG and non-transgenic normal embryos, PG derived cells are present in hematopoietic tissues (fetal liver) at similar levels compared to PG cells in PG ES cell chimeras. After transplantation of chimeric fetal liver from PG aggregation chimeras into lethally irradiated adult mice, PG derived cells contribute substantially (>50%) to the hematopoietic system of recipients, and are represented in major lineages of the peripheral blood, including T- and Blymphocytes, macrophages and erythroid cells, indicating that in aggregation chimeras, PG cells form functional fetal liver hematopoietic stem cells (HSC). Primary embryonic PG cell derived hematopoietic stem cells also persist in adult PG aggregation chimeras; indicative of the presence of PG derived HSC. Analyses of immune function of these cells will be performed. Preliminary lineage differentiation analyses indicate that PG-derived cells both of aggregation chimera and ES cell origin exhibit biased, low contribution to T lymphocytes. If substantiated by outcomes of functional assays, we will employ gene array analyses on PG cells (PG hematopoietic cell types sorted from chimeras or hematopoietic transplant recipients) to identify gene expression patterns associated with these differences in cell differentiation and function. 74 28. Stuti Mehta, Christine Williamson, Simon Ball, Jo Peters [email protected] Role of transcriptional events in the establishment of imprints at the Gnas cluster Imprinted genes are found in clusters and their parent specific expression is regulated by Imprinting Control Regions (ICR). The ICR at the Gnas cluster lies at the promoter of Nespas. Nespas is a non-coding paternally expressed transcript that runs anti-sense to the maternally expressed transcript Nesp. The ICR controls expression of all transcripts in the cluster; and uniquely, exerts indirect control on the expression of downstream Gnas, through the promoter of Exon1A. The Exon1A promoter lies about 2.5 kb upstream of Gnas start site. Thus, the ICR and the Exon1A promoter must interact. I am interested in understanding how, during development; transcriptional events at the cluster contribute to establish this interaction on the paternal allele. The ICR and the Exon1A promoter are both methylated in the female germline and remain unmethylated in sperm. By bisulfite modification and sequencing, I studied the effect of two paternally inherited targeted mutations of the ICR on the methylation status of the Exon1A promoter. One mutant harbours a deletion of the ICR, while the other mutant carries an insertion which truncates the anti-sense Nespas transcript to ~100bp of its length. On paternal inheritance, each of these mutations of Nespas led to gain of methylation at the Exon1A promoter. This was seen at 10.5 dpc, and the methylation pattern was seen retained in new born animals. However, the Exon1A promoter was unmethylated in sperm of the Nespas truncation animals, showing that the gain of ectopic methylation at the Exon1A promoter is a post-fertilization event in this mutant. Nesp has the furthest upstream start site in the cluster and transcribes through the entire cluster, including the Exon1A promoter. Both the mutations of Nespas lead to full expression of Nesp from the paternal allele, as seen by real time PCR. Based on these results, I hypothesise that ectopically expressed Nesp has a role in methylation of the Exon1A promoter in Nespas mutants. I propose that on wildtype paternal alleles, the principal ICR indirectly influences methylation at the minor ICR via repression of Nesp. 75 29. Ania Migdalska [email protected] A genetic screen for genes that regulate imprinting Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are caused by a deficiency of imprinting of paternal or maternal chromosome 15q11-q13, respectively. Genomic imprinting of the PWS/AS domain is regulated through a bipartite cis-acting imprinting center (PWS-IC/AS-IC) within and upstream of the SNRPN promoter. Recently a mouse ES cell line was developed which contains the fluorescent marker EGFP knocked into the Snrpn promoter (Genes Dev. 2006 Oct 15;20(20):2859-70). We are interested in using these Snrpn-EGFP ES cells to screen for genes that regulate imprinting. Genes that regulate imprinting are important not only because of their role in diseases such as PWS and AS, but also because loss of imprinting is a frequent event in tumourigenesis. My project involves ES cell culture, the use of transposons, gene targeting, flow cytometry and the generation and characterization of knockout and knockin mice. 76 30. Adele Murrell, Yoko Ito, Santiago Uribe Lewis, Kathryn Woodfine, Raffaella Nativio, Joanna Huddleston [email protected] Genomic imprinting models for long and short term epigenetic memory in cancer Imprinted genes are associated with CpG rich regions that become methylated on one parental allele. These sequences are known as differentially methylated regions (DMRs) and for each imprinted locus at least one DMR is acquired (established) in the parental germline and subsequently maintained in somatic cells after fertilisation. In addition to the above mentioned germline DMRs, there are somatic DMRs that acquire methylation after fertilisation. Somatic and germline DMRs have different susceptibilities to global and tissue specific reprogramming and we are currently examining genomic and epigenetic features of DMRS to determine the mechanisms behind genome and epigenome instability in both childhood and adult onset cancer. We have recently shown that loss of imprinting of IGF2 and H19 in the germline that leads to congenital disease is associated with different methylation profiles at the DMRs compared to that in Wilms tumour, and adult cancers such as breast and colorectal cancer. We have also shown that the methylation state of the somatic DMR0 at the IGF2 gene is subject to demethylation with advanced age. Further analysis to assess whether somatic DMRs are more susceptible to global demethylation events in cancer has been carried out by comparing the methylation states of LINE1 elements and somatic DMRs at the IGF2 locus. These findings suggest that somatic DMRs and LINE1 elements are temporally demethylated. In childhood cancers such as Wilms Tumour somatic DMRs undergo methylation changes independent of global demethylation and there is no correlation between methylation states of LINE1 elements and DMRs. In adult onset cancers IGF2 DMR0 hypomethylation is an early event and is more frequent than LINE 1 hypomethylation. We have data suggesting that mouse cancer models show similar changes in susceptibilities to changes in DNA methylation at the IGF2 locus as humans and that these changes occur predominantly in the somatic DMRs. Preliminary evidence that the mechanisms that underlie the susceptibility to reprogramming are higher order chromatin structures and proteins that specifically bind methylated DMRs to protect against demethylation will be presented. 77 31. Pablo Navarro, Andrew Oldfield, Mélanie Makhlouf, Philip Avner, Claire Rougeulle [email protected] Toward the understanding of Xist regulation X-chromosome inactivation in mammals is a highly dynamic process and the early mouse development is characterized by several waves of inactivationreactivation. X-inactivation is strictly dependent on the Xist gene, which produces a non-coding RNA (ncRNA) that coats the chromosome in cis and induces its silencing. Only high levels of Xist RNA can induce X-inactivation, which implies a tight control of Xist expression in order to ensure the dynamics of X-inactivation. Part of this regulation is mediated by Tsix, a transcript antisense to Xist, which represses Xist accumulation in cis. We have demonstrated that Tsix is not directly involved in the transcriptional control of Xist expression but rather in its programming, through the induction of complex chromatin modifications within the Xist locus. In addition, Tsix acts as a long-range regulator of the Xinactivation center chromatin through the modulation of H3K27 tri-methylation levels and distribution. We present evidence suggesting that the polymerase transcribing Tsix is itself likely to act as a boundary element to constrain H3K27 methylation to a specific region. We are now focusing on trans-acting factors that are involved in the control of Xist expression, both in mouse and in human. 78 32. Verity F Oliver, W S Cutfield, H L Miles, P L Hofman, I M Morison. [email protected] Does in vitro fertilisation influence epigenetic programming? In vitro fertilisation (IVF) potentially provides a profoundly abnormal environment for an embryo. Studies with mice, sheep and cattle have indicated that the culture environment of the embryo can affect the imprinting of genes and the phenotype of the animal. Approximately 2% of human births worldwide are conceived using IVF. Recent studies have suggested that IVF causes a small but increased risk of epigenetic imprinting aberrations such as Angelman syndrome and Beckwith-Wiedemann syndrome. Given that mosaicism for the imprinting defect has been observed in Angelman syndrome and BeckwithWiedemann syndrome, we hypothesised that low-level, mosaic imprinting defects may be present in phenotypically normal individuals conceived using IVF. DNA samples from peripheral blood were obtained from 69 IVF-conceived pre-pubertal children and 71 matched controls. DNA methylation of CpG sites within the H19, SNRPN and KvDMR1 loci was accurately quantified using methylation-sensitive restriction digest followed by real-time quantitative PCR (MSQ-PCR). Global DNA methylation was also examined by using MSQ-PCR on satellite-2 repeats. No differences in the percentage of methylation between the IVF-conceived and control children were observed at the examined CpG sites. Detailed analysis using bisulphite sequencing is in progress. Current data support the MSQ-PCR results and indicates no difference in methylation between the IVF-conceived and control children. Further investigation into other methylated genes that may be affected by IVF is currently being undertaken. Methylated promoter arrays using an anti-methyl DNA antibody (meDIP) is also being used to identify genes that may be differentially methylated between the IVF-conceived and control children. We concluded that low-level epigenetic imprinting errors are not a common occurrence in children conceived using IVF. Our data provide reassurance that IVF-associated imprinting errors are sporadic and rare. 79 34. Shilpa Pathak, Neelam Kedia, Madhurima Saxena, Ryan D'Souza, Manjit Gill-Sharma, Rahul Upadhyay, Nafisa Balasinor [email protected] Effect of tamoxifen treatment on global and Insulin-like growth factor 2-H19 locus specific DNA methylation in rat spermatozoa, its association with embryo loss and role of estrogen signaling in imprint acquisition. Male-specific genomic imprints are set up during spermatogenesis transmitting epigenetic information to the successive generation and are required for successful embryo development. Insulin-like growth factor2 (Igf2) is paternally expressed imprinted gene promoting early embryo growth. Igf2 exhibits coordinate expression with neighboring H19 gene and its paternal specific expression is dependent on methylation of imprinting control region (ICR) located downstream to Igf2. A significant increase in post-implantation loss with concurrent decrease in the transcript expression of Igf2 in resorbed fetuses was observed following tamoxifen treatment to adult male rats. Resorbed embryos also showed loss of methylation at the CpG island at Igf2-H19 locus concluding imprinting errors in resorbed embryo. Igf2 expression is associated with differential methylation of ICR, which is a primary imprint mark, inherited through gametes. Hence, our results suggested that imprinting errors in the male germ line due to disrupted DNA methylation during spermatogenesis probably lead to hypomethylation at Igf2-H19 ICR in the spermatozoa. Therefore, the objective of the study was to investigate effect of tamoxifen treatment on global DNA methylation and methylation status at Igf2-H19 ICR in the spermatozoa Our bisulfite sequencing analysis at Igf2-H19 locus revealed significant hypomethylation with tamoxifen treatment indicating the effect of tamoxifen in the establishment and /or maintenance of methyl imprint during spermatogenesis. Secondly, the study also showed significant correlation between methylation at Igf2-H19 ICR in spermatozoa and post implantation embryo loss. However, global methylation evaluation of spermatozoa by flow cytometry and bisulfite sequencing analysis of long interspersed nucleotide element 1 did not show any association either with resorption or tamoxifen treatment suggesting locus specific effect of tamoxifen. This signifies Igf2-H19 ICR methylation in spermatozoa as a predictive factor to assess embryonic development. In-silico analysis of rat Igf2-H19 ICR sequence showed presence of estrogen receptor b (ERb) binding sites. Since tamoxifen is a selective estrogen receptor modulator, we have evaluated role of ERb in imprint acquisition at ICR in the male germline by chromatin immunoprecipitation and characterized its association with the members of DNA methylation machinery by immunoprecipitation and confocal approach. Our results suggest plausible role of estrogen in imprint acquisition at Igf2-H19 locus. In conclusion, these results confirm the contribution of paternal epigenome to embryogenesis. 80 35. Charlotte Proudhon, Bourc'his [email protected] Reiner Schultz, Rebecca Oakey, Déborah Toward the identification of new imprinted genes Imprinted expression in mammals relies on DNMT3L, a DNA-methyltransferase homolog specific to this clade. This catalytically inactive DNA-methyltransferase is required to set up differential methylation marks at imprinting control regions (ICRs) in male and female gametes. Biochemical studies showed that DNMT3L interacts with DNMT3A and locally stimulates its de novo methylating activity on target sequences. Dnmt3L-/- females are viable but produce oocytes specifically lacking maternal methylation marks. Using this mutant model, we derived embryos lacking specifically maternal germline imprints and embryos devoid of parental germline imprints altogether, respectively by fertilization and by parthenogenetic activation of Dnmt3L-/- oocytes. These embryos die around 9.5dpc with a similar phenotypic presentation. Phenotypic assessment and comparative expression profiling by microarray at 8.5dpc enabled us to show that maternal imprints have a major impact on early developmental pathways, in agreement with the overwhelming number of imprints derived from the maternal germline compared to the paternal one. In an attempt to identify new imprinted genes, we combined a genome-wide DNA methylation analysis to the expression profile of the maternal imprint-free embryos in comparison to age-matched wildtype embryos. To obtain a map of autosomal and X-linked regions controlled by maternal methylation, a Methylated-DNA ImmunoPrecipitation (MeDIP) coupled with a T7 based linear amplification was performed on female 7.5dpc embryos and the methylation-enriched material was hybridized onto CpG island-pluspromoter microarrays. This study is likely to provide some major insights into the estimation of the real number of imprinted genes in the mouse genome, their genomic characteristics and their functions. Finally, our aim is to identify most of the existing imprinted genes and generate a bank of potential candidates for the increasing number of identified human diseases associated to a parent-of-origin effect. 81 36. Schaefer Christopher, Aravin Alexei, Bourc'his [email protected] Hannon Gregory, Déborah Epigenetic cooperation in DNA methylation in the mammalian germ line Retrotransposon activity is controlled by several layers of repressive mechanisms in the mammalian germ line. Transcriptional silencing though DNA methylation is established in prenatal male germ cells under the control of the DNA-methyltransferase co-factor Dnmt3L. Piwi-interacting small RNAs (piRNAs) provide a level of post-transcriptional silencing through RNA degradation, and potentially cooperate in DNA methylation targeting to retrotransposons. Dnmt3L deficiency results in the reactivation of several classes of retrotransposons and leads to impaired spermatogenesis in mutant mice. A similar phenotype is observed in piRNA-deficient males that are mutant for Miwi2 or Mili, two members of the Piwi family. We analyzed by genetic means the cumulative effects of Dnmt3L and Piwi proteins on retrotransposon methylation and mobilization in Dnmt3L/Miwi2 double-mutants. Consequences of retrotranscript accumulation on the production of retrotransposon-associated piRNAs were moreover examined in Dnmt3L mutant germ cells. This study provides insight into the cooperative role of DNA-methyltransferases and the piRNA pathway in de novo DNA methylation and retrotransposon control in the mammalian germ line. 82 37. Hirosuke Shiura, Takafusa Hikichi, Kenji Nakamura, Toshiaki Hino, Kanako Oda, Rika Suzuki-Migishima, Takashi Kohda, Tomoko KanekoIshino, Fumitoshi Ishino [email protected] Disruption of imprinted regulation in Meg1/Grb10 cluster results in pre- and postnatal growth retardation Most imprinted genes form gene clusters in certain chromosomal regions. It has been thought that mouse Meg1/Grb10 is a single imprinted gene located on proximal chromosome 11. We have recently found that at least two neighboring genes, Cobl at 3’ side and Ddc at 5’side, are imprinted and show parent-originspecific expression, forming the Meg1/Grb10 imprinted gene cluster. To elucidate whether the maternally methylated DMR residing on a brain-specific promoter of Meg1/Grb10 (Meg1-DMR) regulates expression of all the imprinted genes in this region, we generated Meg1-DMR deletion mice. When the deletion was maternally inherited, the expression patterns of these imprinted genes were normal. In contrast, when paternally inherited, maternally expressed Meg1/Grb10 and Cobl became biallelic and paternal expression of Ddc was significantly reduced. These indicate that the Meg1-DMR deletion is comparable to the DMR methylation in terms of genomic function. In the mice with the paternal deletion, pre- and postnatal growth retardation was observed. Meg1-DMR deletion mice with and without neomycin resistant gene showed slightly different growth correlated to the expression level of Meg1/Grb10. These results indicate that Meg1/Grb10 overexpression is responsible for the growth retardation of the mice with both paternal Meg1-DMR deletion and maternal duplication of proximal chromosome 11. In human, it has been reported that the duplications of 7p11-13 containing GRB10 cause Silver-Russell syndrome (SRS) characterized by severe pre- and postnatal growth retardation. Our results strongly suggest that the overexpression of GRB10 is the major cause of growth retardation of the SRS patients with duplication of 7p11-13. 83 38. Jessica Stringer, Marilyn Renfree, Andrew Pask, Geoff Shaw [email protected] The imprint status and expression of GRB10 in the tammar wallaby, Macropus eugenii. Imprinting is presumed to have first evolved at least 130 million years ago after the divergence of therian mammals from the monotremes. Eutherian GRB10 is an imprinted gene encoding a growth suppressor protein that binds to the INS and IGF1 receptor and inhibits the growth-promoting activities of INS and IGF-I and II (O'Neill 1996; Liu and Roth, 1995; Morrione et al., 1996). In the mouse, disruption of the maternal copy of GRB10 results in overgrowth of both the embryo and the placenta (Charalambous et al. 2003). This gene has never been investigated in marsupials. This project examines and compares to that of INS, the imprint status and the expression of GRB10 in placental and neonatal tissues of the tammar wallaby, Macropus eugenii. The sequence of GRB10 has been characterised and expression studies in post-natal issues are underway. Although marsupials deliver an altricial young, INS is clearly important for growth and is imprinted in the marsupial placenta (Ager et al., 2007). It will be of interest therefore to determine the role of GRB10 in early development in this marsupial mammal and its relationship, if any, to INS. Ager, E., Suzuki, S., Pask, A., Shaw, G., Ishino, F. and Renfree, M. B. (2007) Insulin is imprinted in the placenta of the marsupial, Macropus eugenii Dev Biol 309317–328 Charalambous, M., Smith, F. M., Bennett, W. R., Crew, T. E., Mackenzie, F. and Ward, A. (2003)Disruption of the imprinted Grb10 gene leads to disproportionate overgrowth by an Igf2-independent mechanism. PNAS 1008292-8297.s O'Neill, T., Rose, D. W., Pillay, T. S., Hotta, K., Olefsky, J. M. and GustafsonT. A. (1996) Interaction of a GRB-IR Splice Variant (a Human GRB10 Homolog) with the Insulin and Insulin-like Growth Factor I Receptors. J Biol Chem 27122506 13. Liu, F. and Roth R. A. (1995). Grb-IRA SH2-domain-containing protein that binds to the insulin receptor and inhibits its function. Proc Natl Acad Sci USA 921028710291. Morrione, A., Valentinis, B., Li, S., Ooi, J.Y.T., Margolis, B. and Baserga, R. (1996) Grb10A new substrate of the insulin-like growth factor 1 receptor. Cancer Res 561365-67. 84 39. Shunsuke Suzuki, Ryuichi Ono, Takanori Narita, Andrew Pask, Geoff Shaw, Takashi Kohda, Jennifer Graves, Yuji Kohara, Fumitoshi Ishino, Marilyn Renfree, Tomoko Kaneko-Ishino [email protected] Retrotransposon silencing by DNA methylation can drive mammalian genomic imprinting Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10) is a retrotransposonderived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii), but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus), suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5' region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation. 85 40. Keiji Tanimoto, Hitomi Matsuzaki [email protected] Possible role of CTCF binding in the establishment and/or maintenance of methylation imprinting in the H19 ICR at heterologous beta-globin loci in YAC transgenic mice Imprinted expression at the H19/Igf2 locus is regulated by allele-specific methylation of the imprinting control region (ICR) located 5' to the H19 gene. To test whether the ICR activity is autonomous, we previously introduced a H19 ICR DNA fragment into a heterologous genomic context, i.e. 3’ end of the locus control region in the human beta-globin yeast artificial chromosome (YAC), and established YAC TgM lines. In these mice, paternally transmitted transgenic ICR was more heavily methylated, and CTCF transcription factor was preferentially recruited to the maternally inherited transgenic ICR in nucleated erythrocytes. In accord with this result, paternally inherited transgenic beta-like globin genes were more abundantly expressed than that on the maternal allele. These results demonstrated that the ICR could autonomously recapitulate genomic imprinting within a normally non-imprinted locus. An unexpected observation, however, was that hyper-methylation of the transgenic ICR was not observed in male germ cells, suggesting that the ICR can establish methylation imprinting after fertilization. In the current work, we examined the role of CTCF in establishment and/or maintenance of the post-fertilization methylation imprinting at heterologous loci. We generated beta-globin YAC TgM lines carrying mutant ICR fragment that bears mutations in all four CTCF binding sites but leaving the methylation target CpG motifs intact (4xMutNature Genetics 2003; 3366-9). We present data on the methylation status of the mutant ICR and its effect on expression of the beta-globin genes, and discuss possible role of CTCF binding in the parent-of-origin specific methylation marking at heterologous genomic loci. 86 41. Shinichi Tomizawa, Hisato Kobayashi, Toshiaki Watanabe, Hiroyuki Sasaki [email protected] Methylation status and small RNA mapping of the 15 imprinted differentially methylated regions (DMRs) in the mouse germline Imprinted genes in mammals are associated with differentially methylated regions (DMRs) that are methylated on either the maternal or the paternal chromosome. In mice, at least fifteen DMRs are known to acquire gametespecific DNA methylation in the parental germline (3 in spermatogenesis and 12 in oogenesis). We previously determined the extent of parental-origin-specific differential methylation at the 15 germline DMRs in 12.5-dpc embryos by bisulfite sequencing (Kobayashi et al., Cytogenet. Genome. Res. 113:130-137, 2006). Here, we report the extent of differential methylation at the 15 DMRs between sperm and oocyte determined by bisulfite sequencing. Furthermore, since RNAdirected DNA methylation has been described in plants, we studied whether any small RNAs are produced from or targeted to the 15 DMRs in gonocytes and oocytes. We used the small RNA sequences that we determined in our previous studies (Kuramochi-Miyagawa et al., Genes Dev. 22, 908-917, 2008; Watanabe et al., Nature, in press). We here discuss the results obtained from these studies. 87 42. Susannah Varmuza, Keith Latham, Zhiming Han, Jacqueline Chow, Mellissa Mann, Michael Golding, Anastasia Kuzmin [email protected] The PcG gene Sfmbt2 is paternally expressed in extraembryonic tissues The earliest target of genomic imprinting during development is the trophoblast, the extraembryonic tissue that gives rise to the placenta. The effects of genomic imprinting on trophoblast are most dramatically observed in parthenogenetic embryos, which lack a paternal genome. Parthenogenetic (or gynogenetic) embryos develop readily into blastocysts, indicating that they are able to establish trophoblast. However, during and after implantation, significant embryo loss occurs, in large part because trophoblast proliferation and development are not sustained. This failure to maintain trophoblast tissues is reflected in the difficulty with which trophoblast stem cells (TS cells) can be established from parthenogenetic embryos; four parthenogenetic TS cell lines were obtained, but at a much reduced frequency (approx. 1/35) in comparison with fertilized TS cell lines. In order to investigate the molecular underpinnings of genomic imprinting on early embryo development, we performed microarray analysis of androgenetic, gynogenetic and fertilized blastocysts. Among the genes that displayed significant down-regulation in gynogenetic embryos is the PcG gene Sfmbt2. Sfmbt2 maps to the proximal region of Chr 2, a known imprinted domain for which no imprinted genes have yet been identified. Two polymorphisms between Mus domesticus and Mus castaneus that created strain specific restriction sites within the 3’UTR allowed us to establish that Sfmbt2 is expressed exclusively from the paternal allele in early embryos, yolk sac, and placenta, but from both alleles in older embryos and somatic tissues. Expression of Sfmt2 is more robust in extraembryonic tissues than in any other tissue assayed. A CpG island near the transcriptional start is differentially methylated on the maternal allele in placenta. Preliminary evidence indicates that at least four, and possibly six additional genes in the neighbourhood are also imprinted in placenta. This represents the identification of a new imprinted domain in the mouse. 88 43. Kazuki Yamazawa, [email protected] Masayo Kagami, Tsutomu Ogata Silver-Russell syndrome and the IGF2–H19 domain: molecular and clinical studies in bodies and placentas Genomic imprinting is relevant to the acquisition of placental tissues during mammalian evolution. Silver-Russell syndrome (SRS) characterized by growth failure and dysmorphic features is frequently caused by hypomethylation (epimutation) of the H19-DMR. In this regard, although molecular and clinical studies have extensively been performed for SRS patients themselves, such studies have not been carried out for placentas. Thus, we performed methylation analysis of the H19-DMR in 60 Japanese SRS patients, identifying 20 epimutation positive and 40 epimutation negative patients. Methylation patterns were comparable between leukocytes and placentas in both epimutation positive and negative patients. Epimutations resulted in virtually no IGF2 expression and biallelic slight H19 expression in the leukocytes and obviously reduced IGF2 expression of paternal origin and nearly normal H19 expression of maternal origin in the placentas. Epimutation positive patients had characteristic body phenotype and small placentas with hypoplastic chorionic villi, and epimutation negative patients had somewhat small placentas with hypoplastic chorionic villi or massive infarction. Furthermore, significant correlations were identified between the H19-DMR methylation index and the body and placental sizes and between the placental weight and the body size in the epimutation positive patients, whereas such correlations were not detected for the head circumference. These results suggest (1) characteristic phenotype and reduced IGF2 expression in the epimutation positive placentas, (2) similarities and differences in the epigenetic control of the IGF2–H19 domain between leukocytes and placentas, (3) a positive role of the IGF2 expression level, as reflected by the methylation index, in the determination of body and placental growth in epimutation positive patients, except for the brain where IGF2 is expressed biallelically, (4) involvement of placental dysfunction in prenatal growth failure, and (5) relevance of both epigenetic factor(s) and environmental factor(s) to SRS in epimutation negative patients. In addition, the first case of maternal uniparental disomy for chromosome 11 was identified through this study. We would also present this case with literature review. 89