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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
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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.”
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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.”
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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
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_______________________________________________________________
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.”
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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
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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
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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