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Thursday June 3/Morning
NUCLEAR REPROGRAMMING, CHROMOSOME INACTIVATION & IMPRINTING
Derek Goto
The
second session of the symposium was chaired by Adrian Bird (University
of Edinburgh, UK) and featured seven speakers.
The first two talks were on nuclear reprogramming, followed by
two talks related to imprinted genes and (after the coffee break) three
talks on sex chromosome compensation/inactivation.
Rudolf
Jaenisch (Whitehead Institute/MIT) was the first speaker and explained
how nuclear cloning of terminally differentiated cells has a very low
efficiency and that a major obstacle is the requirement for epigenetic
reprogramming. Jaenisch
described how they have developed a system for cloning mature neurons
with genetic and epigenetic markers and demonstrated that the nuclei of
postmitotic neurons are able to reenter the cell cycle.
He also presented data on the potential for reprogramming in
cancer cells based on nuclear transfer experiments using a range of
tumors as donor cells. He
concluded with some unanswered questions and future goals: what is the
best adult cell to use as a donor in nuclear transfer? And might
reprogramming in principle be possible without use of an oocyte at all.
Azim Surani (Wellcome/CRC Institute, UK) then explained that during germ
cell differentiation, there is a critical period of specification after
onset of competence correlated with repression of the somatic program.
He reported the identification of a RIZ family SET domain
protein, Blimp1, that is differentially expressed in single primordial
germ cells and somatic neighbours.
Using a GFP-tagged protein and in situ hybridisation
Surani and coworkers showed
that the presence or absence of this protein marks the change from
competence to initiation.
Wolf
Reik (Babraham Institute, UK) described his analysis of the link between
imprinting and epigenetic regulation of higher order chromatin structure
at the Igf2/H19 locus.
Wolf showed data from a restriction digest/ligation/PCR strategy
that argued for a physical interaction between differentially methylated
regions located some distance apart at the locus.
This interaction would then result in a transcriptionally
quiescent loop forming between the interacting regions and he proposed a
model where this loop may act as a simple epigenetic switch for Igf2.
Interaction between the upstream DMR1 and downstream H19 regions
forces Igf2 within the loop for paternal silencing, whereas
interaction between the downstream DMR2 and H19 regions maternally would
result in Igf2 “sliding out” of the loop and becoming
expressed.
Denise
Barlow (GmbH of the OAW, Austria) gave the following talk and introduced
the role of non-coding RNAs in genomic imprinting.
Denise described the large, 108kb Air non-coding RNA that is
expressed paternally and shown to have a role in silencing of three
imprinted genes including Igf2r in mouse.
She explained how Air is a repressor of Igf2r expression
and resistant to paternal silencing, but is itself repressed on the
maternal chromosome by methylation of its CpG island promoter.
Humans are generally thought to lack imprinted expression of Igf2r.
She explained how the human locus does have all the structural
and epigenetic features that would support imprinted control, including
a CpG island that has potential to act as a promoter.
She then presented results from a series of transient assays and
transgenes demonstrating that this promoter is functional and able to be
expressed under some circumstances.
In the last part of her talk, Barlow made a direct comparison
between Air and Xist (X inactivation transcript), putting forward the
argument that both cis-acting non-coding RNAs share similar
features and thus are likely to act through highly related mechanisms.
Mitzi
Kuroda (Harvard University) gave the first of three talks on sex
chromosome dosage compensation, providing insight into the mechanism of
targeting dosage compensation in flies.
She presented results from a series of experiments showing that a
complex using non-coding RNAs is involved in up-regulation during
compensation, and that this is initially targeted to where the
non-coding RNAs are being transcribed. Kuroda's
talk was followed by presentations from Jeannie Lee (Howard Hughes
Medical Institute, Boston) and Edith Heard (Curie Institute, France),
which provided the audience with both sides of a current controversial
debate in the field of mammalian X-inactivation: does the paternal
genome arrive pre-imprinted in the sperm so that the X chromosome is
inactive, or is there no pre-imprinting so that the X chromosome is
actually active during fertilisation and inactivated at the 4-cell
stage? The first speaker,
Jeannie Lee, explained that there are two forms of X chromosome
inactivation during mammalian development after fertilisation, one that
involves random inactivation in the embryo proper, and one that requires
an imprint so that the paternal X chromosome remains inactivated in the
placenta tissues. In
addition to the known imprint signal on the maternally inherited X
chromosome to resist inactivation, the paternally inherited X chromosome
could carry a predisposition to inactivation.
Lee argued that an imprint signal is on the male chromosome and
that it is placed there during male meiotic sex chromosome inactivation
(MMSI). This would mean
that the male X chromosome arrives ‘pre-inactivated’ at
fertilisation and stays inactive in the placenta tissues.
In support of this she showed data that transcription of cot-1
sequences is absent from the 2 cell stage onwards and that this
correlates with the presence of Xist RNA.
In
contrast, Heard put forward the argument that the paternal X chromosome
does not arrive inactivated and thus a maternal imprint preventing the
maternal X chromosome inactivation is the true imprint.
She supported this argument with immunofluorescence and RNA FISH
data demonstrating a lack of Xist coating and expression of X-linked
genes in the 2-cell stage. This was followed by inactivation and Xist coating at the
4-cell stage that was then correlated with expected changes in histone
modifications. Both
speakers appeared to agree on what was occurring from the 4-cell stage
onwards regarding paternal inactivation.
The critical point was whether the paternal X chromosome is
already inactive at the zygote or 2-cell stage (consistent with
pre-inactivation). In
addition to the RNA FISH evidence for gene activity on the paternal X at
the 2-cell stage, she also presented new, unpublished results in support
of paternal X inactivation simply being due to paternal Xist expression
and not pre-inactivation.
Other
Dispatches
Symposium
69 Live
Symposia
Past (a bit of history and photographs from previous Symposia)
Online
Symposium Volumes (searchable database of past Symposia volumes and
currently received manuscripts) |
Derek Goto
(Martienssen lab)
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