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Sunday, June 6/Afternoon
EPIGENETIC TWISTS
Catherine
Kidner
David Haig described epigenetics as
'anything that doesn't look like conventional genetics'. This meeting
has shown that recent work in epigenetics has been so fruitful that now
we are close to a ‘conventional epigenetics’. This afternoon's session,
chaired by Bruce Stillman, dealt with phenomena both very old (paramutation)
and very new (the
hothead
mutation) that are still definitely unconvential epigenetics.
A context for the variety of
epigenetic effects discussed was provided by the first talk. David Haig
of the department of Organismic and Evolutionary Biology at Harvard
discussed how the meanings of 'epigenetic' have evolved. The earliest
modern use of the term was by Conrad Waddington in 1942, he defined it
as the causal mechanisms of development. The term was taken up by
developmental biologists and used to describe both the unravelling of
information and genetic interactions in the growing organism and the
influences such as other organisms, physical surroundings, temperature
and even gravity that cause variation.
After the discovery of DNA the term
was revived by Nanney to refer to self perpetuating states of the
cytoplasm as opposed to a totally DNA-based source for the phenotype. At
a number of meetings in the late fifties the term and the phenomena to
which it referred were debated. It was eventually conceded that the
nucleus was the main source of heritable information although 'not
everything inherited is genetic', and there was discussion of a role for
non-sequence based aspects of inheritance due to changes associated with
modifications of DNA. Our current use of the term stems from Robin
Holliday who first used it in 1979 to refer to non-sequence changes in
DNA and in 1987 published 'The inheritance of epigenetic defects' in
Science.
The definition of epigenetics as
anything that appears non-mendelian is well suited to the subject of the
second talk of the afternoon. Michel George of the University of Leige
described the strange behavior of the
callipyge
(CLPG) mutation in sheep. This mutation arose in the ram
Solid Gold in Oklahoma
in the 1980’s. It was noticed that his offspring had particulary well
developed buttocks, hence the name. This is a very desirable trait in
sheep and the lineage attracted the interest of animal breeders. The
callipyge
phenotype showed an interesting polar over dominance: the phenotype was
only apparent on inheritance of the mutant allele from the father, in
the absence of a maternally inherited mutation. George presented
evidence that this effect may derive from a combination of imprinting
and miRNA regulation.
By positional cloning, the mutation
was localized to the DLK1-GTL2 domain, an area of about 1MB that
includes 4 protein encoding genes and genes for 4 large noncoding RNAs.
This region is rich in pri-miRNA genes. The mutation itself is an A to G
substitution in a conserved 12bp sequence that affects levels of
expression of the protein coding genes - ectopic expression of DLK1 is
responsible for the hypertrophic muscle phenotype. Imprinting of the
paternal chromosome blocks the expression of the protein coding genes
but allows the expression of the noncoding RNA genes. The loss of the
effect in homozygote mutants suggests that the noncoding RNAs are
imprinted in the maternal allele and downregulate the protein coding
transcripts. Computational analysis has suggested that one of the pri
miRNAs may encode a regulator of DLK1 transcript.
callipyge
is a very recent phenomenon compared to paramatution of the B locus in
maize described by Vicky Chandler of the University of Arizona, Tuscon.
Study of this effect dates back to the early days of the term 'epigenetics'.
Plants carrying B-I produce copius dark anthocyanin pigments, while
those carrying B' produce little anthocyanin. B-I reverts to B' at a
frequency of 0.1 to 10%. Paramutaion refers to the ability of B' to
convert B-I alleles to B'. This effect is notable for its stability and
penetrance. Sequencing of the locus revealed that B' and B-I alleles
both had seven tandem repeats of a 853bp sequence 100kb upstream of the
CDS for the B gene. In alleles not susceptible to the paramutation
(most B alleles) there was only a single 853bp sequence. The extra
repeats were required both for the high expression levels in B-I and the
paramutation effect of B'. Methylation occurs in paramutaion but is a
response to the silencing rather than a cause of it. Most excitingly
Chandler described experiments that randomly inserted the repeats into
the genome of a neutral line and showed that this could confer
susceptibility even though the transgenes were unlinked to the B locus.
This supports an RNA mediated effect or long distance direct pairing.
The two following talks dealt with
prions, which might be seen to fit the 'cytoplasmic inheritance'
definition of epigenetics current in the 1950's. Reed Wickner of NIH,
Bethesda, described the characteristics of these proteins and their
behavior: The effect is non-chromosomal, 'cures' are reversible,
overproduction of the protein leads to more prions and the prion
phenotype requires both the chromosomal gene and the phenotypic prion.
He also described four different forms of prion, two bad, two good:
those which form toxic amyloids, such as BSE; those that form inactive
amyloids such as [URE3] which he described in detail; those that from
active amyloids (Het-s); and a new form - the self activation enzyme
such as b,
'crippled growth' in
Podospora anseria and
PaASK1,
a MAP kinase kinase kinase.
The N terminus of Ure2P controls
prion [URE3] formation. The crystal structure of the amyloid showed
that the N terminal domain formed
b
sheet filaments surrounded by the
‘blobs’ of the C terminal domain.
Wickner also described experiments by Eric Ross in his lab which showed
that it is the amino acid composition rather than sequence that is
important for prion formation.
A role for prions in normal
development was suggested by Kausik Si, of Columbia University. He has
studied the role of the neuronal isoform of Cytoplasmic Polyadenylation
Element Binding protein (CPEB) in activating transcription of genes
required for synaptic facillitation. Like many prions, the N' terminus
of the protein is rich in glutamine and aspargine. Assays in yeast using
the b
gal system showed that the protein exists in two distinct metastable
forms, the active form being dominant. Using GFP fusions he showed that
the active form is produced by overexpression, together with seratonin
signaling, and exists as self perpetuating aggregates.
The most
intriguing talk of the afternoon was Bob Pruitt of Purdue University's
presentation of
hothead (hth), an
Arabidopsis
mutation that causes organ fusion and changes in DNA sequence.
Homozygotes revert to wild type at a frequency from 1 to 10%. This
reversion is due not to 'classical epigenetic' effects, such as
methylation of DNA or modifications in histone proteins, but to
reversion of the DNA sequence to the sequence of the plant's wild type
progenitors. Analysis of the effect of
hth
on a genome wide polymorphic background produced by a cross between two
different ecotypes showed that the reversion was not limited to the HTH
locus. SNPs were converted to parental at the same frequency as the
hth
mutation itself. This effect did, however, depend upon the
hth/hth
genotype. Conversions have been detected only in reproductive tissue and
appear to occur preferentially in the male. The mechanism of this effect
remains wholely unknown. Pruitt suggested that sequence information
might be retained by RNA templates cytoplasmically inherited from the
wild type progenitors and amplified within the
hth/hth
plant. The discussion of this exciting effect was unfortunately cut
short by the need to clear the hall for the next lecture but hopefully
discussion over the evening will have suggested several possible
explanations for this most un-mendelian of epigenetic effects.
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