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Transcript
 Indian Academy of Sciences
PERSPECTIVES
Epigenetics comes of age in the twentyfirst century
ROBIN HOLLIDAY*
12 Roma Court, West Pennant Hills, N.S.W. 2125, Australia
Origins of epigenetics
In the nineteenth century most leading biologists believed
that development and heredity were one and the same
problem. Even as late as 1896, the first edition of E. B.
Wilson’s famous book had the title The cell in development and inheritance, even though, in the absence of the
recognition of Mendel, almost nothing was known about
the mechanism of heredity. It is extremely likely that
Mendel’s work was ignored for 30 years precisely because
heredity was linked to development (Sandler and Sandler
l985). When his work was rediscovered at the beginning
of the twentieth century, the discipline of genetics was
launched. Mendel’s genius was to unravel the laws of
inheritance without knowing anything about the process
of development, and most geneticists followed this path
thereafter. In this context, it is interesting to note that
T. H. Morgan, the leader of the famous school of Drosophila geneticists, started his career as an embryologist,
and his last book was on embryology and genetics
(Morgan l934). However, he did not include Drosophila
development as a major topic of his research. For the
most part, the embryologists and developmental biologists immersed themselves in their own studies, and did
not think about the way genes might control the unfolding of the developmental programme. J. B. S. Haldane
was one of the few geneticists who was interested in the
way genes controlled metabolism, following the earlier
work of Garrod (1909) on inborn errors of metabolism. In
the 1940s, Beadle and Tatum formulated their famous
concept of one gene one enzyme, which proved to be
largely correct.
There were two leading geneticists who also studied
embryology, and who fully realized that sooner or later
the two disciplines would coalesce. One was Ernst
Hadorn, who studied development in Drosophila, and the
other was C. H. Waddington, who first introduced the
term epigenetics (Waddington 1939). In general terms,
this was the study of the way genes controlled and directed the developmental programme. It was derived from
*E-mail: [email protected].
the early dispute between the ‘preformationists’ and
those who believed in epigenesis. The former asserted
that the sperm and fertilized egg already contained a
preformed organism with all major features represented.
In contrast, epigenesis was the process whereby more and
more complex structures emerged during development.
Obviously, this view is correct, so the term epigenetics
had validity from the outset.
DNA methylation
In development there are many branching processes,
whereby a given cell gives rise to two independent cell
types. Such a process can be regarded as a developmental
switch in which one daughter gains a new phenotype, or
potential phenotype, whereas the other retains its original
phenotype. This is a stem cell situation, but in other cases
both daughter cells may have phenotypes different from
each other and also different from the parental cell. There
is also the stability of differentiated cells, so that lymphocytes, fibroblasts and keratinocytes, and so on, retain a stable
karyotype through many divisions. These cells contain the
same chromosomes, the same genes, and the same DNA
sequences, but their patterns of gene expression are different. They can be said to have different epigenotypes.
The concept of switching of gene activities, and the
somatic inheritance of a given set of active and inactive
genes, provided the basis for a new epigenetic mechanism, namely DNA methylation. 5-Methylcytosine (5-mC)
is produced by an enzyme, DNA methyltransferase,
which was known to add a methyl group to the carbon
at the 5 position of the pyrimidine ring soon after
replication. In mammalian DNA 2–5% of cytosines are
methylated, but not at random because CpG doublets are
the major site of methylation, and these doublets are
depleted in relation to the cytosine and guanine contents.
Holliday and Pugh (1975) and Riggs (1975) independently proposed that 5-mC may have an important role in
controlling gene expression during development in higher
organisms. Riggs was particularly interested in X chromosome inactivation in female mammals, where early in
development one X chromosome is permanently inacti-
Journal of Genetics, Vol. 81, No. 1, April 2002
1
Robin Holliday
vated in every cell, whilst the other remains active. In
eutherian mammals this requires a random switch and
subsequent maintenance of the active and inactive states.
Holliday and Pugh discussed these fundamental features
in the wider context of development. Both publications
proposed that the hemimethylated DNA after replication
is a substrate for a maintenance methylase, which does
not act on unmethylated DNA. Neither of these early
papers referred to Waddington’s term epigenetics. The
connection between DNA methylation and epigenetics
was made explicit in 1987 when I published a paper in
Science titled ‘The inheritance of epigenetic defects’
(Holliday l987). This was concerned in part with a longstanding dispute in somatic cell genetics, where it had
been argued on the one hand that inherited variation in
mammalian cells was not, or need not be, due to gene
mutations, and on the other hand that mammalian cells
were essentially similar to microbial systems and mutations could be induced and studied in much the same
way. As is often the case in such controversies, both
viewpoints turned out to be correct, because it had become
apparent that gene inactivation could not only be due to
mutation, but also to DNA methylation. This was demonstrated by the use of the potent demethylating agent 5azacytidine (or 5-azadeoxycytidine) which reactivated at
high frequency genes silenced by DNA methylation. The
term epimutation had been used earlier to describe
heritable changes in phenotype which are not due to any
alteration in DNA sequence (Holliday 1984). Also, it had
been proposed that heritable changes in DNA methylation might be important in carcinogenesis (Holliday
1979). The 1987 paper in Science elaborated on the
possible role of epigenetic defects or epimutations in
cancer and ageing, and also discussed evidence that they
might be transmitted from one generation to the next, a
topic that was explored in much more detail by Jablonka
and Lamb (1995).
A major advance in the study of epigenetics was the
discovery of genomic imprinting in mammalian gametes.
It became clear that the paternal and maternal gametes
have different information which is superimposed on the
chromosomal DNA of one parent and not the other. The
two genomes complement each other to produce a normal
zygote, whereas zygotes arising from two paternal nuclei
or two maternal nuclei produce abnormal embryos. Many
studies have been done that link imprinting to differences
in DNA methylation in male and female gametes. It is
not known in detail how long imprinting persists in the
developing organism, but it is certain that in the
formation of gametes for the next generation the normal
pattern of imprinting is reimposed.
Much information about DNA methylation and the
epigenetic control of gene activity is now available in
plants (Martienssen and Colot 2001). Also, in the last few
years there has been an explosion of interest in the
2
inactivation and methylation of tumour suppressor genes
during tumour progression. In the years since 1995 there
have been over 1400 publications on this topic. Another
very important advance comes from studies that relate
DNA methylation to changes in chromatin proteins, and
particularly the acetylation and deacetylation of histones
(Rountree et al. 2001).
In many of these studies the term epigenetics has been
used, but it is notable that in the area of mammalian
development a key role for DNA methylation has not yet
been revealed. There are, nevertheless, many reasons for
believing that DNA methylation does play a crucial role
in unravelling the developmental programme, although
that may not be the common view amongst developmental biologists. One reason for this was the frequent
assertion that Drosophila DNA contained no 5-mC, but
this belief has recently been shown to be mistaken
(Gowher et al. 2000; Lyko et al. 2000).
Other epigenetic mechanisms, and the
definition of epigenetics
DNA methylation is a major epigenetic mechanism, but it
is not the only one. For example, the changes in DNA
that occur in the assembly of immunoglobulin genes in
cell lineages of the immune system should be regarded as
an epigenetic mechanism, which in this case depends on
the creation of new DNA sequences. In my view, the
alternative splicing of gene transcripts that occurs in
different cell types is also an epigenetic mechanism,
because it results in the formation of particular polypeptide chains and proteins appropriate for a specialized cell.
There are regulatory proteins that stably repress or induce
particular genes without any involvement of DNA methylation, but it is not usually clear whether these gene
activities, or inactivities, are stably inherited through
mitosis.
Whether or not a particular process or mechanism should
be reparded as epigenetic ultimately depends on the definition of epigenetics, which was the subject of a series of
letters in Science (Wu and Morris 2001). It is difficult to
find a single definition, and in l994 I suggested two,
namely:
(i) The study of the changes in gene expression
that occur in organisms with differentiated cells, and
the mitotic inheritance of given patterns of gene
expression.
(ii) Nuclear inheritance that is not based on differences in DNA sequence.
In contrast, Wu and Morris suggest a single definition:
The study of changes in gene function that are
mitotically and/or meiotically heritable and that do
not entail a change in DNA sequence.
Journal of Genetics, Vol. 81, No. 1, April 2002
Epigenetics comes of age in the twentyfirst century
I consider this to be far too narrow a definition because it
does not encompass changes in somatic cells that should
surely be regarded as epigenetic. It does not include an
example I have just mentioned, the rearrangement of
DNA sequences to form antibody genes. It excludes all
changes in phenotype induced by a morphogen, hormone
or growth factor that is not followed by cell division. The
induction of vitellogenin by the hormone oestradiol is a
case in point, where it has been shown that the induction
is preceded by specific demethylation of DNA at the
oestradiol response element (Saluz et al. 1986). Surely this
is an epigenetic change where no cell division is involved?
In fact, Wu and Morris’s definition excludes all changes
in post-mitotic cells, including neurons. I believe it is
unjustifiable to exclude the processing of information in
nondividing cells of the brain, including the imprinting of
parental images in young animals and many other examples of learning. Indeed, I have suggested that long-term
memory could be based on an epigenetic switch involving DNA methylation (Holliday 1999). This is an invalid
use of the term epigenetic according to Wu and Morris.
On the matter of definitions, epimutation has already
been mentioned, but we also need the term epimutagens
(agents that induce epimutations) and therefore epimutagenesis. An epimutator is a cell strain in which epimutations occur at unusually high frequency, one of which
has already been discovered (Holliday and Ho 1998).
The term epigenotype is particularly useful, because
it covers the situation common to all higher organisms:
their somatic cells, with rare exceptions, have the same
genotype, but they have different epigenotypes. That is,
they have different patterns of gene expression that give
rise to the phenotypes of all the specialized cells in the
organism.
Conclusions
What is certain is that the rules governing epigenetic
processes are completely different from those governing
classical genetics. The geneticists of the twentieth
century confirmed Mendel’s results and successfully
uncovered the basic mechanism of heredity in a wide
variety of eukaryotes, and all the variations that can be
imposed on that mechanism. It is a fairly safe prediction
that the twentyfirst century will reveal many epigenetic
mechanisms, and the way they govern or influence the
developmental programmes of animals and plants. The
relative importance of the epigenetic transmission of
information from one generation should also become
apparent, as well as its possible role in evolution. Will it
merely comprise a set of rather unusual examples of nonMendelian inheritance? Or will it open new possibilities
about the way environmental influences might alter the
phenotypes of subsequent offspring, as Jablonka and
Lamb (1995) have argued? There are many cases of human
inheritance that are at present not understood, and these
may be imprecisely labelled as examples of ‘multifactorial inheritance with incomplete penetrance’. Could these
ill-understood types of inheritance be due to epigenetic
information superimposed on the normal genomic DNA?
Finally, it must be said that the sequencing of the human
genome can only be the beginning of a very long set of
further studies. The term epigenomics has already come
into use, and this will encompass the inclusion of the fifth
base 5-mC in future studies of DNA sequences (Beck et al.
1999). Not only that, because it will be necessary to
examine the DNA of many cell types. We can expect
some tracts of DNA to have unchanged patterns of DNA,
for example where transposable elements have been silenced by DNA methylation, but others will have sequence
specificity depending on cell type. To unravel all these
differences will still be an enormous undertaking, even
with many improvements in the best techniques that are
currently available.
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