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Repeat-Induced Gene
Silencing in Fungi
Eric U. Selker∗
Institute of Molecular Biology
University of Oregon
Eugene, Oregon 97403
I. Introduction
II. Discovery and Basic Features of RIP and MIP
III. De Novo and Maintenance Methylation Associated with MIP
and RIP
IV. Consequences of RIP and MIP
V. Concluding Remarks
References
I. INTRODUCTION
The notion that the structure and behavior of an organism are determined primarily by the structure and behavior of the organism’s genome is well accepted.
Lamarckism aside, it is time to contemplate the complementary possibility, namely,
that the structure and behavior of a genome reflect, in part, the structure and behavior of the organism. Consider prokaryotes. The relatively compact genomes
of bacteria and their viruses presumably reflect the economy required for success
in extremely competitive environments. If one considers a thermophilic organism, it is not hard to imagine that the base composition of its genome reflects
an adaptation to the extreme environment. The same reasoning should apply to
* To whom correspondence should be addressed: E-mail: selker@molbio.uoregon.edu; Telephone:
(541) 346-5193; Fax: (541) 346-5891.
Advances in Genetics, Vol. 46
Copyright 2002, Elsevier Science (USA).
All rights reserved.
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eukaryotes, but presumably some organisms need to be more fastidious “genome
keepers” than others. For example, fungi such as Neurospora crassa that rely on
their ability to rapidly exploit nutrient opportunities (Davis, 2000) may place
a premium on maintaining an uncluttered genome. In contrast, organisms with
large genomes composed substantially of transposable elements, such as higher
plants and animals, must have lifestyles that can tolerate untidy genomes. An
appreciation of the forces acting on genomes should give us a better understanding of the variation in eukaryotic genomes, such as the dramatic size differences
between the genomes of Arabidopsis (∼120 Mb; Arabidopsis Genome Initiative,
2000) and other higher plants (typically >1000 Mb), and between those of the
pufferfish (∼400 Mb; Elgar et al., 1996) and mammals (∼3000 Mb).
Of course genomes are not simply products of their physical environments. Evidence for the importance of “selfish” DNA (e.g., transposons) in evolution is undeniable. Moreover, the existence of genome defense systems, such
as repeat-induced point mutation (RIP) in Neurospora, methylation-induced premeiotically (MIP) in Ascobolus immersus, and posttranscriptional gene silencing
(e.g., RNAi) in plants, animals, and fungi, suggests that organisms developed measures to counter perturbations of their genomes. If we wish to study the control of
genome structure, it makes sense to look first to organisms that appear to be fastidious genome keepers. Toward this end, I will briefly review RIP and MIP here.
Early, more extensive reviews of these phenomena are still pertinent (Rossignol
and Faugeron, 1994; Selker, 1990). It is noteworthy that additional processes
that should influence genome structure have been more recently discovered in
Neurospora, including a form of meiotic silencing (Aramayo and Metzenberg,
1996) similar to transvection in Drosophila and a form of posttranscriptional gene
silencing (“quelling”). The latter is the topic of Chapter 9.
II. DISCOVERY AND BASIC FEATURES OF RIP AND MIP
RIP was discovered in 1986 in the course of research on the control of methylation using a chromosomal region in Neurospora called zeta-eta (ζ –η; Selker
and Stevens, 1985), which incidentally is now known to be a natural relic of RIP
(Grayburn and Selker, 1989). Because of clues that the methylation of ζ –η was related to a tandem duplication, we were careful to select single-copy transformants
to analyze the potential of various sequences to induce DNA methylation (Selker
et al., 1987b; Selker and Stevens 1985, 1987). Both unique and repeated copies
of ζ –η sequences induced methylation in vegetative cells. Surprisingly, however,
we found that duplicated sequences, independent of their origin or methylation
state, displayed extreme instability in the sexual phase of the life cycle (Selker
et al., 1987a). Whereas single-copy sequences were stable throughout the life
cycle, tandemly repeated sequences frequently suffered deletions, and those that
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were not deleted almost invariably showed evidence of de novo methylation and
sequence alterations. Unlinked duplications were also found to be modified, although at lower frequencies; typically ∼50% of sexual spores showed alterations
of unlinked duplications. Genetic and molecular analyses revealed that the sequence alterations were all G:C to A:T polarized transition mutations and that
they occurred in the stage of the sexual phase in which the dikaryotic cells, which
form by fusion of cells of opposite mating types, proliferate prior to nuclear fusion
and meiosis (Cambareri et al., 1989; Selker et al., 1987a). We proposed that a
mechanism (RIP) specifically targets repeated sequences for sequence alterations.
Proof that duplications, per se, trigger RIP, and that inactivation occurs in a pairwise manner, came from carefully controlled genetic experiments in which the
stability of a sequence was compared in strains differing only in the presence or
absence of a second copy of the sequence elsewhere in the genome (Selker and
Garrett, 1988). Single copies of a sequence at unlinked sites in separate strains
were stable, but they became sensitive to RIP when they were united in a nucleus
by crossing. It is important to note that every sizable (e.g., >1 kb) duplication
that we know is subject to RIP, unlike the case for some other genome defense
systems such as quelling (Cogoni et al., 1996). Nevertheless, every duplication
escapes RIP at some frequency (typically less than 1% for a tandem duplication
or approximately 50% for an unlinked duplication). Thus duplicated sequences
are never “immune” but are not necessarily eliminated immediately. Even duplications of chromosomal segments containing numerous genes are sensitive to RIP
(Bhat and Kasbekar, 2001; Perkins et al., 1997). Because RIP reliably destroys both
copies of duplicated genes, it has been used by numerous Neurospora researchers to
specifically inactivate endogenous genes (e.g., see Arganoza et al., 1994; Barbato
et al., 1996; Chakraborty et al., 1995; Chang and Staben, 1994; Connerton, 1990;
Connerton et al., 1991; da Silva et al., 1996; Ferea and Bowman, 1996; Fincham,
1990; Fincham et al., 1989; Foss et al., 1991; Glass and Lee, 1992; Grad et al., 1999;
Kuldau et al., 1998; Marathe et al., 1990; Nelson and Metzenberg, 1992; Perkins
et al., 1997; Plesofsky-Vig and Brambl, 1995; Selker et al., 1989; Zhou et al. 1998).
After the discovery of RIP, the occurrence of similar, or identical, gene
silencing mechanisms was sought in a number of fungi including members of
at least the following genera: Saccharomyces, Schizosaccharomyces, Aspergillus,
Schizophillum, Coprinus, Ustilago, Gibberellia, Sordaria, Podospora, Magnaporthe,
Cochliobolus, and Ascobolus. MIP, a process similar to RIP, but with important
differences, was promptly found and characterized by Faugeron, Rossignol, and
colleagues in the filamentous ascomycete Ascobolus immersus (Barry et al., 1993;
Faugeron et al., 1990; Goyon et al., 1988; Rhounim et al., 1992). Evidence of a
version of MIP was also found in Coprinus cinereus (Freedman and Pukkila, 1993).
RIP has not yet been demonstrated outside of Neurospora crassa but sequences
showing hallmarks of RIP (e.g., numerous C to T mutations especially at CpA
dinucleotides) or a similar process have been found in several fungi including
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Magnaporthe grisea (Nakayashiki et al., 1999), Aspergillus fumigatus (Neuveglise
et al., 1996), Podospora anserina (Graia et al., 2001; Hamann et al., 2000), Fusarium
species (Hua-Van et al., 1998; M.-J. Daboussi, personal communication) as well
as several species of Neurospora (Kinsey et al., 1994; Zhou et al., 2001). Like
RIP, MIP detects linked and unlinked sequence duplications during the period
between fertilization and karyogamy and inactivates them in a pairwise manner
(Faugeron et al., 1990; Fincham et al., 1989; Selker and Garrett, 1988). Unlike
RIP, MIP relies exclusively on DNA methylation for inactivation; no evidence of
mutations has been found in sequences inactivated by MIP.
III. DE NOVO AND MAINTENANCE METHYLATION ASSOCIATED
WITH MIP AND RIP
Although DNA methylation is, by definition, integral to MIP, it is not yet known
whether the methylation itself actually occurs premeiotically. The alternative
possibility is that an unidentified imprint is established premeiotically, which then
directs de novo methylation later, i.e., during or after meiosis. Support for the direct
possibility has come from the observation that an Ascobolus gene that shows strong
sequence similarities to known methyltransferases, masc1, plays a role in MIP
(Malagnac et al., 1997). Crosses heterozygous for masc1 show reduced frequencies
of MIP, especially when the duplication is in the nucleus with the defective masc1
allele. Crosses homozygous for defective masc1 are sterile. Negative results from
efforts to detect methyltransferase activity of the product of masc1 may be a
reflection of its dependence on one or more accessory factors, such as a factor
required for recognition of repeated sequences. Interestingly, deletion of masc1
does not result in any obvious reduction in DNA methylation in vegetative cells.
Similarly, mutations in a gene of Neurospora that appears to be a homolog of
masc1 does not result in reduced DNA methylation (M. Freitag and E. U. Selker,
unpublished).
Evidence for a distinction between methylation induced in the sexual
phase and that induced as the vegetative phase of the life cycle of fungi comes
from studies in Neurospora. Methylation is frequently, but not invariably, found
in sequences mutated by RIP. This is consistent with the possibility that methylation is involved in the mechanism of RIP. For example, the C-to-T mutations of
RIP might be the result of deamination of methylated cytosines (Selker, 1990).
On the other hand, methylation of the modified sequences could simply be a
consequence of RIP. Indeed, early studies with the ζ –η region demonstrated that
this relic of RIP can induce methylation de novo in vegetative cells (Miao et al.,
2000; Selker et al., 1987b, 1993). Thus there are two nonexclusive possible explanations for the methylation found associated with sequences subjected to RIP:
(1) it could reflect methylation established during RIP (and perhaps integral
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to its mechanism) and then maintained by some sort of copying mechanism;
(2) it could reflect the creation, by RIP, of signals triggering methylation de novo.
To test the first possibility, a set of methylated am alleles generated by RIP were
demethylated, either by treatment with the methylation inhibitor 5-azacytidine,
or by cloning followed by targeted gene replacement, and then assayed for their
potential to trigger de novo methylation (Singer et al., 1995b). The alleles showing the highest level of mutagenesis by RIP became immediately remethylated,
demonstrating that they had become capable of triggering methylation, as found
previously with some relics of RIP such as ζ –η (Cambareri et al., 1991; Selker
and Stevens, 1987). Several alleles with lower levels of mutation did not become
remethylated, however. This, and a similar observation made with the bacterial
antibiotic resistance gene hph located between copies of am that had been subjected to RIP (Irelan and Selker, 1997), told us that: (1) methylation is induced
in the sexual phase of Neurospora, consistent with the possibility that methylation is involved in the mechanism of RIP; (2) the trigger for this methylation
is different from that operating in vegetative cells; (3) methylation of some sequences is dependent on preexisting methylation, i.e., Neurospora is capable of
some form of maintenance methylation. Interestingly, this continuously propagated methylation in Neurospora, and that resulting from MIP in Ascobolus, is
not limited to symmetrical sites. Thus maintenance methylation can occur by a
mechanism other than that originally proposed by Riggs (1975) and Holliday and
Pugh (1975) based on the symmetry of methylated sites (e.g., 5 CpG3 /3 GpC5 )
in higher eukaryotes. This raises some fascinating questions, such as: (1) To what
extent does methylation at nonsymmetrical sites depend on methylation at symmetrical sites? (2) Does the methylation found at symmetrical sites in higher and
lower eukaryotes depend simply on the methylation status at those sites, as in the
original maintenance methylation model? With respect to the latter possibility,
it should be noted that the occurrence of “spreading” of methylation in animals
(Arnaud et al., 2000; Doerfler et al., 1990; Turker, 1999) and fungi (Miao et al.,
2000) suggests that methylation of a site can promote methylation in its vicinity.
Results of preliminary experiments suggest that Neurospora and Ascobolus may
have somewhat different mechanisms for propagating methylation. Methylation
in Ascobolus may simply depend on a framework of methylation at symmetrical
sites (G. Faugeron, V. Rocco, A. Hanguehard, A. Grégoire, B. Margolin, J.-L.
Rossignol, and E. U. Selker, unpublished), but this does not seem to be the case
in Neurospora (B. Margolin and E. U. Selker, unpublished). One possibility is that
maintenance methylation in Neurospora, and perhaps in other organisms, depends
on modifications of chromatin such as acetylation or methylation of histones.
Control of de novo methylation is not yet well understood in any eukaryote. Although the extent to which principles of methylation discovered in one
system will be applicable to others is not yet clear, it is obvious that Neurospora
offers an excellent system for investigating de novo methylation. Sequences that
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reproducibly trigger de novo methylation can be generated simply from any sequence by RIP and such sequences can be dissected, modified, and tested to
identify the underlying principles. The most extensive example of this approach
used the 1.6-kb ζ –η region (Grayburn and Selker, 1989; Miao et al., 1994, 2000;
Selker et al., 1993). Tests were carried out to assess the methylation potential of a
variety of fragments of this region, as well as chimeras between ζ –η sequences and
the homologous unmutated allele, theta (θ ). Synthetic variants were also tested.
Contructs were integrated precisely in single copy at the am locus on linkage
group VR and/or the his-3 locus on linkage group IR to control for possible confounding effects of random integration including differences in copy number, chromosomal location, and arrangement of the transforming DNA. Some conclusions
from this work include: (1) the ζ –η region contains at least two nonoverlapping
methylation signals; (2) different fragments of the region can induce different
levels of methylation; (3) methylation induced by ζ –η sequences can spread far
into flanking sequences; (4) fragments as small as 171 bp can trigger methylation;
(5) methylation signals behave similarly, but not identically, at different chromosomal sites; (6) mutation density, per se, does not determine whether sequences become methylated; (7) both A:T-richness and high densities of TpA
dinucleotides, typical attributes of methylated sequences in Neurospora, appear
to promote de novo methylation but neither is an essential feature of methylation signals. An important general conclusion is that de novo methylation of ζ –η
sequences does not simply reflect the absence of signals that prevent methylation; rather, the region contains multiple, positive signals that trigger methylation (Miao et al., 2000). Recent tests of synthetic sequences have identified a
variety of simple sequences that can promote de novo methylation in Neurospora
(H. Tamaru and E. U. Selker, in preparation).
Classical genetic approaches to identify elements of the methylation machinery are starting to pay off. Mutant hunts in Neurospora have already implicated
five genes involved in DNA methylation (Foss et al., 1993, 1995, 1998; Tamaru
and Selker, 2001; M. Freitag, A. Hagemann, and E. U. Selker, in preparation).
One, dim-2, the only eukaryotic gene currently known in which mutations appear to eliminate DNA methylation, encodes a DNA methyltransferase (MTase;
Kouzminova and Selker, 2001). The dim-2 MTase is apparently responsible for
methylation at both symmetrical and asymmetrical sites and is required for both
de novo and maintenance methylation. Curiously, dim-2 does not play a role in
RIP; duplicated sequences are mutated in dim-2 strains, as usual, but the mutated
sequences are not methylated (Kouzminova and Selker, 2001). The Neurospora
genome sequence is nearly complete, and only one other potential DNA MTase
has been found. This MTase homolog does not appear to be involved in methylation in vegetative cells (M. Freitag and E. U. Selker, unpublished). The Ascobolus
masc1 gene is the closest known homolog of this potential MTase gene. Thus it
will be interesting to learn whether mth plays a role in RIP.
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IV. CONSEQUENCES OF RIP AND MIP
The most common outcome of RIP is gene inactivation due to nonsense and/or
missense mutations. In addition, RIP can generate functional, or partially functional alleles (e.g., see Barbato et al., 1996; Fincham, 1990; Glass and Lee, 1992).
RIP frequently makes just C-to-T or G-to-A changes on a given strand and prefers
certain sites (e.g., CpA dinucleotides). Because of peculiarities of codon usage in
Neurospora, C-to-T changes on the coding strand are not as serious as C-to-T
changes on the noncoding strand (Singer et al., 1995a, 1995b; Watters et al.,
1999). Some potentially functional alleles are not expressed, however, due to
methylation resulting from RIP. The methylation resulting from RIP and MIP
causes a transcriptional block by a mechanism that remains largely unexplored
(Barry et al., 1993; Rountree and Selker, 1997). There are several clues, however.
Results of nuclear run-on assays using extracts from Neurospora strains bearing
various methylated sequences demonstrated that the block is at the level of transcript elongation; initiation of transcription appeared unperturbed (Rountree and
Selker, 1997). Considering that methylation does not inhibit transcription in vitro,
such as in nuclear extracts, methylation must be having its effect by an indirect
mechanism. Two nonexclusive possibilities are being examined: (1) the inhibitory
effect results from effects of hypothetical methyl-DNA binding proteins, perhaps
similar to those described in higher eukaryotes (e.g., see Hendrich and Bird,
1998); (2) the inhibitory effect results from modifications of chromatin, such as
acetylation or methylation of histones. Methyl-DNA binding proteins have been
detected in Neurospora (G. Kothe, M. Rountree, and E. U. Selker, unpublished),
and there is evidence for chromatin modifications associated with methylation.
For example, in Neurospora, the histone deacetylase inhibitor Trichostatin A was
found to cause derepression of a gene silenced by methylation, repression of a gene
that is activated by methylation of an adjacent transposon, and selective loss of
DNA methylation (Selker, 1998), indicating that DNA methylation and protein
acetylation are connected in one or more ways (see Dobosy and Selker, 2001).
One can only speculate about the long-term consequences of RIP and
MIP. On the one hand, there is good evidence that these processes successfully
control transposable elements. The Neurospora genome contains a variety of sequences related to known transposons, but virtually all of them show evidence of
RIP, and active transposons are absent from most Neurospora strains (Cambareri
et al., 1998; Kinsey et al., 1994; Margolin et al., 1998; Kinsey, 1989; E. U. Selker,
unpublished). On the other hand, it seems likely that RIP and MIP limit the
evolution of new functions through gene duplication and divergence. Aside from
the rDNA, the structurally similar functional genes that have been noted in Neurospora appear to lack segments that are long enough and sufficiently similar to
be subject to RIP. Conceivably, sequences altered by RIP might provide useful
raw material for the evolution of new genes, but I am unaware of evidence that
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this has occurred. A survey of genomic sequences that show the hallmarks of RIP
has revealed that most are methylated (T. Wolf, B. Margolin, and E. U. Selker,
unpublished). It is not known whether such sequences drift, eventually lose their
methylation and become functional, or are lost by deletion.
V. CONCLUDING REMARKS
The discovery of RIP provided dramatic evidence that related sequences can interact efficiently regardless of their relative locations in a genome and provided
the first example of a genome defense system. To date, RIP and MIP are the only
examples of genome defense systems that almost certainly rely on DNA:DNA
interactions. Other genome defense systems based in some way on sequence
homology have come to light more recently, including transcriptional and posttranscriptional gene silencing in plants, quelling in Neurospora and RNAi in
animals. These processes do not appear as potent as RIP and MIP in that they
do not inactivate all duplications and they do not typically result in permanent
silencing. This may be responsible for their broader distribution in nature. The realization that organisms have genome defense systems that can silence and modify
selfish DNA should cause us to reconsider the oft-repeated dogma that experience
cannot be inherited.
Acknowledgments
I thank Michael Freitag and Jeanne Selker for comments on the manuscript. Work in my laboratory
is supported by a grant from the National Institutes of Health (GM35690).
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