Cytosine
methylation
in gene-silencing
mechanisms
Paul S. Chomet University Cytosine a number involvement duplicate
of California,
Berkeley,
Opinion
in Cell
Introduction
Biology
1991,
in the and are
3:438-443
In a number of animal systems, evidence for the direct involvement of methyfation came from studies in which reporter gene DNA was methylated in vitro and then reintroduced into cells. In most cases, genes that were methylated in vitro were not expressed in transient assay systems. Recent studies with plants have also shown that in vitro cytosine methylation can lower gene expression
In the DNA of eukaryotes, 5-methyl cytosine (5-mC) is extensively used as a fifth nucleoside. In mammalian cells 36% of all cytosines are methylated, primarily in CpG sites; in plant cells, methyfated cytosine can account for up to 30% of all cytosines in both CpG and CpNpG sites [1,2]. The potential importance of 5-mC has arisen from studies over the past 10 years that have linked DNA methylation with repression of gene activity in many organisms (for review, see [3*]). A large body of experimental evidence, particularly from transfection studies with in L&W methylated DNA demonstrates that 5-mC af fects promoter function and is used in other mechanisms that lead to gene silencing. The parameters involved in the control and establishment of the methylated state (de nova methylation) are still largely unknown. Recent technological advances have given us a more precise view of methylation in vivo [4**,5*]. Other studies with a more generalized view of the genome have firmly established a link between the state of methylation and a particular chromatin structure [6**]. Approaching the role of DNA methylation from a number of angles should yield an integrated understanding of 5-mC action in which methylation plays a key role in higher-order chromatin changes. The purpose of this review is to present the complexity of DNA methyiation in the genome and its involvement in promoter inactivation, de no210 methyiation of repetitive sequences and X-inactivation. A model linking gene inhbition, methyIation and chromatin structure (formulated by E Selker [7ma]> is discussed. modulates
USA
methylation is associated with gene-silencing mechanisms of eukaryotic organisms. Recent studies directed at of methylation in promoter inactivation, X-chromosome sequence inactivation and in chromatin structure changes, presented.
Current
How methylation
California,
i&91.
Site-specific methylation studies involving both vital and eukaryotic gene promoters have shown that methylation of only one or a few cytosines at CpG sites can alter transcriptional activity. In most cases where expression is affected, these methylated sites are part of binding sequences for transcription factors. Conversely, methylated sites outside such a region do not significantly affect gene expression. For example, methylation at a central cytosine within the adenovirus major late-gene transcription factor (MLTF) binding sequence strongly inhibits binding and in vitro transcription, whereas methylation of a cytosine only six base pairs away has no measurable effect [lO*]. In some studies, demethylation by 5-azacytidine can reverse transcriptional inhibition, suggesting that tramacting factors necessary for expression are available to, but do not interact with, the methylated form of the gene [ 11,12*]. These studies indicate that the effect of CpG site-specific methylation (or possibly CpNpG in plants) on gene expression results directly from the interruption of binding of particular transcription factors to regulatory regions in the gene. One explanation for the methylation-mediated alteration in protein-DNA binding affinity stems from the fact that the addition of a methyl group to cytosine provides a new hydrophobic site. Such an interaction may be analogous to interactions of prokaryotic DNA-binding proteins with the methyl group of thymine [13]. Furthermore, 5-mC is likely to result in unfavorable steric hinderance between the protein and functional groups in the DNA [ 14*]. This steric explanation, however, does not account for the entire effect of gene repression by methylation. All transcription factors, for example, are not affected by methyiation in the same manner or to the same extent. This was shown clearly for the methyfation-independent binding of the mammalian transcription factor, Spl [ 151.
gene expression
Many studies have established a correlation between undermethylation and unimpeded gene expression. The overall conclusion is that the methyiation state of DNA can affect gene expression directly. Primarily, the 5’ end of a gene is usually the methylation-atfected region. This indicates that expression of a specitic gene is repressed through DNA-transcription-factor protein interactions rather than the general transcription machinery.
Abbreviations !i-mC-5-methyl
438
cytosine;
MlTF--major
late-gene
@ Current
transcription
Biology
factor;
RIP-repeat-induced
Ltd ISSN 0!855*74
point
mutation.
Cytosine
It is possible, therefore, that only certain classes of regulatory proteins are directly affected by methylation. In addition, it is known that changes in chromatin structure, evidenced by changes in DNase I sensitivity and histone distribution and acetyiation, are associated with DNA methylation changes [ 12*,16,6**]. Chromatin structure changes suggest an alternative mechanism of gene repression involving cytosine methylation [ 17**]. De nova methylation
In contrast to our understanding of the direct involvement of methylation on gene expression through transcription factor interactions, little is known about the controls governing DNA methyiation. It has been well established that integrated viral DNA in animals can be methylated de nouo in a highly specific manner. However, if only a few, key 5-mC residues can repress gene activity, why are long stretches of methylated regions found? One possible explanation is through a spreading effect of methylation over short distances. Toth et al. [ 17.1 investigated parameters for the establishment of a DNA-methylation pattern in the viral E2A promoter after integrating the viral genome into hamster cell lines. A stable, specific pattern of DNA methylation across the promoter region could occur only after in vitro methylation of a site prior to transfection. A newly methylated region of the promoter was established through many cell generations. These data suggest a cooperatively of methylation at adjacent sites. It is possible that this spread of methylation was either accompanied or perhaps directed by higherorder chromatin changes. Particular DNA sequences may contain information that defines a’e not)0 methylation patterns. A 214 bp fragment near the Thy-l promoter, when transformed into mouse embryos or embryonic stem cells, protects itself and surrounding sequences from methylation [ 18*]. Constitutively hypomethylated regions rich in CpG dinucleotides are normally found interspersed in a non-random fashion in the genome (CpG islands). The 5’ end of the 7@1 gene is normally contained within such a CpG island, indicating that the 214 bp sequence can direct the normal hypomethylated CpG island pattern independent of its chromosomal context, This sequence may have the ability to block methyfation spreading. Moreover, the presence of a sequence-specific binding factor might account for the lack of methylation in this region, as was proposed by Razin and Riggs [ 191 for determinator proteins. Involvement inactivation
of methylation
in X-chromosome
X chromosomes in mammals incorporate methylation in a dosage-compensating mechanism that overrides the usual patterning of hypomethylated CpG islands on one of the X chromosomes. This results in the inactivation, with associated methylation, of one of the X chromosomes of the female. The initiation of inactivation is thought to occur at a site unique to the X chromosome, designated the X-inactivation center [ 201. To explain why
methylation
in gene-silencing
mechanisms
Chomet
only one of two (or more) X chromosomes is inactivated, it is hypothesized that the X-inactivation center on one X chromosome receives a signal early in development that blocks its function, while the center on the remaining X chromosome initiates the inactivation process (see [21*] for recent comments>. New protocols using the polymerase chain reaction have made it possible to show that female-specific methylation of an Xlinked gene (PGK 1, encoding phosphoglycerate kinase) occurs in 5.5-6.5~day-old embryos, about the time of X inactivation and heterochromatization [5*]. That methyfation directly contributes to X-inactivation is demonstrated by the reactivation of genes on the inactive X chromosome using 5azacytidine [22]. Such a result does not preclude the likelihood that methyfation is only one of the events that must occur during heterochromatization of the inactive X chromosome. Once inactivation is established, almost all of the possible CpG sites in the PGK-1 promoter on the inactive X chromosome are methylated and lack protein-DNA footprints. On the other hand, the same sites on the active X chromosome in the same cells were shown to be unmethylated and to have protein-DNA contacts, further suggesting that chromatin-configuration changes occur in the methylated state. This gene-silencing mechanism maintains the inactive state of the X chromosome throughout normal mammalian development [23]. Convincing evidence indicates that DNA methyiation plays a critical role in the maintenance of X-chromosome inactivity (for review, see [24-l ). It would appear that mammalian genomes employ methylation to assist in inactivating gene function and to stabilize this inactive state. Furthermore, this mechanism overrides the methylation patterns associated with autosomal chromosomes. Methylation to duplicate
and gene inactivation sequences
is targeted
Plant genomes have the ability to recognize and modify unlinked duplicate DNA sequences. The presence of a transgene, such as a nopaline synthase T-DNA construct, was shown to affect the state of methylation (in the promoter region) and expression of a second, related transgene (octopine synthase T-DNA construct> in the same genome [25*]. Similarly, multiple, but not allelic, copies of a T-DNA insertion showed a low level of transgene expression and increased methylation of the T-DNA sequence [26-l. Furthermore, in both cases the effect was reversible; after meiotic segregation any one copy of the transgene was highly expressed with reduced methylation. Multicopy-related suppression of gene expression may be due to competition for limiting transcriptional factor(s); consequently, the unbound DNA sequence(s) could become methyiated. An increase in transgene copy number can also suppress endogenous gene expression. This effect, termed co-suppression, was identified in transgenic plants that harbor a chimeric chalcone synthase gene (or the gene encoding dihydroflavanol 4-reductase, both of which are involved in anthocyanin pigment formation) [27**]. Both the transgene and the endogenous gene were transctip-
439
440
Nucleus and aene expression . tionallv inactivated. In some cases. the inactivation was somatically reversible, producing a variegated phenotype. Particular variegated patterns were often heritable. I
Inactivation of transposable element expression has been correlated with the methy-lation state of the element. The Activator (AC) and Suppressor-mutator (Spnz) elements undergo (transposase) gene inactivation associated with methylation near the transcription start site [28*-301. This inactivation is reversible and can occur at a heritable time and frequency during development. Further element methylation is correlated with the suppression of the element’s functions and with further stability of the inactive state (PS Chomet, unpublished data) [28-l. Over a number of plant generations, stable hrpermethylated inactive elements can be observed. Because transposable element sequences are usually repetitive in the genome it may be that the expression of the element, and the phenomenon of co-suppression, reflect a general mechanism of gene suppression and methylation in plants. The reason that duplicate sequences are targeted for methylation is not known. An attractive but quite speculative view may tie methylation of duplicated regions to dominant position-effect variegation. This phenomenon is observed in Drosophila, where transcription of two copies of the brou~ gene, one copy near a rearrangement cc&) and one copy in a normal chromosomal context (tram), is suppressed [31*9]. This trans-inactivation is known to be mediated through a local chromosome effect and is likely to be dependent on somatic pairing of the duplicate regions and their immediate flanking sequence. Furthermore, it is known that genes af fecting position4fect variegation are involved in heterochromatin formation [ 32**]. Interestingly, the instability of the a&ted genes in plants (transposase genes and transgenes) is similar in phenotype to genes influenced by position&Ect variegation. It will be essential to determine if these phenomena share similarities at the level of chromatin structure.
Form
Methylation
and chromatin
structure
Sequences in Neurospora are also know to direct their own de nozlo pattern of methylation. Duplicated sequences introduced into Neuroqm-a are altered by the repeat-induced point mutation (RIP) process and are subsequently heavily methylated (for review see [33-l ). Similarly, gene duplications in the fungus, ~~cobofzcs, are detected and premeiotically methylated at cytosine residues [34]. No single region of an RIP-altered sequence directs its own methylation, but an RIP-altered sequence can direct methylation to adjacent sequences. These observations suggest that the 5-mCs act in a collective manner and may reflect higher-order chromatin changes. These and other results led Selker to propose a model (Fig. 1) that relates alterations in gene expression to methylation and chromatin structure. The presence (Form I-open) or absence (Form II-collapsed) of sequence-dependent DNA-binding proteins dictates the chromatin configuration. A chromosomal region would be in Form I when occupied by various DNA-binding (non-histone) proteins. Loss of binding over a given span would spontaneously result in Form II chromatin. A recent study by Tazi and Bird [6**] demonstrates the presence of alternate chromatin structures. The characteristics of hypomethylated CpG islands compared to bulk chromatin suggest CpG island chromatin to be in a more relaxed configuration than the compact bulk chromatin. Site-specific methylation was also associated with increased DNase I resistance (or endonuclease resistance) again linking methylation with a less accessible chromatin form [ 12*,16]. To explain the role of methylation in alternating between the chromatin configurations, Selker postulates non-sequence-specific proteins that are capable of recognizing methylated versus unmethylated DNA as well as recognizing the alternate chromatin fomls. It is the restricted combinations of proteins that identify the partic-
I
Fig.
1. A
model
of
alternative
chro-
matic states proposed by Selker PI. Chromatin exists in two basic forms. In Form I, a chromosomal open by the presence scription factors and specific shapes). factors,
region is held of bound tranother sequence-
DNA-binding proteins (irregular In the absence of these bound the DNA, organized in nucleo-
somes (0). spontaneously the Form II state
collapses
into
Cytosine
ular state of methyiation or chromatin conformation (i.e. protein classes 1 and 2 only bind unmethylated Form II chromatin) involved in the shift between chromatin Form I and Form U (Fig. 2). In Drmophih, where the genetically controlled assembly of chromatin formation is at least partially understood, it is known that many genes encoding non-histone chromatin proteins can influence the formation of heterochromatin. Heterochromatin in other organisms is as sociated with methylation (i.e. inactivated X in mammals). Futhermore, heterochromatic regions, when placed near genes in Drosophila and mammals, can inactivate the gene in a variegated fashion (position4Tect variegation). It has been postulated that loci that modify this effect encode these non-histone proteins which assemble into a multimeric complex that constitutes heterochromatin (for overview, see [32-l >. Although Drosophila does not have 5-mC, it is likely that general mechanisms of chromatin assembly are universal as a sequence motif found in a heterochromatin protein (encoded by the gene Suua1(2)5) is conserved in animals and plants
Binding of all possible
Loss
methylation
in gene-silencing
mechanisms
Chomet
(35’1. Furthermore, another heterochromatin-associated protein (encoded by the gene Suvut(3)7) contains a novel arrangement of widely spaced zinc-linger motifs, which may help in packaging the chromatin fibers into heterochromatin [ 36.1. Proteins that bind preferentially to methylated DNA might also be involved in the process of higher-order chromatin formation or stabilization [16,37-l. Methylation, in concert with a host of chromosomal proteins, could then provide the cell with stability and heritability of the chromatin state, thereby providing the cell with ‘historical’ information. That methyfation aids in somatic cell stability is a likely possibility and is extensively addressed by Riggs [ 241. Another functional aspect of the active chromatic structure is that it is structurally distinct from the bulk DNA Organisms characterized by large genomes may use this configuration to ‘highlight’ certain promoter sequences as condensed chromatin is likely to be hidden from sequence-specific DNA-binding proteins. It has been shown that methyiated sites in chromatin are approximately 10 times less susceptible to nuclease attack
of protein binding
AOOA
Fig. 2. A model proposing a role for DNA methylation in the alternation of chromation forms. Methylated DNA (solid black lines and unmethylated DNA (broken lines) can be organized as Form I or II chromatin, although methylated Form I and unmethylated Form II chromatin are transitional states. Only DNA in Form II chromatin is subject to new methylation after replication. Loss of protein binding allows the collapse of chromatin Form I to Form II. Reversal of states, Form II to Form I, is accomplished by a subset of proteins (A, A). If reversal to Form I does not occur before DNA replication, the DNA is methylated and is further locked into Form II, as only a subset of the Form II-binding proteins (A) are capable of binding methylated DNA. A transition of Form II to Form I is mediated by a class of proteins A, allowing an additional protein class (0) to bind methylated DNA in Form I. Loss of DNA methylation, resulting from DNA replication without subsequent maintenance of methylation while in Form I, allows more classes of proteins to bind and thereby maintain the Form I configuration.
441
442
Nucleus
and gene expression
.
t.ha~~is naked DNA 116.3~1, suggesting (but not ~roving) that methylate&chiom&n is~ncti&lly hidden. While Selker’s model is preliminary, future studies will clearly need to address the integration of methylation, chromatin structure, and gene expression. Only then will the link between mechanisms employing methyiation be fully realized and the functional nature of methylation be completely understood. Acknowledgements 1 wish to thank RR Dawe. B Klceckener, for critical readings of the manuscript.
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methylated-DNA-binding protein was identified. Digestion of nuclear chromatin with the merhylation-sensitive and .insensitive &o&isomer pair, M.@ and HpaU, showed methyiated CpG sites are not accessible to M@ digestion, suggesting that a protein may be complexing with the methyiated CpGs in viw.
PS Chomet, Plant Biology 111 Genetics and Plant USA
Department, University of California Berkeley, Biology Building, Berkeley, California 94720,
methylation
in gene-silencing
mechanisms
Paul S. Chomet University Cytosine a number involvement duplicate
of California,
Berkeley,
Opinion
in Cell
Introduction
Biology
1991,
in the and are
3:438-443
In a number of animal systems, evidence for the direct involvement of methyfation came from studies in which reporter gene DNA was methylated in vitro and then reintroduced into cells. In most cases, genes that were methylated in vitro were not expressed in transient assay systems. Recent studies with plants have also shown that in vitro cytosine methylation can lower gene expression
In the DNA of eukaryotes, 5-methyl cytosine (5-mC) is extensively used as a fifth nucleoside. In mammalian cells 36% of all cytosines are methylated, primarily in CpG sites; in plant cells, methyfated cytosine can account for up to 30% of all cytosines in both CpG and CpNpG sites [1,2]. The potential importance of 5-mC has arisen from studies over the past 10 years that have linked DNA methylation with repression of gene activity in many organisms (for review, see [3*]). A large body of experimental evidence, particularly from transfection studies with in L&W methylated DNA demonstrates that 5-mC af fects promoter function and is used in other mechanisms that lead to gene silencing. The parameters involved in the control and establishment of the methylated state (de nova methylation) are still largely unknown. Recent technological advances have given us a more precise view of methylation in vivo [4**,5*]. Other studies with a more generalized view of the genome have firmly established a link between the state of methylation and a particular chromatin structure [6**]. Approaching the role of DNA methylation from a number of angles should yield an integrated understanding of 5-mC action in which methylation plays a key role in higher-order chromatin changes. The purpose of this review is to present the complexity of DNA methyiation in the genome and its involvement in promoter inactivation, de no210 methyiation of repetitive sequences and X-inactivation. A model linking gene inhbition, methyIation and chromatin structure (formulated by E Selker [7ma]> is discussed. modulates
USA
methylation is associated with gene-silencing mechanisms of eukaryotic organisms. Recent studies directed at of methylation in promoter inactivation, X-chromosome sequence inactivation and in chromatin structure changes, presented.
Current
How methylation
California,
i&91.
Site-specific methylation studies involving both vital and eukaryotic gene promoters have shown that methylation of only one or a few cytosines at CpG sites can alter transcriptional activity. In most cases where expression is affected, these methylated sites are part of binding sequences for transcription factors. Conversely, methylated sites outside such a region do not significantly affect gene expression. For example, methylation at a central cytosine within the adenovirus major late-gene transcription factor (MLTF) binding sequence strongly inhibits binding and in vitro transcription, whereas methylation of a cytosine only six base pairs away has no measurable effect [lO*]. In some studies, demethylation by 5-azacytidine can reverse transcriptional inhibition, suggesting that tramacting factors necessary for expression are available to, but do not interact with, the methylated form of the gene [ 11,12*]. These studies indicate that the effect of CpG site-specific methylation (or possibly CpNpG in plants) on gene expression results directly from the interruption of binding of particular transcription factors to regulatory regions in the gene. One explanation for the methylation-mediated alteration in protein-DNA binding affinity stems from the fact that the addition of a methyl group to cytosine provides a new hydrophobic site. Such an interaction may be analogous to interactions of prokaryotic DNA-binding proteins with the methyl group of thymine [13]. Furthermore, 5-mC is likely to result in unfavorable steric hinderance between the protein and functional groups in the DNA [ 14*]. This steric explanation, however, does not account for the entire effect of gene repression by methylation. All transcription factors, for example, are not affected by methyiation in the same manner or to the same extent. This was shown clearly for the methyfation-independent binding of the mammalian transcription factor, Spl [ 151.
gene expression
Many studies have established a correlation between undermethylation and unimpeded gene expression. The overall conclusion is that the methyiation state of DNA can affect gene expression directly. Primarily, the 5’ end of a gene is usually the methylation-atfected region. This indicates that expression of a specitic gene is repressed through DNA-transcription-factor protein interactions rather than the general transcription machinery.
Abbreviations !i-mC-5-methyl
438
cytosine;
MlTF--major
late-gene
@ Current
transcription
Biology
factor;
RIP-repeat-induced
Ltd ISSN 0!855*74
point
mutation.
Cytosine
It is possible, therefore, that only certain classes of regulatory proteins are directly affected by methylation. In addition, it is known that changes in chromatin structure, evidenced by changes in DNase I sensitivity and histone distribution and acetyiation, are associated with DNA methylation changes [ 12*,16,6**]. Chromatin structure changes suggest an alternative mechanism of gene repression involving cytosine methylation [ 17**]. De nova methylation
In contrast to our understanding of the direct involvement of methylation on gene expression through transcription factor interactions, little is known about the controls governing DNA methyiation. It has been well established that integrated viral DNA in animals can be methylated de nouo in a highly specific manner. However, if only a few, key 5-mC residues can repress gene activity, why are long stretches of methylated regions found? One possible explanation is through a spreading effect of methylation over short distances. Toth et al. [ 17.1 investigated parameters for the establishment of a DNA-methylation pattern in the viral E2A promoter after integrating the viral genome into hamster cell lines. A stable, specific pattern of DNA methylation across the promoter region could occur only after in vitro methylation of a site prior to transfection. A newly methylated region of the promoter was established through many cell generations. These data suggest a cooperatively of methylation at adjacent sites. It is possible that this spread of methylation was either accompanied or perhaps directed by higherorder chromatin changes. Particular DNA sequences may contain information that defines a’e not)0 methylation patterns. A 214 bp fragment near the Thy-l promoter, when transformed into mouse embryos or embryonic stem cells, protects itself and surrounding sequences from methylation [ 18*]. Constitutively hypomethylated regions rich in CpG dinucleotides are normally found interspersed in a non-random fashion in the genome (CpG islands). The 5’ end of the 7@1 gene is normally contained within such a CpG island, indicating that the 214 bp sequence can direct the normal hypomethylated CpG island pattern independent of its chromosomal context, This sequence may have the ability to block methyfation spreading. Moreover, the presence of a sequence-specific binding factor might account for the lack of methylation in this region, as was proposed by Razin and Riggs [ 191 for determinator proteins. Involvement inactivation
of methylation
in X-chromosome
X chromosomes in mammals incorporate methylation in a dosage-compensating mechanism that overrides the usual patterning of hypomethylated CpG islands on one of the X chromosomes. This results in the inactivation, with associated methylation, of one of the X chromosomes of the female. The initiation of inactivation is thought to occur at a site unique to the X chromosome, designated the X-inactivation center [ 201. To explain why
methylation
in gene-silencing
mechanisms
Chomet
only one of two (or more) X chromosomes is inactivated, it is hypothesized that the X-inactivation center on one X chromosome receives a signal early in development that blocks its function, while the center on the remaining X chromosome initiates the inactivation process (see [21*] for recent comments>. New protocols using the polymerase chain reaction have made it possible to show that female-specific methylation of an Xlinked gene (PGK 1, encoding phosphoglycerate kinase) occurs in 5.5-6.5~day-old embryos, about the time of X inactivation and heterochromatization [5*]. That methyfation directly contributes to X-inactivation is demonstrated by the reactivation of genes on the inactive X chromosome using 5azacytidine [22]. Such a result does not preclude the likelihood that methyfation is only one of the events that must occur during heterochromatization of the inactive X chromosome. Once inactivation is established, almost all of the possible CpG sites in the PGK-1 promoter on the inactive X chromosome are methylated and lack protein-DNA footprints. On the other hand, the same sites on the active X chromosome in the same cells were shown to be unmethylated and to have protein-DNA contacts, further suggesting that chromatin-configuration changes occur in the methylated state. This gene-silencing mechanism maintains the inactive state of the X chromosome throughout normal mammalian development [23]. Convincing evidence indicates that DNA methyiation plays a critical role in the maintenance of X-chromosome inactivity (for review, see [24-l ). It would appear that mammalian genomes employ methylation to assist in inactivating gene function and to stabilize this inactive state. Furthermore, this mechanism overrides the methylation patterns associated with autosomal chromosomes. Methylation to duplicate
and gene inactivation sequences
is targeted
Plant genomes have the ability to recognize and modify unlinked duplicate DNA sequences. The presence of a transgene, such as a nopaline synthase T-DNA construct, was shown to affect the state of methylation (in the promoter region) and expression of a second, related transgene (octopine synthase T-DNA construct> in the same genome [25*]. Similarly, multiple, but not allelic, copies of a T-DNA insertion showed a low level of transgene expression and increased methylation of the T-DNA sequence [26-l. Furthermore, in both cases the effect was reversible; after meiotic segregation any one copy of the transgene was highly expressed with reduced methylation. Multicopy-related suppression of gene expression may be due to competition for limiting transcriptional factor(s); consequently, the unbound DNA sequence(s) could become methyiated. An increase in transgene copy number can also suppress endogenous gene expression. This effect, termed co-suppression, was identified in transgenic plants that harbor a chimeric chalcone synthase gene (or the gene encoding dihydroflavanol 4-reductase, both of which are involved in anthocyanin pigment formation) [27**]. Both the transgene and the endogenous gene were transctip-
439
440
Nucleus and aene expression . tionallv inactivated. In some cases. the inactivation was somatically reversible, producing a variegated phenotype. Particular variegated patterns were often heritable. I
Inactivation of transposable element expression has been correlated with the methy-lation state of the element. The Activator (AC) and Suppressor-mutator (Spnz) elements undergo (transposase) gene inactivation associated with methylation near the transcription start site [28*-301. This inactivation is reversible and can occur at a heritable time and frequency during development. Further element methylation is correlated with the suppression of the element’s functions and with further stability of the inactive state (PS Chomet, unpublished data) [28-l. Over a number of plant generations, stable hrpermethylated inactive elements can be observed. Because transposable element sequences are usually repetitive in the genome it may be that the expression of the element, and the phenomenon of co-suppression, reflect a general mechanism of gene suppression and methylation in plants. The reason that duplicate sequences are targeted for methylation is not known. An attractive but quite speculative view may tie methylation of duplicated regions to dominant position-effect variegation. This phenomenon is observed in Drosophila, where transcription of two copies of the brou~ gene, one copy near a rearrangement cc&) and one copy in a normal chromosomal context (tram), is suppressed [31*9]. This trans-inactivation is known to be mediated through a local chromosome effect and is likely to be dependent on somatic pairing of the duplicate regions and their immediate flanking sequence. Furthermore, it is known that genes af fecting position4fect variegation are involved in heterochromatin formation [ 32**]. Interestingly, the instability of the a&ted genes in plants (transposase genes and transgenes) is similar in phenotype to genes influenced by position&Ect variegation. It will be essential to determine if these phenomena share similarities at the level of chromatin structure.
Form
Methylation
and chromatin
structure
Sequences in Neurospora are also know to direct their own de nozlo pattern of methylation. Duplicated sequences introduced into Neuroqm-a are altered by the repeat-induced point mutation (RIP) process and are subsequently heavily methylated (for review see [33-l ). Similarly, gene duplications in the fungus, ~~cobofzcs, are detected and premeiotically methylated at cytosine residues [34]. No single region of an RIP-altered sequence directs its own methylation, but an RIP-altered sequence can direct methylation to adjacent sequences. These observations suggest that the 5-mCs act in a collective manner and may reflect higher-order chromatin changes. These and other results led Selker to propose a model (Fig. 1) that relates alterations in gene expression to methylation and chromatin structure. The presence (Form I-open) or absence (Form II-collapsed) of sequence-dependent DNA-binding proteins dictates the chromatin configuration. A chromosomal region would be in Form I when occupied by various DNA-binding (non-histone) proteins. Loss of binding over a given span would spontaneously result in Form II chromatin. A recent study by Tazi and Bird [6**] demonstrates the presence of alternate chromatin structures. The characteristics of hypomethylated CpG islands compared to bulk chromatin suggest CpG island chromatin to be in a more relaxed configuration than the compact bulk chromatin. Site-specific methylation was also associated with increased DNase I resistance (or endonuclease resistance) again linking methylation with a less accessible chromatin form [ 12*,16]. To explain the role of methylation in alternating between the chromatin configurations, Selker postulates non-sequence-specific proteins that are capable of recognizing methylated versus unmethylated DNA as well as recognizing the alternate chromatin fomls. It is the restricted combinations of proteins that identify the partic-
I
Fig.
1. A
model
of
alternative
chro-
matic states proposed by Selker PI. Chromatin exists in two basic forms. In Form I, a chromosomal open by the presence scription factors and specific shapes). factors,
region is held of bound tranother sequence-
DNA-binding proteins (irregular In the absence of these bound the DNA, organized in nucleo-
somes (0). spontaneously the Form II state
collapses
into
Cytosine
ular state of methyiation or chromatin conformation (i.e. protein classes 1 and 2 only bind unmethylated Form II chromatin) involved in the shift between chromatin Form I and Form U (Fig. 2). In Drmophih, where the genetically controlled assembly of chromatin formation is at least partially understood, it is known that many genes encoding non-histone chromatin proteins can influence the formation of heterochromatin. Heterochromatin in other organisms is as sociated with methylation (i.e. inactivated X in mammals). Futhermore, heterochromatic regions, when placed near genes in Drosophila and mammals, can inactivate the gene in a variegated fashion (position4Tect variegation). It has been postulated that loci that modify this effect encode these non-histone proteins which assemble into a multimeric complex that constitutes heterochromatin (for overview, see [32-l >. Although Drosophila does not have 5-mC, it is likely that general mechanisms of chromatin assembly are universal as a sequence motif found in a heterochromatin protein (encoded by the gene Suua1(2)5) is conserved in animals and plants
Binding of all possible
Loss
methylation
in gene-silencing
mechanisms
Chomet
(35’1. Furthermore, another heterochromatin-associated protein (encoded by the gene Suvut(3)7) contains a novel arrangement of widely spaced zinc-linger motifs, which may help in packaging the chromatin fibers into heterochromatin [ 36.1. Proteins that bind preferentially to methylated DNA might also be involved in the process of higher-order chromatin formation or stabilization [16,37-l. Methylation, in concert with a host of chromosomal proteins, could then provide the cell with stability and heritability of the chromatin state, thereby providing the cell with ‘historical’ information. That methyfation aids in somatic cell stability is a likely possibility and is extensively addressed by Riggs [ 241. Another functional aspect of the active chromatic structure is that it is structurally distinct from the bulk DNA Organisms characterized by large genomes may use this configuration to ‘highlight’ certain promoter sequences as condensed chromatin is likely to be hidden from sequence-specific DNA-binding proteins. It has been shown that methyiated sites in chromatin are approximately 10 times less susceptible to nuclease attack
of protein binding
AOOA
Fig. 2. A model proposing a role for DNA methylation in the alternation of chromation forms. Methylated DNA (solid black lines and unmethylated DNA (broken lines) can be organized as Form I or II chromatin, although methylated Form I and unmethylated Form II chromatin are transitional states. Only DNA in Form II chromatin is subject to new methylation after replication. Loss of protein binding allows the collapse of chromatin Form I to Form II. Reversal of states, Form II to Form I, is accomplished by a subset of proteins (A, A). If reversal to Form I does not occur before DNA replication, the DNA is methylated and is further locked into Form II, as only a subset of the Form II-binding proteins (A) are capable of binding methylated DNA. A transition of Form II to Form I is mediated by a class of proteins A, allowing an additional protein class (0) to bind methylated DNA in Form I. Loss of DNA methylation, resulting from DNA replication without subsequent maintenance of methylation while in Form I, allows more classes of proteins to bind and thereby maintain the Form I configuration.
441
442
Nucleus
and gene expression
.
t.ha~~is naked DNA 116.3~1, suggesting (but not ~roving) that methylate&chiom&n is~ncti&lly hidden. While Selker’s model is preliminary, future studies will clearly need to address the integration of methylation, chromatin structure, and gene expression. Only then will the link between mechanisms employing methyiation be fully realized and the functional nature of methylation be completely understood. Acknowledgements 1 wish to thank RR Dawe. B Klceckener, for critical readings of the manuscript.
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methylated-DNA-binding protein was identified. Digestion of nuclear chromatin with the merhylation-sensitive and .insensitive &o&isomer pair, M.@ and HpaU, showed methyiated CpG sites are not accessible to M@ digestion, suggesting that a protein may be complexing with the methyiated CpGs in viw.
PS Chomet, Plant Biology 111 Genetics and Plant USA
Department, University of California Berkeley, Biology Building, Berkeley, California 94720,
