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Genomic Imprinting: Imprinting with and without methylation
WOLF REIK AND NICHOLAs D. ALLEN WOLF REIK AND NICHOLAS D. ALLEN
GENOMIC IMPRINTING
GENOMIC IMPRINTING
Imprinting with and without methylation Methyltransferase-deficient mice reveal that DNA methylation is required for the somatic-cell maintenance of parental imprinting, which alters the expression of a gene according to the parent from which it was inherited. Genomic imprinting is an epigenetic mechanism by which some genes in an organism are expressed or repressed solely according to the parent from which they were inherited. Although the number of genes regulated by imprinting is unknown, some imprinted genes have recently been identified and have been found to provide developmentally important functions for fetal growth, viability and behaviour after birth [1]. Amongst other things, having this genetic mechanism leads to the singular requirement of needing both mothers and fathers for mammals to come into existence. One key problem in imprinting is to understand its molecular mechanism. Of the possible epigenetic modifications of DNA that could be used to establish and maintain allelic differences, methylation of cytosine in the dinucleotide CpG by the enzyme methyltransferase has received the most attention, ever since the discovery of allele-specific methylation of some transgenes in mice [1]. More recently, allele-specific methylation differences have been found for each of the three endogenous imprinted genes that have so far been analysed in detail; allele-specific differences in timing of DNA replication and of chromatin compaction have also been found [2-4]. Methylation is indeed important Until now, the significance of any of these allelic modifications has remained open to speculation. But the importance of CpG methylation to imprinting has now been demonstrated directly, in a simple and clean genetic experiment, by En Li and colleagues [5]. In brief, these workers showed that the imprinting of three known imprinted genes is altered in methyltransferasedeficient mice that were created using homologous gene recombination in embryonic stem cells. Importantly, the imprinting was altered in a manner predicted from the observed allele-specific methylation patterns in these genes. Thus, at least for the somatic maintenance of imprinting, an intact DNA-methylation system is necessary. This is a significant result, and comes as a welcome confirmation of their expectations to those who have studied parental methylation for the past several years. The genes studied in methyltransferase-deficient mice by Li et al. [51 were: the maternally repressed insulinlike growth factor 2 gene (Igf2); its paternally repressed neighbour on chromosome 7, H19, and the paternally repressed insulin-like growth factor 2 receptor gene (Igf2r) on chromosome 17. Each of these genes has a characteristic methylation pattern (Fig. 1). H19 probably
has the most conventional allele-specific methylation pattern, as methylation within the promoter correlates with repression of transcription from the gene. The H19 promoter constitutes a small CpG-rich 'island'; together with some sites upstream of the promoter and in the body of the gene, it is more heavily methylated on the repressed paternal allele than on the active maternal one [6-8]. The paternally repressed Igf2r gene also has a paternally methylated promoter region but, less conventionally, it has an intronic CpG-rich region that is highly methylated on the expressed maternal allele [7,91. Igf2, by comparison, is an unconventional gene altogether: it lacks methylation in its promoters but has a paternally more methylated region upstream of the first promoter [10], as well as a more recently discovered intronic region that is also methylated preferentially on the expressed paternal allele (R. Feil, J. Walter, N.D.A and W.R., unpublished observations). To our knowledge, preferential methylation of expressed copies of genes has been observed only with imprinted genes, perhaps indicating the presence in these genes of transcriptional silencers that can be suppressed by methylation [9]. When the allelic differences in methylation are abolished in embryos with methyltransferase deficiency, the effects are predictable: the otherwise silent paternal H19 allele is now expressed, whereas the paternal Igf2 and maternal Igf2r alleles become silenced [5]. (However, Li et al. [51 only demonstrated loss of methylation for the Igf2r intronic region and the H19 gene.) A twist in the tale Using mutant methyltransferase alleles of different 'strengths', Li and co-workers [5] found that, although expression of both H19 and Igf2 is affected by reduced methylation levels, Igf2r requires a greater reduction in methyltransferase activity for its maternal allele to be silenced. In mutant embryos homozygous for the weaker allele, n, genomic DNA was substantially demethylated but significant enzyme activity remained. For the stronger allele, s, most - but possibly not all residual activity was abolished. Nevertheless, some methylation does remain in s/s embryos, perhaps indicating that the s allele is not completely null, or that there are additional methyltransferase genes. The different susceptibilities of the Igf2, H19 and Igf2r genes to altered programming by the n and s alleles could have a simple, easily testable explanation: the Igf2r intron might have a more dense or extensive CpG-rich region than the H19 promoter. A more interesting explanation, however, could reflect different
© Current Biology 1994, Vol 4 No 2
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Current Biology 1994, Vol 4 No 2
Fig. 1. Imprinted genes are differentially methylated on maternal and paternal chromosomes. Methylation of Igf2 (upstream of the gene and in an intronic region) is associated with expression, whereas for H19 methylation is associated with transcriptional repression. In the Igf2r gene, methylation of the maternally inherited allele in an intron is present in the unfertilized egg, whereas paternal methylation in the promoter region arises postzygotically. In methyltransferase mutant embryos, methylation of H19 and the Igf2r intron is decreased 5]. For the Igf2r intron, the mutant s allele of the methyltransferase gene leads to more extensive demethylation than the weaker n allele. The other regions have not yet been analysed in the mutants, but are expected also to be undermethylated.
methylation requirements for the three genes. Thus, if we consider the known genesis of the methylation patterns, we find that the known allelic differences in the H19 and Igf2 genes are not present in the parental germ cells but are first detected around the blastocyst stage of development; in embryonic stem cells, allelic differences are observed only after stem-cell differentiation (171 and R. Feil, J. Walter, N.D.A. and W.R., unpublished observations). In contrast, some of the intronic methylation on the maternal allele of the Igf2r gene is already present in oocytes - and may therefore constitute a primary imprinting signal - and this methylation is rapidly expanded during preimplantation development [7,91.
Although there is some residual methyltransferase activity in the mutant embryos at the blastocyst stage, inherited from the oocyte, it is nevertheless surprising that mutant embryos do not die sooner: embryos that have either two paternal genomes (androgenones) or two maternal genomes (parthenogenones) die significantly earlier in gestation. A better indication of the effects of mutant methyltransferase alleles might be found by examining n/n or s/s embryos derived directly from homozygous mutant embryonic stem cells, for example by aggregating mutant cells with tetraploid host embryos that serve as carriers but that do not themselves contribute extensively to the cells of the embryo [151.
This rapid post-zygotic phase of methylation of the maternal Igf2r allele may still be able to progress normally in methyltransferase mutant embryos, as oocytes contain extraordinarily high levels of maternally inherited methyltransferase activity - up to 15000 times as much as in somatic cells - which would be reduced by a factor of only two in the mutant embryos (as their mothers are heterozygotes) and which remains high until the blastocyst stage 111-131. Imprinting of the Igf2r intronic region would therefore be expected to require only a 'maintenance' methyltransferase activity in postimplantation development, whereas the more susceptible H19 and gf2 genes would require methylation de novo, possibly around implantation at the time of differentiation of the epiblast. The methyltransferase n allele might be less severely compromised in its ability to perform maintenance methylation than in methylation de novo, as the enzyme prefers hemimethylated DNA to non-methylated DNA as a substrate.
Aberrant imprinting may well contribute to the death of the mutant embryos, even though death comes later than in embryos with a monoparental chromosome constitution. But, for the imprinted genes under discussion, we know that having excess copies of the H19 gene does not kill at embryonic stages but later in gestation; fetuses that lack Igf2r die before or soon after birth; and deficiency of Igf2 simply makes mice smaller than their wild-type littermates. Of course, it is likely that other, as-yet unknown, imprinted genes are also deregulated in methyltransferase-deficient embryos and could therefore contribute to the phenotype.
Do methyltransferase-deficient mice die as a result of aberrant imprinting? Mutant embryos homozygous for either n or s alleles die in utero at early post-implantation stages -- day 11 and day 10 of development, respectively - having completed gastrulation and early organogenesis [141.
The most likely cause of death in the mutant embryos is the general deregulation of a large number of genes. Although the brevity of the paper by Li and co-workers [51 is commendable, we anxiously await data addressing the possible deregulation of non-imprinted genes. Of particular interest will be the genes carried on the inactive of the two X chromosomes in females, which are subject to X-inactivation; many of these, like some of the known imprinted genes, have methylated CpG-rich islands. The issue of inactivation of one of the two X chromosomes is linked to that of imprinting, as the paternal X chromosome is preferentially inactivated in extra-embryonic tissues; this probably results from the
DISPATCH
imprinting of the Xist gene, which is maintained only in the very early embryo [161. Another factor that might affect the methyltransferasedeficient mice is the widespread de novo methylation of the genome that normally occurs at around gastrulation and that affects a large number of genes. Is the regulation of all these genes no longer tight without DNA methylation? Global loss of methylation might also have consequences for ordered chromatin structure: the generation of mice lacking methyl-cytosine-binding proteins [17] is eagerly awaited, so that the effects on imprinting can be assessed and their phenotype can be compared with that of the methyltransferase-deficient mutants. Prospects The study by Li et al. [51 justifies previous prejudices that methylation is important in the somatic maintenance of imprinting. Although methylation is now shown to be required, we are left with some very challenging problems. We are still no closer to understanding the nature of the primary imprinting events that occur in the germline. The role of methylation in the germline is difficult to address using the current mutant embryonic stem cells or mice, partly because of the oocyte's inheritance of huge amounts of methyltransferase. This problem might potentially be solved by making conditional germline-specific methyltransferase mutations, so that methyltransferase function could be provided for the duration of development to adulthood but eliminated from the process of oogenesis. The issue is of particular relevance in the case of the Igf2r gene, in which the intronic sites become methylated during oocyte growth. Clearly it would be very interesting to produce an oocyte that lacked methyltransferase: in practice this may prove difficult to achieve, but if successful it would allow us to address the molecular nature of the germline imprint. A second issue that could be addressed more easily by the generation of conditional mutants is the control of the tissue specificity of imprinting, which is being found increasingly - the best example is the lack of imprinting of the Igf2 gene in the leptomeninges and choroid plexus of the brain. It is to be hoped that the n/n and s/s mutant mice represent the beginning of a genetic analysis of the effects of methylation on imprinting. Finally, methylation can only be considered a part of the molecular story of imprinting. Even with a detailed knowledge of some of the allelic methylation differences that exist in imprinted genes, we cannot explain the specificity with which they arise and must invoke other 'imprinting control genes', if only to direct methyltransferase activity to the right place at the right time. Clearly, new strategies must be developed to address these more complex problems. Amongst the candidates for 'imprinting control genes' are the modifier genes that have been shown genetically to determine DNA methylation patterns of some transgenes in a fashion that varies according to the genetic background
of the organism [18-201. It will be important to know at which stages of development these genes act, and whether or not some of the imprinted genes are among their endogenous targets. References 1.
2. 3. 4. 5.
6. 7. 8. 9.
10.
SURANI A, REIK W (EDS): Genomic imprinting in mouse and man. Semin Dev Biol 1992, 3:73-160. BIRD AP: Imprints on islands. CurrBiol1993, 3:275-277. SURANI MA: Genomic imprinting: silence of the genes. Nature 1993, 366:302-303 KITSBERG D, SELIG S, BRANDEIS M, SIMON , KESHET I, DRISCOLL DJ, NICHOLLS RD, CEDAR H: Allele-specific replication timing of imprinted gene regions. Nature 1993, 364:459-463. LI E, BEARD C, JAENISCH R: Role of DNA methylation in genomic imprinting. Nature 1993, 366:362-365. FERGUSON-SMITH AC, SASAKI H, CATTANACH BM, SURANI MA:
Parental-origin-specific epigenetic modification of the H19 gene. Nature 1993, 362:751-754. BRANDEIS M, KAFRI T, ARIEL M, CHAILLET' JR, RAZIN A, CEDAR H: The ontogeny of allele-specific methylation associated with imprinted genes in the mouse. EMBOJ 1993, 12:3669-3677. BARTOLOMEI MS, WEBER AL, BRUNKOW ME, TILGHMAN SM: Epigenetic mechanisms underlying the imprinting of the mouse H19 gene. Genes Dev 1993, 7:1663-1673. STOGER R, KUBICKA P, LIU C-G, KAFRI T, CEDAR H, BARLOW D: Maternal-specific methylation of the imprinted mouse IgJ2r locus identifies the expressed locus as carrying the imprinting signal. Cell 1993, 73:61-71. SASAKI H, JONES PA, CHAILLET JR, FERGUSON-SMITH AC, BARTON
SC, REIK W, SURANI MA: Parental imprinting: potentially active chromatin of the repressed maternal allele of the mouse insulin-like growth factor II (Igf2) gene. Genes Dev 1992, 6:1843-1856. 11.
MONK M, ADAMS RLP, RINALDI A: Decrease in DNA methylase
activity during preimplantation development in the mouse. Development 1991, 112:189-192. 12. HOWLETT SK, REIK W: Methylation levels of maternal and paternal genomes during preimplantation development. Development 1991, 113:119-127. 13. CARLSON LL, PAGE AW, BESTOR TH: Properties and localisation of DNA methyltransferase in preimplantation mouse embryos: implications for genomic imprinting. Genes Dev 1992, 6:2536-2541. 14. LI E, BESTOR TH, JAENISCH R: Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 1992, 69:915-926. 15. NAGY A, GOCZA F, MERENTEA DIAS E, PRIDEUX VR, IVANYI E, MARKKULA M, ROSSANT J: Embryonic stem cells alone are able to
support fetal development in the mouse. Development 1990, 110:815-821. 16.
KAY GF, PENNY GD, PATEL D,
ASHWORTH A, BROCKDORFF N,
RASTAN S: Expression of Xlst during mouse development suggests a role in the initiation of X chromosome inactivation. Cell 1993, 72:171-182. 17.
LEWIS DJ, MEEHAN RR, HENZEL WJ, MAURER-FOGY I, JEPPESON P,
KLEIN F, BIRD A: Purification, sequence and cellular localisation of a novel chromosomal protein that binds to methylated DNA. Cell 1992, 69:905-914. 18. SAI'IENZA C, PAQUETTE J, TRAN TH, PETERSON A: Epigenetic and genetic factors affect transgene methylation imprinting. Development 1989, 107:165-173. 19.
ALLEN ND, NORRIS ML, SURANI MA: Epigenetic control of trans-
gene expression and imprinting by genotype-specific modifiers. Cell 1990, 61:853-860. 20.
ENGLER P, HAASCII D, PINKERT CA, DOGLIO L, GLYMOUR M, BRINSTER R, STORB U: A strain-specific modifier on mouse chro-
mosome 4 controls the methylation of independent transgenic loci. Cell 1991, 65:939-947.
Wolf Reik and Nicholas D. Allen, Laboratory of Developmental Genetics and Imprinting, AFRC Babraham Institute, Cambridge CB2 4AT, UK.
147
GENOMIC IMPRINTING
GENOMIC IMPRINTING
Imprinting with and without methylation Methyltransferase-deficient mice reveal that DNA methylation is required for the somatic-cell maintenance of parental imprinting, which alters the expression of a gene according to the parent from which it was inherited. Genomic imprinting is an epigenetic mechanism by which some genes in an organism are expressed or repressed solely according to the parent from which they were inherited. Although the number of genes regulated by imprinting is unknown, some imprinted genes have recently been identified and have been found to provide developmentally important functions for fetal growth, viability and behaviour after birth [1]. Amongst other things, having this genetic mechanism leads to the singular requirement of needing both mothers and fathers for mammals to come into existence. One key problem in imprinting is to understand its molecular mechanism. Of the possible epigenetic modifications of DNA that could be used to establish and maintain allelic differences, methylation of cytosine in the dinucleotide CpG by the enzyme methyltransferase has received the most attention, ever since the discovery of allele-specific methylation of some transgenes in mice [1]. More recently, allele-specific methylation differences have been found for each of the three endogenous imprinted genes that have so far been analysed in detail; allele-specific differences in timing of DNA replication and of chromatin compaction have also been found [2-4]. Methylation is indeed important Until now, the significance of any of these allelic modifications has remained open to speculation. But the importance of CpG methylation to imprinting has now been demonstrated directly, in a simple and clean genetic experiment, by En Li and colleagues [5]. In brief, these workers showed that the imprinting of three known imprinted genes is altered in methyltransferasedeficient mice that were created using homologous gene recombination in embryonic stem cells. Importantly, the imprinting was altered in a manner predicted from the observed allele-specific methylation patterns in these genes. Thus, at least for the somatic maintenance of imprinting, an intact DNA-methylation system is necessary. This is a significant result, and comes as a welcome confirmation of their expectations to those who have studied parental methylation for the past several years. The genes studied in methyltransferase-deficient mice by Li et al. [51 were: the maternally repressed insulinlike growth factor 2 gene (Igf2); its paternally repressed neighbour on chromosome 7, H19, and the paternally repressed insulin-like growth factor 2 receptor gene (Igf2r) on chromosome 17. Each of these genes has a characteristic methylation pattern (Fig. 1). H19 probably
has the most conventional allele-specific methylation pattern, as methylation within the promoter correlates with repression of transcription from the gene. The H19 promoter constitutes a small CpG-rich 'island'; together with some sites upstream of the promoter and in the body of the gene, it is more heavily methylated on the repressed paternal allele than on the active maternal one [6-8]. The paternally repressed Igf2r gene also has a paternally methylated promoter region but, less conventionally, it has an intronic CpG-rich region that is highly methylated on the expressed maternal allele [7,91. Igf2, by comparison, is an unconventional gene altogether: it lacks methylation in its promoters but has a paternally more methylated region upstream of the first promoter [10], as well as a more recently discovered intronic region that is also methylated preferentially on the expressed paternal allele (R. Feil, J. Walter, N.D.A and W.R., unpublished observations). To our knowledge, preferential methylation of expressed copies of genes has been observed only with imprinted genes, perhaps indicating the presence in these genes of transcriptional silencers that can be suppressed by methylation [9]. When the allelic differences in methylation are abolished in embryos with methyltransferase deficiency, the effects are predictable: the otherwise silent paternal H19 allele is now expressed, whereas the paternal Igf2 and maternal Igf2r alleles become silenced [5]. (However, Li et al. [51 only demonstrated loss of methylation for the Igf2r intronic region and the H19 gene.) A twist in the tale Using mutant methyltransferase alleles of different 'strengths', Li and co-workers [5] found that, although expression of both H19 and Igf2 is affected by reduced methylation levels, Igf2r requires a greater reduction in methyltransferase activity for its maternal allele to be silenced. In mutant embryos homozygous for the weaker allele, n, genomic DNA was substantially demethylated but significant enzyme activity remained. For the stronger allele, s, most - but possibly not all residual activity was abolished. Nevertheless, some methylation does remain in s/s embryos, perhaps indicating that the s allele is not completely null, or that there are additional methyltransferase genes. The different susceptibilities of the Igf2, H19 and Igf2r genes to altered programming by the n and s alleles could have a simple, easily testable explanation: the Igf2r intron might have a more dense or extensive CpG-rich region than the H19 promoter. A more interesting explanation, however, could reflect different
© Current Biology 1994, Vol 4 No 2
145
146
Current Biology 1994, Vol 4 No 2
Fig. 1. Imprinted genes are differentially methylated on maternal and paternal chromosomes. Methylation of Igf2 (upstream of the gene and in an intronic region) is associated with expression, whereas for H19 methylation is associated with transcriptional repression. In the Igf2r gene, methylation of the maternally inherited allele in an intron is present in the unfertilized egg, whereas paternal methylation in the promoter region arises postzygotically. In methyltransferase mutant embryos, methylation of H19 and the Igf2r intron is decreased 5]. For the Igf2r intron, the mutant s allele of the methyltransferase gene leads to more extensive demethylation than the weaker n allele. The other regions have not yet been analysed in the mutants, but are expected also to be undermethylated.
methylation requirements for the three genes. Thus, if we consider the known genesis of the methylation patterns, we find that the known allelic differences in the H19 and Igf2 genes are not present in the parental germ cells but are first detected around the blastocyst stage of development; in embryonic stem cells, allelic differences are observed only after stem-cell differentiation (171 and R. Feil, J. Walter, N.D.A. and W.R., unpublished observations). In contrast, some of the intronic methylation on the maternal allele of the Igf2r gene is already present in oocytes - and may therefore constitute a primary imprinting signal - and this methylation is rapidly expanded during preimplantation development [7,91.
Although there is some residual methyltransferase activity in the mutant embryos at the blastocyst stage, inherited from the oocyte, it is nevertheless surprising that mutant embryos do not die sooner: embryos that have either two paternal genomes (androgenones) or two maternal genomes (parthenogenones) die significantly earlier in gestation. A better indication of the effects of mutant methyltransferase alleles might be found by examining n/n or s/s embryos derived directly from homozygous mutant embryonic stem cells, for example by aggregating mutant cells with tetraploid host embryos that serve as carriers but that do not themselves contribute extensively to the cells of the embryo [151.
This rapid post-zygotic phase of methylation of the maternal Igf2r allele may still be able to progress normally in methyltransferase mutant embryos, as oocytes contain extraordinarily high levels of maternally inherited methyltransferase activity - up to 15000 times as much as in somatic cells - which would be reduced by a factor of only two in the mutant embryos (as their mothers are heterozygotes) and which remains high until the blastocyst stage 111-131. Imprinting of the Igf2r intronic region would therefore be expected to require only a 'maintenance' methyltransferase activity in postimplantation development, whereas the more susceptible H19 and gf2 genes would require methylation de novo, possibly around implantation at the time of differentiation of the epiblast. The methyltransferase n allele might be less severely compromised in its ability to perform maintenance methylation than in methylation de novo, as the enzyme prefers hemimethylated DNA to non-methylated DNA as a substrate.
Aberrant imprinting may well contribute to the death of the mutant embryos, even though death comes later than in embryos with a monoparental chromosome constitution. But, for the imprinted genes under discussion, we know that having excess copies of the H19 gene does not kill at embryonic stages but later in gestation; fetuses that lack Igf2r die before or soon after birth; and deficiency of Igf2 simply makes mice smaller than their wild-type littermates. Of course, it is likely that other, as-yet unknown, imprinted genes are also deregulated in methyltransferase-deficient embryos and could therefore contribute to the phenotype.
Do methyltransferase-deficient mice die as a result of aberrant imprinting? Mutant embryos homozygous for either n or s alleles die in utero at early post-implantation stages -- day 11 and day 10 of development, respectively - having completed gastrulation and early organogenesis [141.
The most likely cause of death in the mutant embryos is the general deregulation of a large number of genes. Although the brevity of the paper by Li and co-workers [51 is commendable, we anxiously await data addressing the possible deregulation of non-imprinted genes. Of particular interest will be the genes carried on the inactive of the two X chromosomes in females, which are subject to X-inactivation; many of these, like some of the known imprinted genes, have methylated CpG-rich islands. The issue of inactivation of one of the two X chromosomes is linked to that of imprinting, as the paternal X chromosome is preferentially inactivated in extra-embryonic tissues; this probably results from the
DISPATCH
imprinting of the Xist gene, which is maintained only in the very early embryo [161. Another factor that might affect the methyltransferasedeficient mice is the widespread de novo methylation of the genome that normally occurs at around gastrulation and that affects a large number of genes. Is the regulation of all these genes no longer tight without DNA methylation? Global loss of methylation might also have consequences for ordered chromatin structure: the generation of mice lacking methyl-cytosine-binding proteins [17] is eagerly awaited, so that the effects on imprinting can be assessed and their phenotype can be compared with that of the methyltransferase-deficient mutants. Prospects The study by Li et al. [51 justifies previous prejudices that methylation is important in the somatic maintenance of imprinting. Although methylation is now shown to be required, we are left with some very challenging problems. We are still no closer to understanding the nature of the primary imprinting events that occur in the germline. The role of methylation in the germline is difficult to address using the current mutant embryonic stem cells or mice, partly because of the oocyte's inheritance of huge amounts of methyltransferase. This problem might potentially be solved by making conditional germline-specific methyltransferase mutations, so that methyltransferase function could be provided for the duration of development to adulthood but eliminated from the process of oogenesis. The issue is of particular relevance in the case of the Igf2r gene, in which the intronic sites become methylated during oocyte growth. Clearly it would be very interesting to produce an oocyte that lacked methyltransferase: in practice this may prove difficult to achieve, but if successful it would allow us to address the molecular nature of the germline imprint. A second issue that could be addressed more easily by the generation of conditional mutants is the control of the tissue specificity of imprinting, which is being found increasingly - the best example is the lack of imprinting of the Igf2 gene in the leptomeninges and choroid plexus of the brain. It is to be hoped that the n/n and s/s mutant mice represent the beginning of a genetic analysis of the effects of methylation on imprinting. Finally, methylation can only be considered a part of the molecular story of imprinting. Even with a detailed knowledge of some of the allelic methylation differences that exist in imprinted genes, we cannot explain the specificity with which they arise and must invoke other 'imprinting control genes', if only to direct methyltransferase activity to the right place at the right time. Clearly, new strategies must be developed to address these more complex problems. Amongst the candidates for 'imprinting control genes' are the modifier genes that have been shown genetically to determine DNA methylation patterns of some transgenes in a fashion that varies according to the genetic background
of the organism [18-201. It will be important to know at which stages of development these genes act, and whether or not some of the imprinted genes are among their endogenous targets. References 1.
2. 3. 4. 5.
6. 7. 8. 9.
10.
SURANI A, REIK W (EDS): Genomic imprinting in mouse and man. Semin Dev Biol 1992, 3:73-160. BIRD AP: Imprints on islands. CurrBiol1993, 3:275-277. SURANI MA: Genomic imprinting: silence of the genes. Nature 1993, 366:302-303 KITSBERG D, SELIG S, BRANDEIS M, SIMON , KESHET I, DRISCOLL DJ, NICHOLLS RD, CEDAR H: Allele-specific replication timing of imprinted gene regions. Nature 1993, 364:459-463. LI E, BEARD C, JAENISCH R: Role of DNA methylation in genomic imprinting. Nature 1993, 366:362-365. FERGUSON-SMITH AC, SASAKI H, CATTANACH BM, SURANI MA:
Parental-origin-specific epigenetic modification of the H19 gene. Nature 1993, 362:751-754. BRANDEIS M, KAFRI T, ARIEL M, CHAILLET' JR, RAZIN A, CEDAR H: The ontogeny of allele-specific methylation associated with imprinted genes in the mouse. EMBOJ 1993, 12:3669-3677. BARTOLOMEI MS, WEBER AL, BRUNKOW ME, TILGHMAN SM: Epigenetic mechanisms underlying the imprinting of the mouse H19 gene. Genes Dev 1993, 7:1663-1673. STOGER R, KUBICKA P, LIU C-G, KAFRI T, CEDAR H, BARLOW D: Maternal-specific methylation of the imprinted mouse IgJ2r locus identifies the expressed locus as carrying the imprinting signal. Cell 1993, 73:61-71. SASAKI H, JONES PA, CHAILLET JR, FERGUSON-SMITH AC, BARTON
SC, REIK W, SURANI MA: Parental imprinting: potentially active chromatin of the repressed maternal allele of the mouse insulin-like growth factor II (Igf2) gene. Genes Dev 1992, 6:1843-1856. 11.
MONK M, ADAMS RLP, RINALDI A: Decrease in DNA methylase
activity during preimplantation development in the mouse. Development 1991, 112:189-192. 12. HOWLETT SK, REIK W: Methylation levels of maternal and paternal genomes during preimplantation development. Development 1991, 113:119-127. 13. CARLSON LL, PAGE AW, BESTOR TH: Properties and localisation of DNA methyltransferase in preimplantation mouse embryos: implications for genomic imprinting. Genes Dev 1992, 6:2536-2541. 14. LI E, BESTOR TH, JAENISCH R: Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 1992, 69:915-926. 15. NAGY A, GOCZA F, MERENTEA DIAS E, PRIDEUX VR, IVANYI E, MARKKULA M, ROSSANT J: Embryonic stem cells alone are able to
support fetal development in the mouse. Development 1990, 110:815-821. 16.
KAY GF, PENNY GD, PATEL D,
ASHWORTH A, BROCKDORFF N,
RASTAN S: Expression of Xlst during mouse development suggests a role in the initiation of X chromosome inactivation. Cell 1993, 72:171-182. 17.
LEWIS DJ, MEEHAN RR, HENZEL WJ, MAURER-FOGY I, JEPPESON P,
KLEIN F, BIRD A: Purification, sequence and cellular localisation of a novel chromosomal protein that binds to methylated DNA. Cell 1992, 69:905-914. 18. SAI'IENZA C, PAQUETTE J, TRAN TH, PETERSON A: Epigenetic and genetic factors affect transgene methylation imprinting. Development 1989, 107:165-173. 19.
ALLEN ND, NORRIS ML, SURANI MA: Epigenetic control of trans-
gene expression and imprinting by genotype-specific modifiers. Cell 1990, 61:853-860. 20.
ENGLER P, HAASCII D, PINKERT CA, DOGLIO L, GLYMOUR M, BRINSTER R, STORB U: A strain-specific modifier on mouse chro-
mosome 4 controls the methylation of independent transgenic loci. Cell 1991, 65:939-947.
Wolf Reik and Nicholas D. Allen, Laboratory of Developmental Genetics and Imprinting, AFRC Babraham Institute, Cambridge CB2 4AT, UK.
147