Genomic Imprinting in Mice: Its Function and Mechanism'

BIOLOGY OF REPRODUCTION 54, 273-278 (1996) Genomic Imprinting in Mice: Its Function and Mechanism'

Philip A. Leighton, Jennifer R. Saam, Robert S. Ingram, and Shirley M. Tilghman 2

Howard Hughes Medical Institute and Departmentof Molecular Biology Princeton University, Princeton, New Jersey 08544

ABSTRACT

Genomic imprinting is an epigenetic phenomenon by which the two parental alleles of a gene are differentially expressed. Although Downloaded from https://academic.oup.com/biolreprod/article/54/2/273/2761811 by guest on 30 September 2021 the function of genomic imprinting is not clear, it has been proposed that it evolved in mammals to regulate intrauterine growth. This proposal is consistent with experiments that were designed to reveal the mechanism and impact of genomic imprinting in a region of mouse chromosome 7 that contains four imprinted genes: Mash-2 (a transcription factor) and H19 (a noncoding RNA) are maternally expressed, whereas Insulin-2(lns-2) and Insulin-like growth factor2(lgf-2) are paternally expressed. Two targeted disruptions at the locus were generated in mice; these support the hypothesis that the function of the H19 gene is to set up the imprinting of both Igf-2 and Ins- 2.H19transcription on the maternal chromosome precludes transcription of the other two genes by a mechanism that involves competition for a common set of enhancers. On the paternal chromosome the H19 gene is silenced by DNA methylation, thus permitting the use of enhancers by the other genes.

INTRODUCTION allelic inactivation by imprinting. The function presumably Genomic imprinting is an epigenetic phenomenon by must confer selective advantage to the organism, as it must which the two parental alleles of a gene are differentially counterbalance the obvious attendant risk of hemizygosity. expressed [1, 2]. The existence of genomic imprinting in Several explanations have been proposed for the acquisi- mice was first deduced from nuclear transplantation exper- tion of genomic imprinting in eutherian mammals, includ- iments in which diploid zygotes were generated with ex- ing protection against parthenogenesis and ovarian tumors clusive genetic contribution from either mothers or fathers as well as chromosome loss [11-14]. To date the most com- [3, 4]. Neither kind of uniparental embryo was able to sus- pelling model has been provided by Haig and his col- tain normal development, and the conclusion was drawn leagues [15], who suggest that imprinting evolved in mam- that the lethality was due to the absence or overexpression mals because of the conflicting interests of maternal and of one or more gene products as the result of the unequal paternal genes within a litter. In mammals, which are pri- expression of genes in the maternal and paternal genomes. marily nonmonogamous, the mother provides significant As of 1995 there are twelve genes known to be subject maternal resources to the offspring both during intrauterine to genomic imprinting in mammals, three that are mater- life and during suckling after birth. Successful passage of nally expressed and nine that are paternally expressed (Ta- paternal genes into the next generation is best ensured by ble 1). However, on the basis of the mapping of imprinted having the embryos consume maternal resources, even if regions in the mouse genome using classical genetics [5], it by so doing the fitness of her future litters is compromised. is evident that Table 1 contains only a subset of the im- The mother's interests are best served by distributing her printed genes in the genome. For most genes that have been resources more equitably among litters. tested, there is conservation of imprinting between mice Haig's model predicts that paternally expressed genes and humans, although there is at least one exception, the will promote embryonic growth whereas maternal genes Insulin-like growth factor 2 receptor (Igf-2r), which is ma- will act to restrain the use of maternal resources. In so do- ternally expressed in mice but biallelic in humans [6-8]. ing, this model is in remarkably good agreement with the Some of the imprinted genes are organized in clusters, and function of at least three imprinted genes: Insulin-like two of the clusters, on mouse chromosomes 7 and 17, con- growthfactor2 (Igf-2), H19, and Igf-2r. Mouse embryos are tain both maternally and paternally expressed genes [9, 10]. exquisitely sensitive to the levels of the paternally expressed growth factor insulin-like growth factor II (IGFII). A complete loss of function of the gene encoding the THE FUNCTION OF GENOMIC IMPRINTING growth factor, Igf-2, leads to a 40% reduction in birth weight With a growing list of imprinted genes in hand, the chal- [16]. Igf-2r, which is maternally expressed, encodes a bi- lenge is now to determine the function and mechanism of functional protein that acts as a sink for the growth factor. Fetuses that lack the receptor are approximately 30% larger 1 Supported by a grant from the National Institutes of Health (GM51460). 2 than normal, have elevated circulating levels of IGFII, and Correspondence: Shirley M. Tilghman, Department of Molecular Biology, Princeton University, Lewis Thomas Laboratory, Room 127, Washington Road, Princeton, NJ 08544. die around birth [17, 18]. It has been argued that the lethality FAX: (609) 258-3345; e-mail: [email protected] is the consequence of a gain of function of the effective

273 274 LEIGHTON ET AL.

TABLE 1. Chromosomal location if imprinted Gene Expression In mouse In human References Igf-2 paternal distal 7 11p15.5 [16, 47 481 H19 maternal distal 7 11p15.5 [28, 49, 50] Insulin-2 paternal distal 7 not tested [241 Mash-2 maternal distal 7 not tested [22] Snrpn paternal central 7 15q13-14 [51, 521 Znf-127 paternal central 7 15q13-14 R. Nicholls, personal comm. IPW paternal not tested 15q13-14 [451 Igf-2r maternal proximal 17 not imprinted 16, 7, 81

Mas paternal proximal 17 not tested 191 Downloaded from https://academic.oup.com/biolreprod/article/54/2/273/2761811 by guest on 30 September 2021 Xist paternal x X [53, 54] U2afbp-rs paternal proximal 11 not tested [551 Peg-I/Mest paternal proximal 6 not tested [561

. ______._ _n- ~11tr\~n the~ distnl~t"I1 ~ nnrtinn Cu1 1 nf1 mnlm11 o rchrnmnnmo1 1 11 1 1 7 thath t Itnntains tI. Mash-2 Ins-2 IXf2 H19 four imprinted genes (Fig. 1). The Mash-2 gene, one of a family of helix-loop-helix proteins, is expressed from the Mat Pat Pat Mat maternal chromosome in spongiotrophoblasts of the mouse placenta [21-23]. The Insulin-2gene (Ins-2), which lies ap- 100kb ---" -- - I 1. -- ... - II I I...1. "1 1 - I "~ .. I ~ [ l. alllla.ly AJtJ- JVJ llThI 11 IJIUJIfLJ 1 Jt'l CA- I ly FIG. 1. An imprinted region on the distal end of nnouse chromosome 7. The po- pressed in the yolk sac of the mouse embryo but biallelically sitions of the imprinted genes Mash-2, Ins-2, Igf- (Igf2 on figure), and H19 on expressed in the pancreas [24]. The paternally expressed mouse chromosome 7 [10, 221 are indicated. Mat, maternally expressed; Pat, pa- Igf-2 gene lies immediately downstream from Ins-2 [16]; and ternally expressed. at the end of the cluster lies the H19gene, an unusual gene concentration of circulating IGFII, orn the basis of the ob- that codes for a nontranslatable RNA [25-27]. Its expression servation that the lethality can be abrmutation []18, 19]. As will be dis- The clustering of four imprinted genes raised the possibility cussed below, the maternally exprn2ssed H19 gene also that imprinting, like X chromosome inactivation, is controlled functions to lower the concentration of IGFII by suppress- by a signal or signals that can act over large distances and ing its transcription on the maternal chromosome [20]. The multiple genes. Although the Igf-2 and H19 genes are ex- implication of these experiments is that imprinting of all pressed from different parental chromosomes, it seemed three genes is functioning to finely reregulate the expression likely that their imprinting was mechanistically linked, as evi- of the Igf-2 gene. denced by the observation that their temporal and tissue-specific patterns of expression are essentially identical [29, 30]. A MODEL FOR THE MECHANIS ;M OF GENOMIC We proposed that the mechanism relied on a competition be- IMPRINTING tween their promoters for a set of shared regulatory elements such as enhancers (Fig. 2) [31, 32]. To begin to dissect the molecular rmechanism of imprint- The original model proposed that the competition was ing, we have focused on a region of approximately 300 kb biased differently on the two parental chromosomes by epigenetic mark(s) placed there during gametogenesis. A search for candidates for the epigenetic mark focused from -T__ 9 the outset on parent-specific DNA methylation, as it was possible to envisage a scenario in which the sperm and egg Ins-2 Igf2 H19 chromosomes could be transmitted with different methylation patterns and that those patterns could be propagated through many somatic cell divisions through the activity of ) DNA methyltransferase, a hemi-methylase that acts at rep- lication forks to reassemble fully methylated DNA. FIG. 2. The enhancer competition model to explain the opposite imprinting of H19 The best candidate for the epigenetic mark at the H19/ and Igf-2(1gf2 on figure)/Ins-2. The genes are indicalted by the boxes, with the hor- izontal arrows indicating the transcribed alleles. The two H193' endoderm enhanc- Igf-2/Ins-2 locus is a region of extensive paternal-specific ers are designated by filled circles. The positions of allele-specific methylation of DNA methylation of the H19 gene and its 5' flank [31, 33]. the H19 gene are indicated by the CH3 symbol. The curved arrows leading from the This methylation is thought to act as a negative signal to enhancers indicate the engagement of the enhancers with the H19 gene on the maternal allele and the Ins-2 and Igf-2 genes on the paternal allele. The imprinting block the interaction of the paternal H19 gene with the en- of the Ins-2 gene is restricted to the yolk sac. hancers, thereby making them available to engage the pa- GENOMIC IMPRINTING: FUNCTION AND MECHANISM 275 ternal Igf-2 gene. On the maternal chromosome, where ,,v both genes are undermethylated and there is no sign of an i v / epigenetic mark, it may be that the H19 is preferentially transcribed, either because it has an inherently stronger promoter or because it resides in closer proximity to the reg- / ulatory elements. Evidence in favor of DNA methylation as the epigenetic mark at the locus comes from a targeted disruption of the DNA methyltransferase gene in mice [34]. Embryos that are homozygous for a null mutation in the gene, and therefore Downloaded from https://academic.oup.com/biolreprod/article/54/2/273/2761811 by guest on 30 September 2021 have dramatically reduced levels of DNA methylation, express both alleles of H19 and have silenced both Igf-2 genes [35]. This result is consistent with the enhancer competition model, as the loss of methylation would convert a paternal chromosome into a phenocopy of a maternal chromosome (see Fig. 2). The evidence that what is important is the methylation of sequences surrounding the H19gene itself is based on the observation that the methylation is inherited from sperm and is stably maintained through a period of mouse embryogenesis when the majority of genomic DNA methylation is erased [36]. Furthermore, these sequences are sufficient to confer appropriate imprinting on a transgene [31].

TESTING THE ROLE OF ENHANCERS IN IMPRINTING The enhancer competition model rests on the premise that H19 and Igf-2 utilize the same enhancers. Only two enhancers have been identified in the vicinity of the Igf-2 and H19 genes, at + 9 and + 11 kb relative to the start of transcription of the H19 gene [371. These enhancers displayed endoderm specificity in both tissue culture and transgenic mouse assays. To ask whether they are required for expression of both H19 and Igf-2, a line of mice was generated in which the enhancers were deleted, and the consequence to the expression of H19 and Igf-2 was assessed [38] (Fig. 3A). When the enhancer deletion was inherited from females, there was a dramatic decline in H19 RNA in all tissues that contained cells of endodermal origin, as illustrated for liver in Figure 3B. The levels of Igf-2 RNA were unaffected by the maternal deletion of the enhancers, consistent with the fact that this allele of Igf-2 is normally silent. When the enhancer deletion was inherited from fathers, FIG. 3. Effect of the H19 enhancer deletion on the expression of H19 and Igf-2. A) however, the levels of Igf-2 RNA declined in exactly the same Organization of the wild-type H19 gene (upper line) and the H19 enhancer deletion chromosome (lower line), with the neomycin resistance gene replacing the 3' en- manner as H19RNA in the maternal heterozygotes, while the doderm enhancers (filled circles). B) Male or female enhancer deletion heterozy- levels of H19 RNA were unaffected (Fig. 3B). Thus the 3' en- gous mice were mated to wild-type animals, and total liver RNA was isolated from doderm enhancers are required equally for expression of the wild-type (+/+) or heterozygous (-/+) neonatal progeny. Each RNA was hybridized to radiolabeled H19 and Igf-2 (lgf2on figure) RNA probes. The migration of the two genes but on different parental chromosomes. H19 and Igf-2 protected fragments is indicated by the arrows on the right. Lanes M, radiolabeled pBR322 digested with Mspl; lanes pH19 and plgf-2, migration of the TESTING THE ROLE OF THE H19 GENE ON Igf-2 full-length probes; lanes H19 and Igf-2, protected fragments from neonatal liver IMPRINTING RNA hybridized with the H19 probe or Igf-2 probe separately. (Adapted from [381.) As noted above, the best candidate for the epigenetic H19 gene on the maternal chromosome is an effective com- mark at the Igf-2/H19 locus that acts to bias the choice for petitor for the enhancers, whereas the methylation on the the enhancers is paternal-specific methylation at the H19 paternal chromosome serves to silence the H19 gene and gene itself. According to this scenario, the unmethylated permits Igf-2 transcription. To test the dependence of Igf-2 276 LEIGHTON ET AL.

A. 3kb

.,~~H pp* '% 4. %'4 o- % % 1% 0. A4-. Downloaded from https://academic.oup.com/biolreprod/article/54/2/273/2761811 by guest on 30 September 2021

FIG. 4. Effect of the H19 deletion on expression of Igf-2. A) Organization of the wild-type H19 gene (upper line) and the H19 gene deletion chromosome (lower line). The filled circles indicate the 3' enhancers. B)Effect of maternal inheritance of the H19 deletion. Heterozygous mutant F1females were mated to B6(CAST-H19) [361 females, and RNA was isolated from livers of neonatal pups. An allele-specific RNAse protection assay that distinguishes between the 129Sv/Ev-derived paternal Igf-2 allele (Pat) and the M. castaneus-derived maternal allele (Mat) was used to assess the expression of gf-2. The lanes designations are M, marker; P, undigested probe; C, M. castaneus RNA, wild-type (+/+) progeny RNA, and heterozygous (+/-) progeny RNA. The B6 lane represents C57BL/6J-derived neonatal liver RNA, which behaves identically to 129Sv/Ev RNA. (Adapted from [20]). imprinting on the H19 gene, a strain of mice was generated the two alleles were detected at equivalent levels (Fig. 4B). that carries a deletion of the paternal-specific methylation Thus the effect of the H19 gene on expression of Igf-2 is domain, including the structural H19 gene itself and 10 kb restricted to those tissues in which Igf-2 is imprinted. of 5' flanking sequence [20] (Fig. 4A). The imprinting of Ins-2 was also disrupted by the H19 Since the paternal allele of H19 is normally silent, it was gene deletion [201. The loss of imprinting of the Ins-2 gene anticipated that paternal inheritance of the H19 deletion can be reconciled with the enhancer competition model if would have no phenotypic consequence. This proved to be the expression of that gene is under the control of the same the case, as both the maternal H19 allele and the paternal regulatory elements that govern H19 and Igf-2 expression allele of Igf-2 were unaffected by the H19 deletion in cis. in yolk sac (see Fig. 2). That is, on the paternal chromosome However, when the H19 deletion was inherited from a fe- the shared enhancers simultaneously activate Igf-2 and Ins- male, the neonatal progeny expressed both paternal and 2 when the H19 gene is in a silent state. In pancreas, on the maternal Igf-2 alleles in all tissues examined (Fig. 4B). Thus other hand, the pancreas-specific enhancers that lie 5' to deletion of the H19 gene and its 5' flanking sequences re- the gene direct biallelic Ins-2 expression [39, 40]. sults in the loss of imprinting of the Igf-2 gene, which lies The fact that the impact of the deletion of the H19 gene 80 kb 5' to the deletion. extends at least 95 kb to the Ins-2 gene raised the possibility The Igf-2 gene normally displays biallelic expression in that Mash-2, which is at least another 200 kb away, was also only two tissues during development, the choroid plexus affected. This is not the case, as neither the enhancer nor the and leptomeninges of the brain [16]. When Igf-2 expression gene deletion affected the imprinted expression of Mash-2 was examined in brains of maternal H19 heterozygotes, [20, 38]. Thus the influence of the H19 gene and its imprint there was no consequence of the deletion of H19 in cis, as does not extend the 200-300 kb distance to Mash-2. GENOMIC IMPRINTING: FUNCTION AND MECHANISM 277

THE POTENTIAL ROLE FOR RNA IN GENOMIC sensitive to the levels of this peptide, as mutations that de- IMPRINTING crease the levels of IGFII affect their growth rate. For ex- The deletion of the H19 gene was a large one, extending ample, mice inheriting a null mutation in Igf-2 from fathers over 10 kb of 5' flanking DNA as well as the gene itself, and are 60% of the normal size at birth and maintain this pro- therefore it is possible that within the deletion lie multiple portional dwarfism throughout adult life [16, 46]. Newborns essential regions required for Igf-2 and Ins-2 imprinting. that inherit the H19 enhancer deletion from fathers were The simplest model is one in which there is a single "im- found to express lower levels of Igf-2 RNA only in endo- printer" that acts on all three genes. We have favored the dermal tissues [38]. Their weight was between 70 and 73% notion that the imprinter is the very strong H19 promoter, that of their wild-type littermates at birth and was 80% at 3 whose activity on the maternal chromosome precludes Igf- wk of age-intermediate in size between mice that were Downloaded from https://academic.oup.com/biolreprod/article/54/2/273/2761811 by guest on 30 September 2021 2 expression. Its inactivation by DNA methylation on the null for Igf-2 and wild-type animals. paternal chromosome is both necessary and sufficient for The biologically active level of IGFII is regulated by two the expression of Igf-2. It is also possible, however, that this maternally expressed genes: through a transcriptional 13-kb domain contains a separate element that blocks the mechanism mediated by H19, which inactivates Igf-2 tran- access of Igf-2 to the enhancers on the maternal chromo- scription on the maternal chromosome, and through the some and that this element is inactivated by DNA methyl- action of the product of the Igf-2r gene, which encodes a ation on the paternal chromosome. binding protein for IGFII that functionally inactivates the Finally, it is possible that the silencing of the maternal growth factor. These two genes, by controlling the expres- Ins-2 and Igf-2 genes requires the product of the maternal sion of a critical growth factor, beautifully fulfill the predic- H19 gene itself. This proposal is derived from reports that tions of Haig's hypothesis for the function of genomic im- the genes for untranslated RNAs similar in overall structure printing in mammals. to H19 have been described in two other imprinted loci. Xist RNA is encoded by a gene that maps to the X chro- ACKNOWLEDGMENTS mosome inactivation center and is exclusively expressed from the paternally inherited inactive X chromosome in The authors are indebted to Drs. Argiris Efstratiadis, Colin Stewart, and Jonathan Eggen- schwiler for contributing to the studies in this review. We also thank the members of the early mouse embryos [41-44]. A noncoding RNA that maps Tilghman laboratory for many enthusiastic discussions during the course of the work. to the Prader-Willi syndrome locus on human chromosome 15 is paternally expressed [45]. The evident conservation of the primary and secondary structure in these RNAs argues REFERENCES that they serve some function in mammals, and their pres- 1. Solter D. Differential imprinting and expression of maternal and paternal genomes. Annu ence in imprinted domains raises the idea that they may Rev Genet 1988; 22:127-146. 2. Efstratiadis A. Parental imprinting of autosomal genes. Curr Opin Genet Dev 1994; function in initiating or maintaining the imprinting of neigh- 4:265-280. boring genes. Further mutations that separate the transcrip- 3. McGrath J, Solter D. Nuclear transplantation in mouse embryos. J Exp Zool 1983; tional role of H19 from its product will be required to test 228:355-362. 4. Surani MAH, Barton SC, Norris ML. Development of reconstituted mouse eggs suggest this intriguing possibility. imprinting of the genome during gametogenesis. Nature 1984; 308:548-550. 5. Beechey CV, Cattanach BM, Searle AG. Genetic imprinting map. Mouse Genome 1990; 87:64-65. THE ROLE OF GENOMIC IMPRINTING 6. Barlow DP, Stoger R, Herrmann BG, Saito K, Schweifer N. The mouse insulin-like growth factor type-2 receptor is imprinted and closely linked to the Tme locus. Nature 1991; IN GROWTH CONTROL 349:84-87. The most striking phenotype associated with the deletion 7. Kalscheuer VM, Mariman EC, Schepens MT, Rehder H, Ropers HH. The insulin-like growth factor type-2 receptor gene is imprinted in the mouse but not in humans. Nature of the H19 gene in maternal heterozygotes at birth was an Genet 1993; 5:74-78. apparently uniform increase in body weight. It was found 8. Xu Y, Goodyer CG, Deal C, Polychronakos C. Functional polymorphism in the parental that at birth, maternal heterozygotes were 27% larger than imprinting of the human IGF2R gene. Biochem Biophys Res Commun 1993; 197:747- 754. their wild-type littermates, a difference that persisted in 9. Villar AJ, Pedersen RA. Parental imprinting of the Mas protooncogene in mouse. Nature adult mice as well [20]. This somatic overgrowth was attrib- Genet 1994; 8:373-379. uted to the gain of function of Igf-2 that occurrs when fe- 10. Zemel S, Bartolomei MS, Tilghman SM. Physical linkage of two mammalian imprinted genes. Nature Genet 1992; 2:61-65. males with the H19 deletion are crossed with males that 11. Varmuza S, Mann M. Genomic imprinting-defusing the ovarian time bomb. Trends carry a disruption in the Igf-2 gene. The double mutants Genet 1994; 10:118-123. were normal in size. Furthermore, no other phenotype was 12. Varmuza S. Gametic imprinting as a speciation mechanism in mammals. J Theor Biol 1993; 164:1-13. apparent, implying that the loss of function of H19 alone 13. Barlow DP. Methylation and imprinting: from host defense to gene regulation? Science has no deleterious effect in mice other than its gain-of-func- 1993; 260:309-310. tion effects on Igf-2 and Ins-2. Thus the H19 gene is the 14. ThomasJH. Genomic imprinting proposed as a surveillance mechanism for chromosome loss. Proc Natl Acad Sci USA 1994; 92:480-482. second paternally imprinted gene whose function involves 15. Moore T, Haig D. Genomic imprinting in mammalian development: a parental tug-of- suppression of the activity of IGFII. Mice are exquisitely war. Trends Gen 1991; 7:45-89. 278 LEIGHTON ET AL.

16. DeChiara TM, Robertson EJ, Efstratiadis A. Parental imprinting of the mouse insulin-like 39. Edlund T. Walker MD. Barr PJ, Rutter WJ. Cell-specific expression of the rat insulin gene: growth factor II gene. Cell 1991; 64:849-859. evidence for role of two distinct 5' flanking elements. Science 1985; 230:912-916. 17. Lau MMH, Stewart CEH, Liu Z, Bhatt H, Rotwein P. Stewart CL. Loss of the imprinted 40. Ohlsson H, Thor S., Edlund T. Novel insulin promoter- and enhancer-binding proteins that IGF2/cation-independent mannose 6-phosphate receptor results in fetal overgrowth and discriminate between pancreatic alpha- and beta-cells. Mol Endocrinol 1991; 5:897-904. perinatal lethality. Genes &Dev 1994; 8:2953-2963. 41. Brown CJ,Hendrich BD, Rupert JL, Lafreniere RG, Xing Y, Lawrence J., Willard HF. The 18. Wang Z-Q, Fung MR, Barlow DP, Wagner EF. Regulation of embryonic growth and Iy- human XISTgene: analysis of a 17 kb inactive X-specific RNA that contains conserved sosomal targeting by the imprinted Igf2/,Mprgene. Nature 1994; 372:464-467. repeats and is highly localized within the nucleus. Cell 1992; 71:527-542. 19. Filson A, Louvi A, Efstradiatis A, Roberston EJ. Rescue of the T-associated maternaleffect 42. Brockdorff N, Ashworth A, Kay GF, McCabe VM,Norris DP, Cooper PJ, Swift S, Rastan in mice carrying null mutations in Igf-2 and Igf2r, two reciprocally imprinted genes. S. The product of the mouse Xist gene is a 15 kb inactive X-specific transcript containing Development 1993; 118:731-736. no conserved ORF and located in the nucleus. Cell 1992; 71:515-526. 20. Leighton PA, Ingram RS, Eggenschwiler J, Efstratiadis A, Tilghman SM. Disruption of 43. Hendrich BD, Brown CJ, Willard HF. Evolutionary conservation of possible functional imprinting caused by deletion of the H19gene region in mice. Nature 1995; 375:34-39 domains of the human and murine XSTgenes. Hum Mol Genet 1993; 2:663-672. 21. Johnson JE, Birren SJ, Anderson DJ. Two rat homologues of Drosophila achaete-scute 44. Kay GF, Penny GD, Patel D, Ashworth A, Brockdorff N, Rastan S. Downloaded from https://academic.oup.com/biolreprod/article/54/2/273/2761811 by guest on 30 September 2021 specifically expressed in neuronal precursors. Nature 1990; 346:858-861. Expression of Xist during mouse development suggests a role in the initiation 22. Guillemot F, Caspary T, Tilghman SM, Copeland NG, Gilbert DJ, Jenkins NA, Anderson of X chromosome inactivation. DJ, Joyner AL, Rossant J, Nagy A. Genomic imprinting of Mlash2, a mouse gene required Cell 1993; 72:171-182. 45. Wevrick R, Kerns for trophoblast development. Nature Genet 1995; 9:235-241. JA, Francke U. Identification of a novel paternally expressed gene in 23. Guillemot F, Nagy A, Auerbach A, Rossant J, Joyner AL. Essential role of Mash-2 in the Prader-Willi syndrome region. Hum Mol Genet 1994: 3:1877-1882. extraembryonic development. Nature 1994; 371:333-336. 46. DeChiara TM, Efstratiadis A, Robertson EJ. A growth-deficiency phenotype in hetero- 24. Giddings SJ, King CD, Harman KW, Flood JF, Carnaghi LR.Allele specific inactivation of zygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature insulin 1 and 2, in the mouse yolk sac, indicates imprinting. Nature Genet 1994; 6:310- 1990; 345:78-80. 313. 47. Giannoukakis N, Deal C, Paquette J, Goodyer CG, Polychronakos C. Parental genomic 25. Pachnis V, Belayew A, Tilghman SM. Locus unlinked to -fetoprotein under the control imprinting of the human IGF2 gene. Nature Genet 1993; 4:98-101. of the murine rafand Rifgenes. Proc Natl Acad Sci USA 1984; 81:5523-5527. 48. Ohlsson R, Nystrom A, Pfeifer-Ohlsson S, Tohonen V. Hedborg F, Schofield P. Flan F. 26. Pachnis V, Brannan CI, Tilghman SM. The structure and expression of a novel gene Ekstrom TJ. IGF2 is parentally imprinted during human embryogenesis and in the Beck- activated in early mouse embryogenesis. EMBOJ 1988; 7:673-681. with-Wiedemann syndrome. Nature Genet 1993: 4:94-97. 27. Brannan C, Dees EC, Ingram RS, Tilghman SM. The product of the H19 gene may 49 Rachmilewitz J, Goshen R, Ariel I, Schneider T, de Groot N, Hochberg A. Parental im- function as an RNA. Mol Cell Biol 1990 10:28-36. printing of the human H19 gene. FEBS Lett 1992; 309:25-28. 28. Bartolomei MS, Zemel S, Tilghman SM. Parental imprinting of the mouse H19 gene. 50. ZhangY,. Tycko B. Monoallelic expression of the human H19 gene. Nature Nature 1991; 351:153-155. Genet 1992: 1:40-44. 29. Lee JE, PintarJ, Efstratiadis A. Pattern of the insulin-like growth factor II gene expression 51. LeffSE, Brannan CI, Reed ML,Ozcelik T, during early mouse embryogenesis. Development 1990; 110:151-159. Francke U, Copeland NG,Jenkins NA. Maternal imprinting of the mouse Snrpn gene and 30. Poirier F, Chan C-TJ.Timmons PM, Robertson EJ, Evans MJ, Rigby PWJ. The murine H19 conserved linkage homology with the human gene is activated during embryonic stem cell differentiation in vitro and at the time of Prader-Willi syndrome region. Nature Genet 1992; 2:259-264. implantation in the developing embryo. Development 1991; 113:1105-1114. 52 Reed ML, Leff SE. Maternal imprinting of human SNRPN, a gene deleted in Prader-Willi 31 Bartolomei MS, Webber AL, Brunkow ME, Tilghman SM. Epigenetic mechanisms un syndrome. Nature Genet 1994; 6:163-167. derlying the imprinting of the mouse H19 gene. Genes &Dev 1993: 7:1663-1673. 53. Brown CJ,Ballabio A, Rupert JL, Lafreniere RG, Grompe M, Tonlorenzi R, Willard HF. A 32. Bartolomei MS, Tilghman SM. Parental imprinting of mouse chromosome 7. Semin Dev gene from the region of the human X chromosome inactivation centre is expressed Biol 1992 3:107-117. exclusively from the inactive X chromosome. Nature 1991; 349:38-44. 33. Ferguson-Smith AC, Sasaki H, Cattanach BM, Surani MA, Parental-origin-specific epi 54. Brockdorff N, Ashworth A, Kay GE, Cooper P, Smith S, McCabe VM, Norris DI', Penny genetic modifications of the mouse H19 gene. Nature 1993; 362:751-755. GD, Patel D, Rastan S. Conservation of position and exclusive expression of mouse Xist 34. LiE, Bestor TH, Jaenisch R. Targeted mutation of the DNA methyltransferase gene results from the inactive X chromosome. Nature 1991; 351:329-331. in embryonic lethality. Cell 1992; 69:915-926. 55. Hayashizaki Y, Shibata H, Hirotsune S, Sugino H, Okazaki Y, SasakiN, Hirose K, Imoto 35. Li E, Beard C, aenisch R. The role of DNA methylation in genomic imprinting. Nature H, Okuizumi H, Muramatsu M, Komatsubara H, Shiroishi T, Moriwaki K. Katsuki M, 1993; 366:362-365. HIatano N, Sasaki H, Ueda T, Mise N, Takagi N, Plass C, Chapman VM.Identification of 36. Tremblay KD, Saam J, Ingram RS, Tilghman SM, Bartolomei MS. Gametic methylation an imprinted U2af binding protein related sequence on mouse chromosome and its maintenance in the imprinted H19 gene. Nature Genet 1995; 9:407-413. 11 using the RLGS method. Nature 37. Yoo-Warren H, Pachnis V, Ingram RS, Tilghman SM. Two regulatory domains flank the Genet 1994; 6:33-40. 56. Kaneko-Ishino T, Kuroiwa mouse H19 gene. Mol Cell Biol 1988; 8:4707-4715. Y, Miyoshi N, Kohda T, Suzuki R, Yokoyama M, Viville S. Barton SC. Ishino F, Surani 38. Leighton PA, SaamJR, Ingram RS,Stewart CL, Tilghman SM.An enhancer deletion affects MA. Pegl/Mest imprinted gene on chromosome 6 identified both H19 and Igf2 expression. Genes &Dev 1995: 9:2079-2089. by cDNA subtraction hybridization. Nature Genet 1995; 11:52-59.