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Cell, Vol. 50, 719-727, August 29, 1987, Copyright 0 1997 by Cell Press Parental Legacy Determines Methylation and Expression of an Autosomal Transgene: A Molecular Mechanism for Parental Imprinting Judith L. Swain: Timothy A. Stewart,t* and Philip Ledert§ * Department of Medicine Duke University Medical Center Durham, North Carolina 27710 r Department of Genetics Harvard Medical School §and Howard Hughes Medical Institute Boston, Massachusetts 02115 We have created a transgenic mouse strain in which an autosomal transgene bearing elements of the RSV LTR and a translocated c-myc gene obeys very unusual rules. If the transgene is inherited from the male parent, it is expressed In the heart and no other tissue. If it is inherited from the female parent, it is not expressed at all. This pattern of expression correlates precisely with a parentally imprinted methylation state evident in all tissues. Methylation of the transgene is acquired by its passage through the female parent and eliminated during gametogenesis in the male. These observations provide direct molecular evidence that autosomal gene expression can depend upon the sex of the parent from which the gene is inherited. They also provide a plausible mechanism for understanding parental imprinting that may be relevant to the failure of parthenogenesis in mammals, the apparent nonMendelian behavior of some autosomal genes, and the role of methylation in gene regulation. nal and maternal genomes has been obtained through genetic experiments. In these, the effects of parental inheritance of specific regions of the genome have been evaluated using intercrosses between mice carrying either Robertsonian or reciprocal translocations of nonhomologous chromosomes (Cattanach and Kirk, 1965; Cattanach, 1966; Searle and Beechey, 1976; Searle and Beechey, 1965; Solter, 1967; Surani, 1967). The implication of these studies is that both maternal and paternal contributions of specific chromosomal regions, and thus specific genes, are necessary for normal embryonic development. These genes must somehow be “imprinted” during inheritance from the parent. The distinctive information imparted by passage of these genes through the male and female parents evidently allows them to act collaboratively in the embryo. This concept of differential expression of paternally and maternally derived genes might explain the failure of successful parthenogenesis in mammals. The molecular basis of parental imprinting is unknown and difficult to understand in mammals. Thus it was particularly fortunate that, in the course of experiments designed to determine the in vivo effect of an activated c-myc oncogene, we produced a transgenic mouse in which the transgene is autosomally inherited, while its expression (which occurs exclusively in the heart) is strictly dictated by its parental origin. Furthermore, this pattern of expression correlates with one of two global patterns of methylation that are differentially imprinted on the transgene during passage through the male and female parents. The study of this unique strain of transgenic mice forms the basis for this report. Introduction Results There is growing evidence, largely from the mouse, that the expression of certain genes is determined by whether the gene is inherited from the male or female parent. This concept, called parental imprinting, is supported by studies demonstrating that both the paternal and maternal genomes are necessary for normal embryonic development (Cattanach and Kirk, 1965; Cattanach, 1966; McGrath and Solter, 1964a; McGrath and Solter, 1966; Mann and Love&Badge, 1964; Renard and Babinet, 1966; Surani et al., 1964; Surani et al., 1966a; Surani et al., 1966b; Surani, 1967). For example, nuclear transplantation experiments in fertilized mouse oocytes indicate that the two parental genomes play complementary roles during embryogenesis. The paternal genome appears to be relatively more important for the development of the extraembryonic tissues, while the maternal genome assumes greater importance for the development of the embryo proper (Barton et al., 1964; Surani et al., 1964). Additional support for the nonequivalence of the pater- Inheritance of the Transgene The recombinant plasmid used to produce the strain of transgenic mice described in these studies is shown in Figure 1. In addition to plasmid sequences, the construct consists of a major portion of the Rous sarcoma virus (RSV) LTR followed by a fusion gene cloned from the SW7 mouse plasmacytoma cell line. The latter contains the c-myc gene translocated into the immunoglobulin locus. Specifically, the plasmacytoma-derived fragment contains the a constant and switch regions of the immunoglobulin heavy chain locus (but not the heavy chain enhancer) and the c-rnyc gene in which the 5’ portion of the untranslated first exon has been deleted. Genomic DNAs from the founder and over 700 descendant mice were subjected to restriction fragment pattern analysis using a probe derived from a portion of the immunoglobulin gene (identified as the a constant region [Cal in Figure 1). The results of these analyses indicated that approximately 10 copies of the construct integrated into a single genomic site where they are arranged in a head-totail, tandem array. Pedigree analyses indicated that the transgene (barring unlikelyx-linked pseudoautosomal be- Summary *Present address: Genentech, 460 Point San Bruno San Francisco, California 94090. Boulevard, South Cell 720 A Figure 1. Diagrammatic Representation of the RSV-S107 Fusion Gene Injected to Produce the Strain of liansgenic Mouse Analyzed in This Study An EcoRl fragment from the mouse 5107 plasmacytoma (Kirsch et al., 1961) containing immunoglobulin and c-myc sequences was ligated into a derivative of the pRSVCAT plasmid (Gorman et al., 1962). The final construct consisted of a 2.2 kb fragment of pBR322, a 437 bp fragment of the 3’ RSV LTR. and a 17 kb fragment containing the immunoglobulin heavy chain and truncated c-myc genes. Vertical hatching withi identifies coding sequences he immunoglobulin region. Disequences within the c-myc gene. agonal hatching identifies cod’ ,mc(p’ The probe used for Souther?-analysis of genomic DNA from the transgenie animals is illustrated and consists of a 1.4 kb EcoRI-Xbal frag ment from the CI constant region of the immunoglobulin gene. Ca, a constant region; R, EcoRI;.X, Xbal. B c-MYC EXON 1 havior) is inherited in an autosomal fashion; that is, it is neither X- or Y-linked since male-to-male and female-tofemale transmission of the gene occurs. A small number of transgenic carriers show a different restriction fragment pattern, suggesting that an internal rearrangement and deletion occurs at low frequency (<0.05%). This rearrangement, which is thereafter stably inherited, may involve a recombination in the immunoglobulin switch region. Myocardial-Specific Expression Since these experiments were initiated to assess the oncogenic effect of the activated c-myc gene in a variety of organ systems (Stewart et al., 1984), it was necessary to identify the organs in which transgene expression occurred. This was done using RNAase protection analysis, which revealed that expression of the transgene was limited to the heart. An example of such an analysis in which RNA samples were taken from the heart, thymus, salivary gland, lung, liver, pancreas, kidney, spleen, brain, and skeletal muscle of a male carrier is shown in Figure 2A. As indicated in the pedigree, the analyzed mouse inherited the transgene from its father. The RNAase protection analysis was performed using a probe derived from the normal, murine c-myc cDNA that detects products of the endogenous c-myc gene as well as a unique product corresponding to the transgene. The probe (Figure 26) extends from a Notl site in the 3’ portion of exon 1 to a Pstl site in the 5’ portion of exon 2 and thus, when annealed to total cellular RNA and digested with RNAase, should yield a protected fragment 365 bases in length corresponding to the normal, processed c-myc mRNA. It should also yield fragments 225 and 145 bases in length, corresponding to the unspliced first and second c-myc exons present in endogenous c-myc precursor mRNA. As expected, these three fragments were observed in all organs tested, whether the animal was (as shown in Figure 2A) or was not (data not shown) a transgenie carrier. In contrast to the normal c-myc gene, the c-myc se- I Not 1 cDNA probe I c-MYC EXON 2 365BP F PS, 1 EXON 1 2’25BP TRANSGENE 175BP EXON 2 145BP Figure 2. RNAase Protection Analysis (A) RNAase protection analysis of RNA prepared from various organs of an RSV-S107 transgenic mouse illustrating myocardial-specific expression of the transgene. The pedigree of the analyzed propositus is shown above the figure. Twenty micrograms of total RNA was incubated with the antisense mRNA probe shown in (B), and RNAase protection analysis was performed as described in Experimental Procedures. Protected fragments of 365 bp corresponding to processed endogenous c-myc mRNA and 225 and 145 bp fragments corresponding to exons 1 and 2 of unprocessed c-rnF are detected in all tissues. A unique 175 bp fragment that corresponds to the processed transgene is detected in the heart. In addition, the amount of the 145 bp fragment (corresponding to exon 2 of c-myc) is increased in the heart compared with other organs. (B) Illustration of the antisense mRNA probe used in the RNAase protection analysis. The probe (open rectangle) encompassed a 365 bp Notl-Pstl fragment of the 3’portion of exon 1 and the 5’portion of exon 2 of the c-myc cDNA. The protected fragments observed are illustrated by hatched rectangles. quence incorporated into the transgene (see Figure 1) carries a truncated first exon. Its transcription is initiated 5’to this truncated first exon, and its spliced transcript protects a unique fragment 175 bases in length when annealed to the cDNA probe shown in Figure 2B. The unspliced precursor of the transcript is a 145 base fragment of the probe corresponding to the second exon of c-myc. This latter fragment is indistinguishable from that protected by endogenous, unprocessed c-myc mRNA. Under the conditions of the assay, the small (30 bp), protected fragment corresponding to the truncated first exon of c-myc is not detectable. Thus, protection of a 175 base fragment is diagnostic for expression of the transgene. It is observed only in the RNA derived from the heart (see Figure 2A). Parental-Specific 721 365 bp- Gene - Expression 12345678 - va e. *. am 145bp- Figure 3. RNAase Protection Analysis of RNA Extracted of Littermates Resulting from a Mating of Heterozygous Carriers from Hearts Transgenic Carrier offspring (heterozygotic and homozygotic not distinguished) are illustrated by half-filled, half-stippled symbols; noncarriers, by open symbols. RNAase protection analysis was performed as described in Figure 2A using the probe illustrated in Figure 28. Although offspring 1 through 7 carry the transgene, only offspring 3 and 7 display the 175 bp protected fragment indicative of transgene expression. The 365, 225, and 145 base long fragments corresponding to processed and unprocessed c-myc mRNA (Figure 28) are present in hearts from both carrier and noncarrier animals. The expression of this fusion gene in tissue of mesodermal origin is consistent with the transgene expression of other strains of mice inheriting RSV fusion genes (Overbeek, et al., 1986). Thus, the tissue specificity is likely to be a property of the RSV LTR rather than the site of integration, though this is certainly not proven. With respect to the physiologic effect of transgene expression, preliminary indications are that the animals do not have an increased incidence of tumor development in any organ including the heart. Since both the endogenous c-myc gene and the transgene can yield an unspliced transcript that protects a 145 base fragment, the increased amount of this fragment seen in the heart could theoretically arise from either or both genes. Note that this product is present in amounts greater than the sum of the unique 175 base transgenic fragment and the 145 base fragment present in the other organs that do not express the transgene (Figure 2A). Assuming that this transcript largely arises from the transgene, it indicates that the relative amount of unspliced transgenic mRNA precursor is greater than spliced tran- script. Alternatively, the transgene may give rise to more than one transcript, for example, one initiating 5’ to the truncated first exon and a second initiating within the first intron. On Northern analysis only a single transcript of 2.3 kb is detected (data not shown), suggesting that transcription initiation starts within the immunoglobulin-derived DNA segment. But transcription at a cryptic promoter in the first intron of c-myc might be indistinguishable from transcript initiation within the immunoglobulin gene switch region. Thus, more than one transcription site may be active in the transgene. Expression of the Ttansgene Depends upon Parental lnherltance In the initial sample of carrier mice assayed, not all expressed the transgene. This variation in expression was at first puzzling and suggested that some additional genetic element(s) governed expression. The initial analysis was further complicated by the fact that the expression phenotype, requiring myocardial analysis, could not be tested until the carrier animal had passed the transgene to several litters to ensure that all possible genotypes were captured. A typical pedigree showing the genotype and phenotype of a litter produced by mating male and female heterozygotes is given in Figure 3. Although 7 of the 8 offspring inherited at least one transgenic locus (as determined by analysis of their DNAs), only 2 of the 7 carriers, a male and a female, expressed it. In both cases, as with all other expressing mice, expression was restricted to the myocardium. To understand the genetic rules that govern expression, a larger group of systematic matings was established; Fl, F2, and further descendant litters were secured and analyzed with respect to genotype and phenotype. Through the analysis of RNA from the hearts of 77 transgenic carriers, the following pattern of expression emerged (Table 1). None of the 42 carrier offspring (males and females) of carrier females mated with noncarrier males expressed the transgene. In contrast, each of the 20 carrier offspring (males and females) of noncarrier females mated with carrier males expressed the transgene. Of the 15 carrier offspring from matings between male and female carrier mice (heterozygote matings), expression was detected in only 6. In cases in which complete organ analyses were carried out, expression was always restricted to the myocardium. Table 1. Influence of Parental Origin of the Transgene on Its Subsequent Expression in Hearts of Transgenic Offspring Status of Transgene in TG Offspring Parent Expression Female Male Expressed Not Expressed TG Non-TG TG Non-TG TG TG 0 20 6 42 0 9 TG, transgenic carrier. Non-TG, nontransgenic carrier. Cell 722 Table 2. Influence of Parental Origin DNA Methylation Status in Transgenic Methylation Pattern in TG Offspring Parent on Its of Transgene Female Male Undermethylated Methylated Combined TG Non-TG TG Non-TG TG TG 0 50 10 59 0 55 0 0 19 TG, transgenic carrier. Non-TG. non-transgenic 12Kb- of the Transgene Offspring carrier. 8.OKb- 4.3Kb3.OKb2.4Kb- 1.4KbFigure 4. Southern rier and Transgenic Analysisof DNAfrom Carrier Matings Offspring of Various Noncar- Genomic DNA was digested with the methylation-sensitive restriction endonuclease Hpall, and Southern analysis was performed as detailed in Experimental Procedures. The probe used is illustrated in Figure 1 and consists of a fragment from the a constant region of the immunoglobulin gene. The pattern observed in offspring from noncarrier crosses is shown in lane 1. The result for offspring carriers with maternally derived transgenes is shown in lane 2; that for offspring carriers with paternally derived transgenes, in lane 3. The pattern in presumed homozygotic carrier offspring is illustrated in lane 4. In animals inheriting the transgene from the female parent (lane 2) large DNA fragments (12 and 8 kb), as well as a 2.4 kb fragment, are detected. In animals inheriting the transgene from the male parent (lane 3) the large fragments are not present and smaller fragments (4.3, 3,2.4, and 1.4 kb) appear. The results indicate that the transgene inherited from the male parent is relatively undermethylated compared with the transgene inherited from a female parent. Animals inheriting a transgenic allele from each parent display a combined pattern. Relationship of Methylation Pattern to Transgene Expression Because expression requires passage of the transgene through the male parent and is observed in the adult as well as the fetal myocardium (data not shown), it is not likely to be the result of prezygotic RNA synthesis (paternal RNA). Furthermore, since information required for expression must be acquired during gametogenesis in the male or shortly after fertilization and since the degree of methylation of cytosine nucleotides appears to correlate with the transcriptional activity of certain genes (reviewed by Razin et al., 1984) and is altered during spermatogenesis (Groudine and Conklin, 1985) methylation seemed a likely mechanism by which to convey this regulatory information. Predictions of this model are easily tested Genomic DNAs extracted from tail samples of carrier mice were digested with Hpall, a restriction endonuclease that cleaves CCGG sequences only when neither cytosine residue is methylated. The resulting transgenic restriction fragment pattern was detected using the standard probe derived from the immunoglobulin portion of the transgene (Figure 1). Three patterns, one of which represented the sum of the other two, could be discerned in mice inheriting the transgene (Figure 4). The first pattern, referred to as methylated, was observed in all carrier offspring of female carriers mated with noncarrier males. It consisted of a 12 kb fragment and less intense 8 and 2.4 kb fragments (Figure 4, lane 2). A second pattern, referred to as undermethylated, was observed in all carrier offspring of carrier males mated to noncarrier females. The 12 and 8 kb fragments were absent and had been replaced by three smaller fragments of 4.3,3.0, and 1.4 kb and a greatly increased signal from the 2.4 kb fragment (Figure 4, lane 3). A third pattern, referred to as combined, was observed in some of the carrier offspring of matings between heterozygote carriers. It consisted of the sum of fragments seen in the other two patterns (Figure 4, lane 4). Offspring from heterozygote matings that display the combined pattern are presumably homozygotic for the transgene, inheriting it from both male and female parents. A summation of the analysis for 193 transgene carriers is shown in Table 2. While the numbers of offspring from heterozygote crosses are small, fewer offspring with undermethylated patterns were observed than would have been expected. These results raise the possibility of transmission distortion, suggesting a deleterious effect of the expressed gene (see below). When DNAs from the transgene carriers were digested with the restriction endonuclease Mspl, an isoschizomer of Hpall that is insensitive to the methylation status of CCGG sequences, only one fragment 1.4 kb in length was observed regardless of which parent donated the transgene (data not shown). These data indicate that, while certain Hpall sites in the transgene are methylated regardless of paternal inheritance, others are methylated only when inherited through the female parent. Furthermore, the methylation pattern observed in DNA derived from the tail was observed in every other somatic organ analyzed, including the heart (see below). It is evidently a global methylation pattern, indifferent to whether or not the gene is actively expressed in a particular organ. The relationship of the methylation pattern to expres- Parental-Specific 723 Table 3. Correlation with Its Myocardial Gene Expression of Methytation Status of the Transgene Expression in Individual Transgenic Mice Status of Transgene Expression -12 Kb- Methylation Status of Transgene Expressed Not Expressed -&OKb- Undermethylated Methylated 23 0 0 36 -43 Kb- -30 Kb-2AKb- sion of the transgene was examined in 61 animals (Table 3). In the 23 transgenic carriers exhibiting the undermethylated pattern, all express the transgene in the heart. In contrast, none of the 36 transgenic carriers exhibiting the methylated pattern express the transgene. No exceptions to these patterns were detected. Expression of the transgene is strictly correlated with its degree of methylation. We have examined nine additional strains of transgenie mice that carry various fusions of the mouse mammary tumor virus LTR and c-myc gene (Stewart et al., 1964); none exhibit parental related expression or methylation patterns of the c-tnyc gene. The methylation pattern of the transgene in any particular animal is not dependent upon the sex of that animal, but rather upon the sex of the animal’s transgenic parent. To demonstrate this further, the methylation pattern of the transgene was examined as inherited from the four possible paternal permutations of sex and methylation genotype (Figure 5). The results demonstrate that both female and male carrier offspring of transgenic males exhibit an undermethylated pattern of the transgene irrespective of the methylation pattern of the male parent (Figures 6A and 58). Likewise, both female and male carrier offspring of transgenic females exhibit a methylated pattern for the transgene irrespective of the methylation pattern of the fe male parent (Figures 5C and 50). Yethylation Pattern Imprinted during Gametogenesis Since the methylation pattern of the transgene is observed in all tissues in the adult animal, it is likely to be imprinted either during gametogenesis in the parent or at an early stage of embryogenesis in the offspring. These possibilities can easily be distinguished in the male parent since about 30% of the testicular cells of adult males are in some stage of gametogenesis (Hecht, 1967). A male animal that inherits the transgene from its female parent would be expected to carry a methylated version of the transgene in every somatic organ. If the maternally inherited pattern were to be altered in the germ line, an undermethylated pattern would begin to emerge in the DNA derived from testicular tissue. Conversely, a male animal inheriting the undermethylated pattern from his father should maintain that pattern in all his somatic organs and transmit it unaltered during gametogenesis. The methylation patterns observed below conform precisely to these predictions. In a male inheriting the transgene from the female parent, all the somatic organs exhibit a methylation pattern (Figure 6). In the testes, however, a fragment of 4.3 kb appears, corresponding to one of the fragments present in animals exhibiting the undermethylated pattern. These -1.4 Kb- 12 Kb 8.0 Kb 4.3 Kb 3.0 Kb 2.4 Kb .!I ,. ‘4 1.4 Kb Figure 5. Southern Analysis Demonstrating Alteration ation Status of the Transgene through Inheritance of the Methyl- Male carriers displaying either the methylated (A) or the undermethylated (B) pattern of the transgene donate an undermethylated transgene to both their male and female carrier offspring. In contrast, female carriers with a methylated (C)or an undermethylated (D) transgene donate a methylated transgene to both their female and male carrier offspring. P 34 12 kb- w 8.0 kb- 4.3 kb- Figure 6. Southern Analysis of Genomic DNA from Various Organs of an Adult Male Tmnsgenic Carrier Mouse That Inherited the More Highly Methylated Form of the Transgene from the Female Parent The methylated form of the transgene is present in sessed by the presence of the 12 and 8 kb bands. In sperm in all stages of development are present, an form of the transgene is detected hy the appearance all tissues as asthe testes, where undermethylated of a 4.3 kb band. Cell 724 data indicate that the female parent-imprinted methylation pattern is being eliminated in the male offspring’s testis during gametogenesis. The methylation state of the transgene in various organs of female carriers was also examined, although the small number of germ cells in the ovary makes it difficult to evaluate the role played by the ovary in imprinting. Females inheriting the transgene from their fathers display the undermethylated pattern in all somatic organs as well as in the ovary (data not shown). Females inheriting the gene from their mothers exhibit the methylated pattern in all organs including the ovary (data not shown). Since the number of mature eggs in the female is small compared with the amount of tissue in the ovary and since the second meiotic division does not occur until after fertilization, we feel that the sensitivity of this assay is not adequate to detect a possible shift from the undermethylated to the methylated pattern during gametogenesis in the female. The question of germ-line versus zygotic imprinting in the female therefore awaits resolution, possibly by hormonally inducing superovulation and collecting sufficient oocytes for DNA analysis. Discussion A Molecular Explanation for Imprinting Phenomena Observed in the Mouse The parental-specific expression of the transgene observed in this strain of mice supports and may explain the molecular basis for previous data demonstrating the nonequivalence of the maternal and paternal genomes. Both the nuclear transfer experiments (McGrath and Solter, 1984a; McGrath and Solter, 1986; Mann and LovellBadge, 1984; Renard and Babinet, 1986; Surani et al., 1984; Surani et al., 1986a; Surani et al., 1986b; Surani, 1987) and the classic genetic experiments involving intercrosses of animals with translocations (Cattanach and Kirk, 1985; Cattanach, 1986; Searle and Beechey, 1978; Searle and Beechey, 1985; Solter, 1987; Surani, 1987) have demonstrated that portions of the genome must be inherited from each parent in order for development of the zygote to proceed to term. Moreover, a specific mouse mutant, hairpin tail (Thp) (Johnson, 1974) has been identified which supports the contention that specific regions of the mouse genome exhibit parental specificity (McGrath and Solter, 1984b). In this mutant, a deletion occurs in the proximal portion of chromosome 17 (Bennett et al., 1975). If the deletion is inherited from the male parent, the offspring are viable. If the deletion is inherited from the female parent, the fetus does not develop to term (Johnson, 1974). Thus it has been hypothesized that certain genes located in the proximal portion of chromosome 17 are required for development and presumably these genes are transcribed only when inherited from the female parent. Additional evidence that parental imprinting influences the expression of genes important in development comes from the analysis of intercrosses of heterozygotes for Robertsonian translocations of chromosomes 11 and 13. Offspring that are parentally disomic or nullisomic for regions on each chromosome are produced (Cattanach and Kirk, 1985). Offspring maternally disomic for the proximal portions of chromosome 11 (and thus paternally nullisomic for the same region) are small compared with normal littermates. In contrast, offspring disomic for the paternal contribution of the same region of chromosome 11 are large compared with their littermates. A similar observation has been made in the distal region of chromosome 2. Although animals either paternally or maternally disomic for the distal portion of chromosome 2 live for only a few days after birth, two distinct phenotypes have been observed. Maternally disomic offspring are hypokinetic and have flat bodies, while paternally disomic offspring are hyperkinetic and have square bodies. These results can be interpreted in terms of a parental effect on gene expression. The data presented here directly demonstrate that parental imprinting of a specific gene completely correlates with its subsequent expression. Parental Imprinting Effects a Transgene Methylation Pattern Universal in all Tissues, yet Expression Is Restricted to the Myocardium The physical mechanism of the demonstrated parental imprinting is as yet unknown. Numerous studies are available to suggest a close correlation of gene expression with methylation status (see Razin, 1984, for review), but exceptions do exist, and thus the precise role of methylation is yet to be determined. 5Azacytidine has been used to block methylation successfully and subsequently alter gene expression, further suggesting that the expression of certain genes is dependent, at least in part, upon the degree of methylation of cytosine nucleotides (Jones, 1984; Jaenisch et al., 1985). Previous studies have demonstrated that certain genes expressed constitutively in the embryo are protected from de novo methylation during spermatogenesis, while genes that are not expressed undergo methylation during gametogenesis in the male (Groudine and Conklin, 1985). The data in this report clearly demonstrate that the parental origin of a specific gene (i.e., the transgene) governs the degree of methylation of that gene. The undermethylated form of the transgene is expressed, while the more methylated form is not. Transgenic alleles inherited from the male parent were undermethylated in comparison to those inherited from the female parent. The correlation between parental origin, methylation status, and expression (as assessed by mRNA content) of the transgene is absolute, suggesting that undermethylation of the transgene plays a role in its subsequent expression. Other physical properties of the genome, possibly depending on methylation, may be involved in parental imprinting. These include the presence of DNAase hypersensitivity sites and the presence of specific conformations such as Z-DNA (Conklin and Groudine, 1984). These properties could be governed in part by the methylation states of the regulatory sequences of specific genes. On the other hand, it is evident that methylation status alone does not determine the expression of the transgene since the male-imprinted pattern of transgene methylation oc- Parental-Specific 725 Gene Expression curs throughout the organism in the heart. Undermethylation sufficient, for the subsequent but expression occurs only may be necessary, but not expression of the gene. A Methyiation Pattern is imprinted in the Female and Eliminated in the Male Germ Line Parental imprinting of the transgene occurs in the germ cells of the male. This is documented by the presence of undermethylated forms of the transgene in the testes of males exhibiting a more highly methylated form of the transgene in all other tissues. Although not proven, it is anticipated that imprinting of the methylation pattern also occurs during gametogenesis in the female. Our studies suggest that four separate mechanisms must occur during gametogenesis to yield the results observed in this strain of transgenic mouse. In the oocyte the transgene either must be maintained in the methylated state or, if not already methylated, must undergo de novo methylation before fertilization. In the male germ cells the transgene must be maintained in the undermethyiated state or become undermethyiated either through a passive mechanism (such as inhibition of a maintenance methylase) or through the action of a specific demethylase. While de novo and maintenance methyiases have been identified in mammalian systems (see Razin, 1984, for a review), no specific demethylase has as yet been identified. is imprinting Dependent upon Transgenic Sequences or the Site of integration? The question arises as to whether the parental-specific expression observed in these mice is determined by the site of integration of the transgene, by the specific sequences in the transgenic construct, or by a combination of these elements. To determine whether this property is conveyed with the inserted fragment or is a property of the chromosomal site of integration, we tested another transgenie strain into which an identical DNA fragment had been introduced. The results of these experiments, still preliminary because of sample size, indicate that the degree of methylation of sequences within the transgene does vary to a certain extent with parental origin. But in contrast to the original strain of mice, exceptions to the established pattern of parental imprinting occur. Such results suggest that the paternal imprinting effect may be an easily damaged or “leaky” property of the transgene. This property may be subject to additional influences such as other control elements operating at the site of integration or alterations of transgene structure that might occur as a consequence of the illegitimate recombination. A Possible Explanation for the Behavior of Certain Human Alleles The results of this study may help to explain the pattern of inheritance noted for certain human diseases. For example, the genetic abnormality responsible for Huntington’s disease has been localized to chromosome 4 (Gusella et al., 1983). This disorder normally displays an autosomal dominant mode of inheritance with high penetrance, but an exception does exist. The juvenile form of Huntington’s disease appears to be inherited preferentially (but not exclusively) from the male parent (Merritt et al., 1989). Diabetic alleles within the HLA complex appear to be preferentially transmitted by the father, a finding that might account for the increased occurrence of diabetes in their offspring (Vadheim et al., 1986). Another disease demonstrating parental influence of inheritance is the infantile form of myotonic muscular dystrophy. Although the locus for myotonic dystrophy has been localized to a region of chromosome 19 (Bartlett et al., 1967) the congenital form of the disease is almost exclusively inherited from the female parent (Roses et al., 1979). While these exceptions might not be afielic with the major forms of these inherited disorders, they might also represent human manifestations of parental-specific gene expression. The occurrence of hydatidiform moles in humans is the clearest example of the aberrant fate of human extraembryonic tissue whose genome is derived entirely from the male parent (Wake et al., 1984). Experlmentel lkansgenic Procedures Mice and Vector Constructlons The plasmid used for the pronuclear injection consists of a portion of the 3’ LTR of the RSV fused to a DNA fragment from the mouse S107 plasmacytoma(Kirsch etal., 1961). In thisplasmacytoma, a 12;15chromosomal hanslocation removed most of the first (noncoding) c-mt+c exon and the two normal c-rn~z promoters, and juxtaposed (5’ to 5’) the truncated c-m gene to the switch sequence of the immunoglobulin heavy chain gene. A 17 kb EcoRl fragment of this rearranged c-myc gene that includes the region from the EcoRl site in the immunoglobulin a constant region to the EcoRl site 6 kb 3’ to the c-m gene was ligated into a derivative of the pRSVCAT plasmid described by Gorman et al. (1962). The pRSVCAT plasmid containing the RSV LTR sequences was digested with EcoRl to delete a 3’ portion of the RSV sequence and all chloramphenicol acetyttransferase and SV40 sequences. The 17 kb EcoRl fragment from the S107 plasmacytoma was then ligated into the EcoRl site that is 3’ of the RSV sequence in the pRSVCAT derivative. The final construct (termed RSV-SW) consists of a 2.2 kb EcoRI-Accl fragment of pBR322, a 437 bp Pvull-EcoRI fragment containing a portion of the 3’ RSV LTR, and the 17 kb fragment containing the fused immunoglobulin heavy chain gene and the truncated c-myc gene. The RSV-S107 plasmid was linearized at a unique Kpnl site 3’to the c-w gene and injected into one of the pronuclei of fertilized one-cell mouse eggs derived from a C57BII6.J x CD-1 mating. The injected eggs were transferred into pseudopregnant foster females and allowed to develop to term. A male founder animal was identified through Southern blot analysis of DNA extracted from the tail. Over 700 descendants of this founder were bred, largely to CD-1 mice, and gene typed. DNA and RNA Isolation DNA was isolated from a short segment of the tail or from organs by a modification of the method of Davis et al. (1960). The resultant nucleic acid pellet was resuspended in 300 ul of a 10 mM Tris (pH 7.4) 0.1 mM EDTA solution. RNA was isolated by homogenizing tissue in a Polytron homogenizer using a buffer containing 4 M guanidine isothiocyanate, 2.5 mM sodium citrate (pH 7) and 0.1 M 6-mercaptoethanol. Total RNA was isolated by the method of Chirgwin et al. (1979) by centrifugation through a 5.7 M cesium chloride, 25 mM sodium acetate cushion. DNA and RNA Analysis DNAs were digested with the appropriate restriction endonuclease, electrophoresed on 0.7% agarose, transferred to nitrocellulose or nylon filters by the method of Southern (1975) and hybridized to a 1.4 kb Cell 726 EcoRI-Xbal fragment containing the a constant region from the RSVS107 plasmid. The relationship of the probe to the vector used for injection is shown in Figure 1. RNA was analyzed for expression of the transgene by the RNAase protection assay (Melton et al., 1984). The probe used consisted of a 1.9 kb BamHI-Pstl fragment of the mouse c-myc cDNA (containing portions of the first and second exons of c-myc) cloned into pGEM-3 (Promega, Madison, WI). The vector was linearized at a unique Nob site in the first exon of c-myc, and 32P-labeled antisense transcripts to portions of the first and second exon of c-rnyc were synthesized using SP6 polymerase and [3zP]UTP by the previously described method (Melton et al., 1984). Total cellular RNA was hybridized to the radiolabeled antisense c-myc RNA probe for at least 6 hr at 50% in a reaction mixture containing 20 ug of total RNA, 10s cpm antisense probe, 50% formamide, and 4 mM PIPES buffer (pH 6.7). Single-stranded RNA was digested with a solution containing RNAase A and RNAase Tl, and the RNAases were inactivated with proteinase K and SDS as described by Melton et al. (1984). The samples were extracted with phenol, precipitated with ethanol, resuspended in 90% formamide, and electrophoresed on an 8 M urea, 6% acrylamide gel. The gels were dried on Whatman 3MM paper and exposed to Kodak XAR-5 X-ray film with an image intensifier at -70% for 24 to 72 hr. Acknowledgments We are most grateful to Racheal Wallace for her assistance in many of the animal experiments and to Ann Kuo and Twila Jackson for their expert technical assistance. This work was supported in part by grants from E. I. DuPont de Nemours, Inc. (to P L.), American Business Foundation for Cancer Research, Inc. (to P. L.). and a National Institutes of Health Award HL26831 (to J. L. S.). J. L. S. is an Established Investigator of the American Heart Association. P L. is a Senior Investigator of the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. 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Nature 328, 251-254) describe transgenic mouse lines in which, respectively, 1 out of 7 and 4 out of 5 transgenic loci tend to display partially imprinted methytation patterns. In the former case, the transgene, an immunoglobulin/chloramphenicol transacetylase sequence, is not expressed regardless of the transmitting parent. In the latter case, quail troponin I transgenes were evidently not tested for expression. In view of this frequency of legacy-related methylation, it seems likely that the expressionlmethylation we observe depends on our specific insert and on the locus into which it is inserted. The locus is likely to imprint the methylation pattern, but the inserted fragment of DNA must be capable of responding to it.