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Proc Natl Acad Sci U S A. 1996 November 26; 93(24): 13876–13883. | PMCID: PMC19455 |
Copyright © 1996, The National Academy of Sciences of the USA Genetics The structural H19 gene is required for
transgene  imprinting Karl Pfeifer, * Philip A. Leighton, † and Shirley M. Tilghman ‡Howard Hughes Medical Institute and Department of Molecular Biology, Princeton University, Princeton, NJ 08544 Accepted October 2, 1996. The product of the H19 gene is an untranslated RNA
that is expressed exclusively from the maternal chromosome during
mammalian development. The H19 gene and its 5′-flanking
sequence are required for the genomic imprinting of two paternally
expressed genes, Ins-2 (encodes insulin-2) and
Igf-2 (encodes insulin-like growth factor-2), that lie
90 and 115 kb 5′ to the H19 gene, respectively. In this
report, the role of the H19 gene in its own imprinting
is investigated by introducing a Mus spretus H19 gene
into heterologous locations in the mouse genome. Multiple copies of the
transgene were sufficient for its paternal silencing and DNA
methylation. Replacing the H19 structural gene with a
luciferase reporter gene resulted in loss of imprinting of the
transgene. That is, high expression and low levels of DNA methylation
were observed upon both paternal and maternal inheritance. The removal
of 701 bp at the 5′ end of the structural gene resulted in a similar
loss of paternal-specific DNA methylation, arguing that those sequences
are required for both the establishment and maintenance of the
sperm-specific gametic mark. The M. spretus H19
transgene could not rescue the loss of Igf-2 imprinting
in trans in H19 deletion mice, implying a cis
requirement for the H19 gene. In contrast to a previous
report in which overexpression of a marked H19 gene was
a prenatal lethal, expression of the M. spretus
transgene had no deleterious effect, leading to the conclusion that the
20-base insertion in the marked gene created a neomorphic mutation. Normal mammalian development requires the contribution of haploid
genomes from both parents, indicating that the two genomes are not
functionally equivalent ( 1, 2, 3, 4). The nonequivalence is the result of
gamete-specific epigenetic modifications of a number of genes that lead
to the unequal expression of the two parental alleles during
development. To date, 16 such imprinted genes have been identified in
the mouse or human ( 5, 6). The most likely candidate for the gametic mark or imprint is the
methylation of CpG residues in the transcriptional control regions of
imprinted genes. Allele-specific DNA methylation has been observed in
the vicinity of most imprinted genes. In some instances, the
methylation is present on the inactive gene, suggesting a role for DNA
methylation in silencing of the gene ( 7, 8, 9). However, specific
methylation of the active alleles of imprinted genes has been described
as well ( 10, 11, 12). Finally, for two imprinted genes, Igf-2r
and H19, allele-specific methylation has been shown to
originate in the gametes and survive a period of genome-wide
demethylation that occurs shortly after fertilization ( 9, 13). These
residual gametic differences remain the best candidates for heritable
imprinting signals. The strongest case for a requirement for DNA
methylation in maintaining the differential expression of parental
alleles of genes comes from the disruption of imprinting in embryos
homozygous for the loss of the maintenance methylase, DNA
methyltransferase ( 14). The H19 gene lies in a cluster of imprinted genes on distal
chromosome 7 in the mouse, a region syntenic with chromosome 11p15.5 in
humans ( 15, 16). The genes encoding p57KIP2, a cyclin-dependent kinase
inhibitor, and Mash-2, a trophoblast-specific transcription factor, lie
at the telomeric end of the cluster and are maternally expressed ( 17,
18). The two growth factor genes, Ins-2 (encodes insulin-2)
and Igf-2 (encodes insulin-like growth factor-2), lie in the
middle of the cluster and are both paternally expressed ( 19, 20), while
H19 resides at the centromeric end of the cluster and
encodes a maternally expressed RNA ( 21, 22). Recent experiments have established a mechanistic link between the
imprinting of Ins-2, Igf-2, and H19 that is
consistent with a primary role for the H19 gene in the
imprinting of both itself as well as the other two genes. First, an
internally deleted transgene consisting of 14 kb of DNA surrounding the
H19 gene is capable of adopting imprinted expression in
heterologous chromosomal locations ( 7), implying that H19
imprinting is regulated by local signals and does not require either
the Igf-2 or Ins-2 gene. Second, a deletion of
the H19 5′-flanking sequence and structural gene results in
the expression of both Igf-2 and Ins-2 from the
maternal as well as the paternal chromosome ( 23). Thus, the region
surrounding the H19 gene that is required for its own
imprinting is also required for the imprinting of its neighbors. We
have suggested that the mechanistic link between the imprinting of
H19, Igf-2, and Ins-2 results from a competition
between the genes for the use of shared enhancers ( 22, 24, 25). On the
paternal chromosome, this competition is biased in the direction of
Igf-2 and Ins-2 expression by the silencing the
H19 gene via DNA methylation ( 14). In that sense, the
imprinting of Igf-2 and Ins-2 can be said to be
nonautonomous. On the maternal chromosome, the fully unmethylated
H19 gene successfully competes for the
enhancers. This enhancer competition model implies that the regulated event in
imprinting at this locus is the establishment and maintenance of the
paternal-specific methylation of the H19 gene. In this
report, we exploit transgenic mice to begin a genetic analysis of the
methylation and imprinting of the H19 gene. Specifically, we
have investigated the requirement for the H19 structural
gene itself in cis in its own methylation and imprinting. No role for the H19 RNA in trans has been established to
date. The RNA is highly abundant during embryogenesis in mesodermal and
endodermal tissues ( 26, 27). Nevertheless, the only phenotype observed
with the deletion of the H19 gene is the loss of imprinting
of Igf-2 and Ins-2 ( 23). Two experimental
approaches have attributed biological effects to overexpression of
H19 RNA. Hao et al. ( 28) have shown by
transfection that human H19 RNA could suppress the
tumorigenicity of rhabdomyosarcoma and Wilms’ tumor cell lines.
Additionally ectopic expression of a marked H19 transgene in
mice resulted in late embryonic lethality between embryonic day 14 and
birth ( 29). In this report, we also used the transgenic system to
clarify the role of ectopic expression in prenatal lethality. Mice. C57BL/6J, DBA/2J, and SJL mice were purchased from
The Jackson Laboratory. The strain B6(CAST- H19) has been
described previously ( 13). Isolation of the Mus spretus H19 Gene. Genomic
DNA prepared from M. spretus liver was digested to
completion with EcoRI and size-fractionated by sucrose
gradient centrifugation. Fragments of ≈10 kb were isolated and cloned
into Lambda Dash II (Stratagene). The library was screened by the
method of Benton and Davis ( 30) using the 3-kb
EcoRI– SalI fragment spanning the H19
gene, and positively hybridizing phage were purified. The recombinant
phage were subcloned, and the nucleotide sequence of the M.
spretus H19 gene was determined by the chain termination method
( 31) using the Sequenase kit (United States Biochemical). Transgene Constructions and Microinjection. The plasmids used
for generating transgenic mice include 0.8 kb or 4 kb of DNA 5′ to the
structural gene and 8 kb or 11 kb of DNA 3′ to the structural gene
cloned into a pBluescript KS vector (Stratagene). In the construct
M. spretus H19, the 3-kb EcoRI– SalI
fragment spanning the Mus domesticus structural gene is
replaced with the 3-kb EcoRI– SalI M.
spretus DNA. The construct Δ1 H19 has a
deletion of a 697-bp DraIII– BsmI fragment from
+3 to +701 bp that removes the first half of exon 1. The Luc transgene
constructs replace the H19 structural gene from the
DraIII site at +3 bp to the unique SalI site 3′
of the gene with the firefly luciferase gene ( 32). DNA was microinjected into one of the pronuclei of fertilized one-cell
mouse eggs derived from (C57BL/6J × SJL) F 1
intercrosses ( 33). Injected embryos were transferred to the oviducts of
pseudopregnant CD1 females. Founder animals were identified by
digestion of tail DNA with appropriate enzymes and analysis by Southern
blot. RNA Analysis. Total RNA was isolated by LiCl-urea extraction
( 34). An non-allele-specific RNase protection probe for H19
( 29) and allele-specific RNase protection probes for H19 and
for Igf-2 have been described ( 24). A 140-bp XbaI
DNA fragment spanning the 5′ end of the H19–luciferase
fusion gene was subcloned into pBluescript KS (Stratagene), and the
resultant plasmid was linearized with KpnI and treated with
the Klenow fragment of DNA polymerase I to generate a template for
synthesizing a probe specific for H19–luciferase RNA.
Radiolabeled probes were incubated with total RNA at 45°C overnight
and digested with 40 μg of RNase A per ml and 2 μg of RNase T1 per
ml at room temperature for 60 min. The products were separated on 6.0%
or 7.5% acrylamide/7 M urea gels and visualized by autoradiography. Microinjection of the H19 Gene into Mouse
Zygotes. Brunkow and Tilghman ( 29) had previously attempted to
generate stable transgenic lines overexpressing H19 RNA by
microinjecting into mouse zygotes H19 transgenes that had
been marked with a 20-bp oligonucleotide insertion in the first exon of
the gene. This insertion was used to distinguish the transgene from the
endogenous gene. The transgene included a 4-kb segment of
5′-flanking DNA that is selectively hypermethylated on the paternal
chromosome throughout embryogenesis ( 13). The 8 or 11 kb of 3′-flanking
DNA contained two enhancers, each sufficient for expression of the gene
in endodermal cell lines in vitro ( 35). Surprisingly, no
stable lines were obtained that expressed the transgene. Instead,
founder transgenic embryos died late in gestation, between embryonic
day 14 and birth. The period of embryonic lethality was consistent with
the lethal phenotype of mice carrying a maternal disomy of chromosome 7
( 36, 37), leading to the hypothesis that extra copies of the
H19 gene were lethal in mice. In that same study, stably expressing transgenic lines were
successfully generated using an internally truncated structural gene
that carried the same 20-bp insertion in exon 1 (Fig.
, Δ2 H19). However, this
transgene also resulted in prenatal lethality, but with incomplete
penetrance, based on the fact that the surviving pups represented a
minority of the transgenic embryos generated ( 29).
To characterize this phenotype further, we wished to determine which
elements of the transgene were responsible for the late embryonic
lethality. We considered that the phenotype could be due to extra
copies of the H19 regulatory elements, such as the promoter,
enhancers, or a potential imprinting signal; increased dosage and/or
ectopic expression of the H19 gene product itself; or an
aberrant gene product created by the insertion of the oligonucleotide
at +580 bp. To discriminate among these possibilities, three additional
transgenes were generated. In the first two, labeled −4kb Luc and
−0.8kb Luc in Fig. , the structural H19 gene was replaced
with the firefly luciferase gene. These constructs differ only in the
amount of 5′-flanking DNA, and they directly test whether the
structural gene or its 5′ flank is required for the lethality. To
examine the possibility that the oligonucleotide was deleterious, we
cloned the M. spretus H19 gene to serve as a wild-type
allele that could be distinguished from the endogenous
M. domesticus allele in transgenic mice. Sequence comparison
of the M. spretus and M. domesticus genes,
including part of the promoters and the entire structural gene,
revealed >99% sequence identity between alleles (data not shown).
However, the small number of base changes was sufficient to distinguish
the two at the DNA level using a BamHI polymorphism at +2585
bp in exon 5 and at the RNA level by an allele-specific RNase
protection assay ( 22). The three transgenes were injected into zygotes, and the presence of
transgenic pups was examined at embryonic day 13 and weaning. In
contrast to the marked transgene, transgenic pups were identified at
the same high frequency at both times (Table 1).
Furthermore, these transgenes expressed luciferase and M. spretus
H19 RNAs in an appropriate manner in endodermal cells (see below).
Together, these experiments demonstrate that neither extra copies of
the H19 regulatory domain nor overexpression of wild-type
H19 RNA in endoderm interferes with normal development.
Rather—and intriguingly—the lethal effect of the original transgene
is probably due to the alteration of the gene product brought about by
the 20-bp insertion.
Appropriate Expression and Imprinting of M. spretus
H19 Transgenes. Transgenic lines were established for all
constructs by mating founders to DBA/2J mice. As summarized in Table
2, of the seven M. spretus lines
analyzed, five expressed the transgene in neonatal liver when inherited
from mothers. The two silent transgenes were present at one or two
copies in the genome, possibly reflecting a sensitivity of low copy
transgenes to position-dependent silencing.
| Table 2Expression and methylation of H19 and
luciferasetransgenes |
Having established that the transgenes were expressed in five lines, we
next examined whether the transgenes could be silenced by passage
through a paternal genome. As shown in Fig.
A for two of three independent
M. spretus transgene lines, the M. spretus RNA
was readily detected when the transgene was inherited through the
maternal germ line but not when inherited through the paternal germ
line. Furthermore, the silent transgene in the progeny of a male could
be reactivated by passage through the female germ line (Fig.
B), a hallmark of imprinting. The three lines that
displayed imprinted expression of M. spretus H19 carried
either four or five copies of the transgene. Two additional lines, both
containing only two copies of the transgene each, displayed no
parent-of-origin differences in expression (data not shown).
At the endogenous H19 locus, the silent paternal
H19 allele is hypermethylated from −5 kb through the
structural gene, while the maternal allele is almost completely
unmethylated in the same region ( 7). To determine if the imprinted
M. spretus transgene also assumed a differentially
methylated state dependent on the parental origin, neonatal liver
genomic DNA from animals inheriting the transgene paternally or
maternally were digested with HpaII, which is sensitive, and
its isoschizomer MspI, which is insensitive, to cytosine
methylation. As shown in Fig. B, a
transgene-specific 2-kb BamHI fragment spanning the 5′-most
flanking region of the transgene is almost completely resistant to
HpaII digestion at the three clustered sites within it when
inherited through a father. In contrast, the same transgene maternally
inherited is almost completely digested with HpaII. In Fig.
C, a 2.5-kb BamHI fragment that is further
downstream in the gene and is not transgene-specific displays the same
kind of extensive paternal-specific methylation of most copies of the
transgene, as indicated by the preponderance of the fully methylated
2.5-kb band in the HpaII lane, whereas the intensity of the
band in progeny of females is greatly reduced. The residual signal in
that band can be accounted for by the endogenous paternal
H19 gene. Consistent with what is observed with the
endogenous gene, hypermethylation in the 3′ flank of the
gene is weak or undetected (data not shown), indicating that marking of
the 5′ flank and gene body by methylation is sequence-specific
and does not reflect generalized methylation of exogenous sequences.
Lack of Imprinting of Luciferase Transgenes. The luciferase
transgenes provided an opportunity to test the function of the 5′ flank
and the H19 structural gene itself on the regulated
expression and imprinting of H19. Consistent expression of
the luciferase transgenes was observed in neonatal endodermal tissues
with all lines in which the transgene was present at greater than one
copy per genome. Overall, expression correlated well with copy number
of the transgene, and the RNA levels were not noticeably reduced by the
3.2-kb truncation of 5′-flanking DNA in −0.8k-b Luc (data not shown).
The two nonexpressing lines carrying luciferase genes were both present
at single copy in the genome, consistent with the silence of low copy
M. spretus H19 transgenes (Table 2). The expression of maternally and paternally inherited
H19–luciferase transgenes in neonatal liver was compared in
six lines carrying −4-kb Luc and the three carrying −0.8-kb Luc, as
illustrated in Fig. . No parental-specific
difference in the levels of expression was noted in any instance. Two
additional generations of backcrossing were undertaken for three −4-kb
Luc lines; however, the expression of the transgene remained
independent of its parental origin (data not shown).
Three −4-kb Luc lines were analyzed for evidence of parent-specific
DNA methylation in the 5′ flank, using the experimental strategy
described above. Representative results are presented in Fig.
. In contrast to the results obtained for the
M. spretus H19 transgene, there were no differences in the
pattern of DNA methylation between the maternally and paternally
inherited transgenes. Rather, the transgenes were almost completely
unmethylated, as is evident from the decrease in intensity of the
transgene-specific 2-kb BamHI fragment detected with probe I
following HpaII digestion. Thus, by the criteria of both
expression and CpG methylation, replacement of the H19 RNA
coding sequences results in loss of imprinting of the transgene, with
the transgene adopting the “maternal” mode of expression from
both chromosomes.
Defining the Requirements for H19 Imprinting. The striking difference between the imprinted expression of the
M. spretus H19 transgene and the absence of imprinting of
the luciferase transgenes could result from the loss of critical DNA
sequences, or from interference by foreign firefly sequences. We had
previously shown that bases +680 to +1660 are dispensable for
imprinting of the internally deleted Δ2 H19
transgene ( 7). To test other sequences within the gene, we created a
deletion of the first 700 bp of the structural gene,
Δ1 H19 (Fig. ). The deletion encompasses a highly
conserved domain within exon 1 that is differentially methylated on the
parental chromosomes ( 7, 21). When transiently transfected into the
human hepatoma cell line, Hep3B, Δ1 H19 RNA was
expressed at ≈5% of the level of M. spretus H19,
presumably because the shorter transcript is less stable. Δ1 H19 was microinjected into zygotes and lines were
established by crossing to DBA/2J. As expected from the transient
transfection results, Δ1 H19 RNA was not detected by
RNase protection or Northern analysis in the high background of the
endogenous H19 RNA expression (data not shown).
Therefore, we used the DNA methylation status of the transgenes
inherited from both parents as a means to assess the imprinting status
of the transgene. Three multicopy lines containing
Δ1 H19 were examined for methylation of
HpaII sites in the 5′-flanking region and at the promoter
(Table 2). As shown in Fig. for a representative line, the digestion
patterns obtained were indistinguishable for transgenes inherited
through the paternal and the maternal germ lines. Furthermore, like
−4-kb Luc, they resembled the pattern of the female-inherited M.
spretus transgene, which was strongly expressed. Thus, the
deletion of 700 bp of the 5′ end of the H19 gene was
sufficient to eliminate parental-specific DNA methylation. Transgene Methylation in Sperm. The paternally inherited copy
of the endogenous H19 gene acquires its
methylation during gametogenesis, whereas the female germ line
maintains the gene in an unmethylated state ( 9, 13). That difference is
retained during embryogenesis, including a period between fertilization
and blastocyst when the majority of the genome is demethylated ( 38,
39). The failure of the luciferase and Δ1 H19
transgenes to maintain paternal methylation of their 5′ flank
could reflect a failure to methylate the transgenes during
spermatogenesis or a failure to maintain the methylation during
embryogenesis. To discriminate between these possibilities, the status
of the transgene methylation was examined in testes DNA, which is
composed almost entirely of sperm DNA. As shown for a nontransgenic animal in Fig. ,
the 8-kb BamHI fragment spanning the 5′-distal portion of
the flank of the endogenous H19 gene is largely
methylated in sperm at all HpaII and HhaI sites
contained within it. The two imprinted M. spretus H19 lines
are also heavily methylated in sperm, as can be seen by the intensity
of the 2-kb transgene-specific band in the HpaII- and
HhaI- digested lanes (Fig. ) and the absence of the fully
digested 0.6-kb product.
The luciferase and Δ1H19 lines are inherited from
sperm in a slightly less methylated state, as is evident from the
appearance of fully digested products in both the HpaII and
HhaI digestions using the 5′-specific probe I (Fig. ).
However, in each case, a substantial fraction of the transgenes remain
fully methylated within the 5′ region. The methylation patterns of the
M. spretus H19, luciferase, and Δ1H19
transgenes also appear essentially identical further downstream, as
assessed using probe II (data not shown). Thus the luciferase and
Δ1H19 transgenes are inherited in a mostly
methylated state but cannot retain their methylation through
embryogenesis. Mutations Disrupting Imprinting Act Only in Cis. The foregoing
experiments establish a requirement for either the DNA sequences within
the H19 gene itself, or the RNA product, in transgene
imprinting. The experiments were performed in a wild-type
H19 genetic background, implying that the
endogenous H19 RNA is unable to rescue in trans
the loss of imprinting of the mutant transgenes. Previously we reported
that the H19 structural gene and its 5′ flank were required
for the imprinting of Igf-2 and Ins-2 as well
( 23). To test whether the loss of imprinting of Igf-2 could
be complemented by supplying H19 RNA in trans, we examined
the effect of maternally inherited M. spretus H19 transgene
expression on the expression of Igf-2 in H19
−/+ heterozygotes. Transgene line S21 was crossed to an
H19 deletion homozygote, and then female transgenic progeny
were crossed to B6(CAST- H19) males carrying the Igf-2
Mus castaneus allele. An allele-specific Igf-2 RNase
protection assay detected both the maternal M. domesticus
and the paternal M. castaneus alleles of Igf-2
RNA in neonatal liver in both transgenic and nontransgenic progeny
carrying the H19 deletion (Fig.
A), despite the fact that the
wild-type M. spretus H19 RNA was strongly expressed (Fig.
B). Thus, the transgene does not rescue the loss of
Igf-2 imprinting, implying that the chromosomal deletion of
H19 is acting in cis to cause misregulation of
Igf-2.
The nonequivalence of the haploid genomes contributed via the egg
and the sperm ( 1, 2, 3, 4) has been attributed to a subset of genes whose
expression is restricted to the maternal or to the paternal alleles.
Using reciprocal translocations to generate maternal and paternal
disomies at a high frequency, a number of chromosomal regions have been
identified in the mouse that must contain such imprinted genes ( 40).
The distal portion of mouse chromosome 7 is such a region. Paternal
disomic mice for the distal end of chromosome 7 die early in gestation
while maternal disomies cause late embryonic lethality ( 36). The early
embryonic lethality observed in Mash-2 null mice could
potentially explain the paternal disomy phenotype, as that gene is
maternally expressed ( 41). The gene(s) responsible for the late
embryonic lethality of maternal disomies is not yet clear. We had
suggested that ectopic expression of H19 might be a
phenocopy of the maternal disomy, based on the lethality we observed
with expression of a marked H19 transgene. In this study,
however, we could provide no evidence for this proposal, as ectopic
expression of wild-type H19 is not detrimental to normal
development. Rather, the lethality in the earlier study is probably due
to a neomorphic mutation generated by the DNA insertion used to mark
the transgene. This study establishes that sequences required for temporally correct
expression of the H19 gene in endodermal tissues map between
−800 bp and +11 kb relative to the start of transcription of the gene
at +1 bp. These transgenic results are consistent with transient
transfection studies ( 35) that had suggested the transcriptional
signals in the 5′ flank of the gene were contained within the first 50
bp. The importance of the two 3′ enhancers in directing
endoderm-specific expression of both the H19 and
Igf-2 genes was recently verified by the targeted deletion
of both enhancers in mice ( 24). In those mice, endoderm expression of
H19 and Igf-2 is ablated on the maternal and
paternal chromosomes, respectively. No single-copy M. spretus H19 or luciferase transgene was
expressed in vivo, suggesting that the constructs lacked
sufficient sequences to insulate the H19 transgene from
position effects. On the other hand, transgenes present at three or
more copies were uniformly expressed, with an approximate correlation
between copy number and expression level. Whether it is the duplication
of the enhancers themselves or another element acting as an insulting
element that leads to expression of the transgene in two or more copies
remains to be determined. In any case, these transgenes allowed us to
begin a dissection of the sequences required for imprinting of the
H19 gene. The first expressed imprinted gene identified in mice was not an
endogenous gene, but a foreign transgene ( 42), and
transgenic mice have served as a model system for examining the
molecular mechanism for genomic imprinting. Essentially, two classes of
imprinted transgenes have been investigated. In the first class are
transgenes that show imprinting in a position-dependent manner, as
manifested by hypermethylation of the maternal allele ( 43, 44, 45, 46, 47, 48).
Imprinting of these transgenes is particularly susceptible to the
genetic background of the mouse, a property which has been exploited to
map genetic modifiers of transgene imprinting ( 47, 48). In the second
class is the transgene RSVIgmyc, which is a fusion of
elements of the Rous sarcoma virus long terminal repeat, the
immunoglobulin heavy chain locus, the mouse c- myc gene, and
the plasmid vector pBR322 ( 42). This hybrid fragment is consistently
imprinted in a position-independent manner, with expression exclusively
from the hypomethylated paternal allele ( 49, 50). Mutational analysis
has begun to identify key sequences required for its imprinting ( 50). To date, the only endogenous gene that displays consistent
imprinting behavior as a transgene is the H19 gene.
Bartolomei et al. ( 7) had demonstrated in two independent
lines that an internally truncated version of the H19 gene,
Δ2 H19, displayed maternal-specific expression and
paternal methylation. Thus, unlike all other transgenes studied to
date, the H19 transgene is methylated when inherited from
fathers, not mothers, in keeping with the methylation of the
endogenous gene itself. This observation has been extended
in this study by using the full-length and unmarked M. spretus
H19 transgene, which is imprinted in three of five lines that
express the gene. The two exceptions were present at just two copies in
the genome, suggesting that the transgenes lack the full complement of
imprinting signals normally provided at the endogenous
locus. In fact, Tremblay et al. ( 13) have recently
identified CpG dinucleotides in the 5′ flank of the H19 gene
that lie outside the limits of the M. spretus transgene and
display properties that might be expected of a gametic mark. That is,
these sites are methylated in sperm but not in eggs and retain their
methylation during embryogenesis. Those missing sequences, if
important, can be replaced with multiple copies of the sequences
between −4 and +11 kb. The fact that the two-copy transgenes are well
expressed implies that the imprinting signals can be separated from the
transcriptional regulatory elements that are required for
high-level expression of the gene. Even in multiple copies, the imprinting of the M. spretus
H19 transgene is incompletely penetrant. In 5% of transgenic pups
where the transgene was inherited from the male, the transgene was
expressed at levels equal to that seen with maternal inheritance (data
not shown). In these pups, methylation of the transgene mimicked that
seen with maternally inherited transgenes. These pups represent either
a failure to establish the gametic imprint, an early misreading of the
imprinting signal, or a failure to maintain it, as the loss of
imprinting is complete, rather than intermediate, as would be the case
if there was a cell-by-cell error in interpreting the imprinting mark. A dissection of the imprinting signals on the M. spretus H19
transgene was begun by replacing the structural gene itself with a
luciferase reporter gene. By both the criterion of parental-specific
expression of the transgene and DNA methylation of its 5′ flank, the
fusion gene had lost all imprinting behavior. One can envisage three
possible explanations for the loss of imprinting of the luciferase
transgenes. The least interesting one is that lack of imprinting did
not reflect a requirement for the H19 gene itself, but
rather the foreign luciferase DNA interfered with imprinting signals
that were present on the transgene. This possibility was ruled out by
the lack of methylation imprinting of Δ1H19, where
no foreign DNA was introduced. Therefore either the DNA or the RNA it
encodes is required either to establish the epigenetic mark in the
gametes, and/or to retain that mark in the embryo. The luciferase transgenes were methylated in sperm, although not to the
same degree as the endogenous gene or the imprinted
M. spretus H19 gene. The reduction in methylation was most
evident in the 5′-most region examined, for both HpaII and
HhaI sites. This region contains at least a subset of the
sites of exclusive paternal DNA methylation that survive the
demethylation that occurs in the embryo ( 13). It appears that the
luciferase transgene cannot maintain the methylation it inherits, as
later in development the luciferase 5′ flank had become further
undermethylated on the paternally inherited chromosome. Thus the
structural H19 gene is required to establish its own
transgene imprinting. Previously Leighton et al. ( 23) showed that removal of the
active maternal H19 gene and its flank is sufficient to
completely overcome the silencing of the maternal Igf-2
gene. This result is consistent with the recent demonstration by Penny
et al. ( 51) that removal of the Xist gene, an
RNA-coding gene that maps to the X chromosome inactivation center,
prevents the inactivation of genes on the X chromosome carrying the
deletion. In each case, a genetic conundrum is presented ( 52). Is it
the loss of the DNA sequences or loss of the gene product that results
in the failure to silence the neighboring genes? We showed here that
expression in trans of M. spretus H19 RNA does not rescue
the loss of imprinting of maternal Igf-2, just as the
endogenous RNA does not rescue the loss of imprinting of
the luciferase transgenes. This suggests that, if there is a role for
H19 RNA in either its own silencing or the silencing of
Igf-2, the RNA acts in cis. Unlike a protein, a regulatory
RNA can act locally—at the site of transcription—so that mutations
can have a cis effect, even though the molecular mechanism may involve
a gene product. In this study, we show that a subset of the structural H19
gene itself is required for its imprinting as a transgene. The most
straightforward interpretation of these results is that these sequences
represent DNA regulatory elements required to mark the locus as
paternal and to maintain that mark during embryogenesis. An alternate
interpretation, equally consistent with the experimental results, is
that an RNA product synthesized from the 5′ third of the H19
gene is required to establish its own methylation imprinting. There are
two implications that follow from this interpretation, however. First,
the imprinting of Δ2H19 transgene argues strongly
that if an RNA product is required, it is not the mature, fully
spliced, and folded H19 RNA that accumulates at high levels
in many fetal tissues. Rather, the phenotypes of the two H19
deletions can only be reconciled with a role for the RNA itself by
proposing that the 5′ end of the RNA acts independently of the rest of
the RNA, for example, while the rest of the RNA is still being
synthesized. Second, it is paradoxical that the absence of
H19 RNA in the Luc and Δ1H19 transgenes
is affecting the imprinting of the paternal chromosome, on which the
RNA is transiently and weakly expressed only during spermatogenesis (J.
Saam and S.M.T., unpublished results). This leaves no apparent role for
the RNA on the maternal chromosome, where it is highly expressed.
Distinguishing between a role for H19 as a DNA element and
H19 as a regulatory RNA will be required to fully understand
the regulation of this cluster of imprinted genes on mouse chromosome
7. This work was supported by a grant from the National Institutes of
Health (GM51460). K.P. was a fellow of the Damon Runyon–Walter
Winchell Cancer Fund, and S.M.T. is an Investigator of the Howard
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