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Vol. 12, No. 23, pp. 3693-3702, December 1, 1998
Howard Hughes Medical Institute and Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 USA
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Abstract |
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Top
Abstract
Introduction
Results
Discussion
Materials & Methods
References
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Differentially methylated sequences associated with imprinted genes are proposed to control genomic imprinting. A 2-kb region located 5' to the imprinted mouse H19 gene is hypermethylated on the inactive paternal allele throughout development. To determine whether this differentially methylated domain (DMD) is required for imprinted expression at the endogenous locus, we have generated mice harboring a 1.6-kb targeted deletion of the DMD and assayed for allelic expression of H19 and the linked, oppositely imprinted Igf2 gene. H19 is activated and Igf2 expression is reduced when the DMD deletion is paternally inherited; conversely, upon maternal transmission of the mutation, H19 expression is reduced and Igf2 is activated. Consistent with the DMD's hypothesized role of setting up the methylation imprint, the mutation also perturbs allele-specific methylation of the remaining H19 sequences. In conclusion, these experiments show that the H19 hypermethylated 5' flanking sequences are required to silence paternally derived H19. Additionally, these experiments demonstrate a novel role for the DMD on the maternal chromosome where it is required for the maximal expression of H19 and the silencing of Igf2. Thus, the H19 differentially methylated sequences are required for both H19 and Igf2 imprinting.
[Key Words: Differentially methylated domain; DNA methylation; H19; Igf2; genomic imprinting]
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Introduction |
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Top
Abstract
Introduction
Results
Discussion
Materials & Methods
References
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The imprinted, maternally expressed mouse H19 gene is
located in proximity to a number of imprinted genes on the distal
portion of mouse chromosome 7 (Bartolomei et al. 1991
; Caspary et al. 1998). Other genes in the region include the
paternally expressed insulin-like growth factor 2 (Igf2) and insulin 2 (Ins2) genes and the
maternally expressed p57KIP2, Kvlqt1, and
Mash2 genes (DeChiara et al. 1991; Giddings et al. 1994;
Guillemot et al. 1995; Hatada and Mukai 1995; Gould and Pfeifer 1998).
A conserved cluster of imprinted genes is found on human chromosome
11p15.5 in the Beckwith-Wiedemann syndrome critical region (Reid et
al. 1997). A second cluster of imprinted genes resides in the
Prader-Willi and Angelman syndrome critical regions on human chromosome
15, with the syntenic region located centrally on mouse chromosome 7 (Nicholls et al. 1998). Given the intriguing clustering of imprinted
genes, it has been proposed that the imprinting of individual genes is
dependent upon their linkage to other imprinted genes (Barlow 1997;
Bartolomei and Tilghman 1992, 1997).
The interdependence of imprinted genes has been demonstrated clearly
for the mouse H19 and Igf2 genes. The mouse
H19 gene is highly expressed, does not encode a protein
product, and is located 75 kb from the gene encoding the fetal
mitogenic protein IGFII (Zemel et al. 1992). It has been shown that the
expression of these two genes is, in part, dependent upon competition
for two endodermal-specific enhancers that are located +9 and +11 kb
relative to the start of H19 transcription (Yoo-Warren et al. 1988). Deletion of these enhancers on the maternal chromosome results
in a loss of H19 expression in endodermal tissues, whereas deletion of the enhancers on the paternal chromosome results in the
corresponding loss of Igf2 expression (Leighton et al. 1995b). Additionally, deletion of the H19 structural gene and 10 kb of upstream flanking sequence from the maternal allele leads to expression of the normally repressed Igf2 gene, indicating that the
enhancers which initially supported maternally derived H19
expression were free to enhance the expression of Igf2
(Leighton et al. 1995a).
Whereas the imprinted expression of Igf2 is dependent upon
linkage to the H19 locus, the imprinting of H19
appears to be autonomous. This idea is supported by H19
transgenic experiments in which constructs harboring 4 kb of upstream
flanking sequence, an internally deleted H19 structural gene
and the 3' endodermal enhancers are imprinted similarly to the
endogenous gene (Bartolomei et al. 1993; Pfeifer et al. 1996; Elson and
Bartolomei 1997). We have proposed that this autonomous regulation is
governed by paternal-specific DNA methylation present at the endogenous
and transgenic loci (Bartolomei et al. 1993). The 7 kb of
paternal-specific methylation observed in somatic tissues and sperm
includes 4 kb of upstream flanking sequence and the H19
structural gene (Bartolomei et al. 1993; Brandeis et al. 1993;
Ferguson-Smith et al. 1993). The importance of methylation for the
repression of the paternal allele of H19 is underscored by
experiments in which imprinted gene expression was characterized in
mice that were deficient for the DNA methyltransferase gene
Dnmt1 (Li et al. 1993). When analyzed prior to their death, Dnmt1 null mice expressed both alleles of H19,
suggesting that DNA methylation is at least required to maintain
H19 imprinting. To demonstrate that DNA methylation could also
serve a causative role in the marking of the parental alleles and the
setting of the parental imprint, we assayed DNA methylation of
H19 during embryogenesis, with an emphasis on preimplantation
development as this is the time when the embryo undergoes a period of
generalized demethylation (Monk et al. 1987; Sanford et al. 1987). A
2-kb region located from 
2 to 4 kb relative to the start of
transcription is methylated exclusively on the paternal allele
throughout development, suggesting that this region is crucial to
determining the imprinted expression of H19 (Tremblay et al.
1995, 1997). When this region was deleted from the original imprinted
H19 transgene, the new transgenes were expressed and
hypomethylated regardless of parental origin (Elson and Bartolomei 1997).
To determine the role of the 2-kb differentially methylated domain (DMD) at the endogenous H19 locus, we have generated mice lacking the DMD. When the DMD deletion allele is transmitted to the progeny from the father, the normally repressed paternal H19 allele is activated and the expression of the linked paternal Igf2 gene is reduced coordinately. In contrast, transmission of the mutant H19 allele by the mother results in reduced expression of the H19 gene with a concomitant activation of the maternal Igf2 allele, revealing a novel regulatory role for this region. Whereas these experiments prove that the DMD is necessary for silencing the paternal H19 allele, they also show that the DMD is essential on the maternal chromosome for the exclusive expression of H19 and the silencing of Igf2. We conclude that the DMD is required on both parental alleles for the reciprocal imprinting of H19 and Igf2.
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Results |
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Top
Abstract
Introduction
Results
Discussion
Materials & Methods
References
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Targeted disruption of the H19 upstream differentially methylated domain
The region from approximately 2 to 4 kb relative to the
start of H19 transcription is methylated in sperm,
unmethylated in oocytes, and preferentially methylated on the paternal
allele throughout development (Tremblay et al. 1995, 1997; Olek and
Walter 1997). To test the role of this DMD at the endogenous locus, we deleted most of the DMD by gene targeting in embryonic stem (ES) cells
and generated mice with the deletion. As shown in Figure 1b, a
targeting vector was constructed in which 1.6 kb of the DMD was
replaced by the neomycin resistance (neor) gene
flanked by loxP sites. The deletion removes 48 of the
CpG dinucleotides that we have proposed to be essential for conferring imprinted expression (Tremblay et al. 1995, 1997). The remaining DMD
sequence includes five differentially methylated CpG dinucleotides located 5' to the targeted deletion.
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Because the function of a putative imprinting regulatory element was
being tested, it was important to eliminate any new regulatory elements
introduced by the neor gene cassette. Therefore,
following the identification of correctly targeted cells lines, two
independent clones were chosen for a second electroporation with a
vector encoding Cre recombinase to derive clones that deleted the
neor gene (Fig. 1c,d). Cells (with and without the
neor gene) were injected into C57BL/6J
host blastocysts and mice inheriting the targeted allele were selected
for subsequent breeding. The mutant mice were maintained by breeding to
C57BL/6J mice. For analysis of allelic imprinting
patterns, the heterozygous DMD mutant mice were mated with a strain of
mice [B6(CAST-H19), (Tremblay et al. 1995)] in which the
portion of distal chromosome 7 harboring the imprinted genes of
interest was derived from Mus musculus castaneus. Heterozygous
and homozygous DMD mutant mice were obtained in the predicted Mendelian
ratio and were viable and fertile.
Paternal inheritance of the DMD deletion
To determine the effect of the DMD deletion on the expression of
imprinted genes, mice that inherited the mutant allele
(H19
DMD) from the father were first
tested for H19 expression (Fig. 2a). When
the livers from neonatal heterozygous mice were analyzed by RNase
protection, the normally silent paternal H19 allele (Fig. 2a,
lanes 8-10) was activated to a level of ~60% of that observed for
the maternal wild-type allele, whereas H19 expression from the
maternal allele was unaffected (Fig. 2a, lanes 11-13). The analysis of
other tissues showed that the level of activation of the mutant
paternal H19 allele varied according to tissue type, with gut
derivatives exhibiting a moderate level of activation, whereas in
muscle derivatives activation was nearly equivalent to that for liver
(data not shown). These results indicate that deletion of the DMD
eliminated sequences that were repressive to H19 gene
expression. The deletion did not, however, completely activate
H19 expression to levels observed for the wild-type maternal allele.
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Because transcription of the Igf2 and H19 genes is
linked (Leighton et al. 1995a,b), the effect of the DMD deletion on
Igf2 expression was examined. Paternal transmission of the
mutant H19DMD allele caused
repression of the paternally inherited Igf2 gene (Fig. 2b, cf.
lanes 10-12 and lanes 7-9). When the level of paternal allele
expression in the liver of neonatal heterozygous mice was compared to
that of wild-type littermates, a 66% reduction in Igf2 RNA
was observed (Fig. 2d). As noted for H19, the reduction in
Igf2 expression varied according to tissue (data not shown). Consistent with the decrease in Igf2 expression, the weights
of the heterozygous littermates were on average 93% that of their wild-type littermates. Whereas reduction of Igf2 expression in liver is concordant with experiments proving that H19 and
Igf2 share enhancers, it is striking that the level of
activation of H19 from the mutant paternal allele was
equivalent roughly to the reduction of Igf2 expression on the
same allele.
Maternal inheritance of the DMD deletion
The effect of maternal transmission of the DMD mutation was also
tested for H19 and Igf2 expression. Surprisingly,
transmission of the mutant H19DMD
allele through the maternal germ line resulted in the reduced expression of the H19 gene (Fig. 2a, cf. lanes 4-7 with lanes 1-3). When quantified by RNase protection, the expression of
H19 in neonatal livers was approximately half that observed in
wild-type littermates (Fig. 2c). To determine if Igf2 was
affected by the maternally derived DMD mutation, allelic Igf2
expression levels were assayed. The normally silent maternal
Igf2 allele was activated to about one-third the level of the
wild-type paternal allele and expression from the wild-type paternal
allele was unaffected (Fig. 2b, lanes 2-6). Additionally, when
compared to their wild-type littermates, the mice that inherited the
mutant allele maternally were on average 17% larger, which is
consistent with activation of maternal Igf2. Thus, a reduction
in the level of H19 expression on the mutant maternal allele
was accompanied by an activation of the maternally derived
Igf2 gene and a slight increase in weight. Because the
coordinated expression of H19 and Igf2 was also
observed upon paternal transmission of the mutation, these results
indicate that deletion of the DMD resulted in a true competition for
the endodermal enhancers. These results additionally demonstrate that the DMD has a previously unsuspected positive regulatory function for
the exclusive expression of the maternal H19 allele.
Methylation analysis of the targeted H19 alleles
We have proposed that the DMD harbors an imprinting mark in the form
of paternal-specific methylation (Tremblay et al. 1995, 1997). Because
not all of the differentially methylated sequences at the H19
locus were removed by the DMD deletion, it was of interest to determine
the effect of the deletion on the methylation of the remaining CpG
dinucleotides. The CpG dinucleotides located in the promoter-proximal
region and the 5' portion of the H19 structural gene are
preferentially methylated on the paternal allele late in gestation
[see Fig. 3d, sites between 500 and +501 bp (Bartolomei et al.
1993; Brandeis et al. 1993; Ferguson-Smith et al. 1993; Tremblay et al.
1997)]. In contrast, the 3' portion of the
H19 structural gene is equally methylated on both alleles [see Fig. 3d, sites downstream of +501 bp (Ferguson-Smith et al. 1993)], as are sites 5' of the 2-kb DMD [Fig. 3d, upstream of 4000 bp (Tremblay et al. 1997)]. To determine whether the
methylation of the H19 promoter and structural gene was
affected by the mutation, neonatal liver DNA from reciprocal
heterozygotes was digested with PvuII and StuI and
the methylation-sensitive restriction enzyme HpaII and
subjected to Southern analysis (Southern 1975). The wild-type M. castaneus allele lacks a PvuII site, which enabled the
distinction between the mutant C57BL/6 H19
allele (3.2 kb) and the wild-type M. castaneus H19 allele (3.4 kb) (Bartolomei et al. 1993). When the mutation was transmitted to the
progeny by the mother, the methylation of the H19 promoter and
structural gene appeared unaffected (Fig. 3b, cf. lanes 4 and 6). In
contrast, the deletion of the DMD on the paternally inherited allele
resulted in the hypomethylation of the HpaII sites in the
promoter region (Fig. 3b, PvuII/StuI
fragment in lanes 8 and 10). Similarly, a HhaI site in the
promoter was not methylated on the mutant paternal allele (data not
shown). These results were consistent with methylation analysis in
homozygous mutant animals in which the promoter was hypomethylated on
both alleles (Fig. 3b, lane 13). Thus, deletion of the DMD from the
paternal allele was accompanied by a loss of methylation in the
sequences surrounding the promoter, causing the mutant paternal allele
to resemble the wild-type maternal allele (Fig. 3d).
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Next, we examined the methylation status of upstream CpG dinucleotides
that remained on the mutant allele (Fig. 3a, sites between 4500 and
500 bp). The HhaI site located immediately 5' to the
deletion and the two HhaI sites located between the promoter
and the DMD were analyzed using SacI polymorphic fragments (Fig. 3a). In wild-type animals, the sites were hypermethylated on the
paternal allele but hypomethylated on the maternal allele (Fig. 3c,
lanes 5,6,9,10). However, the mutant
H19DMD allele was hypermethylated
whether it was maternally or paternally transmitted (Fig. 3c, 
DMD
SacI fragment, lanes 7 and 8 and 11 and 12, respectively).
This hypermethylation of the mutant allele was also evident in
homozygous mutant animals (Fig. 3c, lanes 15,16). The remaining
HpaII sites in this upstream region were also hypermethylated
on both mutant alleles (data not shown). Thus, whereas the DMD
deletion was associated with the hypomethylation of cytosine residues
surrounding the promoter, CpG dinucleotides located upstream from the
promoter were partially methylated on the mutant alleles (Fig. 3c and
data not shown). Taken together, these results indicate that the
methylation status of the mutant alleles reflected neither the maternal
nor paternal wild-type pattern (Fig. 3d). Rather, the mutant allelic
methylation pattern was intermediate between the two wild-type parental
alleles and no longer parental-specific.
We also compared the methylation status of sperm DNA isolated from
wild-type and homozygous mutant adult males generated from F1
heterozygous intercrosses. The methylation state of the H19 promoter, structural gene, and remaining 5' sequence was unchanged with the removal of the DMD sequence. Specifically, the wild-type and
DMD sperm DNA were similarly unmethylated in the H19
promoter region (Fig. 3b, lanes 14 and 15) and were similarly
methylated at HhaI sites immediately 5' and 3' of the
deleted sequence (data not shown). These data indicate that removal of
DMD sequence did not perturb the acquisition of the sperm-specific
methylation pattern in the remaining H19 sequence.
Analysis of H19DMDneo alleles
The experiments described above used mice in which the
neor gene was excised. To determine if the
perturbation of imprinted gene expression was caused solely by the
absence of the DMD or if the spacing change imposed by the deletion was
responsible for altered gene-expression patterns, mice in which the
neor gene remained at the H19 locus were
examined. Because the size of the neor gene was
similar to that of the deleted DMD fragment, inclusion of the
neor gene preserved spacing of the H19
upstream elements. As observed for the
H19DMD alleles, both the H19
and Igf2 genes were expressed on the maternal and paternal
H19DMDneo alleles in neonatal liver
(Fig. 4). The neor gene did not
affect H19 expression levels on either parental allele (Fig.
4a, data not shown), supporting the proposed role of the DMD as both a
positive and negative regulator of H19 gene expression.
However, the presence of the neor gene caused a less
dramatic increase in the expression of Igf2 on the mutant
maternal allele (Fig. 4b, lanes 2,3). That is, in the presence of the
neor gene, Igf2 was activated to ~6%
that of the wild-type paternal allele, whereas in the absence of the
neor gene, Igf2 expression on the mutant
maternal allele increased to an average of one-third the level of the
wild-type paternal allele (Fig. 4b, lanes 1-3 and 4-6, respectively).
These results suggest that the neor gene interfered
with the activation of Igf2 on the mutant maternal allele and
are consistent with studies of regulatory elements at other loci
demonstrating that the neor gene regulatory elements
affected gene expression (Fiering et al. 1995).
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The imprinting of the neor gene was also assessed in
RNA isolated from livers of heterozygous and homozygous
H19DMDneo neonatal mice. Northern
blot analysis showed that the neor expression levels
were similar in the maternal and paternal
H19DMDneo heterozygous mutants and
twofold higher in the homozygous mutants (data not shown),
demonstrating neor expression was not imprinted.
Thus, in contrast to previous experiments in which the
neor gene was used to replace the H19
transcription unit and was imprinted (Ripoche et al. 1997), the
sequences remaining at the H19DMD
locus were not sufficient to imprint the neor gene.
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Discussion |
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Top
Abstract
Introduction
Results
Discussion
Materials & Methods
References
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We proposed previously that paternal-specific DNA methylation in the
region located from 2 to 4 kb relative to the start of
H19 transcription is involved in the imprinting of the mouse H19 gene (Tremblay et al. 1997). Absence of this region in a
normally imprinted mouse transgene results in parental-independent
expression and hypomethylation of the derivative transgene (Elson and
Bartolomei 1997), indicating that the DMD is crucial for suppression of
a paternally transmitted transgene. This element also has unique properties in Drosophila where it acts as a silencer, despite the absence of DNA methylation in the Drosophila genome (Lyko et al. 1997).
To test the role of the DMD at the endogenous H19 gene locus,
we have deleted most of this region using gene-targeting technology. Paternal transmission of the mutant allele resulted in activation of
H19 expression, a concomitant reduction in Igf2
expression, and a reduction in methylation of the remaining CpG
dinucleotides at the H19 locus (Fig. 5).
These results are consistent with the transgenic experiments and
support the hypothesis that the DMD represses transcription of the
paternally derived H19 allele. The negative regulatory role of
the paternally derived DMD is likely caused by its hypermethylation
that could either act directly by preventing the binding of factors
that establish a transcriptionally competent state or could act
indirectly through the methyl-CpG-binding protein MeCP2, or other
unknown proteins with analogous activities, which subsequently recruits
histone deacetylases and represses transcription (Jones et al. 1998b;
Nan et al. 1998).
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A new finding of this study was the observed decrease in H19
expression together with the concomitant activation of Igf2
upon maternal transmission of the mutant allele, suggesting that the DMD also influences transcription of H19 on the maternal
allele positively (Fig. 5). Indeed, chromatin studies of the
H19 locus have shown that the DMD is hypersensitive to
nucleases exclusively on the maternal chromosome, supporting the
concept of a novel maternally derived positive role for this element
(Hark and Tilghman 1998). A number of mechanisms could lead to this
positive regulatory function. First, the unmethylated DMD might bind
transcription factors that promote the expression of H19. In
the case of the mutant maternal allele, the decreased expression of
H19 may be caused by the loss of positive transcriptional
elements rendering H19 less effective in utilizing the
endoderm-specific enhancers. These enhancers are then free to drive the
expression of Igf2. Thus, although the DMD is not required for
transgenic H19 expression (Pfeifer et al. 1996; Elson and
Bartolomei 1997), possibly because of the absence of the competing
Igf2 gene, it appears to be essential for the optimal and
exclusive expression of H19 at the endogenous locus. Second,
the maternally derived DMD may form a unique chromatin configuration
that promotes transcriptional activity. Third, the DMD could act as a
selector that directs transcription either toward H19 or
Igf2. Fourth, the DMD may function to repress Igf2 transcription on the maternal allele, thereby indirectly affecting H19 expression. Thus, in the absence of the DMD on the
maternal allele, Igf2 expression is derepressed, which reduces
access of the enhancers to H19. Finally, Tilghman and
colleagues have suggested that the DMD acts as a domain boundary or a
chromatin insulator which isolates the H19 promoter and
endodermal enhancers and blocks the Igf2 gene from accessing
these enhancers on the maternal chromosome (Webber et al. 1998).
Originally identified in Drosophila, boundary elements
insulate a gene and its regulatory elements from position effect
variegation and can block gene expression when placed between a gene
and its enhancers (Kellum and Schedl 1991, 1992). The proposal that the
DMD functions as a domain boundary in mouse is supported by experiments
in which Igf2 is preferentially expressed on maternally derived chromosomes in which the H19 endoderm enhancers were
removed from their normal location and placed between the H19
and Igf2 genes and upstream of the DMD (Webber et al. 1998).
Although these latter experiments could be explained by distance
effects or the placement of the enhancers in a new chromatin
environment, taken together, the gene-targeting experiments would argue
in favor of the DMD acting as a domain boundary. Formal proof of this
model will require the demonstration that the DMD insulates gene
expression at a heterologous locus.
The experiments described in this report support the original model
proposing that the reciprocal imprinting of the H19 and Igf2 genes is mediated by a competition for the shared set of endoderm enhancers [Fig. 5 (Bartolomei and Tilghman 1992)]. Mice harboring the DMD deletion express H19 and Igf2 from
the mutant chromosome, with enhanced expression of one gene accompanied
by a coordinate decrease in the expression of the other gene. The canonical example for this type of regulation is the promoter competition in the chicken 
-globin gene complex, where the switch from the embryonic 
-globin to the adult -globin is achieved through a competition for the -globin enhancer (Choi and Engel 1988; Foley and Engel 1992). Similar to the compensatory expression changes observed in our mutant mice, mutations that attenuated adult
-globin expression were accompanied by an increase in the expression of -globin (Foley and Engel 1992). Recent experiments by Jones and colleagues indicate that the competition by H19
and Igf2 promoters for the endoderm enhancers may not be
mediated strictly by the promoters and DMD alone (Jones et al. 1998a). In experiments in which the H19 transcriptional unit was
replaced with the luciferase gene, luciferase was
expressed at variable levels on the paternal allele, whereas the
expression and imprinting of Igf2 was maintained at wild-type
levels. One interpretation of these experiments is that the RNA-coding
portion of the H19 gene is also required for linked
competition of these genes.
Our study does not address the mechanism by which H19 and
Igf2 share enhancer elements at the cellular level. For
example, in the paternal mutant heterozygote, H19 and
Igf2 may be expressed simultaneously from the mutant allele.
Alternatively, each cell makes a choice: some cells may exclusively
express Igf2 from the mutant paternal allele and other cells
may exclusively express H19. It is also possible that each
cell expresses both genes from the mutant paternal allele but only one
gene is expressed at a given time. The latter is analogous to the
flip-flop model of gene regulation that has been proposed to explain
how distal control elements allow the simultaneous expression of 
-
and -globin in early development (Wijgerde et al. 1995). Future
studies of our mutants at the cellular level will discriminate between
these possibilities.
Finally, we have determined that deletion of the DMD on one chromosome
does not appear to affect the expression or methylation of the genes on
the wild-type chromosome. Unlike experiments showing that Igf2
transgenes can transactivate the endogenous Igf2 gene and lead
to Beckwith-Wiedemann-related symptoms (Sun et al. 1997), the
activation of either H19 or Igf2 on the mutant
chromosome does not affect the expression of their counterparts on the
wild-type chromosome. Furthermore, after three generations of breeding
the mutant mice, the only observed phenotypes in animals
with alterations in H19 and Igf2 expression are
subtle size effects that are consistent with the changes in
Igf2 expression. No phenotypic consequences reminiscent of
Beckwith-Wiedemann have been noted. Additionally, homozygous mutant
animals have no apparent phenotype, possibly because the total
expression of H19 and Igf2 in the neonatal livers of
homozygous animals is similar to that of wild-type animals (data not shown).
In conclusion, we have demonstrated that the DMD has multiple roles in regulating the imprinting of the H19 gene. Our hypothesis that the differential methylation serves as the allelic mark is strengthened by the observation that deletion of the DMD on both chromosomes renders them indistinguishable by the criteria employed in these studies. As expected, the DMD mediates a repressive effect on the transcription of the paternally derived chromosome, presumably through its hypermethylation. Additionally, we have shown that the DMD permits the exclusive expression of H19 on the maternal chromosome, possibly through the binding of positive regulatory factors, a unique chromatin configuration, inhibition of Igf2, or through the assembly of a domain boundary which prevents access of Igf2 to the enhancers. Future studies will elucidate the factors responsible for the multiple roles of this complex element.
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Materials and methods |
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Top
Abstract
Introduction
Results
Discussion
Materials & Methods
References
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Preparation of the targeting vector
A phage clone containing 13.5 kb of 5' flanking and 1.8 kb of
H19 gene sequence was isolated from a 129Sv/J
mouse genomic library in the Lambda FIX II vector (Stratagene). The
phage insert was subcloned as two fragments into Bluescript II KS
(Stratagene); a NotI/KpnI fragment
corresponding to 9.8 kb of 5' flanking H19 sequence
(p9.8N/K) and a KpnI/NotI
fragment containing the remaining 5' flanking and H19 gene
sequence (p5.5K/N). The KpnI site is 3.7 kb
relative to the start of H19 transcription and is the 5'
boundary of the DMD-targeting event. To initiate construction of the
targeting vector, an EcoRI/XhoI
fragment containing a 2-kb PGK-neo loxP flanked cassette was
subcloned into Bluescript II KS and the XhoI site was
eliminated subsequently (ploxPneo-BS). p5.5K/N was then
digested with HindIII, blunted with Klenow, and digested with
NotI to generate the 3.9-kb
HindIII/NotI H19 fragment that
serves as the 3' arm of the targeting vector. This fragment was
subcloned into NotI/SmaI-digested
ploxPneo-BS to generate pneo3'H19. To assemble the target vector the
following were simultaneously ligated: a 6.6-kb BamHI/KpnI
5' H19 fragment (from p9.8N/K), a 5.9-kb
KpnI/NotI fragment including
loxP-neo cassette and 3.9-kb H19 sequence (from
pneo3'H19), and NotI/BamHI digested
Bluescript II KS. Finally, a 2.3-kb SalI fragment containing a
diphtheria toxin A cassette (McCarrick et al. 1993) was ligated to the
XhoI linearized target vector. The final targeting vector
contains a total of 10.1 kb of homology to the H19 locus (Fig. 1b).
Targeted disruption of the DMD region in ES cells
The vector was linearized at a unique NotI site prior to
electroporation into ES cells. E14.1 ES cells (Kuhn et al. 1991) were
grown on neomycin-resistant mouse embryonic fibroblasts. ES cells
(1.5 × 107/ml) were collected in 0.8 ml of
phosphate-buffered saline and electroporated with a pulse of 250 V/500 mF (Gene Pulser, Bio-Rad) with 25 µg of
linearized targeting vector. Following a 24-hr recovery, the medium was
adjusted to 200 µg/ml G418. After 8-10 days growth, G418-resistant colonies were isolated and expanded, and DNA was prepared. The DNA was digested with EcoRV (5'-end
confirmation) or StuI (3'-end confirmation) and size
fractionated on 0.75% and 1.0% agarose gels, respectively. DNA was
transferred to nitrocellulose (Southern 1975) and hybridized to
nick-translated external probes (Rigby et al. 1977). The
EcoRV/EcoRI probe was used for
5'-end confirmation and the
BamHI/StuI probe was used for
3'-end confirmation (Fig. 1b).
The neor cassette was removed by transiently
transfecting two independent DMDneo ES cell lines with
25 µg of a plasmid encoding the Cre recombinase (Sauer and
Henderson 1990). Correctly excised clones were verified by digestion
with StuI or EcoRV, as described above, and by a 350-bp PCR product that was amplified using primers that flank the
DMD mutation. The forward primer was
5'-ATCCAGGAGGCATCCGAATT-3' and the reverse primer was
5'-GTGTCACAAATGCCTGATCC-3'.
Cells from targeted ES cell clones (with and without the
neor gene) were injected into C57BL/6J
blastocysts, and the blastocysts were transferred to pseudopregnant
female mice. To determine if germ-line transmission of the mutant
allele had occurred, chimeras were bred with C57BL/6J
mice, and DNA was isolated from tail biopsies of progeny. The Southern
blot and PCR analyses described above were used to genotype the mice.
To analyze allelic expression and methylation patterns, the
heterozygous mutant mice were bred to the B6(CAST-H19) strain
of mice (Tremblay et al. 1995). These mice have M. castaneus
H19 and Igf2 alleles on a C57BL/6
background. For F1 hybrid mice, the maternal parent is
designated first.
RNA isolation and analysis
Total RNA was prepared from various staged mouse tissues by the
lithium chloride method (Auffray and Rougeon 1980). The RNase protection assays that were used to detect H19 (Bartolomei et al. 1991; Brunkow and Tilghman 1991), Igf2 (Leighton et al.
1995a), and rpL32 (Dudov and Perry 1984) gene expression were
performed as described previously. Products were resolved on a 7.0%
acrylamide/7 M urea gel.
For quantitation, RNase protection gels were exposed to storage
phosphor screens that were scanned on a Storm 840 PhosphorImager (Molecular Dynamics). Band intensities were calculated using ImageQuant Version 1.0 (Molecular Dynamics). After pseudocolor enhancement of the
image, bands of interest were traced using the freehand drawing tool.
The median pixel values of individual segment boundaries were used as
the background values. In all cases, RNase protection assays of the
deletion allele produce one protected fragment, whereas assays of the
wild-type M. castaneus allele produce two protected fragments.
In samples in which biallelic expression was observed, the value of the
mutant protected fragment was quantified relative to the two fragments
corresponding to the wild-type B6(CAST-H19) allele. The
levels of H19 RNA in DMD maternal heterozygotes and Igf2 RNA in DMD paternal heterozygotes were quantified
relative to wild-type levels using rpL32 as an internal control.
DNA isolation and methylation analysis
DNA was isolated from tissues and sperm as described previously
(Bartolomei et al. 1993). Genomic DNA (10 µg) was digested with
PvuII and StuI in combination with HpaII or
MspI to analyze the methylation of the H19 structural
gene or with SacI and HhaI to analyze the methylation
of upstream sequences. The probes used for the respective analyses were
the 2.5-kb EcoRI-StuI (RSt) fragment and the 0.9-kb
SacI-KpnI (ScK) 5' fragment (Fig. 3a).
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Acknowledgments |
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We thank J. Richa and the University of Pennsylvania Transgenic Core Facility for the production of chimeric mice. We thank M. Malim, S. Liebhaber, A. Webber, K. Tremblay, and members of the lab for critical reviews of the manuscript. We are grateful to A. Hark and S.M. Tilghman for communication of results prior to publication. This work was supported by U.S. Public Service grant GM51279 and the Howard Hughes Medical Institute. J.L.T. was supported by National Research Service Award postdoctoral fellowship GM18458.
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked `advertisement' in accordance with 18 USC section 1734 solely to indicate this fact.
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Footnotes |
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Received September 3, 1998; revised version accepted October 8, 1998.
1 Corresponding author.
E-MAIL bartolom{at}mail.med.upenn.edu; FAX (215) 573-6434.
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References |
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Top
Abstract
Introduction
Results
Discussion
Materials & Methods
References
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a common motif for the imprinting mechanism.
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Genes & Dev.
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2203-2213- vs. -globin gene transcription: Genetic confirmation of promoter competition during globin gene switching.
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  A. L. Fortier, F. L. Lopes, N. Darricarrere, J. Martel, and J. M. Trasler Superovulation alters the expression |
) The endodermal enhancers located at +9 and +11
kb. (b) From top to bottom: the linearized
targeting vector, the endogenous H19 locus, the targeted
H19 locus (H19
).
Molecular sizes (in kb) are indicated to the right. Of the 85 G418-resistant clones analyzed, four were correctly targeted to the
H19 locus.
