Download Epigenetic and genetic factors affect transgene

165
Development 107, 165-168 (1989)
Printed in Great Britain @ The Company of Biologists Limited 1989
Epigenetic and genetic factors affect transgene methylation imprinting
CARMEN SAPIENZA*, JEAN PAQUETTE, THU HANG TRAN and ALAN PETERSON
Ludwig Institute for Cancer Research, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1
* To whom correspondence should be addressed
Summary
In some lines of transgenic mice, the methylation ofMspl
sites within or adjacent to the transgene locus is affected
by the sex of the parent from which the transgene is
inherited. These differences are consistent with a role for
DNA methylation in genome imprinting. In a previous
report, we noted that in one such line, all offspring of
females exhibited hypermethylation of the transgene
while only some offspring of males carried a hypomethylated transgene. In this report, we provide evidence
that this phenomenon is controlled by at least two
factors, one of which acts in cis and is dependent on the
transgene locus, and one of which acts in trans and is
supplied by the maternal genome. We also provide
evidence that there are genetic differences between
inbred mouse strains in the trans-acting factor.
Introduction
tions, gel eletrophoresis, in vitro 32P-labelling of hybridization
probes, hybridization and autoradiography were performed
as described in Sapienza et al. 1987. The derivation of the 379
troponin 1 transgenic line is described in Hallauer et al. 1988.
Embryos used for injection of DNA were the result of matings
between B6D2Fi mice. C57BL/6J, DBA/2J and B6D2F!
mice were obtained from The Jackson Laboratory, Bar
Harbor, ME.
The methylation state of some transgene loci changes in
a predictable manner depending upon their maternal or
paternal inheritance (Reik et al. 1987; Sapienza et al.
1987; Swain et al. 1987; Hadchouel et al. 1987). A direct
relationship between this type of 'methylation imprinting' and imprinting as defined by pronuclear transplantation (McGrath and Solter, 1984; Surani et al. 1984;
Solter, 1988) or genetic experiments (Cattanach and
Kirk, 1985; Searle and Beechey, 1985; Solter, 1988) has
not been established. However, these observations are
consistent with a role for differential DNA methylation
in the process of genome imprinting.
We have previously demonstrated (Sapienza et al.
1987) that particular Mspl sites within a quail troponin I
transgene always became methylated in the somatic
tissues of offspring that inherited the locus from a
female. However, when the same locus was inherited
from a male, some, but not all, offspring exhibited
transgene hypomethylation in their somatic tissues
(Sapienza et al. 1987).
In this report, we demonstrate that the transgene
methylation phenotype observed in the somatic tissues
of offspring of transgenic males is affected by both the
transgene methylation phenotype exhibited by the
male, and the strain background of the non-transgenic
female. These data imply that both cw-acting and transacting elements are involved in the establishment of the
methylation imprint.
Materials and methods
All tissue DNA preparations, restriction endonuclease diges-
Key words: genome imprinting, transgenic mice, mouse
genetics.
Results
A transgenic mouse line, designated line 379, was
established by microinjection of a 7-1 kb DNA fragment
which contained the entire coding sequence of the quail
troponin I gene, as well as 375 bp of pBR322 at the 5'
end and 275 bp of pBR322 at the 3' end (Fig. 1 and
Sapienza et al. 1987). This fragment contains sequences
sufficient to direct the expression of the quail gene in
fast twitch muscle fibers of the mouse (Hallauer et al.
1988). As previously reported (Sapienza et al. 1987) this
transgene locus, consisting of 15-20 copies of the
injected sequence, displayed gamete-of-origin dependent methylation changes at some Mspl sites within the
transgene array. While the hypermethylated phenotype
appeared consistent when the transgene was inherited
from a female, inheritance of the transgene from males
did not always result in hypomethylation of the transgene (Sapienza et al. 1987).
Blot hybridization analysis of more than 200 transgenic offspring from crosses between non-transgenic
females and males hemizygous for the transgene locus
has revealed three apparently distinct transgene methylation phenotypes. Representatives of these pheno-
166
E
C. Sapienza and others
B
MMM
M
MM
M
M B S
I kb
Fig. 1. Partial restriction endonuclease cleavage map of the
micro-injected DNA fragment containing the quail troponin
I gene. The open box shows the position of the
BamHl-Kpnl fragment used as a hybridization probe in
Figs 2-4. E: EcoRl, B: BamHl, M: Mspl, S: Sail.
types are shown in Fig. 2. Individuals 64, 427, 429, and
431 represent the 'low' transgene methylation pattern,
characterized by prominent 0-87 kb and 1-3 kb bands,
the absence of a 1-35 kb band, and less prominent and
approximately equally intense 2-9 kb and 3-2 kb bands.
The 'intermediate' phenotype is represented by individuals 433, 434, 435, 437 and 298 and is characterized
by a prominent 3-2 kb band, a faint 1-35 kb band and
the continued presence of the 0-87 kb band. The 'high'
phenotype is shown by individuals 438, 439, 442, and 56
and is characterized by a virtual absence of the 0-87 kb
band, a prominent 3-2 kb band, and 1-3 kb and 1-35 kb
bands which are of equal intensity. Hybridization of the
same probe used in Fig. 2 to Mspl-cleaved DNAs
results in the appearance of only the 0-87 kb fragment
(data not shown). Additional matings and hybridization
analyses revealed no evidence of either somatic or
germline loss of transgene sequences (data not shown).
Which of the three phenotypes appears in the off-
spring of transgenic males mated to non-transgenic
females depends on both the methylation phenotype of
the transgenic male, and the strain background of the
non-transgenic female. This is illustrated by the crosses
in Fig. 2.
When mated to DBA/2J females, male 64 (low
phenotype) gave rise to offspring that displayed the low
somatic transgene methylation pattern, identical to his
own. However, when mated to C57BL/6J females, his
offspring displayed the intermediate pattern. Because
all of these offspring were sired by the same male, these
differences most likely arise as a consequence of differences in the genetic background of the non-transgenic
female. Similarly, male 56 (high phenotype) gave rise to
offspring that displayed one of two transgene methyl.ation phenotypes. When mated to C57BL/6J females,
his offspring displayed a high methylation phenotype,
identical to his own. However, when mated to DBA2/J
females, his offspring displayed the intermediate
phenotype (Fig. 2 and data not shown). The difference
between these two classes of offspring again appears to
reflect differences in the genotype of the non-transgenic
female.
To determine whether such differences were genetic,
and based upon single or multiple loci, a series of
crosses was analyzed. Transgenic males with the high
methylation phenotype were mated to B6D2Fi females,
and the methylation phenotype of the offspring assayed. Fig. 3 shows some of the results obtained. The
apparent strain-specific differences observed in Fig. 2
B6D2
427 429 431 433 434 435 437 438 439 442
298
3.22.9 —
1.9 —
1.3—
•
*
ft
1.90.87
I
1.3Fig. 2. Blot hybridization analysis of transgene methylation
phenotype of individuals resulting from crosses between
non-transgenic females and transgenic males. Open symbols
are non-transgenic individuals, half-filled symbols are
individuals that are hemizygous for the transgene. DNA
prepared from tail biopsies was cleaved with Hpall and
BamHl. All restriction endonuclease digestions were
checked for completion of digestion by incubating 0-75 fig of
a test plasmid with an aliquot of the sample digest.
Digestions were judged to be complete when the cleavage
pattern of the test plasmid incubated with the sample was
identical to the cleavage pattern of the test plasmid alone.
0.87Fig. 3. Blot hybridization analysis of transgene methylation
phenotype of individuals resulting from crosses between
B6D2F! females and transgenic males. Lanes under halffilled triangles contain DNA samples extracted from the
skin of day 14 embryos. DNAs were cleaved with Hpall
and BamHl as in Fig. 2.
Transgene methylation imprinting in mice
d98
>*
64
(A
^
I fill I I *
3.22.9-
1.9-
1.3 —
0.87 —
Fig. 4. Blot hybridization analysis of transgene methylation
phenotype of tissues of a high phenotypc male (male 98,
see also Fig. 3) and low phenotype male (male 64, see also
Fig. 2). DNAs were cleaved with HpaU and BamHl as in
Fig. 2.
segregate in the ova of Fi females, giving rise to
offspring with either the high phenotype or the intermediate phenotype within the same litter. Of 36 offspring sired by high methylation phenotype males
mated to B6D2Fj females, 17 exhibited the intermediate phenotype and 19 showed the high phenotype
(Fig. 3 and data not shown). This ratio does not differ
significantly from 1:1 and is consistent with an allelic
difference at a single genetic locus.
To determine whether the differences observed between the offspring of high and low methylation phenotype males (male 98 and male 64, respectively) reflected
differences in the methylation state of the transgene in
the gametes of each male, we analyzed the methylation
phenotype of tissues derived from each primary germ
layer (Hogan et al. 1986) of each individual, including
testes. Fig. 4 shows that within each individual, all
somatic tissues analyzed exhibit the same transgene
methylation phenotype. Thus the difference in the
somatic methylation phenotype is consistent between
all somatic tissues of the two males. However, no
difference was observed in the methylation phenotype
of their testes. Therefore, the transgene methylation
phenotype of their gametes does not correlate with the
differences observed between their offspring.
Discussion
Analysis of the transgene methylation phenotype of
more than 200 offspring sired by transgenic males has
revealed that the methylation phenotype of offspring is
167
controlled by both an epigenetic factor and a genetic
factor. The epigenetic factor reflects the methylation
phenotype of the sire; those sires with the low somatic
phenotype giving rise to offspring with either the low or
intermediate phenotype; and those sires with the high
somatic phenotype giving rise to offspring with either
the high or the intermediate phenotype. Which of the
phenotypes appears among their progeny is dependent
on the inbred strain background of the non-transgenic
female to which the male was mated.
These data imply that the process of transgene
methylation imprinting involves both cw-acting elements, which reflect the methylation state of the
transgene in the previous generation, and trans-acting
factors, which are supplied by the maternal genome
after fertilization. Our data also demonstrate that there
are genetic differences among inbred strains in the
trans-acting factor. The trans-acting factor that gives
rise to these phenotypes is apparently controlled by a
single genetic locus because the segregation ratio of
phenotypes produced by Fi females is 1:1.
In order to account for the segregation of these
phenotypes from diploid Fi ova, the expression of this
locus must either be subject to allelic exclusion (if
expression begins prior to meiosis in the oocyte) or
expression must not begin until the completion of
meiosis, i.e. after formation of the second polar body,
at fertilization. The existence of a transacting factor
that does not operate until after fertilization is consistent with data obtained on the somatic methylation
pattern of endogenous loci, which indicate that postfertilization changes take place (Sanford et al. 1987).
The cw-acting element that operates within this
system must logically be due to either the transgene
itself or its integration site. One must presume that
integration site has some effect because different lines
that carry the same transgene may behave differently,
with respect to whether a gamete of origin methylation
effect is observed (Reik et al. 1987; Sapienza et al.
1987), but these data do not eliminate the possibility
that sequences endogenous to the transgene also have
an effect. In this regard, it is interesting to note that
four of seven lines transgenic for the same quail
troponin I sequences displayed gamete-of-origin dependent methylation changes (Sapienza et al. 1987;
McGowan et al. 1989 and unpublished) while only one
of eight lines transgenic for an immunoglobulinchloramphenicol acetyltransferase construct showed
such characteristics (Reik et al. 1987).
Because we are unable to demonstrate transgene
methylation differences in the gametes of males that
give rise to offspring with different transgene methylation phenotypes, it seems unlikely that the m-acting
signal that gives rise to these differences is methylation
of the transgene per se, unless the sites that dictate these
differences are not revealed by our assay (Bird, 1986).
While this is possible, it does not explain how such
differences are established at an allele that is identical
by descent within all individuals.
In some respects, these differences are reminiscent of
the differences observed in the inheritance of the fragile
168
C. Sapienza and others
X-linked mental retardation syndrome in the human,
where only some individuals who inherit the fragile X
chromosome express the mental retardation phenotype
(Camerino et al. 1983; Sherman et al. 1985). In a recent
model (Laird, 1987), this variability has been proposed
to arise from a ds-acting mutation, which leads to a
local block in the reactivation of a previously inactivated fragile X. A similar mechanism may explain the
variability in transgene methylation phenotype that we
observe.
One conclusion that may be drawn from this study is
that comparisons between different methylation
imprinting studies are difficult, if not impossible, in the
absence of genetic analyses. All published reports on
gamete-of-origin dependent changes in transgene methylation have been carried out on non-inbred mouse
populations (Reik et al. 1987; Sapienza et al. 1987;
Swain et al. 1987; Hadchouel et al. 1987). While some of
these transgene loci seem relatively immune to strain
background effects (Swain et al. 1987), others (Hadchouel et al. 1987) apparently show the same strain
dependence. Because such differences sometimes affect
the expression of transgenes (Swain et al. 1987), this
caveat is not restricted to studies on genome imprinting,
but may be relevant to any investigation involving the
expression of exogenous DNA sequences in transgenic
mice.
We are grateful to Catherine Italiano, Mira Puri, Irene
Tretjakoff and Susan Gauthier for technical assistance, Susan
Caluori and Monique Roger for typing the manuscript and
Linda Sapienza and Robert Derval for artwork.
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{Accepted 31 May 1989)