Download Autosomal and X-chromosome imprinting

Development 199(1 Supplement, 63-72
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63
Autosomal and X-chromosome imprinting
BRUCE M. CATTANACH and COLIN V. BEECHEY
MRC Radiobiology Unit, Chilton, Didcol, Oxon. 0X11 ORD, UK
Summary
Mouse genetic studies using Robertsonian and reciprocal
translations have shown that certain autosomal
regions of loci are subject to a parental germ line
imprint, which renders maternal and paternal copies
functionally inequivalent in the embryo or later stages of
development. Duplication of maternal or paternal copies
with corresponding paternal/maternal deficiencies in
chromosomally balanced zygotes causes various effects.
These range from early embryonic lethalities through to
mid-fetal and neonatal lethalities, and in some instances
viable young with phenotypic effects are obtained. Eight
to nine chromosomal regions that give such imprinting
effects have been identified. Six to seven of these regions
are located in only three chromosomes (2, 7 and 17). The
two other regions are located in chromosomes 6 and 11.
Maternal and paternal disomies for each of four other
chromosomes (1, 5, 9 and 14) have been recovered with
different frequencies, but the possibility that this may be
due to imprinting has yet to be supported by follow-up
studies on regions of the chromosomes concerned. No
clear evidence of genetic-background modifications of
the imprinting process have been observed in these
mouse genetic experiments.
The mammalian X chromosome is also subject to
imprinting, as demonstrated by the non-random,
paternal X-inactivation in female mouse extra-embryonic tissues and in the somatic cells of marsupial females.
There is also the opposite bias towards inactivation of the
maternal X in the somatic cells of female mice. On the
basis that both X-chromosome inactivation and autosomal chromosome imprinting may be concerned with
gene regulation, it is suggested that evidence from
X-chromosome inactivation studies may help to elucidate factors underlying the imprinting of autosomes.
The relevant aspects of X-inactivation are summarized.
Introduction
mapped are located in the imprinting regions defined by
the genetic experiments. It has therefore been concluded that imprinting may be much more widespread
throughout the genome than indicated in the imprinting
map of the mouse. Moreover, a variation in expression
of transgenes in cells of different tissues and organs
correlated with overall changes in DNA methylation
has been noted, and observed to be dependent upon
genetic background (Sapienza, 1989). This, together
with the recognised variability in the penetrance and
expression of certain human conditions thought to be
influenced by imprinting effects, has suggested that the
imprinting process itself is subject to genetic modification (Sapienza, 1989). If true, this could further
reduce the likelihood of imprinting effects being easily
detected in the mouse genetic experiments.
This paper therefore reviews the mouse genetic data
with additional emphasis given to the question of
genetic background effects. An update of the imprinting map also identifies a new imprinting region, and a
series of data which may also demonstrate a further
consequence of imprinting is presented. In addition,
certain aspects of X-chromosome inactivation that
indicate imprinting effects in this chromosome are
discussed. It is suggested that autosomal chromosome
The genetic data describing chromosome imprinting
phenomena in the mouse have been the subject of a
number of recent reviews (Searle and Beechey, 1985;
Cattanach, 1986; Cattanach and Beechey, 1990).
These have shown the development of an imprinting
map which has defined regions of autosomes in which
one or more genes appear to be modified as they pass
through the germ lines of one, or other, or both
parents, with the result that the maternal and paternal
copies become functionally different in the zygote.
Differential DNA methylation has been suggested as
a mechanism by which these functional differences
could be brought about (Surani et al. 1988). This view
has been supported by a number of studies which have
demonstrated that certain transgenic insertions show
differing methylation and expression levels according to
parental route of transmission (Reik et al. 1987;
Sapienza et al. 1987; Swain et al. 1987; Hadchouel et al.
1987). The concept implied is that the transgenes reflect
the behaviour of the endogenous sequences in the
region of their site of integration. However, a high
proportion of the transgenes studied show such
imprinting effects and none of the four that have been
Key words: imprinting, parental origin effects,
X-activation.
64
B. M. Cattanach and C. V. Beechev
and X-chromosome imprinting are manifestations of
the same phenomenon, and that evidence from
X-inactivation studies may help to elucidate factors
underlying the imprinting of autosomes.
Methods
The genetic methods which have identified the mouse
imprinting regions have been described in previous
reviews (Searle and Beechey, 1985; Cattanach, 1986;
Cattanach and Beechey, 1990). Suffice to say here
that by intercrossing heterozygotes for Robertsonian
translocations that exhibit high rates of non-disjunction,
animals can be produced which have two homologues
of a selected chromosome from one parent and none
from the other. The maternal or paternal disomy (with
corresponding paternal or maternal nullisomy, respectively) can be identified with appropriate marker genes
(Fig. 1). Similarly, intercrosses of heterozygotes for
reciprocal translocations can generate animals with two
copies of regions proximal or distal to the translocation
breakpoints from one parent, and none from the other.
Again, such duplications (with corresponding deficiencies) can be identified with marker genes. All such
animals are chromosomally balanced and differ genetically from normal mice only by the parental origins of
their two copies of a particular chromosome or
chromosome region. A failure of a disomy or duplication from one parent to complement normally a
corresponding nullisomy or deficiency from the other
parent constitutes the genetic evidence for the occurrence of chromosome imprinting.
Established autosomal chromosome imprinting
effects
Fig. 2 illustrates the current status of the imprinting
map and it can be seen that most of the chromosome
complement has now been investigated. Only chromosome 12 and parts of chromosomes 7, 8, 10 and 18
remain to be studied; and for each of these untested
regions, translocations (indicated in italics) are available that could be used for complementation analyses.
The chromosomes of main interest are chromosomes 2,
6, 7, 11 and 17 and, as will be discussed, chromosomes
1, 5, 9 and 14 may also show imprinting phenomena.
Three somewhat overlapping but clearly established
categories of imprinting effect have been found with the
main imprinting regions. First, there is the category in
which a complementation type (maternal or paternal
duplication) dies very early in development. Three
examples of this type of effect have been observed
(Fig. 2); with maternal duplication of proximal chromosome 2, with maternal duplication of proximal chromosome 6, (Beechey and Cattanach, 1989), and with
paternal duplication of distal chromosome 7 (Beechey
and Searle, 1987). In each case eye colour markers have
been available, and this has permitted screening for
complementation among the zygotes from about day 11
non-disjunction
plus complementation
paternal disomy 11
(homozygous vt)
maternal disomy 11
(homozygojs +)
normal proeeny
(heterozygous vt)
Fig. 1. Production of animals with maternal and paternal
disomy. Heterozygotes for Robertsonian translocations,
e.g. Rb(U.13)4Bnr shown above, often exhibit high levels
of non-disjunction for the chromosome arms involved.
Therefore, when intercrossed and non-disjunction occurs in
both parents, gametes bearing the complementary products
may fuse (illustrated for chromosome 11) with the result
that chromosomally balanced, disomic young are produced.
These can be readily detected when one parent is
homozygous for a marker gene (vestigial tail, vt in the
illustration); marked young are then either maternal or
paternal disomies according to which parent carries the
marker. A similar number of unmarked young will be
disomies of the reciprocal types which can be distinguished
from their normal sibs in test-matings by demonstrating
they do not carry the relevant marker gene. The size
effects attributable to maternal and paternal disomy 1J are
illustrated. In a similar manner, intercrosses using
reciprocal translocations can identify regions of
chromosomes showing imprinting effects (Cattanach and
Kirk, J985).
of gestation, when eye pigmentation is first detectable.
The absence of the marked young has indicated that
each of the above genotypic classes dies before or
shortly after implantation (Cattanach and Beechey,
1990). Translocations (indicated in italics in Fig. 2) are
available which could further define the limits of the
imprinting regions of proximal chromosome 2 and distal
chromosome 7. Because of the severity of these
imprinting effects, their occurrence in embryonic stages
Autosomal and X-chromosome imprinting
10
M
12 13
11
P
RWBw
RblBnr
M
14 15
RMBnr
P
4 4 4
16
17
4 «
T1MC
Fu
Tm
T47H
i
: |
*h
T1M
C
TI9Ad
19
P HI
M
i
i
If
18
65
T!2Ad
T11H
TSJH
T27H
T1IH
a
T24H
T11H
T3BH
TISn
I
•
I
I
l
I]
M
M
I I
Irani
normal complementation
established Imprinting regions
differential recovery
untested
Fig. 2. Autosomal chromosome imprinting map of the mouse. Map of the mouse autosomal chromosomes showing regions
of normal and defective complementation (imprinted regions). Chromosomes showing normal complementation or giving
differential recoveries of their maternal (M) or paternal (P) disomy classes are shown only once. Chromosomes showing
imprinting are displayed separately, the maternal (M) and paternal (P) copies being indicated. Symbols for Robertsonian
translocations (e.g. RbJBnr) that have been used to identify imprinted chromosomes are shown above the centromeres;
symbols for reciprocal translocations (e.g. TJ3H) used to localise the regions are shown at their breakpoint positions.
Reciprocal translocations that may be used to localise further the' imprinting regions are given in italics.
that are difficult to investigate, and the rarity of
recovery of maternal/paternal duplications for the
proximal regions of chromosomes, they cannot be used
effectively to provide information on possible genetic
background effects. It is worth noting, however, that
the proximal chromosome 6 lethality was observed in
two separate studies with two different Robertsonian
translocations (one of which is of feral origin) and with
marker genes derived from unrelated stocks. The
imprinting effect is not therefore dependent upon a
single genetic background.
A second category of imprinting effect is observed as
mid-fetal to early neonatal death. This has been seen
with three regions (Fig. 2); at mid-gestation with
maternal duplication for distal chromosome 7 (Beechey
and Searle, 1987), at a later stage of fetal to early
neonatal development with maternal duplication for
proximal chromosome 7 (Beechey and Searle, 1987),
and at about the time of birth with a maternal deletion
(not balanced by paternal duplication) of a proximal
part of chromosome 17 (Johnston, 1974, 1975). As yet,
little is known about any strain-dependent variabilities
in the chromosome 7 late lethalities. Both, however,
were detected with two different reciprocal translocations and with marker genes derived from two
different stocks.
The chromosome 17 phenomenon is somewhat
different from all others described so far but can be
interpreted as an imprinting effect. The basic observation is that the hairpin tail (7Vip) deletion can only be
maintained by transmission through the male; when it is
inherited through the female the deletion behaves as a
dominant lethal (Johnston, 1974; 1975). This has been
interpreted to mean that a maternal copy of a gene
located in the region of the deletion is required for
normal development; the same reasoning would suggest
that the paternal copy is not functional. Deletion
mapping has allowed the region responsible, denoted T
maternal effect (Tme), to be identified (Winking and
Silver, 1984). Variability in the abnormality leading to
the lethality has not been documented, but it is known
that the lethality may occur before birth, or a day or two
thereafter, and in some exceptional circumstances
maternally-derived Thp young have survived to adulthood (Lyon, personal communication). It is not clear,
however, whether this is due to a strain-dependent
66
B. M. Cattanach and C. V. Beechev
Table 1. Recovery of maternal and paternal duplications for distal regions of chromosome 2
Translocation
Marked
parent
Number of
young
% maternal
duplication
% paternal
duplication
M
P
M
P
M
P
-
124
158
73
74
76
147
128
481
326
21)9
90
12.1
15.2
12.3
17.6
5.3
8.3
18.0
15.6
9.2
15 3
-
15.3
10.1
9.6
13 5
6.6
15.2
23.4
15.2
10.4
17 7
-
T(2.4)13H
T(2:3)24H
T(2.9)11H
T(2.11)3OH
a)
b)
T(2.S)26H
T(2:4)lSn
T(2:16)2SH
M = maternally marked: P=paternally marked
variability in the imprinting process or only to a
variation in the ability of mice of different genetic
backgrounds to survive the effects.
Phenotypic abnormalities characterise the third category of imprinting effect seen in mouse genetic
experiments. Three examples of this type of phenomena have been found (Fig. 2); with maternal and
paternal duplication for a distal region of chromosome
2 (Cattanach and Kirk, 1985); with maternal and
paternal duplication for the proximal region of chromosome 11 (Cattanach and Kirk. 1985); and, very recently,
with paternal duplication for a distal region of
chromosome 17. The latter finding has not been
reported previously (Cattanach and Beechey, unpublished observations).
The distal chromosome 2 imprinting effect has been
most amenable to investigation, partly because there
are a number of reciprocal translocations with breakpoints in chromosome 2 (Fig. 2), and partly because the
frequencies of occurrence of the pertinent complementation types that arise from the normal adjacent-1
disjunction is relatively high (approximately 15%).
Both factors provide opportunities for detecting genetic
background effects. The basic observation is that two
apparently opposite, anomalous phenotypes occur with
maternal and paternal duplication of a distal region of
the chromosome. With maternal duplication, the young
are hypo-kinetic and show a flat-sided, arch-backed
phenotype. They fail to suckle and consequently die
within 24h. In contrast, with the corresponding
paternal duplication, the young are overtly hyperkinetic and show a square-bodied, flat-backed phenotype. These young typically survive for several days but,
usually, their behavioural abnormality becomes progressively worse and this ultimately results in their
death.
These chromosome 2 imprinting effects have been
observed with all translocations with breakpoints
proximal to that of TISn, but not with one translocation
(T28H) which has its chromosome 2 breakpoint located
more distally (Fig. 2). This localises the imprinting
region between the TISn and T28H breakpoints. Two
other translocations are known which have breakpoints
between TISn and T28H (shown in italics in Fig. 2),
and studies with these may further define the exact
region subject to imprinting.
Table 1 summarizes the frequencies with which the
hypo- and hyperkinetic classes have been detected in
studies with a series of different chromosome 2
translocations. It can be seen that, generally, the
recovery varied little among the different translocations; the biggest variation was instead found in
separate studies with one translocation (T30H) carried
out on different genetic backgrounds. More significant,
perhaps, has been the consistent observation that
whereas the hyperkinetic class invariably dies within
4-5 days of birth with T24H, T30H and T26H, it can
survive for up to 10 days with TISn and, not
infrequently, into adulthood with T11H. Clearly,
survival does not correlate with the location of the
translocation breakpoints in the chromosome (Fig. 2),
nor, ultimately, does it appear to be dependent upon
genetic background. Thus, recent work has shown that
the genetic background that appeared to reduce the
recovery of the hyperkinetic class with T30H (Table 1,
test a cf. test b) has not been found to reduce the
survival of the hyperkinetic class with Tl 1H (15/17 cf.
12/17) (Cattanach, unpublished observations). The
reason for the variability in survival of the hyperkinetic
paternal duplication type within the different translocations remains an enigma.
Opposite phenotypes also characterise the chromosome 11 imprinting region (Fig. 2). Here, the effect
appears to be restricted to growth and, ultimately, adult
size; maternal duplication is invariably associated with a
small size (approximately 70% of normal) and paternal
duplication with large size (approximately 130% of
normal). Growth rates from birth to adulthood have
appeared normal, and prenatally the size differences of
the two complementation classes have been distinguished in fetuses as early as 13.5 days of gestation,
with placental sizes being correlated. A significant
difference in fetal size was not detected at 12.5 days,
which might suggest that the size difference may have
been initiated only a little earlier. The chromosome 11
effect has been observed principally in studies using the
Autosomal and X-chromosome imprinting
67
Table 2. Recovery of maternal and paternal disomies involving chromosomes 1, 5, 9 and 14
Chromosome
1
5
Translocation
Test
number
Rb(1.3)lBnr
Rb(5.15)3Bnr
Disomy
Number of
marked young
Total
young
%
marked
Ratio
M:P
1
M
P
11
7
631
512
1.74
1.37
1.3:1
2
M
P
12
1
589
540
2.04
0.19
10.7:1
Tease and
Cattanach, 1986
3
M
P
19
14
Cattanach. 1988
M
P
42
22
2.48
1.78
2.12
1.20
1.2:1
Total
766
788
1986
1840
1
M
P
M
P
M
P
3
9
2
12
0.52
1.38
0.53
2.29
0.51
1.79
1:2.7
5
21
580
654
380
522
960
1176
Total
Reference
1.8:1
1:4.3
Tease and
Cattanach, 1986
1:3.5
9
Rb(9.14)6Bnr
1
M
P
4
15
811
917
0.49
1.64
1:3.5
14
Rb(9.14)6Bnr
1
M
P
5
14
469
1229
1.07
1.14
1:1.1
2
M
P
1:6.6
M
P
811
917
1280
2146
0.25
1.64
Total
2
15
7
29
0.55
1.35
1:2.5
Lyon, 1983a
Lyon, 1983a
M, maternal disomy: P, paternal disomy.
Rb4Bnr Robertsonian translocation, which is of feral
origin, but it has also been detected with the T30H
reciprocal translocation; and the latter finding has
localised the region involved to the proximal part of the
chromosome. Two different markers, derived from
stocks of different genetic backgrounds were used in the
former studies. Therefore, there is as yet no indication
that chromosome 11 imprinting varies with genetic
background.
The last example of anomalous complementation
phenotypes has been detected only very recently.
Paternal duplication of the region of chromosome 17
distal to T138Ca (Fig. 1) is associated with a size
reduction (approximately 70% of normal); no clear
effect of any kind has yet been observed with the
reciprocal, maternal duplication genotype (Cattanach
and Beechey, unpublished observations). Although the
gene markers used have not permitted the early
detection of the complementation classes, size differences among the young have not been noted at birth, in
contrast to the situation found with chromosome 11
imprinting. Rather, the size difference was only noted
in the third week of life after which it was maintained at
about 70% of normal through to adulthood. It has not
yet been established if the size effect with distal
chromosome 17 imprinting is limited to the one
generation, as is the case for both the distal chromosome 2 and proximal chromosome 17 imprinting
effects.
Other possible examples of autosomal
chromosome imprinting
Two other phenomena in the mouse could be regarded
as possible examples of imprinting. One of these has
been recognised (Cattanach and Beechey, 1990)
through differential recoveries of maternal and paternal
disomies, or duplications for certain chromosome
regions in crosses between heterozygotes for Robertsonian or reciprocal translocations. Tables 2 and 3
summarize the pertinent data. The first example can be
found in the work of Lyon and Glenister (1977) who
noted a consistent shortage of marked young representing paternal, as opposed to maternal, duplication
for the region of chromosome 17, proximal to the
T138Ca breakpoint (Fig. 2). They suggested this represented a further manifestation of the Tme (Tl'p) effect
associated with this region of the chromosome,
although the abnormalities associated with the Tme
lethality were not directly observed. A complicating
factor in these studies was the presence of / haplotypes
which would have distorted the segregation ratios in
one of the reciprocal crosses. However, similar observations have since been made for chromosomes 1, 5, 9
and 14 in various studies using Robertsonian translocations (Fig. 2, Table 2).
In each of the three experiments with chromosome 1,
maternal disomies were more numerous than paternal
disomies. This differed significantly in magnitude in the
68
B. M. Cattanach and C. V. Beechev
Table 3. Recovery of maternal and paternal duplications for the distal regions of chromosomes I, 5, 9 and 14
Chromosome
Translocation
Test
number
Duplication
Number of
marked young
Total
young
/o
marked
Ratio
M:P
Reference
1
T(l:13)70H
1
M
P
6
4
14
42
43.0
95
45:1
Searle eial. 1971
Beechey. unpublished data
5
T(5;13)264Ca
1
M
P
16
72
6.3
11 1
1.1.8
Searle and Beechey. 197S
2
M
P
1
8
12
6
67
60
17 9
10 0
1.8:1
Searle eial. 1971
3
M
P
15
34
120
208
12 5
16 4
1:1.3
Beechey. unpublished data
Cattanach and Beechev
(unpublished data)
9
T(9:17)138Ca
1
M
P
12
11
148
S7
S.I
12.6
1:1.6
Cattanach and Beechev
(unpublished data)
14
T(14.l5)6Ca
1
M
P
10
5
124
44
81
11.4
1.1.3
Cattanach and Beechev
(unpublished data)
M. maternal; P. paternal.
three studies (^=6.04; P=0.049), but overall there was
a significant difference in the frequency of the two
disomic classes (/T = 5.29; P=0.011). With chromosome
5 there was a smaller and non-significant variation
between the results of repeat experiments (^=0.25;
P=0.62) and, here, there was a significant excess of
paternal disomies over maternal disomies (^7 = 7.41;
P=0.33xl0~ 2 ). The one result involving chromosome
9 also showed a significant excess of paternal disomies
over maternal disomies (P=0.035). And, finally, with
chromosome 14 there was again significant variation
between the results of repeat experiments 0^=4.47;
P=0.035) but, overall, a significant excess of paternal
disomies over maternal disomies (/f=5.35; P=0.01).
No phenotypic abnormalities or indications of impaired
development were noted for any of the disomic classes
discussed.
Some reciprocal translocation data pertaining to the
distal regions of chromosomes J, 5, 9 and 14 are also
available and these are presented in Table 3. Consistent
with the observed excess of maternal disomy 1 young in
the Robertsonian translocation studies (Table 2), results with T(l;13)70H intercrosses have suggested that
maternal duplication for the region of chromosome 1
distal to the breakpoint may be recovered more
frequently than the reciprocal class. This might localise
a responsible factor to this region of the chromosome
(not shown in Fig. 2) but the data are limited. That a
region of chromosome 13 might be responsible for the
effect observed can be discounted as no differential
recovery of disomy 13 young has been found in
Robertsonian translocation studies (Tease and Cattanach, 1986).
At variance with the disomy 5 findings (Table 2) are
the results from a series of studies with T(5;13)264Ca
(Table 3) which, overall, showed no significance excess
of paternal duplication for the distal region of the
chromosome over the equivalent maternal class
(;rf=0.076; P=0.78), and indicated heterogeneity be-
tween experiments (^=2.87; P=0.24). This negative
result might be interpreted to mean that a factor
responsible for the excess of paternal disomy 5
(Table 2) is located more proximally in the chromosome but, alternatively, it could simply mean that the
reciprocal translocation data do not confirm the
Robertsonian translocation findings. The same conclusions could be drawn from the reciprocal translocation data for the distal regions of chromosomes 9 and
14 (Table 3). Although there was in each case an excess
of the paternal duplication class over the reciprocal
maternal duplication class consistent with the disomy
findings (Table 2), the differences were not significant
(for chromosome 9, P=0.26; for chromosome 14,
P=0.54).
It is difficult to evaluate the significance of the
differential recoveries of the chromosomes 1, 5, 9 and
14 maternal and paternal disomies when supporting
evidence is not available from the reciprocal translocation studies. Further data with T27H might establish
the phenomenon for the distal region of chromosome 1,
but for chromosomes 5, 9 and 14 evaluation of the
disomy data must await the results of reciprocal
translocation studies upon the proximal regions of the
chromosomes; and, because of the rarity of the
requisite meiotic (adjacent-2) disjunction, this data will
be hard to obtain. Should the validity of the differential
recovery phenomena be established it is likely that prenatal or neo-natal semi-lethality will be the cause. This
would be consistent with the results of Lyon and
Glenister (1977) with T138Ca, which showed a shortage
of young with paternal duplication for the proximal
region of chromosome 17, apparently due to the Tme
(T'p) imprinting effect. It is pertinent to note that
neither in the T138Ca study, nor in the recent T1'1' work
(Lyon, personal communication), was any impairment
of post-natal development noted among the survivors of
the neo-natal lethal class. The failure to detect defects
in numerically deficient disomy classes is not therefore
Autosomal and X-chromosome imprinting
at variance with the concept that regions of the
chromosomes concerned may be subject to chromosome imprinting. As yet the available differential
recovery data are not convincing, however.
The other possible type of imprinting effect recognised in the mouse is that based on the observation that
the semi-dominant gene, fused, (Fu) shows a differential expression according to parental origin. Thus, the
penetrance and expression is lower when the gene is
transmitted by the mother than by the father, and this is
influenced by genetic background (Reed, 1937). A
recent brief report has further suggested that it is the
penetrance of the maternally-derived gene that is
subject to genetic background modification (Agulnik
and Ruvinsky, 1988), but the results are not consistent
with the earlier work. Significantly, the Fu gene is
located proximally on chromosome 17 near to Tme and
within the region of differential recovery defined by the
T138Ca studies (Lyon and Glenister, 1977). The
phenomenon, even if attributable to imprinting, does
not therefore extend the range of imprinting regions in
the mouse beyond those demonstrated by the various
genetic studies. In this connection, it might be noted
that in man, numerous examples of differential expression of semi-dominant genes according to parental
origin have been cited as possible examples of
imprinting, and most of these tend to locate in or near
regions of the human chromosome map homologous to
the mouse imprinting regions (Reik, 1989; Cattanach
and Beechey, 1990).
Conclusions on autosomal imprinting
There are now at least 8 autosomal regions in which
clearly-established imprinting effects have been identified, and these act upon at least three different stages
of development. The number of such regions could be
extended to 9 if the two distal chromosome 7 lethalities
are regarded as distinct, separate phenomena. Irrespective of the exact number, it is evident that the
distribution of the imprinting regions over the autosomal chromosome complement is non-random; 6 (or
7) of the 8 (or 9) regions locate to only 3 chromosomes
(chromosomes 2, 7 and 17), the others being on
chromosomes 6 and 11. This situation could be
modified somewhat if the differential recovery effects
noted with the disomies for chromosomes 1, 5, 9 and 14
were established as imprinting phenomena, but as yet
the evidence is not convincing. The fact that the closely
linked Tlip and Fu loci show evidence of imprinting,
suggests that more than one locus is subject to
imprinting in each of the imprinting regions currently
only crudely defined by the breakpoints of the
translocation studies. No convincing evidence of genetic background effects has been found for any of the
imprinting phenomena distinguished in the mouse
genetic experiments. This contrasts with the findings
made in the transgenic work (Sapienza, 1989). Further
investigation with a broader range of mouse strains may
69
be needed to determine if the imprinting process is
subject to genetic background modification.
X chromosome imprinting
In addition to the imprinting phenomena seen with the
mouse autosomal chromosome translocations there is
also the earlier evidence of X chromosome imprinting
(Lyon and Rastan, 1984). The best known example is
the preferential inactivation of the paternal X in the
extra-embryonic membranes of female mice (Takagi
and Sasaki, 1975; West et al. 1977; Harper et al. 1982),
which indicates that the maternal and paternal X
chromosomes have received some imprint to permit
their distinction in the zygote. The non-randomness is
not rigidly fixed, however. Patroclinous XO females do
not have their single (paternal) X inactivated in these
tissues (Frels and Chapman, 1979; Papaionnou and
West, 1981); parthenogenetic diploid embryos may
have one X inactivated in both embryonic (Kaufman et
al. 1978) and extra-embryonic tissues (Rastan et al.
1980) even though both their X chromosomes are of
maternal origin; and androgenic diploid moles with two
paternal X chromosomes inactivate only one X (Tsukahara and Kajii, 1985). The imprint to distinguish
maternal and paternal X chromosomes may therefore
only confer an enhanced probability of inactivation of
the paternal X, rather than the maternal X, in extraembryonic tissues (Lyon 19836).
A further example of non-random X inactivation is
found in marsupials (Vandeberg et al. 1987). Rather
than the random X inactivation that occurs in the
somatic cells of eutherian females, paternal X inactivation is the rule in the somatic cells of marsupial
females. Less well known, however, is the small but
consistent non-random element in the inactivation of
the X in the somatic cells of female mice. This was
initially observed in a large series of studies using an
X-autosome translocation that caused autosomal gene
variegation attributable to X inactivation (Cattanach
and Perez, 1970). The level of variegation differed
between females derived from reciprocal crosses in a
way which would suggest that the maternal X is more
likely to become inactivated. An equivalent difference
in expression of the X-linked gene Ta in reciprocal
crosses (Dun and Fraser, 1959; Fraser and Kindred,
1960) can be similarly interpreted. The same type of
finding has since been made with the X-linked genes,
brindled (Mobr) (Falconer et al. 1982), phosphoglycerate kinase (Pgk-1) (Bucher and Krietsch, 1988;
Cattanach and Biicher, unpublished observations) and
then very recently with glucose-6-phosphate dehydrogenase (Gpdx) (Peters, personal communication). This
phenomenon in somatic cells is therefore similar to that
in the extra-embryonic membranes and to that in the
somatic cells of marsupials, but the bias is in the
opposite direction, and because of the substantial
random element it is relatively hard to detect. Another
possible example of X-chromosome imprinting is the
earlier time in development at which the maternal Pgk-
70
B. M. Cattanach and C. V. Beechev
1 allele is thought to be expressed, relative to that of the
paternal allele (Krietsch et al. 1982). In all such
situations, an imprinting of the maternal or paternal
chromosomes to influence their behaviour in the zygote
is indicated.
There are also a number of other points regarding Xinactivation that may be pertinent to the imprinting of
the chromosome.
First, X-inactivation appears to be dependent upon a
single locus, or inactivation centre. A variety of data
has established this in the mouse (reviewed by
Cattanach, 1975); and in man (Therman et al. 1974,
1979; Tabor et al. 1983). In the mouse the putative
inactivation centre, Xce, has different allelic forms
which govern the probability of the X in which they are
located becoming the inactive one in somatic cells
(Johnston and Cattanach, 1981). The evidence on
whether they modify the non-random X-inactivation in
extra-embryonic tissues is, unfortunately, conflicting
(Rastan and Cattanach, 1983; Biicher et al. 1985).
Second, inactivation of the X is achieved by a spread
of inactivation from the centre to contiguous regions of
the X, and into attached autosomal regions (Lyon,
1968; Cattanach, 1975; Russell, 1983).
Third, the spread of inactivation is reversible such
that with age, or time, inactivated loci may be
reactivated (Cattanach, 1974; Wareham et al. 1987;
Brown and Rastan, 1988). Reactivation of certain loci
also typifies the paternal X inactivation of marsupials
(Cooper et al. 1977; Johnston et al. 1978).
As a final point, it may be noted that experiments to
search for genetic control of X-inactivation and position
effect variegation in the mouse identified only the Xchromosomal (Xce) system of control (Cattanach and
Isaacson, 1965, 1967; Falconer and Isaacson, 1972;
Falconer et al. 1982). As yet, no evidence of genetic
background modifications have been found.
X-chromosome imprinting as a model for
autosome imprinting
The term "chromosome imprinting' was originally
coined by Crouse (1960) to define the mechanism that
led to the selective paternal chromosome eliminations
that occur in the soma and germ line of the fly, Sciara. It
has also been used in relation to the genetic inactivation
and heterochromatic behaviour of the paternal chromosome set in coccids (Brown and Nur, 1964) and, more
recently, it has been applied to differential DNA strand
modification in yeasts and allelic exclusion in higher
plants (this volume). It is a moot point as to whether all
imprinting effects, including the autosomal and X
chromosome examples in the mouse, represent diverse
manifestations of the same basic phenomenon. However, it would be surprising if more than one mechanism
for identifying maternal and paternal chromosomes
and, thence, regulating gene dosage, were to have
evolved within a species (mouse), or within a class
(mammals). As such, information on imprinting and
inactivation of the X chromosome could help to
female o«m ceO
male germ cofl
preferential pelemal X-lnactrvalion
exira embryonic tissue
'
rwwborns/aduGs/aged animals
Fig. 3. Imprinting and inactivation of an X-chromosome in
female mouse embryonic and extra-embryonic tissues. X
inactivation is shown as being mediated by a controlling
locus, or inactivation centre, Xce, which is subject to a
paternal germ line imprint (heavy print). The consequence
is that the paternal X is preferentially inactivated in extraembryonic tissues. In the somatic cells of the embryo,
either X may be inactivated, and this is achieved by a
spread of inactivation in both directions from the Xce
locus. With age or time there is a progressive reversal of
the inactivation such that reactivation of previously
inactivated loci occurs. In this illustration, the imprinting is
indicated as taking place in male germ cells; it could
equally well occur in female germ cells (Lyon and Rastan.
1984). Other aspects of X-inactivation are considered in the
text.
elucidate factors underlying autosomal chromosome
imprinting effects (see Fig. 3). Thus: (1) in each
imprinting region there may be a controlling locus
which receives the maternal/paternal germ line
imprint; (2) according to the imprint the controlling
locus may modify adjacent gene loci in the zygote to
render them inactive; (3) the modification may be
achieved by a spread of inactivation such that only one
or a series of adjacent loci may be inactivated; (4) the
inactivation may not be permanent and could therefore
last for only a short period in development and be
absent at later ages; (5) the imprinting may only
influence the probability with which one or other
parental copy may be active, and therefore not all
maternal/paternal disomies or duplications may give
major, easily detectable effects; (6) there could be
Autosomal and X-chromosome imprinting
different alleles at each controlling locus which
have different sensitivities to imprinting and therefore
different probabilities of inactivating adjacent loci.
Resolution of how autosomal imprinting effects are
brought about must await identification of loci subject
to the imprinting processes. It is remarkable, however,
that so many of the established and putative examples
of imprinting appear to concern growth (Cattanach and
Beechey, 1990); in mammals, there may be a specific
need for the regulation of genes for growth factors and
this may be achieved by chromosome imprinting.
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