ArticlePDF Available

Maternal epigenetics and methyl supplements affect agouti gene expression in A/a mice

Authors:

Abstract

'Viable yellow' (Avy/a) mice are larger, obese, hyperinsulinemic, more susceptible to cancer, and, on average, shorter lived than their non-yellow siblings. They are epigenetic mosaics ranging from a yellow phenotype with maximum ectopic agouti overexpression, through a continuum of mottled agouti/yellow phenotypes with partial agouti overexpression, to a pseudoagouti phenotype with minimal ectopic expression. Pseudoagouti Avy/a mice are lean, healthy, and longer lived than their yellow siblings. Here we report that feeding pregnant black a/a dams methyl-supplemented diets alters epigenetic regulation of agouti expression in their offspring, as indicated by increased agouti/black mottling in the direction of the pseudoagouti phenotype. We also present confirmatory evidence that epigenetic phenotypes are maternally heritable. Thus Avy expression, already known to be modulated by imprinting, strain-specific modification, and maternal epigenetic inheritance, is also modulated by maternal diet. These observations suggest, at least in this special case, that maternal dietary supplementation may positively affect health and longevity of the offspring. Therefore, this experimental system should be useful for identifying maternal factors that modulate epigenetic mechanisms, especially DNA methylation, in developing embryos.
9490892-6638/98/0012-0949/$02.25 Q FASEB
/ 382d 0001 Mp 949 Wednesday Aug 05 09:00 AM LP–FASEB 0001
Maternal epigenetics and methyl supplements affect
agouti gene expression in A
vy
/a mice
GEORGE L. WOLFF,*
,1
RALPH L. KODELL,
STEPHEN R. MOORE,
AND CRAIG A. COONEY
§
*Division of Biochemical Toxicology,
Division of Biometry and Risk Assessment,
§
Division of
Molecular Epidemiology, National Center for Toxicological Research/Food and Drug Administration,
Jefferson, Arkansas 72079, USA; *Departments of Biochemistry/Molecular Biology and Pharmacology/
Interdisciplinary Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205,
USA; and
The Bionetics Corporation, Jefferson, Arkansas 72079, USA
ABSTRACT
‘Viable yellow’ (A
vy
/a) mice are larger,
obese, hyperinsulinemic, more susceptible to cancer,
and, on average, shorter lived than their non-yellow
siblings. They are epigenetic mosaics ranging from a
yellow phenotype with maximum ectopic agouti over-
expression, through a continuum of mottled agouti/
yellow phenotypes with partial agouti overexpression,
to a pseudoagouti phenotype with minimal ectopic
expression. Pseudoagouti A
vy
/a mice are lean,
healthy, and longer lived than their yellow siblings.
Here we report that feeding pregnant black a/a dams
methyl-supplemented diets alters epigenetic regula-
tion of agouti expression in their offspring, as indi-
cated by increased agouti/black mottling in the di-
rection of the pseudoagouti phenotype. We also
present confirmatory evidence that epigenetic phe-
notypes are maternally heritable. Thus A
vy
expres-
sion, already known to be modulated by imprinting,
strain-specific modification, and maternal epigenetic
inheritance, is also modulated by maternal diet.
These observations suggest, at least in this special
case, that maternal dietary supplementation may pos-
itively affect health and longevity of the offspring.
Therefore, this experimental system should be useful
for identifying maternal factors that modulate epi-
genetic mechanisms, especially DNA methylation, in
developing embryos.Wolff, G. L., Kodell, R. L.,
Moore, S. R., Cooney, C. A. Maternal epigenetics and
methyl supplements affect agouti gene expression in
A
vy
/a mice. FASEB J. 12, 949957
Key Words: maternal diet · methyl supplementation · DNA
methylation · epigenetic regulation of gene expression · yellow
mice
T
HE MATERNAL REPRODUCTIVE TRACT
, arguably, is the
environment most critical to the developing mam-
malian embryo. Its metabolic and physiologic char-
acteristics modulate the zygote’s development
through all embryonic stages until birth. Indeed, the
conditions in the embryo’s immediate milieu seem to
determine many characteristics and susceptibilities of
the adult organism (14).
Mammalian development is dependent on DNA
methyltransferase (MTase)
2
and its product 5-meth-
ylcytosine (5MC) to help establish, define, or stabilize
the various cell types that constitute the developing
embryo (5, 6). In mammals, 5MC is a major epige-
netic mechanism, with some 5MC patterns being in-
herited epigenetically (68). DNA MTase requires S-
adenosylmethionine (SAM) (9) and uses zinc as a co-
factor (10).
Synthesis of the chief methyl donor SAM is depen-
dent on dietary folates, vitamin B
12
, methionine, cho-
line, and betaine (11), which are also available as nu-
tritional methyl supplements. In the human maternal
diet, folic acid is important for the prevention of neu-
ral tube birth defects, where it may act via methyl
metabolism (12, 13) and possibly via 5MC. Little is
known about how maternal dietary methyl supple-
ments affect epigenetic regulation of the developing
mammalian embryo or whether high levels of certain
methyl supplements are toxic. Cooney (11) proposed
that such supplements could affect gene expression
and 5MC levels in adults and that the level of gene-
specific 5MC in young mammals could affect their
adult health and longevity.
The mouse agouti alleles, A
w
and A, regulate the
alternative production of black (eumelanin) and yel-
low (pheomelanin) pigment in individual hair folli-
cles. Transcription of the gene occurs only in the skin
during the short period when the yellow subapical
band is formed at the beginning of each hair growth
cycle. This cyclic expression results in the ‘agouti’
coat pattern.
1
Correspondence: National Center for Toxicological Re-
search, 3900 NCTR Road, Jefferson, AR 720799502, USA. E-
mail: GWOLFF@NCTR.FDA.GOV
2
Abbreviations: EM, eumelanic mottling; HS diet, contains
half of the supplement level in the MS diet; IAP, intracisternal
A particle; LTR, long terminal repeat; 5MC, 5-methylcytosine;
MS diet, standard methyl-supplemented diet; SAM, S-adeno-
sylmethionine; 3SZM diet, contains 31 as much methyl
supplement as MS diet plus zinc plus methionine; MTase,
DNA methyltransferase.
950 Vol. 12 August 1998 The FASEB Journal WOLFF ET AL.
/ 382d 0001 Mp 950 Wednesday Aug 05 09:00 AM LP–FASEB 0001
TABLE 1. Proportions of high and low degrees of eumelanic
mottling (EM) among A
vy
/a offspring from a/a dams of two inbred
strains fed methyl-supplemented diets
Strain and diet
High EM
a
% (N)
Low or no EM
b
% (N)
Strain VY
NIH-31 (control) 42.6 (75) 57.4 (101)
MS 59.8 (98)
c
40.2 (66)
3SZM 65.8 (52)
c
34.2 (27)
Strain YS
NIH-31 (control) 65.6 (99) 34.4 (52)
MS 66.3 (67) 33.7 (34)
3SZM 78.4 (69)
d
21.6 (19)
a
Pseudoagouti / almost pseudoagouti / heavily mottled / 1/2
of the mottled pups.
b
Slightly mottled / clear yellow / 1/2 of the
mottled pups.
c
P õ 0.001 for VY strain and MS diet compared to
control NIH-31 diet, as well as for the trend, NIH-31 õ MS õ
3SZM.
d
P õ 0.05 for YS strain with 3SZM diet compared to MS
or NIH-31 diet and for the trend NIH-31 õ MS õ 3SZM.
Due to mutations in the regulatory region of the
agouti locus, mice bearing the dominant ‘viable yel-
low’ (A
vy
), ‘IAPyellow’ (A
iapy
), or ‘hypervariable yel-
low’ (A
hvy
) alleles synthesize much more pheo-
melanin than eumelanin. These mutations arose
through spontaneous insertions of single intracister-
nal A particle (IAP) sequences in different regions of
the agouti gene, all preceding the first coding exon
(1, 2, 14). In these yellow mice, the gene is under
control of the IAP promoter/enhancer and is tran-
scribed continuously in essentially all tissues. This re-
sults not only in yellow hair but also in obesity, hy-
perinsulinemia, diabetes, increased somatic growth,
and increased susceptibility to hyperplasia and tu-
morigenesis (15).
In mice of the non-agouti genotype, a/a, no pheo-
melanin is synthesized, except in hair follicles in the
ears and perineal area, due to insertions of non-IAP
retroviral sequences in the intron preceding the first
coding exon of the agouti gene (16). Because the a
allele produces neither yellow nor pseudoagouti phe-
notypes, it is often used as the second allele in studies
with the dominant yellow mutants.
Expression of A
vy
, A
iapy
, and A
hvy
can be down-reg-
ulated epigenetically. In yellow A
iapy
/a and A
hvy
/a
mice, the proximal IAP long terminal repeat (LTR),
containing promoters and enhancers, is hypomethy-
lated, whereas in pseudoagouti A
iapy
/a mice and their
black A
hvy
/a homologs, the LTR is methylated (1, 2).
Thus, in pseudoagouti mice the IAP promoter is in-
active, allowing the agouti promoters to regulate tran-
scription of the gene. The mouse genotypes A
vy
/a
and A
iapy
/a are expressed in almost identical pheno-
types. Since the agouti protein is continuously and
ectopically expressed in both mutants, it is likely that
in A
vy
/a mice, a regulatory mechanism is operative
similar to that reported for A
iapy
(2) and A
hvy
(1).
A continuous spectrum of variegated patterns of
eumelanic mottling (EM), i.e., agouti/black areas, on
a yellow background characterizes A
vy
/a mice. Their
phenotypes are defined by the degree of EM (Table
1). Thus, a ‘clear yellow’ mouse is at one extreme of
the EM spectrum and the ‘pseudoagouti’ mouse (Fig.
1) occupies the other extreme. The latter resembles
the agouti (A/0) coat color phenotype, does not be-
come obese, is normoinsulinemic, and is less suscep-
tible to tumorigenesis (17).
In A
vy
/a mice, there is partial maternal epigenetic
inheritance of phenotype. In general, maternal epi-
genetic inheritance occurs when the epigenetic phe-
notype and/or allelic expression of the mother is a
determinant of the epigenetic phenotype and/or al-
lelic expression of the offspring. For A
vy
/a dams, the
proportion of pseudoagouti offspring depends on
the mother’s agouti locus epigenetic phenotype. The
data in Table 2 confirm and extend previous obser-
vations (2, 18) that pseudoagouti dams produce a
considerably higher proportion of pseudoagouti off-
spring than do yellow phenotype dams. The epige-
netic phenotype affects not only epigenetic inheri-
tance in the A
vy
/a genotype, but also may confer the
potential for multigenerational inheritance of epi-
genetically determined characteristics (Table 3).
In both A
vy
/a and A
iapy
/a mice, the proportions of
zygotes differentiating into pseudoagouti phenotype
offspring are determined by the gender of the parent
contributing the mutant allele, as well as by the dam’s
strain genome. These gender and strain effects dem-
onstrate, respectively, genomic imprinting and strain-
specific modification of the A
vy
and A
iapy
alleles.
Genomic imprinting occurs when the level of al-
lelic expression in offspring depends on the gender
of the parent contributing the allele (6). Imprinting
is a parental gender effect on gene expression in off-
spring but is neither the inheritance of a parent’s ep-
igenetic somatic characteristics nor the inheritance of
a parent’s somatic allelic imprint (although these
may happen to coincide between parent and off-
spring). The data in Table 2 also confirm and extend
previous observations (18) on genomic imprinting
and strain-specific modification in A
vy
/a mice.
Here we report that differentiation toward the
pseudoagouti phenotype of A
vy
/a embryos is favored
by feeding the mothers methyl-supplemented diets
during pregnancy. Thus, epigenetic regulation of the
A
vy
allele is modulated not only by imprinting, strain-
specific modifier effects, and maternal epigenetic in-
heritance, but also by the maternal diet.
MATERIALS AND METHODS
Animals
Inbred mouse strains YS/WffC3Hf/Nctr-A
vy
and VY/
WffC3Hf/Nctr-A
vy
were used for dietary studies at F
116
-F
120
of
MATERNAL DIET, EPIGENETICS, AND
AGOUTI
GENE EXPRESSION 951
/ 382d 0001 Mp 951 Wednesday Aug 05 09:00 AM LP–FASEB 0001
Figure 1. Strain YS A
vy
/a phenotypes. From left to right: pseudoagouti, almost pseudoagouti, heavily mottled, and slightly mottled.
The heavily and slightly mottled phenotypes are found at opposite ends of a continuum in which each mouse has a unique
pattern. Note the characteristic faint yellow stripes on the right rear quadrant of the almost pseudoagouti mouse; the yellowish
tinge of this mouse is a lighting artifact. The white spot on each mouse is due to recessive spotting (s), for which the YS strain
is homozygous. The very light area on the rump of the heavily mottled mouse is a lighting artifact.
TABLE 2. Proportion of pseudoagouti mice among A
vy
/a offspring produced by yellow A
vy
/a, pseudoagouti A
vy
/a, and black a/a dams given
control diet
DAM
Geno(Pheno)type
SIRE
Geno(Pheno)type
Pseudoagouti A
vy
/a offspring
19721977
a
%N
19911997
%N
Strain VY
b
A
vy
/a (mottled yellow) a/a 1.0* 17/1706 1.0** 31/3015
A
vy
/a (pseudoagouti) a/a 5.3* 15/281 6.3** 51/809
a/a A
vy
/a (mottled yellow) 11.8 272/2296 10.2 237/2323
a/a A
vy
/a (pseudoagouti) 10.1 60/593 9.3 62/669
Strain YS
c
A
vy
/a (mottled yellow) a/a 0.3 2/607 0.2** 5/2579
A
vy
/a (pseudoagouti) a/a 1.2 4/329 2.3** 5/216
a/a A
vy
/a (mottled yellow) 16.8 49/292 7.0 181/2576
a/a A
vy
/a (pseudoagouti) 16.9 72/427 8.4 19/225
a
From ref. 18, with permission.
b
VY/Wf-A
vy
197278; VY/WffC3Hf/Nctr-A
vy
19781997.
c
YS/ChWf-A
vy
197278; YS/ChWffC3Hf/
Nctr-A
vy
19781997. * P õ 0.001 compared to other mating combinations using the same strain and time period. ** P õ 0.0001 compared to
other mating combinations within the same strain and time period.
YS and F
107
-F
111
of VY and for maternal epigenetic inheritance
studies. In dietary studies, black a/a females were date-mated
at 68 wk of age to the same strain of mottled yellow or pseu-
doagouti A
vy
/a males to produce litters of black a/a and vari-
ous phenotypes of A
vy
/a mice. The litters were phenotyped at
8 and 21 days of age. The spectrum of mottling necessitated
the definition of six phenotypic classes (Table 1) for the pur-
pose of grading offspring in dietary studies.
Maternal epigenetic inheritance and imprinting data (Ta-
ble 2) were obtained from phenotypes of offspring produced
by 1) clear yellow, mottled yellow, or pseudoagouti A
vy
/a fe-
males mated by black a/a males, and 2) black a/a females
mated by clear yellow, mottled yellow, or pseudoagouti A
vy
/a
males. The proportions of clear/mottled yellow, pseudo-
agouti, and black phenotypes among their offspring were de-
termined. Maternal epigenetic inheritance and imprinting
data were obtained from animals on control diet only.
Diets
Methyl-supplemented diets were designed to provide sub-
stantially increased amounts of cofactors and methyl donors
for methyl metabolism (11) and, in one diet, to provide
additional zinc, a cofactor for the mouse DNA MTase (10).
The methyl-supplemented diets, viz., standard methyl-sup-
952 Vol. 12 August 1998 The FASEB Journal WOLFF ET AL.
/ 382d 0001 Mp 952 Wednesday Aug 05 09:00 AM LP–FASEB 0001
TABLE 3. Some determinants of epigenetic inheritance
Specific class General category Possible mechanisms
Maternal epigenetic phenotype
(gene specific)*
Possible multigenerational effects
Epigenetic
inheritance
5MC or chromatin marks carried through to the next
generation.
Uterine metabolic or hormonal factors leading to recapitulation
of a specific pattern of gene expression in offspring.
These mechanisms could be influenced by maternal
environmental factors.
Paternal epigenetic phenotype
(gene specific)
Established multigenerational effects
Epigenetic
inheritance
Initiated by nuclear transplantation in early embryos. Altered
cytoplasmic environment in very early development leads to
5MC changes and possibly other chromatin marks.
Once established, these marks are directly or indirectly carried
through to the next generation (33).
Maternal genotype** Strain-specific
modifiers
Specific genes acting in trans to produce gene products that
effect epigenetic silencing, e.g., could be genes for
transcription factors, chromatin factors, metabolism, or
signaling pathways (31, 32, 42, 43, 44, 45).
Gender of parent contributing a
particular allele**
Genomic imprinting Gender-specific marking of germline genomes during germ cell
development. Genomic imprinting can be complete (6, 46) or
partial (3, 18, 47).
Maternal methyl-supplemented
diets*
Possible multigenerational effects
Maternal nutrition MS diet: SAM and SAH levels, membrane fluidity, intracellular
signaling, early embryo growth rate (11, 38).
3SZM diet: Same as for MS diet plus greater zinc saturation of
DNA MTase or other zinc finger chromatin proteins (10, 41,
48).
IAP LTR expression**
Possible multigenerational effects
Transposition,
cis-activation
Gene-regulating DNA sequence that is a target for 5MC or other
epigenetic silencing mechanism (1, 2, 14, 27).
Transgene inactivation
Variety of multigenerational effects
Gene silencing Provides a DNA sequence that is a target for silencing and
becomes epigenetically modified, often by 5MC (31, 43, 44,
45, 49).
Monoallelic expression Gene dosage
adjustment
Sequence copy number in diploid cells (50). Parts of counting
mechanisms to regulate gene dosage such as in X inactivation,
e.g., 5MC, chromatin, and specific RNAs such as Xist (50, 51,
52, 53).
* Observed in A
vy
/0 mice. ** Observed in both A
vy
/0 and A
iapy
/0 mice.
TABLE 4. Composition of dietary methyl supplements
a
MS diet supplement HS diet supplement 3SZM diet supplement
5 g Choline 2.5 g Choline 15 g Choline
5 g Betaine 2.5 g Betaine 15 g Betaine
5 mg Folic acid 2.5 mg Folic acid 15 mg Folic acid
0.5 mg Vitamin B
12
0.25 mg Vitamin B
12
1.5 mg Vitamin B
12
7.5 g L-methionine
150 mg Zinc
a
The above are added to NIH-31 diet to give 1000 g of the respective final diet.
The final total amounts in 3SZM represent, in terms of the NIH-31 levels, Ç9 times
choline, Ç9 times folic acid, Ç60 times vitamin B
12
, Ç3.1 times methionine, and
Ç4.7 times zinc. Betaine not determined in NIH-31. Choline is from choline chlo-
ride, betaine is anhydrous, zinc is from ZnSO
4
·7H
2
O. All componentswere obtained
from Harlan Teklad (Madison Wis.) except betaine which was from Finnsugar Bio-
products (Schaumburg Ill.), and zinc, which was from Fluka (Milwaukee Wis.).
plemented diet (MS), HS (contains half of the supplement
level in the MS diet), and 3SZM (contains 31 as much
methyl supplement as MS diet plus zinc plus methionine),
were prepared by fortifying the control ‘methyl-sufficient’
NIH-31 diet (Table 4).
These supplements contain folic acid, vitamin B
12
, betaine,
and choline in the same proportions but in different absolute
amounts. The relative level of components in HS, MS, and
3SZM supplements is 0.5, 1.0, and 3.0, respectively. 3SZM sup-
plement also contains methionine and zinc (in addition to
threefold the MS level of folic acid, vitamin B
12
, betaine, and
choline). The absolute amounts of all components in the HS,
MS, and 3SZM supplements are given in Table 4. All diets were
pelleted after addition of the supplements and were fed ad
libitum.
Dams were taken off their supplemented diets after giving
birth; however, to avoid health problems in dams or pups from
abrupt diet change after giving birth, the dams were given
MATERNAL DIET, EPIGENETICS, AND
AGOUTI
GENE EXPRESSION 953
/ 382d 0001 Mp 953 Wednesday Aug 05 09:00 AM LP–FASEB 0001
lower dose diets for 1 or 2 wk before being placed solely on
NIH-31 diet again. Two different schedules were followed: #1)
MS diet for 2 wk before first date mating and through preg-
nancy, HS diet for the first week after birth, NIH-31 diet there-
after; #2) 3SZM diet for 2 wk before first date mating and
through pregnancy, MS diet for first week after birth, HS diet
for second week after birth, NIH-31 diet thereafter. Males
were given control diet only.
Statistical analyses
For statistical analysis of the dietary studies, data from animals
representing those with a majority of yellow in their coats
(clear yellow, slightly mottled, and one-half of the mottled
animals) were combined and compared with combined data
from animals with a relatively high degree of EM in their coats
(pseudoagouti, almost pseudoagouti, heavily mottled, and the
other one-half of the mottled mice) (Table 1). The ‘mottled’
category included mice with intermediate degrees of EM that
could not be designated as either ‘lightly’ or ‘heavily’ mottled.
For the statistical analysis, one-half of these mottled mice were
assigned to the ‘high EM’ category and the remaining one-
half to the ‘low or no EM’ category.
To detect a trend in the ratio of high to low degrees of EM
across dietary groups, Rao-Scott tests (19) were conducted sep-
arately for the YS and VY strains. To determine whether the
two strains differed in these ratios, a stratified analysis, using
the Cochran-Mantel-Haenszel test (20), was performed, with
each dietary group serving as a stratum.
Whether methylated diets influenced the number of pups
born or weaned was determined by analyses conducted sepa-
rately for each strain using the SAS procedure GLM for single
factor analysis of variance. Dunnett’s test was used to compare
each diet group to its appropriate control (21).
To analyze maternal epigenetic inheritance and imprinting
data, comparisons of the proportions of pseudoagouti mice
among A
vy
/a offspring were made for each strain between
dams of various phenotypes, using chi-square tests for differ-
ences in proportions (22).
RESULTS
Data from two types of studies are presented here. In
dietary studies, the effects on offspring of maternal
diet during pregnancy were determined. In maternal
epigenetic inheritance and imprinting studies, the ef-
fects on offspring of maternal epigenetic phenotype
and of the gender of the parent contributing the A
vy
allele were determined in animals fed only the con-
trol diet.
Dietary studies
Dams of two inbred mouse strains were fed methyl-
supplemented diets during pregnancy and the de-
grees of EM of their offsprings’ coats were estimated.
Maternal dietary methyl supplementation (Table 4)
increased EM of the offspring in both mouse strains
(Table 1). In strain VY mice, the MS diet produced a
strong effect on offspring phenotype, with the pro-
portion of offspring with high EM increasing from
42.7% with control diet to 59.8% on MS diet
(Põ0.001). In these mice, the 3SZM diet had the ad-
ditional effect of inducing a new phenotype, ‘almost
pseudoagouti’ (see below).
In strain YS mice, the MS diet produced no signif-
icant effect on offspring phenotype, whereas the
3SZM diet increased the proportion of offspring with
high EM from 65.5% on control diet to 78.4% on MS
diet (Põ0.05). The 3SZM diet also induced the new
‘almost pseudoagouti’ phenotype in these mice.
The Rao-Scott test indicated statistically significant
differences in means and trend in phenotypic classi-
fications among the three diet groups for the VY
strain (Põ0.001) and between the 3SZM and other
diet groups in the YS strain (Põ0.05). Methyl supple-
mentation increased the ratio of high to low EM in
the predicted direction (Table 1).
A phenotype not previously observed by the grader
(G.L.W.) in 30 years, designated ‘almost pseudo-
agouti’, was found only in litters from dams fed the
3SZM diet (Fig. 1). These mice have a few thin yellow
lines or tiny spots, mainly in the rump area, on a pseu-
doagouti background and were found in Ç13%
(NÅ10) and Ç20% (NÅ18) of VY and YS offspring,
respectively. Thus, the 3SZM diet greatly increased
the proportion of mice that had ‘almost’ or entirely
agouti coat color patterns.
The Cochran-Mantel-Haenszel test demonstrated a
statistically significant difference between strains in
the proportion of mice with high EM. This propor-
tion was larger in all three dietary groups among YS
mice than among VY mice (Põ0.001) (Table 1).
Despite very high levels of supplementation (es-
pecially in the 3SZM diet, with over 4% w/w supple-
ment), none of the diets exerted any detectable ad-
verse effects on litter size, mortality between birth
and weaning, health of the dams or that of the off-
spring until they were at least 6 wk old, or the ratio
of a/a to A
vy
/a offspring (data not shown).
Maternal epigenetic inheritance and imprinting
studies
Analyses of breeding data from two time periods, sep-
arated by 20 years, revealed partial maternal epige-
netic inheritance and imprinting with respect to ep-
igenetic regulation of the A
vy
allele among animals
on control diets (Table 2).
Maternal epigenetic inheritance (23, 24) may be
due either to the direct inheritance of epigenetic fac-
tors affecting gene expression from dam to offspring
or to recapitulation in the offspring of the dam’s pat-
tern of gene expression and epigenetic allele modi-
fication due to metabolism, hormonal balance, or
other factors in her uterine microenvironment.
The degree of maternal epigenetic inheritance can
be measured by comparing the percentage of pseu-
doagouti offspring from pseudoagouti dams with that
from mottled dams. For example, if the proportion
of pseudoagouti A
vy
/a offspring from pseudoagouti
954 Vol. 12 August 1998 The FASEB Journal WOLFF ET AL.
/ 382d 0001 Mp 954 Wednesday Aug 05 09:00 AM LP–FASEB 0001
dams is P
p
and the proportion of pseudoagouti A
vy
/
a offspring from mottled yellow dams is P
m
, then the
ratio P
p
/P
m
gives a value E, which is a measure of the
degree of maternal epigenetic inheritance (i.e., P
p
/
P
m
ÅE). There is maternal inheritance of epigenetic
phenotype when E ú 1. In strain VY, the percentage
of pseudoagouti A
vy
/a offspring from pseudoagouti
dams was 6.3% in 19911997, whereas the percent-
age of pseudoagouti A
vy
/a offspring from mottled
dams was 1.0% (Table 2). Thus P
p
/P
m
was 6.3/1.0; E
Å 6.3 and there was maternal epigenetic inheritance
(Eú1; Põ0.0001). Therefore, in this case, pseudo-
agouti dams produced about 6.3-fold the proportion
of pseudoagouti offspring as did yellow dams.
Pairwise comparisons between yellow, pseudo-
agouti, and black dams of the proportions of pseudo-
agouti offspring were highly significant (Põ0.001).
For both strains, the proportions of pseudoagouti off-
spring varied for dams of different phenotypes as fol-
lows: black ú pseudoagouti ú yellow.
Genomic imprinting occurs in A
vy
/a mice and can
be measured by comparing the percentage of pseu-
doagouti offspring when the A
vy
allele is paternally in-
herited with that when the A
vy
allele is maternally in-
herited. For example, if the proportion of
pseudoagouti A
vy
/a offspring from sires is P
s
and the
proportion of pseudoagouti A
vy
/a offspring from
dams is P
d
, then the ratio P
s
/P
d
gives a value G, which
is a measure of the degree of genomic imprinting
(i.e., P
s
/P
d
ÅG). There is genomic imprinting when
G x 1.
When comparing mottled yellow parents, the per-
centage in strain VY of pseudoagouti A
vy
/a offspring
from mottled A
vy
/a sires (dam was black a/a) was
10.2% in 19911997, whereas the percentage of
pseudoagouti A
vy
/a offspring from mottled A
vy
/a
dams (sire was black a/a) was 1.0% (Table 2). Thus,
P
s
/P
d
is 10.2/1.0, G
m
à 10, and there is genomic im-
printing (G
m
x 1; Põ0.0001).
When comparing pseudoagouti parents, the per-
centage in strain VY of pseudoagouti A
vy
/a offspring
from pseudoagouti sires (dams were black a/a) was
9.3% in 19911997, whereas the percentage of pseu-
doagouti A
vy
/a offspring from pseudoagouti dams
(sires were black a/a) was 6.3% (Table 2). Thus, P
s
/
P
d
is 9.3/6.3, G
p
à 1.5, and there is genomic imprint-
ing (G
p
x 1; Põ0.02).
That the A
vy
allele is imprinted is indicated by the
greater proportion of pseudoagouti offspring when
the A
vy
allele is contributed by the sire than when
contributed by the dam. The degree of genomic im-
printing depends on epigenetic phenotype, i.e.,
whether the parental gender comparison is of pseu-
doagouti mice or mottled yellow mice. In strain VY
(in 1991 to 1997), G
m
x G
p
(Põ0.001).
Similar calculations can be made using the remain-
ing data in Table 2 from both strains and both time
periods. Thus, there is maternal epigenetic inheri-
tance of alterations in A
vy
expression and an epige-
netic influence on the degree of imprinting at A
vy
.
DISCUSSION
As indicated by increased proportions of the more
strongly mottled phenotypes, methyl-supplemented
diets affect expression of A
vy
. With the control diet,
we confirm important previous observations (18)
about the effects of maternal epigenetic phenotype
and parental gender on A
vy
expression in offspring:
1) the epigenetic phenotype of A
vy
is, in part, mater-
nally heritable, i.e., a pseudoagouti dam is more likely
than a yellow dam to produce pseudoagouti off-
spring; 2) the gene is partly imprinted, i.e., if A
vy
is
derived from the sire, the epigenetic phenotype of
the offspring is much more likely to be pseudoagouti
than when the gene comes from the dam. The cur-
rent data suggest that diet and/or metabolic effects
may modulate IAP LTR expression, strain-specific
modifiers, maternal epigenetic inheritance, and im-
printingall factors in the epigenetic regulation of
transcription of A
vy
.
IAPs are endogenous retrovirus-like transposons
with about 1000 copies each, widely dispersed in the
mouse genome. IAP expression is regulated by DNA
methylation (25, 26) and IAP activation of nearby
genes is regulated by methylation (1, 2) or other ep-
igenetic factors (27).
Similar sequences (HIAPs) are found in the hu-
man genome associated with a number of immuno-
logical diseases (28). IAP expression is associated
with adverse effects in both mice and humans due to
activation of neighboring genes or transcription of
the IAP itself; epigenetic suppression of IAPs and
other repetitive sequences may broadly affect ge-
nome integrity, and thus health (29). In the cases
where IAP expression adversely affects health, certain
diets and supplements may suppress IAP expression
and in turn exert apparent positive effects on health.
Since hair follicle cells develop clonally from single
precursor cells (30), there may be an inverse corre-
lation between the degree of eumelanic mottling and
the developmental stage at which the A
vy
IAP LTRs
in the affected cells were epigenetically down-regu-
lated. For example, if the IAP LTR of a precursor cell
is down-regulated by methylation, the whole clone
will produce eumelanin, except for normal yellow
band production. This results in a large agouti/black
area. If the methylation occurs later during clone for-
mation, the eumelanic areas will be smaller, their spe-
cific size depending on the stage of clone develop-
ment at which the presumed IAP LTR methylation
took place. The few yellow lines and spots that char-
acterize the ‘almost pseudoagouti’ phenotype may
represent a small number of hair follicle cells in
which, for unknown reasons, the IAP LTRs were de-
MATERNAL DIET, EPIGENETICS, AND
AGOUTI
GENE EXPRESSION 955
/ 382d 0001 Mp 955 Wednesday Aug 05 09:00 AM LP–FASEB 0001
methylated relatively late in clone formation. Alter-
natively, they may represent small clones derived
from progenitor cells in which the IAP LTRs were
never methylated or otherwise epigenetically down-
regulated.
Epigenetic regulation of A
vy
expression is initiated
during gametogenesis and development. A
vy
expres-
sion is also partly inherited maternally, suggesting
that either epigenetic information is retained during
gametogenesis and development or, if once lost, is
recapitulated during these processes. This maternal
influence, as well as the capacity to recapitulate, is
evident from the modulation of expression of the pa-
ternal A
vy
allele by the maternal genotype. Paternal
phenotype (yellow or pseudoagouti) has little or no
effect on offspring phenotype, whereas maternal ge-
notype and phenotype exert major effects (18). We
show here that the maternal influence on expression
is influenced strongly by maternal diet, is partly in-
dependent of genotype, and appears to be depen-
dent on reproductive tract microenvironment and
maternal metabolism. By analogy with other se-
quences whose epigenetic regulation is modified by
the maternal genotype (31, 32), regulation of A
vy
ap-
parently is also modulated by strain-specific modifi-
ers. This suggests that strain-specific modifiers may
act via metabolism or at least are dependent on diet
and metabolism for their effects.
Important factors affecting A
vy
penetrance in the
offspring are A
vy
expression in the mother and the
sex of the parent contributing the A
vy
allele. Depend-
ing on the former, there can be large differences in
penetrance among the offspring. Suppression of A
vy
expression in the pseudoagouti dam is partly inher-
ited by some of her offspring (Table 2). This dem-
onstrates that a specific epigenetic alteration of gene
expression, resulting in the pseudoagouti phenotype,
can be passed through the maternal germline to pro-
duce the same specific alterations in the offspring.
Such epigenetic inheritance is not limited to a single
gender, as Roemer et al. (33) reported partial pater-
nal inheritance of epigenetic phenotypes of specific
genes produced by specific embryo manipulations.
This, the first report of an effect of dietary methyl
supplements on gene imprinting and specific gene
expression, demonstrates that diet influences mech-
anisms of epigenetic regulation, imprinting, and de-
velopment. Assays of 5MC in the IAP promoter in
mottled yellow and pseudoagouti A
vy
/a mice are cur-
rently under way to determine whether 5MC is a
likely participant in these epigenetic effects of diet in
A
vy
/a mice.
Others have shown neurodevelopmental or bio-
chemical effects of choline supplementation (34, 35).
Meck et al. (34) demonstrated a lifelong improve-
ment in the memory of rats whose mothers were
given choline supplements during pregnancy. Sev-
eral metabolic parameters in dam and embryo have
been changed by maternal choline supplementation
(35); however, no specific gene effects were identi-
fied. Most other examples of phenotypic change re-
ported in offspring based on maternal treatments re-
flect prevention of pathology caused by dietary
deficiency, e.g., neural tube defects or adverse drug
effects such as those induced by diethylstilbestrol
(36) or alloxan (37).
Cooney (11) proposed that dietary methyl supple-
ments could affect gene expression and 5MC levels
in adult animals and that the level of gene-specific
5MC in young mammals might affect their adult
health and longevity. The present study reveals that
specific methyl supplements in the diets of pregnant
mouse dams can affect the expression of a specific
gene, agouti, even in the offspring. At least in this
special case of A
vy
/a offspring, maternal supplemen-
tation may have beneficial health effects in adult-
hood for the offspring by preventing some of the
health problems associated with ectopic agouti ex-
pression.
The most obvious mechanisms for these effects of
methyl supplements on epigenetic phenotype are
changes in methyl metabolism that extend to the em-
bryos. These changes may affect DNA MTase activity
by increasing the substrate SAM or decreasing the
inhibitor SAH in early embryos, and thus increase the
level of DNA methylation in early embryos.
There is also an interplay with strain background
genome in that MS diet has a clear effect on strain
VY but not on strain YS mice. Again, a likely mecha-
nism would be differences in aspects of methyl me-
tabolism between these two strains. Note that these
two strains also differ in the proportion of EM in off-
spring from dams on control diet. Apparently, one
or more of the MS supplement components are lim-
iting in the control diet for dams of the VY strain, but
not for those of the YS strain.
Both strains respond to 3SZM supplement, which
indicates that one or more of the 3SZM supplement
components are limiting in the control diet for VY
strain mice and in the MS diet for YS strain mice. In
addition, the appearance of the almost pseudoagouti
phenotype in both strains on 3SZM diet indicates that
one or more of the 3SZM supplement components
are limiting with respect to one or more aspects of
methyl metabolism in the MS diet for strain VY mice,
at least for inducing this phenotype in offspring. The
most obvious mechanisms for the effects of 3SZM
supplement are on methyl metabolism and/or on
zinc availability. Saturation of the DNA MTase with
zinc may also affect its activity or specificity and thus
affect the methylation level or pattern on DNA (10).
There are other less direct or obvious mechanisms
for the effects of these supplements on the epigenet-
ics and development of offspring. Choline, directly
from the diet or synthesized via methyl metabolism,
is known to affect cell membrane fluidity, membrane-
956 Vol. 12 August 1998 The FASEB Journal WOLFF ET AL.
/ 382d 0001 Mp 956 Wednesday Aug 05 09:00 AM LP–FASEB 0001
bound enzyme activity, and intracellular signal trans-
duction (38). Likewise, methionine and folates have
essential functions in development including protein
and DNA synthesis (39, 40). Effects on these param-
eters could be important in embryonic development
and epigenetics. Similarly, zinc has numerous bio-
logical functions and could act via other mechanisms
to affect epigenetics in development. For example,
numerous zinc finger proteins besides DNA MTase
are important for cellular differentiation in devel-
opment (41).
In humans, folic acid supplements are important
for preventing some neural tube birth defects. Be-
cause present supplementation levels in humans do
not prevent all such defects, it is useful to know what
supplementation levels are toxic in mammalian de-
velopment. In this study, we found no evidence of
toxicity at high levels of supplementation in mice.
Whereas most mottled yellow A
vy
/a mice become
fat, are hyperinsulinemic, and are more susceptible
to tumor formation, pseudoagouti A
vy
/a mice remain
lean, are normoinsulinemic, and have significantly
lower lindane-associated liver tumor prevalence (17).
Previously unpublished data from the latter study
(17) reveal that, between about 17 and 24 months of
age, only 24% of pseudoagouti A
vy
/a (YS x VY)F
1
fe-
males died compared with 50% of mottled yellow
A
vy
/a and 23% of black a/a female mice; no differ-
ences in mortality between the lindane-treated and
untreated control mice were observed. Thus, by in-
creasing the proportion of the phenotype with a
down-regulated A
vy
allele, the maternal methyl-sup-
plemented diets direct the differentiation of more
A
vy
/a embryos toward a relatively healthier and
longer lived phenotype. These effects on health in
A
vy
mice may be a special case; however, similar ec-
topic expression of other genes in other animals may
also have adverse health effects that remain unrecog-
nized simply because they are not accompanied by
obvious changes in coat color.
Because coat color pattern is indicative of future
health and longevity in these A
vy
/a animals and is
identifiable in mice once the coat appears at 7 days
of age, adult characteristics of this epigenetic phe-
notype can be predicted at 1 wk of age. Therefore,
several generations of animals, categorized by their
predicted relative long-term health and longevity,
can be produced within about 2 years to determine
the multigenerational effects of diet or drugs on rel-
ative health and longevity.
Overall, the dietary supplement effects and the ma-
ternal epigenetic inheritance effect reported here
are moderate. Although the effects are significant,
they apparently did not interfere with normal bio-
logical development or with the high degrees of
methylation, demethylation, and other epigenetic
changes required in the development and growth of
embryos. These moderate effects and the partial ex-
pression and penetrance of A
vy
make this experimen-
tal system with visual markers well suited for study of
the effects of diet and drugs on epigenetic modula-
tion of gene expression. In contrast, systems in which
epigenetic control is complete do not provide the
sensitivity to monitor subtle to moderate changes in
gene expression. The coat color markers reflecting
A
vy
expression in development are important be-
cause, even with PCR techniques, evaluation of mo-
saic phenotypes in animal populations is difficult;
however, coat color markers such as those induced
by A
vy
make identification easy. For these reasons as
well as the maternal epigenetic inheritance of phe-
notype, this A
vy
/a experimental system should be use-
ful for identifying factors that modulate epigenetic
mechanisms, e.g., DNA methylation in developing
embryos and over multiple generations.
We gratefully acknowledge the administrative support and
encouragement of L. A. Poirier, helpful discussions with I.
Pogribny, and helpful discussions and constructive manuscript
suggestions by D. B. Galbraith, D. Hansen, and S. J. James.
Without the outstanding technical support from E. J. Kelley
(animal breeding and recordkeeping), A. Matson and J.
Carson (diet preparation), K. A. Carroll (computerized data-
base), J. Parker (statistical analyses), and K. Otwell and R. Bar-
ringer (digitized phenotype images), this study could not have
been successfully completed. Financial support includes an
appointment to the Postgraduate Research Program admin-
istered by the Oak Ridge Institute for Science and Education
through an interagency agreement between the U.S. Depart-
ment of Energy and the U.S. Food and Drug Administration
(to C.A.C.).
REFERENCES
1. Argeson, A. C., Nelson, K. K., and Siracusa, L. D. (1996) Molec-
ular basis of the pleiotropic phenotype of mice carrying the hy-
pervariable yellow (A
hvy
) mutation at the agouti locus Genetics
142, 557567
2. Michaud, E. J., van Vugt, M. J., Bultman, S. J., Sweet, H. O.,
Davisson, M. T., and Woychik, R. P. (1994) Differential expres-
sion of a new dominant agouti allele (A
iapy
) is correlated with
methylation state and is influenced by parental lineage. Genes &
Dev. 8, 14631472
3. Weichman, K., and Chaillet, J. R. (1997) Phenotypic variation in
a genetically identical population of mice. Mol. Cell. Biol. 17,
52695274
4. Wolff, G. L. (1996) Variability in gene expression and tumor
formation within genetically homogeneous animal populations
in bioassays. Fundam. Appl. Toxicol. 29, 176184
5. Li, E., Bestor, T. H., and Jaenisch, R. (1992) Targeted mutation
of the DNA methyltransferase gene results in embryonic lethal-
ity. Cell 69, 915926
6. Jaenisch, R. (1997) DNA methylation and imprinting: why
bother? Trends Genet. 13, 323329
7. Riggs, A. D., and Pfeifer, G. P. (1992) X-chromosome inactiva-
tion and cell memory. Trends Genet. 8, 169174
8. John, R. M., and Surani, M. A. (1996) Imprinted genes and reg-
ulation of gene expression by epigenetic inheritance. Curr. Opin.
Cell Biol. 8, 348353
9. Adams, R. L. P., and Burdon, R. H. (1985) Molecular Biology of
DNA Methylation, Springer-Verlag, New York
10. Bestor, T. H. (1992) Activation of mammalian DNA methyltrans-
ferase by cleavage of a Zn binding regulatory domain. EMBO J.
11, 26112617
MATERNAL DIET, EPIGENETICS, AND
AGOUTI
GENE EXPRESSION 957
/ 382d 0001 Mp 957 Wednesday Aug 05 09:00 AM LP–FASEB 0001
11. Cooney, C. A. (1993) Are somatic cells inherently deficient in
methylation metabolism? A proposed mechanism for DNA
methylation loss, senescence and aging. Growth Dev. Aging 57,
261273
12. Mills, J. L., Scott, J. M., Kirke, P. N., McPartlin, J. M., Conley,
M. R., Weir, D. G., Molloy, A. M., and Lee, Y. J. (1996) Homo-
cysteine and neural tube defects. J. Nutr. 126, 756S760S
13. Moephuli, S. R., Klein, N. W., Baldwin, M. T., and Krider, H. M.
(1997) Effects of methionine on the cytoplasmic distribution of
actin and tubulin during neural tube closure in rat embryos.
Proc. Natl. Acad. Sci. USA 94, 543548
14. Duhl, D. M. J., Vrieling, H., Miller, K. A., Wolff, G. L., and Barsh,
G. S. (1994) Neomorphic agouti mutations in obese yellow mice.
Nature Genet. 8, 59–65
15. Miltenberger, R. J., Mynatt, R. L., Wilkinson, J. E., and Woychik,
R. P. (1997) The role of the agouti gene in the yellow obese
syndrome. J. Nutr. 127, 1902S1907S
16. Bultman, S. J., Klebig, M. L., Michaud, E. J., Sweet, H. O., Davis-
son, M. T., and Woychik, R. P. (1994) Molecular analysis of re-
verse mutations from nonagouti (a) to black-and-tan (a
t
) and
white-bellied agouti (A
w
) reveals alternative forms of agouti
transcripts. Genes & Dev. 8, 481490
17. Wolff, G. L., Roberts, D. W., Morrissey, R. L., Greenman, D. L.,
Allen, R. R., Campbell, W. L., Bergman, H., Nesnow, S., and
Frith, C. H. (1987) Tumorigenic responses to lindane in mice:
potentiation by a dominant mutation. Carcinogenesis 8, 1889
1897
18. Wolff, G. L. (1978) Influence of maternal phenotype on meta-
bolic differentiation of agouti locus mutants in the mouse. Ge-
netics 88, 529539
19. Fung, K. Y, Krewski, D., Rao, J. N. K., and Scott, A. J. (1994) Tests
for trend in developmental toxicity experiments with correlated
binary data. Risk Anal. 14, 639648
20. Landis, R. J, Heyman, E. R., and Koch, G. G. (1978) Average
partial association in three-way contingency tables: a review and
discussion of alternative tests. Int. Stat. Rev. 46, 237254
21. SAS (1993) SAS/STAT User’s Guide, Vol. 2. SAS Institute, Inc.,
Cary, North Carolina
22. Conover, W. J. (1971) Practical Nonparametric Statistics, Wiley, New
York
23. Jablonka, E., and Lamb, M. J. (1995) Epigenetic Inheritance and
Evolution, Oxford University Press, London
24. Henikoff, S., Matzke, M. A. (1997) Exploring and explaining
epigenetic effects. Trends Genet. 13, 293295
25. Mays-Hoopes, L. L., Brown, A., and Huang, R. C. (1983) Meth-
ylation and rearrangement of mouse intracisternal A particle
genes in development, aging, and myeloma. Mol. Cell. Biol. 3,
13711380
26. Feenstra, A., Fewell, J., Lueders, K., and Kuff, E. (1986) In vitro
methylation inhibits the promoter activity of a cloned intracis-
ternal A particle LTR. Nucleic Acids Res. 14, 43434352
27. Wu, M., Rinchik, E. M., Wilkinson, E., and Johnson, D. K. (1997)
Inherited somatic mosaicism caused by an intracisternal A par-
ticle insertion in the mouse tyrosinase gene. Proc. Natl. Acad. Sci.
USA 94, 890894
28. Jaspan, J. B., Sullivan, K., Garry, R. F., Lopez, M., Wolfe, M.,
Clejan, S., Yan, C., Tenenbaum, S., Sander, D. M., Ahmed, B.,
and Bryer-Ash, M. (1996) The interaction of a type A retroviral
particle and class II human leukocyte antigen susceptibility
genes in the pathogenesis of Graves’ disease. J. Clin. Endocrinol.
Metab. 81, 22712279
29. Yoder, J. A., Walsh, C. P., and Bestor, T. H. (1997) Cytosine
methylation and the ecology of intragenomic parasites. Trends
Genet. 13, 335340
30. Mintz, B., and Bradl, M. (1991) Mosaic expression of a tyrosinase
fusion gene in albino mice yields a heritable striped coat color
pattern in transgenic homozygotes. Proc. Natl. Acad. Sci. USA 88,
96439647
31. Allen, N. D., Norris, M. L., and Surani, M. A. (1990) Epigenetic
control of transgene expression and imprinting by genotype-spe-
cific modifiers. Cell 61, 853861
32. Engler, P., Haasch, D., Pinkert, C. A., Doglio, L., Glymour, M.,
Brinster, R., and Storb U. (1991) A strain-specific modifier on
mouse chromosome 4 controls the methylation of independent
transgenic loci. Cell 65, 939947
33. Roemer, I., Reik, W., Dean, W., and Klose, J. (1997) Epigenetic
inheritance in the mouse. Curr. Biol. 7, 277280
34. Meck, W. H., Smith, R. A., and Williams, C. L. (1988) Pre- and
postnatal choline supplementation produces long-term facilita-
tion of spatial memory. Dev. Psychobiol. 21, 339353
35. Garner, S. C., Mar, M. H., and Zeisel, S. H. (1995) Choline dis-
tribution and metabolism in pregnant rats and fetuses are influ-
enced by the choline content of the maternal diet. J. Nutr. 125,
28512858
36. Walker, B. E., and Haven, M. I. (1997) Intensity of multigener-
ational carcinogenesis from diethylstilbestrol in mice. Carcino-
genesis 18, 791793
37. Spergel, G., Khan, F., and Goldner, M. G. (1975) Emergence of
overt diabetes in offspring of rats with induced latent diabetes.
Metab. Clin. Exp. 24, 13111319
38. Zeisel, S. H. (1996) Choline. A nutrient that is involved in the
regulation of cell proliferation, cell death, and cell transforma-
tion. Adv. Exp. Med. Biol. 399, 131141
39. van Straaten, H. W., Blom, H., Peeters, M. C., Rousseau, A. M.,
Cole, K. J., and Seller, M. J. (1995) Dietary methionine does not
reduce penetrance in curly tail mice but causes a phenotype-
specific decrease in embryonic growth. J. Nutr. 125, 27332740
40. Seller, M. J. (1994) Vitamins, folic acid and the cause and pre-
vention of neural tube defects. Ciba Fndn. Symp. 181, 161173
41. Jurata, L. W., Kenny, D. A., and Gill, G. N. (1996) Nuclear LIM
interactor, a rhombotin and LIM homeodomain interacting pro-
tein, is expressed early in neuronal development. Proc. Natl.
Acad. Sci. USA 93, 1169311698
42. Forejt, J., and Gregorova, S. (1992) Genetic analysis of genomic
imprinting: an Imprintor-1 gene controls inactivation of the pa-
ternal copy of the mouse Tme locus. Cell 70, 443450
43. Koetsier, P.A., Mangel, L., Schmitz, B., and Doerfler, W. (1996)
Stability of transgene methylation patterns in mice: position ef-
fects, strain specificity and cellular mosaicism. Transgenic Res. 5,
235244
44. Martin, D. I., and Whitelaw, E. (1996) The vagaries of variegating
transgenes. Bioessays 18, 919923
45. Sapienza, C., Paquette, J., Tran, T. H., and Peterson, A. (1989)
Epigenetic and genetic factors affect transgene methylation im-
printing. Development 107, 165168
46. Stewart, C. L., Pedersen, R., Rotwein, P., Bestor, T., Rastan, S.,
Hastie, N., Nichols, R., and Mutter, G. (1997) NIEHS/EPA
Workshops. Genomic imprinting. Reprod. Toxicol. 11, 309316
47. Petronis A. (1996) Genomic imprinting in unstable DNA dis-
eases. BioEssays 18, 587590
48. Chuang, L. S., Ng, H. H., Chia, J. N., and Li, B. F. (1996) Char-
acterisation of independent DNA and multiple Zn-binding do-
mains at the N terminus of human DNA-(cytosine-5) methyl-
transferase: modulating the property of a DNA-binding domain
by contiguous Zn-binding motifs. J. Mol. Biol. 257, 935948
49. Hadchouel, M., Farza, H., Simon, D., Tiollais, P., and Pourcel,
C. (1987) Maternal inhibition of hepatitis B surface antigen gene
expression in transgenic mice correlates with de novo methyla-
tion. Nature (London) 329, 454456
50. Shemer, R., Birger, Y., Riggs, A. D., and Razin, A. (1997) Struc-
ture of the imprinted mouse Snrpn gene and establishment of
its parental-specific methylation pattern. Proc. Natl. Acad. Sci. USA
94, 1026710272
51. Marahrens, Y., Panning, B., Dausman, J., Strauss, W., and Jaen-
isch R. (1997) Xist-deficient mice are defective in dosage com-
pensation but not spermatogenesis. Genes & Dev. 11, 156166
52. Panning, B., Dausman, J., and Jaenisch, R. (1997) X chromo-
some inactivation is mediated by Xist RNA stabilization. Cell 90,
907916
53. Jamieson, R. V., Tam, P. P., and Gardiner-Garden, M. (1996) X-
chromosome activity: impact of imprinting and chromatin struc-
ture. Int. J. Dev. Biol. 40, 10651080
Received for publication January 9, 1998.
Revised for publication March 6, 1998.
... Furthermore, genetic polymorphisms associated with diabetes and obesity fail to explain the increase in childhood obesity and type 2 diabetes, both of which are risk factors for CVD [6]. In contrast, there is substantial evidence supporting the involvement of prenatal and postnatal exposure to environmental risk factors in determining life-long disease susceptibility [7][8][9][10][11]. For example, newborns of pregnancies complicated by gestational diabetes mellitus (GDM) are at increased risk of developing type 2 diabetes and CVD in adult life [12]. ...
... For example, in individuals who were prenatally exposed to the Dutch famine during World War II, it was identified 60 years later that they had distinct epigenetic changes in the form of decreased DNA methylation of the Insulinlike growth factor (IGF)2 gene compared to their same sex sibling who were unexposed to maternal undernutrition [193]. Further, studies in animal models which manipulated the maternal diet during pregnancy, and analyzed the epigenetic modifications in the fetus, provided insight into the developmental origins of metabolic diseases [7,194,195]. For example, changes in dietary methyl supplements fed to pregnant mice alters DNA methylation in the fetus and leads to associated phenotypic changes [7,194], indicating that maternal nutritional status influences the long-term health of the fetus. ...
... Further, studies in animal models which manipulated the maternal diet during pregnancy, and analyzed the epigenetic modifications in the fetus, provided insight into the developmental origins of metabolic diseases [7,194,195]. For example, changes in dietary methyl supplements fed to pregnant mice alters DNA methylation in the fetus and leads to associated phenotypic changes [7,194], indicating that maternal nutritional status influences the long-term health of the fetus. There are numerous studies that report epigenetic changes in placenta and cord blood [15,35,36,108,[196][197][198] in association with GDM. ...
Full-text available
Article
Extracellular vesicles are critical mediators of cell communication. They encapsulate a variety of molecular cargo such as proteins, lipids, and nucleic acids including miRNAs, lncRNAs, circular RNAs, and mRNAs, and through transfer of these molecular signals can alter the metabolic phenotype in recipient cells. Emerging studies show the important role of extracellular vesicle signaling in the development and progression of cardiovascular diseases and associated risk factors such as type 2 diabetes and obesity. Gestational diabetes mellitus (GDM) is hyperglycemia that develops during pregnancy and increases the future risk of developing obesity, impaired glucose metabolism, and cardiovascular disease in both the mother and infant. Available evidence shows that changes in maternal metabolism and exposure to the hyperglycemic intrauterine environment can reprogram the fetal genome, leaving metabolic imprints that define life-long health and disease susceptibility. Understanding the factors that contribute to the increased susceptibility to metabolic disorders of children born to GDM mothers is critical for implementation of preventive strategies in GDM. In this review, we discuss the current literature on the fetal programming of cardiovascular diseases in GDM and the impact of extracellular vesicle (EV) signaling in epigenetic programming in cardiovascular disease, to determine the potential link between EV signaling in GDM and the development of cardiovascular disease in infants.
... Sadenosylmethionine, the methyl-donor for DNA methyltransferases, is formed by pathways that metabolize a number of nutrients [methionine, 5-methyltetrahydrofolate, betaine (from choline), vitamin B-12, and vitamin B-6]. For this reason, DNA methylation is influenced by diet (325,(335)(336)(337)(338)(339)(340). Diets high in methyl-group donors increase DNA methylation of specific genes (341) and can result in a permanent change in phenotype [e.g., coat color in the Agouti mouse (342) or twisted tails in Axin fused mice (336,343)]. ...
... Dietary exposures can induce epigenetic changes (335)(336)(337)(338)(339)(340), and perhaps, sensitive windows provide the opportunity to retune metabolism if the infant is born into a dietary environment markedly different from that expected based on the environment in utero (325). The epigenome appears more susceptible to environmental factors during periods of extensive epigenetic reprogramming in early life, particularly during the prenatal, neonatal, and pubertal periods. ...
Full-text available
Article
The ASN Board of Directors appointed the Nutrition Research Task Force to develop a report on scientific methods used in nutrition science to advance discovery, interpretation, and application of knowledge in the field. The genesis of this report was growing concern about the tone of discourse among nutrition professionals and the implications of acrimony on the productive study and translation of nutrition science. Too often, honest differences of opinion are cast as conflicts instead of areas of needed collaboration. Recognition of the value (and limitations) of contributions from well-executed nutrition science derived from the various approaches used in the discipline, as well as appreciation of how their layering will yield the strongest evidence base, will provide a basis for greater productivity and impact. Greater collaborative efforts within the field of nutrition science will require an understanding that each method or approach has a place and function that should be valued and used together to create the nutrition evidence base. Precision nutrition was identified as an important emerging nutrition topic by the preponderance of task force members, and this theme was adopted for the report because it lent itself to integration of many approaches in nutrition science. Although the primary audience for this report is nutrition researchers and other nutrition professionals, a secondary aim is to develop a document useful for the various audiences that translate nutrition research, including journalists, clinicians, and policymakers. The intent is to promote accurate, transparent, verifiable evidence-based communication about nutrition science. This will facilitate reasoned interpretation and application of emerging findings and, thereby, improve understanding and trust in nutrition science and appropriate characterization, development, and adoption of recommendations.
... Studies on the Agouti mouse provide examples of epigenetic transgenerational inheritance; food supplements have been shown to change phenotypic characteristics in five successive generations [4][5][6][7] . ...
Full-text available
Article
Nutritional conditions early in human life may influence phenotypic characteristics in later generations. A male-line transgenerational pathway, triggered by the early environment, has been postulated with support from animal and a small number of human studies. Here we analyse individuals born in Uppsala Sweden 1915–29 with linked data from their children and parents, which enables us to explore the hypothesis that pre-pubertal food abundance may trigger a transgenerational effect on cancer events. We used cancer registry and cause-of-death data to analyse 3422 cancer events in grandchildren (G2) by grandparental (G0) food access. We show that variation in harvests and food access in G0 predicts cancer occurrence in G2 in a specific way: abundance among paternal grandfathers, but not any other grandparent, predicts cancer occurrence in grandsons but not in granddaughters. This male-line response is observed for several groups of cancers, suggesting a general susceptibility, possibly acquired in early embryonic development. We observed no transgenerational influence in the middle generation. Nutritional conditions experienced early in life may influence the disease risk of future children and grandchildren. Here the authors report that food abundance among boys before puberty associates with the relative risk of a range of cancers in grandsons, but not in granddaughters.
... Corroboration of the results we observe in a second and third genetic background is required to test the generality of our results. Strain genetics influence various diet responses, including metabolism, offspring development time and transgenerational phenotypes [44][45][46]. The white mutation in Drosophila has received particular attention as it is now known to influence a wide variety of metabolic pathways [44,47]. ...
Full-text available
Article
Studies in a broad range of animal species have revealed phenotypes that are caused by ancestral life experiences, including stress and diet. Ancestral dietary macronutrient composition and quantity (over- and under-nutrition) have been shown to alter descendent growth, metabolism and behaviour. Molecules have been identified in gametes that are changed by ancestral diet and are required for transgenerational effects. However, there is less understanding of the developmental pathways altered by inherited molecules during the period between fertilization and adulthood. To investigate this non-genetic inheritance, we exposed great grand-parental and grand-parental generations to defined protein to carbohydrate (P:C) dietary ratios. Descendent developmental timing was consistently faster in the period between the embryonic and pupal stages when ancestors had a higher P:C ratio diet. Transcriptional analysis revealed extensive and long-lasting changes to the MAPK signalling pathway, which controls growth rate through the regulation of ribosomal RNA transcription. Pharmacological inhibition of both MAPK and rRNA pathways recapitulated the ancestral diet-induced developmental changes. This work provides insight into non-genetic inheritance between fertilization and adulthood. © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
... Relevant studies in this area include our original observation that monozygotic twins show epigenetic differences (Fraga et al., 2005), understood as the chemical marks such as DNA methylation and histone modifications that regulate gene expression, that might explain different population traits and distinct penetrance of diseases in these people, a finding supported in later studies (Kaminsky et al., 2009), including The NASA Twins Study (Garrett-Bakelman et al., 2019). These questions can be more easily addressed in experimental models where the researcher can intervene, such as the Agouti mice (Wolff et al., 1998) and cloned animals (Rideout et al., 2001), whereas in humans, the investigator has a more passive role, waiting for the right sample to appear. In this re-gard, one of the most documented cases is the Dutch famine at the end of WWII that was associated with less DNA methylation of the imprinted IGF2 gene compared with their unexposed, same-sex siblings (Heijmans et al., 2008). ...
Full-text available
Article
The human face is one of the most visible features of our unique identity as individuals. Interestingly, monozygotic twins share almost identical facial traits and the same DNA sequence but could exhibit differences in other biometrical parameters. The expansion of the world wide web and the possibility to exchange pictures of humans across the planet has increased the number of people identified online as virtual twins or doubles that are not family related. Herein, we have characterized in detail a set of “look-alike” humans, defined by facial recognition algorithms, for their multiomics landscape. We report that these individuals share similar genotypes and differ in their DNA methylation and microbiome landscape. These results not only provide insights about the genetics that determine our face but also might have implications for the establishment of other human anthropometric properties and even personality characteristics.
... For example, it has been suggested that regions of Po-fOm may be vulnerable to stochastic or environmentallysensitive loss of methylation on the usually-methylated allele or gain of methylation on the usually-unmethylated allele at a later time-point, leading to interindividual methylation variation (57,71,102). Similarly, certain IAP elements (a class of ERVK LTR retrotransposon) show methylation variation between isogenic mice (67,68) that in several cases can be influenced by pre-natal environment (103)(104)(105). Whilst transposable elements are usually silenced to prevent insertion events from damaging the genome, recent evidence suggests that methylation variability at IAP elements is partly driven by low-affinity binding of transacting Krüppel-associated box (KRAB)-containing zinc finger proteins (KZFPs) (69) and by sequence variation in KZFP-binding sites (69,106). ...
Full-text available
Article
We analysed DNA methylation data from 30 datasets comprising 3474 individuals, 19 tissues and 8 ethnicities at CpGs covered by the Illumina450K array. We identified 4143 hypervariable CpGs (‘hvCpGs’) with methylation in the top 5% most variable sites across multiple tissues and ethnicities. hvCpG methylation was influenced but not determined by genetic variation, and was not linked to probe reliability, epigenetic drift, age, sex or cell heterogeneity effects. hvCpG methylation tended to covary across tissues derived from different germ-layers and hvCpGs were enriched for proximity to ERV1 and ERVK retrovirus elements. hvCpGs were also enriched for loci previously associated with periconceptional environment, parent-of-origin-specific methylation, and distinctive methylation signatures in monozygotic twins. Together, these properties position hvCpGs as strong candidates for studying how stochastic and/or environmentally influenced DNA methylation states which are established in the early embryo and maintained stably thereafter can influence life-long health and disease.
Article
Objectives: The purpose of this study was to evaluate the effects of maternal high folic acid (FA) supplementation during pregnancy on glucose intolerance in dams and insulin resistance in offspring. Methods: Wistar female rats (n=18) were mated and randomly divided into 3 groups: a control group and 2 experimental groups. Three different feeding protocols were administered during pregnancy: control group-2 mg/kg FA (recommended level FA supplementation); experimental 1 group-5 mg/kg FA (tolerable upper intake level of FA supplementation [ULFolS]); and experimental 2 group-40 mg/kg FA (high FA supplementation [HfolS]). All dams were fed the same FA content diet (2 mg/kg FA) during the lactation period. An oral glucose tolerance test was performed on day 16th of pregnancy. After the lactation period, body weight and food intake of 36 pups were monitored. Dams were euthanized at the end of the lactation period and half of the pups were euthanized at the end of week 7 and the others at the end of week 12. Serum FA, homocysteine, vitamin B12, insulin, glucose, interleukin-6, tumour necrosis factor-alpha, glycated hemoglobin (A1C) and adiponectin levels of mothers and pups were evaluated. The homeostatic model of insulin resistance (HOMA-IR) was used to determine insulin resistance in dams and offspring. Results: According to glucose tolerance test results of dams, blood glucose values at minutes 0, 60, 90 and 120 for the HFolS group were significantly higher compared with the control group (p<0.05). The glycated hemoglobin A1C level in HFolS dams was significantly higher than in the control group (p<0.05). The mean birthweight of the pups in the HFolS group was significantly higher than that of control pups (p<0.05). HOMA-IR values for control and HFolS offspring were similar at weeks 7 and 12 and higher than in ULFolS offspring (p>0.05). Conclusions: In this study, it was determined that the effects of high doses of FA exposure during pregnancy on glucose intolerance in dams and insulin resistance in offspring.
Chapter
Women’s reproductive cancers are a group of cancers that initiate in women’s reproductive system including the cervix, ovaries, uterus, vagina and vulva. There are different risk factors and etiological mechanisms that are involved in oncogenesis of gynecologic malignancies. Exploration of effective chemopreventive and therapeutic approaches that are universally applied in most gynecologic cancers will be critically important and benefit women’s welfare. In the last two decades, novel strategies that target epigenetics-related molecular events have emerged as one of the major cancer prevention and therapies employed by dietary phytochemicals. These bioactive dietary components found in fruits and vegetables such as green tea, soybean, cruciferous vegetables, grapes and certain Asian spices have shown potent inhibitory activities against human cancer including female reproductive cancers. More important, these bioactive dietary compounds can elicit their chemopreventive/therapeutic properties through, at least in part, modulation of epigenetic processes including histone modification, DNA methylation and non-coding RNA expression during carcinogenesis. Given the fact that epigenetic aberrations dynamically contribute to cancer pathogenesis, reversal of abnormal epigenetic codes and their-induced gene expression profiles by bioactive dietary compounds could be an effective approach for gynecologic cancer prevention and therapy. In this review, we will discuss the advances in understanding the epigenetic effects of dietary phytochemicals on gene expression during cancer development, and how these epigenetics diets help to improve cancer prevention and treatment efficacy in major gynecologic cancers.
Chapter
Nutritional intake during key developmental windows in early life has increasingly been linked to long-term health trajectories and the risk of non-communicable disease. The mechanisms by which early-life environment can influence later phenotypes and long-term disease risk has been suggested to include epigenetic processes. Epigenetic processes modulate gene expression without a change in the DNA nucleotide sequence, determining when and where a gene is expressed. Both maternal and paternal diet have been shown to induce epigenetic changes in the offspring, leading to changes in gene expression that can persist throughout the life course and influence later disease susceptibility. This has led in recent years to the identification of epigenetic markers for lifestyle-related disease risk being investigated as potential therapeutic biomarkers. Elucidation of such modifiable epigenetic processes linked to non-communicable disease risk may enable early intervention strategies using targeted approaches through nutrition or pharmacological epigenetic modifiers to improve long-term health.
Article
During the past few decades we have witnessed an era of remarkable growth in the field of molecular biology. In 1950 very little was known of the chemical constitution of biological systems, the manner in which in­ formation was transmitted from one organism to another, or the extent to which the chemical basis of life is unified. The picture today is dramati­ cally different. We have an almost bewildering variety of information detailing many different aspects of life at the molecular level. These great advances have brought with them some breath-taking insights into the molecular mechanisms used by nature for replicating, distributing and modifying biological information. We have learned a great deal about the chemical and physical nature of the macromolecular nucleic acids and proteins, and the manner in which carbohydrates, lipids and smaller mole­ cules work together to provide the molecular setting of living systems. It might be said that these few decades have replaced a near vacuum of information with a very large surplus. It is in the context of this flood of information that this series of mono­ graphs on molecular biology has been organized. The idea is to bring together in one place, between the covers of one book, a concise assess­ ment of the state of the subject in a well-defined field.
Article
This paper reviews and discusses alternative methods for assessing average partial association in three-way contingency tables. Primary attention is directed at the relationship between two of the variables, while controlling for the effects of a set of covariables. One approach is a class of multivariate extensions of the Cochran-Mantel-Haenszel test to sets of (s × r) tables. These statistics are based on expected values and covariances from the multiple hypergeometric distribution for each table. As such they make no underlying assumption regarding second-order interaction, although the absence of such interaction is incorporated within the hypothesis being tested. Alternatively, a log-linear model can be used to investigate average partial association conditional on the assumption of no second-order interaction. If the fit of such a model is supported by the data, then likelihood ratio methods or functional asymptotic regression methods (FARM) can be used to test the significance of correspondingly appropriate parameters. These procedures are all illustrated with a data set relating atomic bomb radiation to the incidence of leukemia adjusted for age at exposure. /// Cet article passe en revue et discute un choix de méthodes en vue d'établir la présence d'association partielle moyenne dans les tableaux de contingence à trois dimensions. L'attention se porte avant tout sur la relation existant entre deux des variables, alors qu'on contrôle les effets d'un ensemble de covariables. Une approche consiste à étendre à plus de deux variables le test de Cochran-Mantel-Haenszel pour des ensembles de tableaux (r × s). Ces statistiques reposent sur des espérances mathématiques et des covariances de la distribution hypergéométrique généralisée concernant chaque tableau. Par là même, elles ne comportent aucune hypothèse sous-jacente sur les interactions du second ordre, bien que l'absence de telles interactions soit incluse dans l'hypothèse à tester. On peut encore passer par un modèle log-linéaire pour explorer l'association partielle moyenne conditionnée par l'hypothèse de non-interaction du second ordre. Si les données se plient à un tel modèle, on peut alors employer soit les méthodes du ratio des vraisemblances, soit les méthodes de régression asymptotique fonctionnelle (FARM) pour tester si les paramètres correspondants sont ou non significatifs. Toutes ces façons de procéder sont illustrées au moyen d'un ensemble de données concernant les radiations de la bombe atomique et leur incidence sur la leucémie, corrélativement à l'âge lors de l'exposition aux radiations.
Article
The results of extensive breeding experiments indicate that the phenotypic differentiation of embryos carrying the viable yellow, A( vy), or mottled, a(m), mutations is influenced to a major extent by the agouti locus genotype and the strain genome of the dam. The A(vy)/a and a(m)/a genotypes are each expressed in a spectrum of coat color phenotypes. These can be grouped into two classes, mottled and pseudoagouti.-In a reciprocal cross of C57BL/6JNIcrWf and AM/Wf-a(m)/a(m) mice, 29.5% of the offspring of C57BL/6 dams were of the pseudoagouti phenotype, whereas no pseudoagouti offspring were produced by AM strain dams.-Mottled yellow A(vy)/a mice become obese and tumor formation is enhanced in these mice in comparison with the lean pseudoagouti A(vy)/a siblings.-In two different reciprocal crosses using four different inbred strains, the proportion of pseudoagouti A(vy)/a offspring differed according to the strain of the dam. Regardless of strain, mottled yellow A( vy)/a dams produced significantly fewer pseudoagouti A( vy)/a offspring than did black a/a dams.-The data suggest that metabolic differentiation of A(vy)/a zygotes into phenotypic classes with different susceptibilities to obesity and tumor formation is influenced to a considerable degree by the metabolic characteristics of the oviductal and uterine environment of the dam.
Article
A single subdiabetogenic dose of alloxan administered to the weanling rat induces a persistent state of latent diabetes which progresses to fasting hyperglycemia by the seventh generation. Initial descendants of alloxan-treated animals have hyperinsulinism which progresses to insulinopenia in later generations. Later generation animals develop ketoacidosis when challenged with a dose of alloxan that has no effect on control animals. The significant sex difference in glucose tolerance rates disappears as the animals become more diabetic and decreased fertility and parity become apparent. One explanation for this data remains the hypothesis of paramutation, induced by alloxan, affecting regulator gene activity. Light microscopy of diabetic animals shows no pathology.
Article
The Thp deletion on mouse chromosome 17 is lethal when inherited from the mother, because it deletes the T-associated maternal effect (Tme) locus, the paternal copy of which is inactivated by genomic imprinting. We have found a paternally nonimprinted Tme variant in crosses of Thp females with Mus m. musculus males. The data are consistent with the existence of a single Tme-unlinked gene, Imprintor-1 (Imp-1), with two alleles, one of which only causes imprinting at the Tme locus. Imp-1 is unlinked to the gene for cation-dependent Man-6-P receptor and acts prezygotically. Although Tme and Igf2r were thought to be identical, they show different patterns of imprinting in interspecies hybrids. The apparent nonequivalence of the Igf2r gene and Tme results in occurrence of viable mice lacking an active Igf2r gene. These mice are bigger at birth than their normal littermates, in accord with the proposed function of the IGF-II/Man-6-P receptor.
Article
Mammalian X-chromosome inactivation is an excellent example of the faithful maintenance of a determined chromosomal state. As such, it may provide insight into the mechanisms for cell memory, defined as the faithful maintenance of a determined state in clonally derived progeny cells. We review here the aspects of X-chromosome inactivation that are relevant to cell memory and discuss the various molecular mechanisms that have been proposed to explain its occurrence, with emphasis on DNA methylation and a recently proposed mechanism that depends on the timing of replication.
Article
Gene targeting in embryonic stem (ES) cells has been used to mutate the murine DNA methyltransferase gene. ES cell lines homozygous for the mutation were generated by consecutive targeting of both wild-type alleles; the mutant cells were viable and showed no obvious abnormalities with respect to growth rate or morphology, and had only trace levels of DNA methyltransferase activity. A quantitative end-labeling assay showed that the level of m5C in the DNA of homozygous mutant cells was about one-third that of wild-type cells, and Southern blot analysis after cleavage of the DNA with a methylation-sensitive restriction endonuclease revealed substantial demethylation of endogenous retroviral DNA. The mutation was introduced into the germline of mice and found to cause a recessive lethal phenotype. Homozygous embryos were stunted, delayed in development, and did not survive past mid-gestation. The DNA of homozygous embryos showed a reduction of the level of m5C similar to that of homozygous ES cells. These results indicate that while a 3-fold reduction in levels of genomic m5C has no detectable effect on the viability or proliferation of ES cells in culture, a similar reduction of DNA methylation in embryos causes abnormal development and embryonic lethality.