Discussion 2: Genes and Personality
Prior to beginning work on this discussion, listen to the WNYC Studios (2012) podcast Inheritancefrom http://www.radiolab.org/story/251876-inheritance/ , and read the Hurley (2013) Trait vs. Fate, the Weaver et al. (2004) Epigenetic Programming by Maternal Behavior, and the Webster (2013) blog post. (BOTH ARTICLES ARE PROVIDED IN THE ATTACHMENTS)
The Great Rat Mother Switcheroohttps://www.wnycstudios.org/podcasts/radiolab/articles/261176-the-great-mother-switcheroo . The recommended sources for this week provide additional information on these topics that may be helpful.
In this weeks required sources, you learned that while genes have a lot to say about who we are, environmental factors, particularly parental behavior, can shape the behavior of offspring on a biochemical level. In your initial post of a minimum of 350 words,
Briefly describe the research that has been done with rat mothers to illustrate this phenomenon. Be specific about the maternal behaviors and correlated biochemical changes to their offspring. This section demonstrates that you understand what research has been conducted and what the results mean, so be sure to go beyond quoting and paraphrasing to explain and interpret the research as you understand it.
Explain the implications of the research on parental behavior and environmental factors as they relate to human personality development.
Based on your understanding of the research cited in the required sources, assess the emerging field of epigenetics, explaining how it is impacting the longstanding nature versus nurture paradigm and evaluating the possible promise and risks with respect to the human experiments.
Be sure to cite all the REQUIRED RESOURCES in your initial post.
Hurley, D. (2013). Trait vs. fate. Discover 34(4), 48-55. Retrieved from http://discovermagazine.com
Weaver, I. C. G., Cervoni, N., Champagne, F. A., DAlessio, A. C, Sharma, S., Seckl, J. R., Meaney, M. J. (2004). Epigenetic programming by maternal behavior. Nature Neuroscience, 7(8), 847-854. http://dx.doi.org/10.1038/nn1276
Webster, M. (2013, January 10). The great rat mother switcheroo(Links to an external site.) [Blog post]. Retrieved from http://www.radiolab.org/story/261176-the-great-mother-switcheroo
WNYC Studios. (2012, November 18). Inheritance(Links to an external site.) [Audio podcast]. Retrieved from http://www.radiolab.org/story/251876-inheritance/
A R T I C L E S
Through undefined epigenetic processes, maternal effects influence
the development of defensive responses to threat in organisms rang-
ing from plants to mammals1,2. In the rat, such effects are mediated by
variations in maternal behavior, which serve as the basis for the trans-
mission of individual differences in stress responses from mother to
offspring35. Mother-pup contact in the rat primarily occurs within
the context of a nest-bout, which begins when the mother approaches
the litter, licks and grooms her pups, and nurses while occasionally
licking and grooming the pups6. There are stable individual differ-
ences in two forms of maternal behaviorLG and ABNover the
first week of lactation610. Such naturally occurring variations in
maternal behavior are associated with the development of individual
differences in behavioral and HPA responses to stress in the offspring.
As adults, the offspring of high-LG-ABN mothers are less fearful and
show more modest HPA responses to stress than the offspring of low-
LG-ABN mothers69. Cross-fostering studies show that the biological
offspring of low-LG-ABN mothers reared by high-LG-ABN dams
resemble the normal offspring of high-LG-ABN mothers (and vice
versa9). These findings suggest that variations in maternal behavior
serve as a mechanism for the nongenomic transmission of individual
differences in stress reactivity across generations4,5,9. The critical
question concerns the mechanisms whereby these maternal effects, or
other forms of environmental programming, are sustained over the
lifespan of the animal.
Maternal behavior in the rat permanently alters the development of
HPA responses to stress through tissue-specific effects on gene
expression. The magnitude of the HPA response to acute stress is a
function of hypothalamic corticotropin-releasing factor (CRF)
release, which activates the pituitary-adrenal system. There are also
modulatory influences, such as glucocorticoid negative feedback that
inhibits CRF synthesis and release, thus dampening HPA responses to
stress11. The adult offspring of high- compared with low-LG-ABN
mothers show increased hippocampal GR expression and enhanced
glucocorticoid feedback sensitivity7,9. Predictably, adult offspring of
high-LG-ABN mothers show decreased hypothalamic CRF expres-
sion and more modest HPA responses to stress7. Eliminating the dif-
ference in hippocampal GR levels abolishes the effects of early
experience on HPA responses to stress in adulthood12, suggesting that
the difference in hippocampal GR expression serves as a mechanism
for the effect of early experience on the development of individual
differences in HPA responses to stress5.
In vivo and in vitro studies suggest that maternal LG and ABN
increase GR gene expression in the offspring through increased sero-
tonin (5-HT) activity at 5-HT7 receptors, and the subsequent activa-
tion of cAMP and cAMP-dependent protein kinase activity1315. Both
the in vitro effect of 5-HT and the in vivo effect of maternal behavior
on GR gene expression are accompanied by an increased hippocam-
pal expression of nerve growth factor-inducible protein A (NGFI-A, a
transcription factor also known as egr-1, krox-24, zenk and zif-268).
The non-coding exon 1 region of the hippocampal GR includes a pro-
moter region, exon 17, containing a binding site for NGFI-A
16
(Fig. 1a). Splice variants of the GR mRNA containing the exon 17
sequence are found predominantly in the brain, and the expression of
GR mRNAs containing the exon 17 sequence is increased in the off-
spring of high-LG-ABN mothers or following manipulations that
increase maternal licking and grooming16 (Weaver, I.C.G. et al., Soc.
Neurosci. Abstr. 697.15, 2001), suggesting that the use of this promoter
is enhanced as a function of maternal care. Although these findings
might explain the increased GR expression in the neonate, we are left
with the question of how the effect of maternal care might persist into
1Douglas Hospital Research Center, 6875 LaSalle Blvd., Montral, Qubec H4H 1R3, Canada. 2McGill Program for the Study of Behaviour, Genes and Environment
and 3Department of Pharmacology and Therapeutics, McGill University, 3655 Sir William Osler Promenade, Montral, Qubec H3G 1Y6, Canada. 4Molecular
Medicine Centre, Edinburgh University, Western General Hospital, Edinburgh EH4 2XU, UK. Correspondence should be addressed to M.J.M.
([emailprotected]) or M.S. ([emailprotected]).
Published online 27 June 2004; corrected 27 July 2004 (details online); doi:10.1038/nn1276
Epigenetic programming by maternal behavior
Ian C G Weaver1,2, Nadia Cervoni3, Frances A Champagne1,2, Ana C DAlessio3, Shakti Sharma1,
Jonathan R Seckl4, Sergiy Dymov3, Moshe Szyf2,3 & Michael J Meaney1,2
Here we report that increased pup licking and grooming (LG) and arched-back nursing (ABN) by rat mothers altered the offspring
epigenome at a glucocorticoid receptor (GR) gene promoter in the hippocampus. Offspring of mothers that showed high levels of
LG and ABN were found to have differences in DNA methylation, as compared to offspring of low-LG-ABN mothers. These
differences emerged over the first week of life, were reversed with cross-fostering, persisted into adulthood and were associated
with altered histone acetylation and transcription factor (NGFI-A) binding to the GR promoter. Central infusion of a histone
deacetylase inhibitor removed the group differences in histone acetylation, DNA methylation, NGFI-A binding, GR expression and
hypothalamic-pituitary-adrenal (HPA) responses to stress, suggesting a causal relation among epigenomic state, GR expression
and the maternal effect on stress responses in the offspring. Thus we show that an epigenomic state of a gene can be established
through behavioral programming, and it is potentially reversible.
NATURE NEUROSCIENCE VOLUME 7 | NUMBER 8 | AUGUST 2004 8 4 7
2
0
0
4
N
a
tu
re
P
u
b
lis
h
in
g
G
ro
u
p
h
tt
p
:/
/w
w
w
.n
a
tu
re
.c
o
m
/n
a
tu
re
n
e
u
ro
sc
ie
n
ce
sites of the exon 17 GR promoter sequence (Fig. 1b,c). A two-way
ANOVA revealed a highly significant effect of Group (F = 55.9,
P < 0.0001) and Region (F = 27.7, P < 0.0001), as well as a significant
Group Region interaction effect (F = 27.7, P < 0.0001).
Importantly, the cytosine residue within the 5 CpG dinucleotide
(site 16) of the NGFI-A consensus sequence (Fig. 1c) is always
methylated in the offspring of low-LG-ABN mothers, and rarely
methylated in the offspring of high-LG-ABN dams. In contrast, the
3 CpG dinucleotide (site 17) remains methylated, regardless of dif-
ferences in maternal care. Dissected hippocampi inevitably contain
glial cells as well as neurons. Considering the pronounced effect of
maternal care on the methylation status of the 5 CpG dinucleotide
of the NGFI-A response element (>90%), the effect of maternal care
must include neuronal as well as glial cells; both populations express
GR23,24 and NGFI-A25 genes.
Cross-fostering reveals epigenetic marking by maternal behavior
Our findings suggest that specific sites within the exon 17 GR pro-
moter are differentially methylated as a function of maternal behav-
ior, but these findings are merely correlational. To directly examine
the relation between maternal behavior and DNA methylation
within the exon 17 promoter, we performed an adoption study in
which the biological offspring of high- or low-LG-ABN mothers
were cross-fostered to either high- or low-LG-ABN dams within 12 h
of birth9. Cross-fostering produced a pattern of exon 17 promoter
methylation that was associated with the rearing mother (F = 4.8,
P < 0.05; Fig. 1d) and thus reversed the difference in methylation at
specific cytosines, notably at the 5 CpG dinucleotide (site 16) of the
Figure 1 Maternal care alters cytosine
methylation of GR promoter. (a) Sequence map
of the exon 17 GR promoter including the 17
CpG dinucleotides (bold) and the NGFI-A binding
region16 (encircled). (b,c) Methylation analysis of
the 17 CpG dinucleotides of the exon 17 GR
promoter region from adult high- and low-LG-
ABN offspring (610 clones sequenced/animal;
n = 4 animals/group; *P < 0.01). (b) Percentage
of cytosine residues that were methylated (mean
s.e.m.) for the first 15 CpG dinucleotides
(*P < 0.05). (c) Percentage of methylated
cytosines (mean s.e.m.) for the 5 (site 16) and
3 (site 17) CpG dinucleotides within the NGFI-A
binding sequence (*P < 0.0001). (d) The effect
of cross-fostering the offspring of high- and low-
LG-ABN mothers on cytosine methylation of the
5 and 3 CpG dinucleotides within the NGFI-A
binding sequence of the exon 17 GR promoter
gene in adult hippocampi (n = 5 animals/group).
L-L: animals born to and reared by low-LG-ABN
mothers; H-H: animals born to and reared by
high-LG-ABN mothers; H-L: animals born to
high-LG-ABN mothers and reared by low-LG-ABN
mothers; L-H: animals born to low-LG-ABN
mothers and reared by high-LG-ABN mothers.
(e) Percentage of cytosine methylation (mean
s.e.m.) of the 5 and 3 CpG dinucleotides within
the NGFI-A binding region of the exon 17 GR
promoter gene in the offspring of high- or low-
LG-ABN mothers (n = 5 animals/group;
P < 0.001) as a function of age. There were no
differences at any postnatal age in level of
cytosine methylation of the 3 CpG (site 17).
A R T I C L E S
adulthood. Gene expression is controlled by the epigenome, which is
comprised of chromatin structure17 and DNA methylation18. We
tested the hypothesis that maternal care alters DNA methylation of
the GR exon 17 promoter, and that these changes are stably main-
tained into adulthood and associated with differences in GR expres-
sion and HPA responses to stress.
RESULTS
Maternal care and methylation of exon 17 promoter
DNA methylation is a stable, epigenomic mark at CpG dinucleotides
often associated with stable variations in gene transcription1820. Two
kinds of changes in DNA methylation are known to affect gene
expression: regional, non-site specific DNA methylation around a
promoter19 and site-specific methylation. Hypomethylation of CpG
dinucleotides of regulatory regions of genes is associated with active
chromatin structure and transcriptional activity18,20. Thus, the
methylation pattern is a stable signature of the epigenomic status of a
regulatory sequence. We focused on the methylation state of the exon
17 GR promoter, which is activated in the hippocampus in offspring
of high-LG-ABN mothers.
To determine whether DNA methylation of specific target sites on
the GR promoter change in response to maternal care, we mapped
differences in the methylation status of individual cytosines within
the CpG dinucleotides of the exon 17 promoter from hippocampal
tissue from the adult offspring of high- and low-LG-ABN mothers.
We used sodium bisulfite mapping21,22, with a particular interest in
the region around the NGFI-A consensus sequence (Fig. 1a). The
results showed significant differences in the methylation of specific
8 4 8 VOLUME 7 | NUMBER 8 | AUGUST 2004 NATURE NEUROSCIENCE
L-L H-H H-L L-H
0
20
40
60
80
100
C
-m
e
th
yl
a
tio
n
(
%
)
L-L H-H H-L L-H
0
20
40
60
80
100
5 CpG dinucleotide 3 CpG dinucleotide
High-LG/ABN
Low-LG/ABN
Age (d)
E20 P1 P6 P21 P90
0
20
40
60
80
100
E20 P1 P6 P21 P90
0
20
40
60
80
100
* * *
C
-m
e
th
yl
a
tio
n
(
%
)
5 CpG dinucleotide 3 CpG dinucleotide
1681 ccc
1741 ctctgctagtgtgacacactt1cg2cg caactc3cgcagttgg4cggg5cg6cggaccacccctg7c
1801 ggctctgc8cggctggctgtcaccct9cgggggctctggctgc10cgaccca11cgggg12cgggct
1861 c13cgag14cggtt ccaagcct15cggagtggg16cggggg17cgggagggagcctgggagaa
11 14 15 16 17 18 19 110 111 2
5 3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Region
C
-m
e
th
yl
a
tio
n
(
%
)
Low-LG/ABN
High-LG-ABN
0
20
40
60
80
100
*
*
**
*
*
*
5 3
CpG dinucleotide
Low LG-ABN
High LG-ABN
*
0
20
40
60
80
100
a
b c
d e
C
-m
e
th
yl
a
tio
n
(
%
)
2
0
0
4
N
a
tu
re
P
u
b
lis
h
in
g
G
ro
u
p
h
tt
p
:/
/w
w
w
.n
a
tu
re
.c
o
m
/n
a
tu
re
n
e
u
ro
sc
ie
n
ce
A R T I C L E S
NGFI-A consensus sequence (Fig. 1d, left panel). Thus, in the
low-LG-ABN offspring that were fostered to high-LG-ABN dams,
methylation of this 5 site within the exon 17 promoter was indistin-
guishable from that of the biological offspring of high-LG-ABN
mothers. Likewise, the methylation of the same 5 CpG dinucleotide
in the biological offspring of high-LG-ABN mothers reared by low-
LG-ABN dams was comparable to that of low-LG-ABN offspring.
There was no effect of cross-fostering at the cytosine within the 3
CpG dinucleotide (site 17; Fig. 1d).
These findings suggest that variations in maternal care directly alter
the methylation status of the exon 17 promoter of the GR gene. Thus
we have demonstrated that a DNA methylation pattern can be estab-
lished through a behavioral mode of programming without germ line
transmission. In parental imprinting, a well-established paradigm of
inheritance of an epigenomic mark, the paternally and maternally
inherited alleles are differentially methylated. These methylation pat-
terns are defined during maturation of spermatocytes and oocytes,
and are transmitted to the offspring through the germ line26.
Timing of the maternal effect on DNA methylation
The maternal care of high- and low-LG-ABN mothers differs only
during the first week of life7,8. Thus, we wondered whether this period
corresponds to the timing for the appearance of the difference in
DNA methylation in the offspring. We used sodium bisulfite mapping
to examine the methylation status of the cytosines within the exon 17
GR promoter during development (Fig. 1e). Statistical analysis of the
data for the 5 CpG (site 16) revealed a highly significant effect of
Group (F = 66.7, P < 0.0001) and Age (F = 21.1, P < 0.0001) as well as
a significant interaction effect (F = 13.7, P < 0.0001). Tukey post-hoc
analysis revealed that the Group effect on methylation status of the
5 CpG (site 16) was significant at P6, P21 and P90 (P < 0.001), but
not at E20 or P1. Just before birth (embryonic day 20; E20) the entire
region was unmethylated in both groups. Strikingly, one day after
birth (postnatal day 1; P1) the exon 17 GR promoter was de novo
methylated in both groups. The 5 and 3 CpG sites of the exon 17 GR
NGFI-A response element in the offspring of both high- and low-
LG-ABN mothers, which exhibit differential methylation later in life,
were de novo methylated to the same extent. These data show that
NATURE NEUROSCIENCE VOLUME 7 | NUMBER 8 | AUGUST 2004 8 4 9
Figure 2 Chromatin immunoprecipitation analysis of the association
between histone H3-K9 acetylation and NGFI-A binding to the exon 17 GR
sequence in hippocampal tissue from adult offspring of high- and low-LG-
ABN mothers (n = 4 animals/group). (a,b) Lanes were loaded with non-
immunoprecipitated input (I), acetylated histone H3-K9 (top) or NGFI-A
(middle) primary antibody immunoprecipitated (A), or non-immune IgG
antibody immuno-precipitated (N) hippocampal extracts). (a) Representative
Southern blot of the amplified exon 17 region from acetyl-histone H3-K9
immunoprecipitated hippocampal tissue (194 bp band) and -actin (171 bp
band) control. (b) Representative Southern blot of the amplified exon 17
region of the GR from NGFI-A immunoprecipitated hippocampal tissue
(194 bp band). DNA loading was controlled using primers specific for the
ubiquitously expressed -actin promoter- region. Exon 1b estrogen
receptor- promoter region, which does not contain NGFI-A recognition
elements (493 bp), amplified from the same NGFI-A immunoprecipitated
hippocampal tissue was run as a control for specificity and showed no
signal. (c) Relative optical density (ROD; mean s.e.m.) of exon 17
sequence amplified from acetyl-histone H3-K9 or NGFI-A
immunoprecipitated hippocampal tissue of adult high- and low-LG-ABN
offspring (n = 4 animals/group; *P < 0.001; **P < 0.0001).
Figure 3 HDAC inhibitior (TSA) eliminates maternal effect on histone
acetylation and NGFI-A binding. (a) Chromatin immunoprecipitation
analysis of the association between histone H3-K9 acetylation and NGFI-A
binding to the exon 17 GR promoter sequence in hippocampal tissue from
vehicle- and TSA-treated (100 ng/ml) adult offspring of high- and low-LG-
ABN mothers (n = 4 animals/group; lane labels as described in Fig. 2).
(b) Relative optical density (ROD; mean s.e.m.) of exon 17 sequence
amplified from acetyl-histone H3-K9 or NGFI-A immunoprecipitated
hippocampal tissue (*P < 0.05; **P < 0.01).
2
0
0
4
N
a
tu
re
P
u
b
lis
h
in
g
G
ro
u
p
h
tt
p
:/
/w
w
w
.n
a
tu
re
.c
o
m
/n
a
tu
re
n
e
u
ro
sc
ie
n
ce
A R T I C L E S
both the basal state of methylation and the first wave of de novo
methylation after birth occur similarly in both groups. Whereas it is
generally accepted that DNA methylation patterns are formed prena-
tally and that de novo methylation occurs early in development, there
is at least one documented example of postnatal de novo methylation
of the Hoxa5 and Hoxb5 genes27. Because similar analyses are not doc-
umented for other genes, it remains unknown whether changes in
methylation are common around birth or whether they are unique to
this GR promoter.
The differences in the methylation status of the exon 17 GR pro-
moter between the two groups developed between P1 and P6, the
period when differences in the maternal behavior of high- and low-
LG-ABN dams are apparent5,8. By P6, the NGFI-A response element
5 CpG dinucleotide (site 16) was effectively demethylated in the
high-, but not in the low-LG-ABN group. The group difference in
CpG dinucleotide methylation remains consistent through to adult-
hood (P90; Fig. 1e). These findings, together with those of the cross-
fostering study, suggest that the group difference in DNA
methylation occurs as a function of a maternal behavior over the
first week of life. The results of earlier studies indicate that the first
week of postnatal life is a critical period for the effects of early
experience on hippocampal GR expression28.
Maternal effects on chromatin structure and NGFI-A binding
The next question concerns the functional importance of such dif-
ferences in methylation. DNA methylation is associated with
changes in chromatin activity states18. Chromatin gates the accessi-
bility of promoters to transcription factors17. Histone acetylation
at the lysine-9 (K9) residue of H3 and H4 histones is a well-estab-
lished marker of active chromatin17,29. Acetylation of the histone
tails neutralizes the positively charged histones, which disrupts his-
tone binding to negatively charged DNA and thus promotes tran-
scription factor binding. We tested the hypothesis that the maternal
effect on DNA methylation results in (i) increased histone acetyla-
tion at the K9 residue of the H3 histone(s) associated with the exon
17 GR promoter and (ii) increased interaction between NGFI-A
and the promoter sequence. We performed a chromatin immuno-
precipitation (ChIP) analysis of histone H3-K9 acetylation and
NGFI-A protein binding to the exon 17 GR promoter in the native
chromatin environment in vivo. Intact hippocampi from adult off-
spring of high- and low-LG-ABN mothers were crosslinked in vivo
by paraformaldehyde perfusion. We then selectively immunopre-
cipitated protein-DNA complexes with either an acetylated H3-K9
histone primary antibody or an NGFI-A primary antibody. The
protein-DNA complexes were uncrosslinked, and the precipitated
genomic DNA was subjected to PCR amplification with primers
specific for the exon 17 GR promoter sequence. There were signifi-
cant Group effects for the association of both histone H3-K9 acety-
lation (t = 2.1, *P < 0.001) and NGFI-A (t = 3.1, **P < 0.0001) with
the exon 17 GR promoter sequence. These results indicated signifi-
cantly greater histone H3-K9 acetylation association and threefold
greater binding of NGFI-A protein to the hippocampal exon 17 GR
promoter in the adult offspring of high- compared with low-LG-
ABN mothers (Fig. 2). Thus, maternal programming of the exon 17
GR promoter involves DNA methylation, histone H3-K9 acetyla-
tion and alterations in NGFI-A binding.
Reversal of maternally mediated epigenetic marking
These findings suggest that maternal care influences hippocampal GR
expression, and thus HPA function in the offspring, through epigenetic
alterations that regulate NGFI-A binding to the exon 17 promoter. A
critical question is whether the impact of early experience is reversible
and whether epigenetic programming is modifiable in adult, post-
mitotic tissues? The generally accepted model is that the DNA methyla-
tion pattern is an irreversible reaction in adult post-mitotic cells.
However, recent data from in vitro experiments suggests that in certain
instances it is possible to induce replication-independent demethylation
of ectopically methylated genes by increasing histone acetylation using
the histone deacetylase (HDAC) inhibitor trichostatin A (TSA)29,30.
Cytosine methylation attracts methylated DNA binding proteins and
HDACs that prevent histone acetylation and thus transcription factor
binding29,30. Activation of chromatin through HDAC inhibition might
trigger DNA demethylation by increasing the accessibility of DNA to
demethylase activity30. We tested the hypothesis that inhibition of
HDACs with TSA would result in increased K9 acetylation of H3-his-
tones associated with the exon 17 GR promoter, DNA demethylation,
NGFI-A binding and reversal of maternal programming of stress
responses in the adult offspring of low-LG-ABN mothers.
We first used ChIP analysis to determine whether histone H3-K9
acetylation and NGFI-A protein binding to the exon 17 GR pro-
moter is altered in the offspring of high- and low-LG-ABN mothers
through intracerebroventricular (i.c.v.) infusion of the adult off-
spring with TSA (100 ng/ml) or vehicle. Statistical analysis revealed
a significant Group Treatment interaction effect for both the his-
tone H3-K9 acetylation (F = 4.93, P < 0.05) and NGFI-A (F = 8.97,
P = 0.01). Post-hoc analysis showed that for both assays, vehicle -
treated offspring of low-LG-ABN mothers showed significantly
(*P < 0.01) less association than any other group. These results indi-
cate greater histone H3-K9 acetylation association and more bind-
8 5 0 VOLUME 7 | NUMBER 8 | AUGUST 2004 NATURE NEUROSCIENCE
Figure 4 TSA effects on cytosine methylation. (a,b) Methylation analysis of
the 17 CpG dinucleotides of the exon 17 GR promoter in hippocampi of
vehicle- and TSA-treated (100 ng/ml) adult offspring of high- and low-LG-
ABN mothers (n = 5 animals/group). (a) Percentage of cytosine residues
that were methylated (mean s.e.m.) for the first 15 CpG dinucleotides
(*P < 0.05). (b) Percentage of methylated cytosines for the 5 (site 16) and
3 (site 17) CpG dinucleotides within the NGFI-A binding region
(*P < 0.001; **P < 0.003).
2
0
0
4
N
a
tu
re
P
u
b
lis
h
in
g
G
ro
u
p
h
tt
p
:/
/w
w
w
.n
a
tu
re
.c
o
m
/n
a
tu
re
n
e
u
ro
sc
ie
n
ce
A R T I C L E S
ing (>3 fold) of NGFI-A protein to the hippocampal exon 17 GR
promoter in the adult offspring of TSA-treated low-LG-ABN moth-
ers compared with the vehicle-treated offspring of low-LG-ABN
mothers (Fig. 3); there were no significant differences between TSA-
treated offspring of low-LG-ABN mothers and either TSA- or vehi-
cle-treated offspring of high-LG-ABN dams. As expected, TSA
treatment did not change histone H3-K9 acetylation or NGFI-A
binding in the adult offspring of high-LG-ABN mothers, because
the GR exon 17 promoter region in the offspring of high-LG-ABN
mothers is normally associated with acetylated histones and highly
bound with NGFI-A.
To determine whether TSA treatment reverses the maternal effect
on methylation within specific CpG dinucleotides on the exon 17
GR promoter, we mapped the differences in methylation using the
sodium bisulfite technique, focusing on the NGFI-A consensus
sequence within the exon 17 region (Fig. 1a). Statistical analysis of
the data across all 17 sites revealed a significant effect of Group
(F = 93.2, P < 0.0001), Treatment (F = 52.8, P < 0.0001) and Region
(F = 30.4, P < 0.0001), as well as a significant Group Treatment
Region interaction (F = 2.1, P = 0.01), Group Treatment interac-
tion (F = 19.9, P < 0.0001), Group Region interaction (F = 4.1,
P < 0.0001) and Treatment Region interaction (F = 2.8,
P < 0.0001). The results again revealed significant differences in the
methylation of a number of regions of the exon 17 GR promoter
sequence (Fig. 4) with significant differences within the 5 CpG (site
16) and 3 CpG (site 17) dinucleotides of the NGFI-A consensus
sequence (Fig. 4b). Statistical analysis of the data from these two
sites revealed a highly significant effect of Group (F = 43.8,
P < 0.0001), Treatment (F = 65.3, P < 0.0001) and Region
(F = 113.3, P < 0.0001), as well as a significant Group Treatment
interaction (F = 16.0, P < 0.0001), Group Region interaction
(F = 37.8, P < 0.0001) and Treatment Region interaction (F = 4.5,
P = 0.04). Post-hoc analysis revealed that TSA treatment signifi-
cantly decreased the degree of cytosine methylation within the 5
(site 16) CpG dinucleotide of the NGFI-A binding region of the
exon 17 GR promoter in the offspring of low-LG-ABN mothers in
comparison to vehicle-treated low-LG-ABN mothers (*P < 0.001).
TSA treatment produced demethylation of the 5 CpG (site 16)
and 3 CpG (site 17) dinucleotides in the offspring of low-LG-ABN
mothers, and hypomethylation of the 3 CpG (site 17) dinucleotide
in the offspring of high-LG-ABN mothers (Fig. 4b). These findings
suggest that TSA treatment can reverse the hypermethylated status of
the exon 17 GR promoter in the offspring of low-LG-ABN mothers.
TSA treatment resulted in a more extensive change in DNA methyla-
tion than maternal care per se, since the 3 CpG (site 17) dinu-
cleotide, which is unaffected by maternal behavior, is partially
demethylated in response to TSA treatment in both cohorts
(Fig. 4b). Also, as in the original study (Fig. 1b), maternal care altered
the methylation status of other CpG dinucleotides in the exon 17
sequence; in the case of sites 1, 2, 5, 12, 14 and 15, these effects were
similarly reversed with central TSA infusion. The significance of
these sites for transcription factor binding is currently unknown and
thus a focus of ongoing studies. Thus, stable DNA methylation
marking by maternal behavior is reversible in the adult offspring
hippocampus by pharmacological modulation of chromatin struc-
ture. While TSA altered the methylation of the both the 5 and 3
CpG sites within the NGFI-A response element, the former appears
to be critical for the effect on NGFI-A binding to the exon 17 pro-
moter. In a previous in vitro study using electrophilic mobility shift
assays (EMSA) with purified recombinant NGFI-A protein31 and
differentially methylated oligonucleotide sequences containing the
NGFI-A response element, we found that methylation of the cyto-
sine within the 5 CpG dinucleotide (site 16) completely eliminated
the binding of NGFI-A, whereas methylation of the cytosine within
the 3 CpG dinucleotide (site 17) only slightly reduced NGFI-A pro-
tein binding (I.C.G.W., M.S. & M.J.M., unpublished data).
Reversal of maternal effect on GR expression
GR gene expression in the hippocampus is increased in the adult
offspring of high- compared with low-LG-ABN mothers7,9. We
suggest that such differences are mediated by the differential
methylation of the 5 CpG dinucleotide (site 16) of the NGFI-A
consensus sequence in the exon 17 GR promoter and the subse-
quent alteration of histone acetylation and NGFI-A binding to the
exon 17 sequence. If the differential epigenetic marking regulates
the expression of the exon 17 GR promoter in high- versus low-LG
offspring, then reversal of the epigenetic marking should be accom-
NATURE NEUROSCIENCE VOLUME 7 | NUMBER 8 | AUGUST 2004 8 5 1
Figure 5 TSA eliminates the maternal effect on hippocampal GR
expression and HPA responses to stress. (a) Top: a representative western
blot showing absolute levels of electrophoresed hippocampal GR
immunoreactivity (IR) from vehicle- and TSA (100 ng/ml)-treated adult
offspring of high- or low-LG-ABN mothers. Molecular weight markers
(SeeBlue, Santa Cruz Biotech) correspond to a single major band at 92
kDa. The middle panel shows the membrane reprobed for -tubulin IR,
illustrating absolute levels of electrophoresed hippocampal protein bound
to the transfer membrane. Molecular weight markers correspond to a single
major band at 60 kDa and the intensity of the signal was similar in all
lanes. The lower panel shows quantitative densitometric analysis (relative
optical density, ROD) of GR IR levels from samples (n = 5 animals/group;
*P < 0.001). (b) Plasma corticosterone responses7 (mean s.e.m.) to a
20-min period of restraint stress (solid bar) in vehicle- and TSA
(100 ng/ml)-treated adult offspring of high- or low-LG-ABN mothers
(n = 10 animals/group; *P < 0.01).
2
0
0
4
N
a
tu
re
P
u
b
lis
h
in
g
G
ro
u
p
h
tt
p
:/
/w
w
w
.n
a
tu
re
.c
o
m
/n
a
tu
re
n
e
u
ro
sc
ie
n
ce
A R T I C L E S
panied by an increase in hippocampal GR expression. This hypoth-
esis is supported by the results (Fig. 5a) showing that hippocampal
GR expression was significantly increased in TSA-treated offspring
of low-LG-ABN mothers to levels that were comparable to those of
either the vehicle- or TSA-treated offspring of high-LG-ABN
mothers. ANOVA revealed highly significant main effects of Group
(F = 7.4, P = 0.01) and Dose (F = 24.8, P < 0.0001), as well as a sig-
nificant Group Dose interaction effect (F = 3.1, P = 0.048). Post-
hoc analysis indicated that 100 ng/ml TSA treatment significantly
increases hippocampal GR expression in the offspring of low-LG-
ABN mothers (vehicle-treated low-LG-ABN vs. 100 ng/ml TSA-
treated low-LG-ABN, *P < 0.001), such that there is no longer a
significant difference in hippocampal GR expression between the
offspring of low- or high-LG-ABN mothers (100 ng/ml TSA treated
low-LG-ABN vs. 100 ng/ml TSA treated h
