The main objectives of these assignments are to: 1) encourage you to read papers and grasp
information from outside the textbook, 2) improve your critical thinking and 3) incrementally
improve your written and verbal communication styles of what you learn.
Each summary should have the following five sections:
1. Introduction: State the essential take home message of the assigned paper and what it
is all about. Do not simply restate the abstract.
2. Key points: Describe the main points in the paper
3. Methods/Experiments: Discuss the experiments or methods used to prove these points
(if applicable)
4. Strengths/Weaknesses: Discuss the strengths and weaknesses of the paper. Indicate
additional points of investigation that you think should be pursued and were not
addressed in the paper
5. Conclusion
Dr. Rola Bekdash
Rutgers University – Newark
Department of Biological Sciences
Epigenetics (21:120:454)
Article Assignments & Guidelines
I.
Article Assignments
We will discuss five articles this semester. These articles address current topics in the field of
Epigenetics and focus on four main themes such as 1) Epigenetics of stress, 2) epigenetics of
neurodegeneration, 3) epigenetics of mental health and 4) epigenetics of cancer. We will
spend time explaining the main physiological concepts and the experimental approaches used
in investigating the role of epigenetic mechanisms in human health and diseases.
Students are expected to read the assigned articles before coming to class and actively
participate in discussions. You will not receive credit for any missed discussion session. You are
also required to submit on Blackboard by the due date a copy of each paper summary. Your
summary should focus on main points of the assigned article and should be 800 – 1000 words.
You will then receive feedback from your instructor on how to improve the structure of your
summary writing. You are required to resend a revised final version of your summary by the
due date to receive credit. Refer to the “Summary Rubric” that I posted on Blackboard under
“Assignments” to understand how you will be assessed and graded.
II. Papers Summaries
You are required to submit on Blackboard in a docx. File a concise summary (first draft) of
selected articles by the due date. No late submission is allowed.
The main objectives of these assignments are to: 1) encourage you to read papers and grasp
information from outside the textbook, 2) improve your critical thinking and 3) incrementally
improve your written and verbal communication styles of what you learn.
Each summary should have the following five sections:
1. Introduction: State the essential take home message of the assigned paper and what it
is all about. Do not simply restate the abstract.
2. Key points: Describe the main points in the paper
3. Methods/Experiments: Discuss the experiments or methods used to prove these points
(if applicable)
4. Strengths/Weaknesses: Discuss the strengths and weaknesses of the paper. Indicate
additional points of investigation that you think should be pursued and were not
addressed in the paper
5. Conclusion
1
Dr. Rola Bekdash
Write 800 – 1000 words, 11 point Ariel Font, double spaced with one inch margin. After you
receive the instructor’s feedback for improvement, you are required to resubmit a revised
version of your summary on blackboard.
III.
Recitations & Paper Summaries
Useful website: https://learn.genetics.utah.edu/content/epigenetics/
Paper Reference
1
2
3
4
5
Felsenfeld, G. A brief history of epigenetics. Cold Spring Harbor perspectives in
Biology. 1, 6:1 (2014).
https://www.ncbi.nlm.nih.gov/pubmed/24384572
Environmental epigenomics and disease susceptibility. Jirtle & Skinner, 2007.
https://www.ncbi.nlm.nih.gov/pubmed/17363974
Pages 1-5, pages 7-8 and conclusion
Histone methylation versus histone acetylation: new insights into epigenetic
regulation. Rice & Allis. Current Opinion in Cell Biology 2001, 13:263–273.
https://www.ncbi.nlm.nih.gov/pubmed/?term=Histone+methylation+versus
+histone+acetylation%3A+new+insights+into+epigenetic+regulation.
Reversal of maternal programming of stress responses in adult offspring through
methyl supplementation: altering epigenetic marking later in life. Weaver et al.,
2005, J. Neuroscience, 25, 11045-11054.
https://www.ncbi.nlm.nih.gov/pubmed/?term=Reversal+of+maternal+programmin
g+of+stress+responses+in+adult+offspring+through+methyl+supplementation%3A
+altering+epigenetic+marking+later+in+life
Epigenetic mechanisms in neurological and neurodegenerative diseases.
Landgrave-Gomez et al., 2015. Frontiers in Cellular Neuroscience, 9, 1-11.
https://www.ncbi.nlm.nih.gov/pubmed/25774124
Subject to changes with prior notice from the Instructor.
2
Epigenetics (21:120:454)
Article Summary – Rubric
Requirement
Introduction
Main points
Experimental approach
Strengths & weaknesses
Conclusion
Excellent Exceeds assignment objectives
Good Meets assignment objectives
20 points
Includes strong thesis statement that
clearly describes the purpose of the
paper.
40 points
Describes the main points of the paper
without including extraneous
information. Doesn’t just repeat what
is stated in the paper but rather
presents these points in an effective
and concise manner.
40 points
Describes in a concise manner but not
in details the main experiments (if
applicable) used. Also, describes the
results of these experiments. Analysis
is comprehensive.
15 points
Thesis statement is clear but not detailed
or descriptive.
10 point
Thesis statement is weak and does not
describe the purpose of the paper.
35 points
Describes the main points of the paper
but includes extraneous information that
are not pivotal.
30 points
Does not describe the main points of the
paper but includes extraneous
information that are not important.
35 points
Describes the main experiments and the
results but does not demonstrate an
understanding of the physiological
significance or the outcomes of these
experiments. Analysis is not
comprehensive or may overlook key
elements.
25 points
Presents some of the strengths and
weaknesses of the paper. Lacks
thoughtful and creative
recommendations for future research.
30 points
Experiments and results are not clearly
described. Lacks a comprehensive
analysis.
15 points
Summary states the concluding thoughts
of the paper.
10 point
Summary lacks key points and insights.
30 points
Presents effectively the main strengths
and weaknesses of the paper and
suggests alternatives to address the
weaknesses. Presents thoughtful and
creative recommendations for future
research.
20 points
Summarizes key points with insights.
1
Poor Assignment objectives minimally met
20 points
Does not identify correctly the main
strengths and weaknesses of the paper. .
Lacks thoughtful and creative
recommendations for future research.
The Journal of Neuroscience, November 23, 2005 • 25(47):11045–11054 • 11045
Development/Plasticity/Repair
Reversal of Maternal Programming of Stress Responses in
Adult Offspring through Methyl Supplementation: Altering
Epigenetic Marking Later in Life
Ian C. G. Weaver,1,2 Frances A. Champagne,1 Shelley E. Brown,3 Sergiy Dymov,3 Shakti Sharma,1 Michael J. Meaney,1,2
and Moshe Szyf2,3
1
3
Douglas Hospital Research Center, Montréal, Québec H4H 1R3, Canada, and 2McGill Program for the Study of Behaviour, Genes, and Environment and
Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec H3G 1Y6, Canada
Stress responses in the adult rat are programmed early in life by maternal care and associated with epigenomic marking of the hippocampal exon 17 glucocorticoid receptor (GR) promoter. To examine whether such epigenetic programming is reversible in adult life, we
centrally infused the adult offspring with the essential amino acid L-methionine, a precursor to S-adenosyl-methionine that serves as the
donor of methyl groups for DNA methylation. Here we report that methionine infusion reverses the effect of maternal behavior on DNA
methylation, nerve growth factor-inducible protein-A binding to the exon 17 promoter, GR expression, and hypothalamic–pituitary–
adrenal and behavioral responses to stress, suggesting a causal relationship among epigenomic state, GR expression, and stress responses
in the adult offspring. These results demonstrate that, despite the inherent stability of the epigenomic marks established early in life
through behavioral programming, they are potentially reversible in the adult brain.
Key words: maternal behavior; rat; hippocampus; glucocorticoid receptor; epigenetics; L-methionine
Introduction
The hypothalamic–pituitary–adrenal (HPA) response to stress is
an inducible defense in mammals that is activated in response to
threat. In primates and rodents, as in nonmammalian species,
there are maternal effects on the magnitude of such defensive
responses in the offspring (Higley et al., 1991; Agrawal, 2001;
Meaney, 2001). In the rat, these effects are mediated by variations
in maternal care such that maternal behavior stably alters the
development of HPA responses to stress in the offspring through
tissue-specific effects on gene expression (Liu et al., 1997; Francis
et al., 1999). The magnitude of the HPA response to stress is a
function of the neural stimulation of hypothalamic
corticotropin-releasing factor (CRF) release, which activates the
pituitary–adrenal system, as well as modulatory influences, such
as glucocorticoid-negative feedback that inhibits CRF synthesis
and release and, thus, dampens HPA responses to stress (De Kloet
et al., 1998). As adults, the offspring of mothers that exhibit increased levels of pup licking/grooming and arched-back nursing
Received Aug. 28, 2005; revised Oct. 12, 2005; accepted Oct. 12, 2005.
This work was supported by a grant from the Canadian Institutes for Health Research (CIHR) (M.J.M., M.S.) and
from the National Cancer Institute of Canada (M.S.). M.J.M. is supported by a CIHR Senior Scientist award, and the
project was supported by a Distinguished Investigator Award (M.J.M.) from the National Alliance for Research on
Schizophrenia and Affective Disorders.
Correspondence should be addressed to either of the following: Dr. Moshe Szyf, Department of Pharmacology
and Therapeutics, McGill University, 3655 Drummond Street, Room 1309, Montréal, Québec, Canada H3G 1Y6,
E-mail: moshe.szyf@mcgill.ca; or Dr. Michael Meaney, Department of Psychiatry, Perry Pavilion, Douglas Hospital
Research Center, 6875 LaSalle Boulevard, Room E-4105.1, Verdun, Montréal, Québec, Canada H4H 1R3, E-mail:
michael.meaney@mcgill.ca.
DOI:10.1523/JNEUROSCI.3652-05.2005
Copyright © 2005 Society for Neuroscience 0270-6474/05/2511045-10$15.00/0
(high LG-ABN mothers) over the first week of life show increased
hippocampal glucocorticoid receptor (GR) expression and enhanced glucocorticoid feedback sensitivity compared with adult
animals reared by low LG-ABN mothers (Liu et al., 1997; Francis
et al., 1999). Predictably, adult offspring of high LG-ABN mothers show decreased hypothalamic CRF expression and more
modest HPA responses to stress (Liu et al., 1997). Eliminating the
difference in hippocampal GR levels abolishes the effects of early
experience on HPA responses to stress in adulthood (Meaney et
al., 1989), 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 stress (Meaney, 2001).
The adult offspring of high LG-ABN mothers are also behaviorally less fearful under conditions of stress than are animals
reared by low LG-ABN dams (Caldji et al., 1998). Cross-fostering
studies show that, on such measures, the biological offspring of
low LG-ABN mothers reared by high LG-ABN dams resemble the
normal offspring of high LG-ABN (and vice versa) (Francis et al.,
1999). These findings suggest that variations in maternal behavior can directly program rudimentary defensive responses to
stress and serve as a mechanism for the nongenomic transmission
of individual differences in stress reactivity across generations
(Fleming et al., 1999; Francis et al., 1999; Meaney, 2001).
Our previous studies suggest that epigenetic mechanisms are
involved in the long-term maternal programming of the offspring’s responses to stress in adulthood (Weaver et al., 2004b).
The exon 17 GR promoter sequence is uniquely expressed in the
brain and exhibits considerable transactivational activity (Mc-
11046 • J. Neurosci., November 23, 2005 • 25(47):11045–11054
Cormick et al., 2000). The adult offspring of low LG-ABN mothers express significantly lower levels of the hippocampal exon 17
mRNA transcript compared with the offspring of high LG-ABN
mothers. In the adult offspring of low LG-ABN mothers, the exon
17 GR promoter is hypermethylated, associated with hypoacetylated histone H3-lysine (K)-9 and reduced binding to nerve
growth factor-inducible protein-A (NGFI-A) (a transcription
factor also known as egr-1, krox-24, zenk, and zif-268), whereas
in the adult offspring of high LG-ABN mothers, the promoter is
hypomethylated, associated with hyperacetylated histone H3-K9
and bound to increased levels of NGFI-A (Weaver et al., 2004b).
Central infusion of the histone deacetylase (HDAC) inhibitor
trichostatin A (TSA) enhanced histone H3-K9 acetylation of the
exon 17 GR promoter in the offspring of the low LG-ABN mothers, increased NGFI-A binding, induced hypomethylation of
CpG dinucleotide sequences in the promoter, and eliminated the
maternal effect on hippocampal GR expression and HPA responses to stress (Weaver et al., 2004b). Although commonly
thought that DNA methylation patterns are plastic during development and fixed in adult postmitotic tissue, these data imply
that demethylation machinery is present in the adult brain and
can gain access to methylated genes by pharmacologically induced alterations in chromatin conformation. If indeed the epigenetic status of the adult genome is truly in a dynamic equilibrium of methylation verses demethylation, it should be possible
to reverse the DNA methylation pattern in the other direction by
inducing hypermethylation.
Dietary methionine is converted by methionine adenosyltransferase into S-adenosyl-methionine (also termed SAM)
(Mudd and Cantoni, 1958; Cantoni, 1975), which serves as the
donor of methyl groups for DNA methylation. The methyldonor SAM has been proposed to cause DNA hypermethylation
by either activating DNA methylation enzymes or inhibiting active demethylation (Detich et al., 2003). Importantly, the synthesis of SAM is dependent on the local availability of methionine
(Cooney, 1993). We reasoned that, if the DNA methylation pattern of the exon 17 GR promoter within the adult offspring remains sensitive to both DNA methylation and DNA demethylation enzymes, then increasing brain methionine levels should
result in DNA hypermethylation and a reversal of the maternal
programming of GR expression and HPA responses to stress. Our
current findings support this hypothesis and suggest that the
methylation status of specific DNA sequences is modifiable even
in postmitotic cells, and, thus, the otherwise stable effects of maternal care on phenotype are potentially reversible.
Materials and Methods
Animals and maternal behavior. The animals used in all studies were
derived from Long–Evans hooded rats born in our colony from animals
originally obtained from Charles River Canada (St. Catharine’s, Québec,
Canada). The animals were mated with males drawn randomly from a
breeding stock maintained in our colony. In cases in which the offspring
of high or low LG-ABN mothers were used in studies, no more than two
animals per group were drawn from any single mother. At the time of
weaning on day 22 of life, pups were housed in same-sex, same-litter
groups of three to four animals per cage until day 45 of life and two
animals per cage from this point until the time of testing, which occurred
no earlier than 100 d of age. All procedures were performed according to
guidelines developed by the Canadian Council on Animal Care and protocol approved by the McGill University Animal Care Committee. Behavioral observations were performed with mothers and their litters
housed in 46 ⫻ 18 ⫻ 30 cm Plexiglas cages that permitted a clear view of
all activity within the cage. Food and water were provided ad libitum. The
colony was maintained on a 12 hr light/dark schedule with lights on at
Weaver et al. • Altering Epigenetic Marking Later in Life
8:00 A.M. Maternal behavior was scored as described previously (Myers
et al., 1989; Champagne et al., 2003). The behavior of each dam was
observed for six 100 min observation periods daily for the first 10 d
postpartum. Observations occurred at regular times each day, with four
periods during the light phase (9:00 A.M., 12:00 P.M., 3:00 P.M., and 6:00
P.M.) and two periods during the dark phase of the light/dark cycle (6:00
A.M. and 9:00 P.M.). Within each observation period, the behavior of
each mother was scored every 4 min (25 observations per period for six
periods per day, for 150 observations per mother per day). All observations were performed by individuals unaware of the origin of the animals.
On occasion, because of disturbances in the animal room, observation
sessions were uncompleted. Although all mothers were observed for exactly the same number of periods, there was some variation across days.
The data were therefore analyzed as the percentage of observations in
which animals engaged in the target behavior. The following behaviors
were scored: mother off pups; mother licking/grooming any pup; and
mother nursing pups in an arched-back posture, a “blanket” posture in
which the mother lays over the pups, or a passive posture in which the
mother is lying either on her back or side while the pups nurse (Myers et
al., 1989). Note that behavioral categories are not mutually exclusive.
For, example, licking/grooming most often occurred while the mother
was nursing the pups. The frequency of maternal licking/grooming and
arched-back nursing across a large number of mothers is normally
and not bimodally distributed (Champagne et al., 2003). Hence, the high
and low LG-ABN mothers represent two ends of a continuum rather
than two distinct populations. To define these populations for the current study, we observed the maternal behavior in a cohort of 32 mothers
and devised the group mean and SD for each behavior over the first 10 d
of life. High LG-ABN mothers were defined as females whose frequency
scores for both licking/grooming and arched-back nursing were ⬎1 SD
above the mean. Low LG-ABN mothers were defined as females whose
frequency scores for both licking/grooming and arched-back nursing
were ⬎1 SD below the mean. Previous reports (Levine, 1994; Meaney,
2001) of licking/grooming behavior suggest that the frequency of licking/
grooming and arched-back nursing are highly correlated (r ⬎ ⫹0.90).
Intracerebroventricular infusions. For intracerebroventricular cannula
placement, 90-d-old (adult) male high and low LG-ABN offspring were
anesthetized, and the area between the ears (across the neck and scapulae) was shaven. The shaven skin was swabbed with iodine, and the animal was mounted and secured in a stereotaxic frame. A cut was made
directly down the midline, and the fascia and muscle were separated to
expose the skull surface, which was cleaned and dried with sterile gauze
and cotton swabs. A stainless-steel guide cannula (22 gauge), 8 mm
length (Plastics One, Roanoke, VA) was aimed at the left lateral ventricle
(1.5 mm posterior to bregma, 2.0 mm lateral to midline, and 3.0 mm
ventral to the brain surface). Guide cannulas were secured with dental
cement and three stainless-steel jeweler’s screws and kept patent with
stainless-steel stylets (30 gauge). The animals were placed under heat
lamps until they regained consciousness, and then they were transferred
to single cages. After a 7 d recovery period, animals received a single
infusion every day for 7 consecutive days as described below. Animals
were removed from their cages and gently held while an infusion cannula
(28 gauge) attached to tubing (polyethylene 20) was lowered into the
guide. A total volume of 2 l of TSA (100 ng/ml in saline), L-methionine
(100 g/ml in saline), or saline vehicle alone was injected (using a Hamilton 10 l microsyringe) through the infusion cannula over a 1 min
period. Infusion cannulas were left in place for an additional 1 min after
infusion. Animals were then returned to their home cage.
Sodium bisulfite mapping. Sodium bisulfite mapping was performed
using the procedure (Frommer et al., 1992) in which sodium bisulfite
solution (3.6 M NaBis/1 mM hydroquinone) was added to the resuspended DNA and incubated (14 h, 55°C). DNA was eluted (QIAquick,
PCR purification kit; Qiagen, Hilden, Germany) in Tris buffer (10 mM),
pH 8.5, and NaOH (3 M) was added to a final concentration of 0.3N
NaOH and incubated (15 min, 37°C). NH4OAc (10 M) was added to a
final concentration of 3 M NH4OA, followed by the addition of tRNA (0.1
mg/ml) and EtOH (2 v/v, 95%), and the bisulfited DNA solution was
cooled (20 min, ⫺20°C). The precipitated solution was then centrifuged
(4°C, 13,200 rpm, 30 min), and the pelleted DNA was lyophilized and
Weaver et al. • Altering Epigenetic Marking Later in Life
resuspended in ddH2O (50 ng/ml). The rat exon 17 GR promoter region
(GenBank accession number AJ271870) of the sodium bisulfite-treated
hippocampal DNA (50 ng/ml) was subjected to PCR amplification using
outside primers (forward, 1646-TTTTTTAGGTTTTTTTAGAGGG1667; reverse, 1930-ATTTCTTTAATTTCTCTTCTCC-1908). The thermocycler protocol involved an initial denaturation cycle (5 min, 95°C),
34 cycles of denaturation (1 min, 95°C), annealing (2 min 30 s, 56°C) and
extension (1 min, 72°C), followed by a final extension cycle (5 min,
72°C), terminating at 4°C. The PCR product (285 bp) was used as a
template for subsequent PCR reactions using nested primers (forward,
1738-TTTTTTTGTTAGTGTGATATATTT-1761; reverse, 1914-TTCT
CCCAAACTCCCTCC-1897). The nested PCR product (177 bp) was
purified (QIAquick, PCR purification kit; Qiagen), and the DNA was
eluted in Tris buffer (10 mM), pH 8.5. The purified product was separated
on an agarose gel (2%). The band corresponding to DNA fragment (177
bp) was cut from the gel, and DNA was extracted (QIAEX, II Agarose Gel
Extraction; Qiagen) and then eluted in Tris buffer (10 mM), pH 8.5. The
PCR product (177 bp) was then subcloned (Original TA cloning kit;
Invitrogen, Carlsbad, CA), transformed into chemically made competent
bacteria, and grown on 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (40 mg/ml)-treated ampicillin (100 mg/ml; Invitrogen, Carlsbad, CA) plates (16 h, 37°C). Ten different clones per plate were mini
prepped and grown (14 h, 37°C) in 2 ml of Lauria broth treated with
ampicillin (100 mg/ml), and the pellet was resuspended in STET (sucrose, Triton X-100, 0.5 M EDTA, and 1 M Tris, pH 8.0). Lysozyme (10
mg/ml; Roche Diagnostics, Mannheim, Germany) dissolved in Tris
buffer (25 mM), pH 8.0, was added to each sample (3 s, 22°C), which was
subsequently boiled (45 s, 100°C). Finally, the recovered DNA was resuspended in 1⫻ Tris (10 mM; pH 8)–EDTA (1 mM) TE/RNase A (10 mg/
ml). Ten plasmids, containing the ligated exon 17 GR promoter DNA
fragment, were sequenced per animal (T7 sequencing kit, United States
Biochemical; Amersham Biosciences), starting from procedure C in the
protocol by the manufacturer. The sequencing reactions were resolved
on a denaturing PAGE (6%) run in 1⫻ Tris– borate–EDTA (3 h, 75 W,
22°C) and visualized by autoradiography.
5-Methylcytosine and neuronal-specific nuclear protein immunohistochemistry. Whole brains were removed from 90-d-old (adult) male offspring by rapid decapitation ⬍1 min after their removal from the home
cage. Coronal sections (16 n) corresponding to stereotaxic levels from
⫺2.30 to ⫺3.80 mm from bregma were thaw mounted (three sections
per slide) onto gelatin-subbed slides and temporarily stored within the
cryostat (⫺20°C). Once sectioning was complete, the slides were vacuum
dried within a desacator (4°C, 14 h) and stored at ⫺80°C. All sections
were processed in parallel. Sections were fixed (10 min, 22°C) in paraformaldehyde (4%), washed with 50% ethanol (30 min), blocked in 10%
FCS plus PBS containing 0.2% Triton X-100 (2 h), and immersed in 2N
HCl (2 h, 37°C). Anti-5-methylcytosine (5-mC) antibody (a kind gift
from Dr. Alain Niveleau, Université Joseph Fourier, Grenoble, France)
diluted 1:500 in 5% serum plus PBS and mouse anti-neuronal-specific
nuclear protein (NeuN) Alexa-Fluor 488-conjugated monoclonal antibody (Chemicon, Temecula, CA) diluted 1:1000 in 5% serum plus PBS
were applied to each section, which were then incubated (1 h, 37°C) and
left to hybridize (40 h, 4°C). Negative control sections for each rat were
produced by omitting the addition of the primary antibodies. Rabbit
anti-mouse IgG Alexa-Fluor 568 (Invitrogen, Carlsbad, CA) diluted
1:600 in 5% serum plus PBS was applied to the 5-mC-labeled sections.
The sections were then dried (2 h) and coverslipped with Aqua Polymount (Polysciences, Warrington, PA). Visualization of 5-mC and
NeuN labeling was performed using a Zeiss (Oberkochen, Germany)
Axioplan 2 Imaging fluorescence microscope, equipped with a highresolution color digital camera and connected to a computer running
Zeiss Axiovision 4.1 Software. The appropriate filter combination and a
63 plan-apochromatic oil-immersion objective was used to capture images of the 5-mC-positive nuclei over the entire area of the dentate gyrus
and CA1, CA2, and CA3 hippocampal regions of Ammon’s horn. These
images were converted to Tiff format and imported into an MCID Elite
image analysis system (Imaging Research, St. Catharines, Ontario, Canada) for quantification. An observer blind to the experimental conditions
performed the analysis.
J. Neurosci., November 23, 2005 • 25(47):11045–11054 • 11047
Chromatin immunoprecipitation assay. In the preparation of fixed tissue, 90-d-old (adult) male high and low LG-ABN offspring were deeply
anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and then transcardially perfused with heparinized saline flush (30 – 60 ml), followed by
paraformaldehyde (4%) in PBS, pH 7.4, for 15 min. After perfusion, all
brains were removed and postfixed in the same fixation solution overnight at 4°C and then transferred to phosphate buffer sucrose (20%) for
48 h. Chromatin immunoprecipitation (ChIP) assays (Crane-Robinson
et al., 1999) were performed following the ChIP assay kit protocol (Upstate Biotechnology, Lake Placid, NY). Hippocampi were dissected from
each rat brain, and chromatin was immunoprecipitated using rabbit
polyclonal anti-acetyl-histone H3 antibody (Upstate Cell Signaling Solutions, Waltham, MA), rabbit polyclonal anti-NGFI-A antibody (Santa
Cruz Biotechnology, Santa Cruz, CA), or normal rabbit IgG nonimmune
antibody (Santa Cruz Biotechnology). One-tenth of the lysate was kept to
quantify the amount of DNA present in different samples before immunoprecipitation (input). Protein–DNA complexes were uncross-linked,
by adding 20 l of NaCl (5 M) to each sample (4 h, 65°C), followed by 10
l of EDTA (0.5 M), 20 l of Tris-HCl (1 M), pH 6.5, and 2 l of Proteinase K (10 mg/ml; 1 h, 45°C). After phenol-chloroform (0.5 v/v) extraction, the free-DNA was ethanol (2 v/v, 95%) precipitated with 5 l of
tRNA (10 mg/ml) and resuspended in 100 l of 1⫻ TE. The rat hippocampal exon 17 GR promoter region (GenBank accession number
AJ271870) of the uncross-linked DNA was subjected to PCR amplification (forward primer, 1750-TGTGACACACTTCGCGCA-1767; reverse
primer, 1943-GGAGGGAAACCGAGTTTC-1926). PCR reactions were
performed after the FailSafe PCR system protocol using FailSafe PCR 2X
PreMix D (EPICENTRE, Madison, WI). The thermocycler protocol involved an initial denaturation cycle (5 min, 95°C), 34 cycles of denaturation (1 min, 95°C), annealing (1 min, 56°C), and extension (1 min,
72°C), followed by a final extension cycle (10 min, 72°C) terminating at
4°C. To control for unequal loading of acetyl-histone H3-K9 immunoprecipitate, the rat hippocampal -actin promoter-␣ region (GenBank
accession number V01217) of the uncross-linked DNA was subjected to
PCR amplification (forward primer, 10-TCAACTCACTTCTCTCTACT-29; reverse primer, 161-GCAAGGCTTTAACGGAAAAT-180).
PCR reactions were performed after the FailSafe PCR system protocol
using FailSafe PCR 2X PreMix L (EPICENTRE) with the same thermocycler protocol as described previously. To control for purity of the
NGFI-A immunoprecipitate, the rat hippocampal exon 1b estrogen receptor (ER)-␣ promoter region (GenBank accession number X98236) of
the uncross-linked DNA was subjected to PCR amplification (forward
primer, 1836-GAAGAAACTCCCCTCAGCAT-1855; reverse primer,
2346-GAAATCAAAACACCGATCCT-2327). PCR reactions were performed following the FailSafe PCR system protocol using FailSafe PCR
2X PreMix A (EPICENTRE). The thermocycler protocol involved an
initial denaturation cycle (5 min, 95°C), 34 cycles of denaturation (1 min,
95°C), annealing (1 min, 60°C), and extension (1 min, 72°C), followed by
a final extension cycle (10 min, 72°C) terminating at 4°C. PCR reactions
on DNA purified from non-immunoprecipitated samples and immunoprecipitated samples were repeated exhaustively using varying amounts
of template to ensure that results were within the linear range of the PCR.
A total of 10 l of the amplified products were separated on an agarose gel
(2%) and post-stained with ethidium bromide to visualize bands corresponding to the exon 17 glucocorticoid receptor promoter (194 bp),
-actin promoter-␣ (171 bp), or exon 1b ER-␣ promoter (493 bp) DNA
fragments. Nucleic acids were transferred by Southern blot (14 h, 22°C)
to positively charged nylon transfer membrane (Hybond-N ⫹; Amersham Biosciences). An oligonucleotide (20 bp) specific for the exon 17
GR promoter sequence (GenBank accession number AJ271870) was synthesized (forward, 1881-TCCCGAGCGGTTCCAAGCCT-1907), as well
as an oligonucleotide (21 bp) specific for the -actin promoter-␣
sequence (GenBank accession number V01217; forward: 95-GTAAA
AAAATGCTGCACTGTC-115) and an oligonucleotide (20 bp) specific
for the exon 1b ER-␣ promoter sequence (GenBank accession number
X98236; forward: 1942-AGAAAGCACTGGACATTTCT-1961). The oligonucleotides were radiolabeled [1 l of T4 polynucleotide kinase
(PNK); Promega, Madison, WI] with 5 l of [␥- 32P]ATP (Amersham
Biosciences) (2 h, 37°C) and then hybridized to the membranes, which
11048 • J. Neurosci., November 23, 2005 • 25(47):11045–11054
were exposed to PhosphorImager screens (Type BAS-III Imaging Plate;
Fujifilm, Tokyo, Japan) overnight at 22°C. The screens were scanned by
PhosphorImager (Storm840; Molecular Dynamics, Sunnyvale, CA) running Storm840 software (Molecular Dynamics). Relative optical density
(ROD) readings were determined using a computer-assisted densitometry program (ImageQuant; Molecular Dynamics). To calculate the final
signal for each sample, the ROD value of the band within the antibody
lane was divided by the ROD value of the band within the input lane. To
control for equal loading between samples, the final signal of the exon 17
GR promoter, amplified from the acetyl-histone H3-K9 immunoprecipitations, was divided by the final signal from the -actin promoter-␣
amplified from the same precipitate.
RT-PCR analysis. Whole brains were removed by rapid decapitation
⬍1 min after the animal’s removal from the home cage. The hippocampal tissue was dissected, snap frozen on dry ice, and stored at ⫺80°C.
Total hippocampal RNA was isolated with the Trizol reagent method
(Invitrogen, Burlington, Ontario, Canada), and the precipitated RNA
was dissolved in RNase-free H2O and quantified (⬃ 1.0 mg/ml) by UV
photospectrometry with absorbance at 260 nm. The overall quality and
yield of the RNA preparation was determined by denaturing agarose
(1%) gel electrophoresis fractionation. In addition, RNA integrity was
confirmed using an Agilent Bioanalyzer (Agilent Technologies, Palo
Alto, CA). cDNA was synthesized in a 20 l reaction volume containing
2 g of total RNA, 40 U of Moloney murine leukemia virus reverse
transcriptase (MBI Fermentas Life Sciences, Burlington, Ontario, Canada), 5 M random primer (Roche Diagnostics), a 1 mM concentration of
each of the four deoxynucleotide triphosphates, and 40 U of RNase inhibitor (Roche Diagnostics). The mRNA was denatured (5 min, 70°C),
the random primers were annealed (10 min, 25°C), and mRNA was
reverse transcribed (1 h, 37°C). The reverse transcriptase was heat inactivated (10 min, 72°C), and the products were stored at ⫺20°C. The rat
hippocampal GR exon 17 region (GenBank accession number AJ271870)
was subjected to PCR amplification (forward primer, 5⬘-TCCCAGGC
CAGTTAATATTTGC-3⬘; reverse primer, 5⬘-TTGAACTCTTGGGG
TTCTCTGG-3⬘). To control for equal loading, the rat hippocampal
-actin exon region (GenBank accession number V01217) was also subjected to PCR amplification (forward primer, 5⬘-GTTGCTAGCCAG
GCTGTGCT-3⬘; reverse primer, 5⬘-CGGATGTCCACGTCACACTT3⬘). The GR exon 17 and -actin amplification were performed in
parallel, using a 25 l reaction mixture containing 1.5 l of synthesized
cDNA product, 2.5 l of 10⫻ PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs,
1 U of Taq polymerase (all from MBI Fermentas Life Sciences), and 0.5
M of each primer. The thermocycler protocol involved an initial denaturation cycle (5 min, 95°C), 20 –30 cycles of denaturation (30 s, 95°C),
annealing (30 s, 60°C), and extension (30 s, 72°C), followed by a final
extension cycle (5 min, 72°C) terminating at 4°C. Samples were removed
every two cycles between 20 and 30 cycles to determine the linear range of
the PCR amplification. Products were separated on an agarose gel (2%)
to visualize bands corresponding to the GR exon 17 (514 bp) or -actin
(470 bp) cDNA fragments. Nucleic acids were transferred by Southern
blot (14 h, 22°C) to positively charged nylon transfer membrane (Hybond-N ⫹; Amersham Biosciences). An oligonucleotide (20 bp) specific
for the GR exon 17 sequence (GenBank accession number AJ271870;
forward, 5⬘-TCCCAGGCCAGTTAATATTTGC-3⬘) was synthesized, as
well as an oligonucleotide (21 bp) specific for the -actin sequence (GenBank accession number V01217; forward, 5⬘-GTTGCTAGCCAGGC
TGTGCT-3⬘). The oligonucleotides were radiolabeled (1 l of T4 PNK;
Promega) with 5 l of [␥- 32P]ATP (Amersham Biosciences) (2 h, 37°C)
and then hybridized to the membranes, which were exposed to PhosphorImager screens (Type BAS-III Imaging Plate; Fujifilm) overnight at
22°C. The screens were scanned by PhosphorImager (Storm840; Molecular Dynamics) running Storm840 software (Molecular Dynamics).
ROD readings were determined using a computer-assisted densitometry
program (ImageQuant; Molecular Dynamics). To control for equal loading between samples, the signal of the GR exon 17 region was divided by
the signal from the -actin region amplified from the same sample.
Western blotting. Whole brains were removed from 90-d-old (adult)
male high and low LG-ABN offspring by rapid decapitation ⬍1 min after
their removal from the home cage. The hippocampi were dissected, snap
Weaver et al. • Altering Epigenetic Marking Later in Life
frozen on dry ice, and stored at ⫺80°C. The hippocampal tissue wholecell extract was prepared using tissue sonicated on ice (20 s pulse at 40°C)
in Tris (20 mM), EDTA (50 mM), and diethyloithiocarbamic acid (0.1
mM; pH 8.0) buffer (100 ml per 50 g of hippocampal tissue) containing
NaCl (0.4 M) and protease inhibitors [3.5 mg of aprotinin, 0.4 mg/ml
4-(2-aminoethyl) benzenesulfonylfluouride, 1 mg/ml leupeptin, and 1
mg/ml pepstatin]. Aliquots of the supernatant were subsequently taken
to determine the levels of protein in the whole hippocampus. Western
blots were performed using equal quantities of protein (40 g) separated
on Novex 4 –12% Tris-glycine PAGE precast gels (Helixx Technologies,
Toronto, Ontario, Canada) with stained molecular markers (SeeBlue;
Invitrogen, Burlington, Ontario, Canada) loaded for reference. Proteins
were then electrophoretically transferred (Towbin et al., 1979) onto nitrocellulose membranes (Amersham Biosciences). The membranes were
blocked (1 h, 22°C) with Carnation dried milk (5%) in Tris-buffered
saline with Tween-20 (TBS-T) [Tris, NaCl, and Tween-20 (0.1%), pH
7.6], washed briefly in TBS-T, and incubated (14 h, 4°C) with anti-rat
GR-␣ monoclonal primary antibodies in blocking buffer (1: 4000; Affinity BioReagents, Golden, CO). Membranes were washed with TBS-T (20
min, 22°C) and then incubated (1 h, 22°C) with horseradish peroxidaseconjugated sheep anti-mouse IgG antibody (1:3000; Amersham Biosciences). After four 15 min washes with TBS-T, the bands were visualized by enhanced chemiluminescence (Amersham Biosciences) and
apposed to autoradiography film (Hyperfilm-MP; Amersham Biosciences) before being developed. A single band was observed at ⬃92
kDa. To verify the accuracy of sample loading, selected blots were incubated (70°C, 30 min) in stripping solution (62.5 mM Tris-HCl, 100 mM
-mercaptoethanol, and 2% SDS) before being blocked (14 h, 4°C) and
reprobed (1 h, 22°C) with an ␣-tubulin monoclonal antibody (1: 5000;
Biodesign International, Kennebunk, ME). A single band was observed at
⬃60 kDa, and the intensity of the signal were similar in all lanes. ROD
readings for the GR-␣ band was determined using a computer-assisted
densitometry program (MCID 4.0; Imaging Research) from samples run
in triplicate on three different blots. For all studies, single blots were
derived from samples from one animal.
HPA response to restraint stress. HPA stress response testing involved
placing 90-d-old (adult) male high and low LG-ABN offspring into Plexiglas restrainers (8.5 ⫻ 21.5 cm; Kent Scientific, Litchfield, CT) for a 20
min period. Prestress blood samples were taken from rats within 30 s of
removal from the cage, and restraint stress was performed during the
light cycle between 12:00 P.M. and 3:00 P.M. with blood sampling (300
l) from the tail vein at 10, 20, 40, 60, and 90 min after the onset of
restraint (Meaney et al., 1989). Plasma (10 l) corticosterone was measured by radioimmunoassay with a highly specific B antiserum (B3-163;
Endocrine Sciences, Tarzana, CA) and [ 3H]corticosterone (101 Ci/
mmol; NEN, Boston, MA) tracer. The antiserum cross-reacts slightly
with deoxycorticosterone (⬃4%) but not with aldosterone, cortisol, and
progesterone (⬍1%). The intraassay and interassay coefficients of variation were 8.8 and 10.4%, respectively. The standard curve 50% effective
concentration was 16 g/dl, and the detection limit of the assay was 0.63
g/dl.
Forced-swim test. Behavioral stress response testing was performed, as
described previously (Porsolt et al., 1977), using 90-d-old (adult) male
high and low LG-ABN offspring. Naive animals were placed in a Plexiglas
cylinder (46 cm high ⫻ 20 cm diameter) filled with water (25 ⫾ 1°C) to
a depth of 30 cm for 15 min. After the 15 min training session, the animals
were removed from the water and allowed to dry (15 min, 32°C) before
being returned to their home cages. After 24 h, the animals were replaced
in the water-filled cylinder, and the total duration of immobility was
recorded during a 5 min test session. Immobile behavior was scored
whenever the animal remained passively afloat without struggling, in a
slightly hunched but upright position with the head slightly immersed
(i.e., the rat makes only those movements necessary to keep its head
above the water). An observer blind to the experimental conditions
scored the behavior. The single 15 min training exposure was sufficient to
produce a relatively constant level of immobility in the subsequent 5 min
test, which was highly reproducible between different groups.
Weaver et al. • Altering Epigenetic Marking Later in Life
J. Neurosci., November 23, 2005 • 25(47):11045–11054 • 11049
significant effect of group (F(1,32) ⫽ 54.2;
p ⬍ 0.0001), treatment (F(1,32) ⫽ 74.3; p ⬍
0.0001), and region (F(1,32) ⫽ 115.3; p ⬍
0.0001), as well as a significant group ⫻
treatment interaction (F(1,32) ⫽ 20.0; p ⬍
0.0001), group ⫻ region interaction
(F(1,32) ⫽ 47.2; p ⬍ 0.000), and treatment ⫻ region interaction (F(1,32) ⫽ 4.5;
p ⫽ 0.04). Post hoc analysis revealed that
the cytosine within the 5⬘ CpG dinucleotide of the NGFI-A consensus sequence is
significantly (*p ⬍ 0.0001) hypermethylated in the adult offspring of low LG-ABN
mothers compared with the offspring of
high LG-ABN dams, and that methionine
treatment significantly (**p ⬍ 0.001) increased cytosine methylation within the 5⬘
CpG dinucleotide in the offspring of high
LG-ABN mothers compared with vehicletreated high LG-ABN mothers. Thus, methionine treatment produced “remethylation” of the 5⬘ CpG (site 16) dinucleotide
in the offspring of high LG-ABN mothers
(Fig. 1c). These findings suggest that meFigure 1. Methionine alters cytosine methylation of GR promoter. a, Sequence map of the exon 17 GR promoter including the
thionine treatment can reverse the hy17 CpG dinucleotides (bold) and the NGFI-A consensus sequence (McCormick et al., 2000) (encircled). b, c, Methylation analysis of
the 17 CpG dinucleotides of the exon 17 GR promoter region from vehicle- and methionine-treated (100 g/ml) adult high and pomethylated status of the exon 17 GR
low LG-ABN offspring (6 –10 clones sequenced per animal; n ⫽ 4 animals per group; *p ⬍ 0.01). b, Percentage of methylated promoter in the adult offspring of high
cytosine residues (mean ⫾ SEM) for the first 15 CpG dinucleotides (*p ⬍ 0.05). c, Percentage of methylated cytosine residues LG-ABN mothers.
Maternal care altered the methylation
(mean ⫾ SEM) for the 5⬘ (site 16) and 3⬘ (site 17) CpG dinucleotides within the NGFI-A consensus sequence (*p ⬍ 0.0001; **p ⬍
0.001). di– div, Confocal photomicrographs of representative 5-mC-positive neurons located within the CA1 hippocampal region status of other CpG dinucleotides in the
of Ammon’s horn from vehicle- and methionine-treated (100 g/ml) adult high and low LG-ABN offspring (n ⫽ 6 animals per exon 17 sequence; in the case of sites 1, 2, 5,
group with 9 sections per animal). Only large round nuclei corresponding to neuronal nuclei (indicated by arrows pointing 6, 7, 8, 9, 10, 12, 13, 14, and 15, the methupward) were included for analysis, and partial or smaller nuclei (indicated by arrows pointing downward) were not included in ylation was similarly reversed with central
the quantification. Scale bar, 50 m.
methionine infusion (Fig. 1b). The significance of these sites for transcription factor
binding is currently unknown and a focus of ongoing studies.
Results
The effects of methionine on the methylation pattern of the
Thus, stable DNA methylation marked by maternal behavior is
exon 17 promoter
reversible in the adult offspring hippocampus by increases in
To determine the effects of methionine treatment, adult offspring
methionine. Methionine altered the methylation of the 5⬘ CpG
of high and low LG-ABN mothers were infused with methionine
(site 16) dinucleotide within the NGFI-A consensus sequence
(100 g/ml) or saline vehicle once a day for 7 consecutive days,
that is critical for the effect on NGFI-A binding to the exon 17
promoter. In a previous in vitro study using electrophilic mobility
and differences in methylation were mapped using the sodium
shift assays with purified recombinant NGFI-A protein (Milbisulfite technique (Comb and Goodman, 1990; Clark et al.,
brandt, 1987) and differentially methylated oligonucleotide se1994), with particular interest in the NGFI-A consensus sequence
quences containing the NGFI-A consensus sequence, we found
(Fig. 1a). Statistical analysis of the data across all 17 CpG sites
that methylation of the cytosine within the 5⬘ CpG (site 16) dinu(Fig. 1b,c) revealed a significant effect of group (F(1,272) ⫽ 93.2;
p ⬍ 0.0001), treatment (F(1,272) ⫽ 52.8; p ⬍ 0.0001), and region
cleotide completely eliminated the binding of NGFI-A. However,
(F(16,272) ⫽ 30.4; p ⬍ 0.0001), as well as a significant group ⫻
methylation of the cytosine within the 3⬘ CpG (site 17) dinucletreatment ⫻ region interaction (F(16,272) ⫽ 2.1; p ⫽ 0.0), group ⫻
otide only slightly reduced NGFI-A protein binding (Weaver et
treatment interaction (F(1,272) ⫽ 19.9; p ⬍ 0.0001), group ⫻ real., 2004a).
gion interaction (F(16,272) ⫽ 4.1; p ⬍ 0.0001), and treatment ⫻
An important question here is whether the effects of methioregion interaction (F(16,272) ⫽ 2.8; p ⬍ 0.0001). The results renine are limited to a subset of genes such as GR or whether they
vealed significant differences in the methylation of a number of
disrupted the DNA methylation patterns across the entire geregions of the exon 17 GR promoter sequence (Fig. 1b) with signome. Surprisingly, results from Affymetrix (Santa Clara, CA)
nificant differences within the 5⬘ CpG (site 16) and 3⬘ CpG (site
microarray analysis performed on hippocampal tissue from a
17) dinucleotides of the NGFI-A consensus sequence (Fig. 1c). As
separate cohort of methionine-treated high and low LG-ABN
reported previously (Weaver et al., 2004b), the cytosine within
offspring showed that the methionine treatment significantly efthe 5⬘ CpG (site 16) dinucleotide is heavily methylated in the
fected only ⬃300 genes, representing ⬍1% of the population of
offspring of low LG-ABN mothers and rarely in the offspring of
genes on the chip (I. C. G. Weaver, M. J. Meaney, and M. Szyf,
high LG-ABN mothers. In contrast, the cytosine within the 3⬘
unpublished observation). These findings suggest an impressive
CpG (site 17) dinucleotide of the NGFI-A consensus sequence is
level of specificity. Although we certainly cannot exclude the posalmost always methylated, independent of maternal care. Statissibility that some component(s) of the modified genes are reletical analysis of the data from these two sites revealed a highly
vant for the effects observed here, it would appear that these
11050 • J. Neurosci., November 23, 2005 • 25(47):11045–11054
results do not emerge as a function of a widespread alteration in
hippocampal gene expression. To further examine whether maternal care or methionine treatment effected global DNA methylation levels, dorsal hippocampal coronal sections from the
methionine- or vehicle-treated adult offspring were coimmunostained using antibodies specific for 5-mC or NeuN to assess
genomic methylation levels. The tissue sections were analyzed
thoroughly over the entire area of the dentate gyrus and CA1,
CA2, and CA3 hippocampal regions of Ammon’s horn. However,
the staining intensity within the different regions remained the
same, regardless of maternal care or methionine treatment. A
representative image of 5-mC staining within the CA1 region
from each treatment group is shown in Figure 1di– div. Neuronal
nuclei were only considered positive for 5-mC if they were round
and intensely fluorescent in strong contrast to the surrounding
tissue. 5-mC labeling intensity and background were consistent
among slides. ANOVA revealed no significant effect of group
(vehicle-treated high LG-ABN vs vehicle-treated low LG-ABN,
F(1,16) ⫽ 0.53, p ⬎ 0.05) or treatment (vehicle-treated low LGABN vs methionine-treated low LG-ABN, F(1,16) ⫽ 0.42, p ⬎
0.05; vehicle-treated high LG-ABN vs methionine-treated high
LG-ABN, F(1,16) ⫽ 0.37, p ⬎ 0.05). These results show that neither maternal care nor methionine treatment affected global
DNA methylation levels. Our findings suggest that alterations of
cytosine methylation in the adult brain through global procedures are surprisingly specific. Methionine alone does not methylate DNA but is converted to the methyl donor SAM in the DNA
methylation reaction. An increase in SAM would change the
DNA methylation pattern of a gene only if the DNA machinery
was present on the gene. Because the DNA is not replicating, it is
not expected that maintenance DNA methyltransferases
(DNMTs), which are normally present in the replication fork,
would be ubiquitously present. It is becoming clear now that
chromatin and DNA methylation enzymes are targeted to specific genes in a regulated process. Thus, the specificity of the effect
of methionine is likely determined by the occupancy of distinct
promoters by DNMTs. It is tempting to speculate that genes involved in crucial regulatory functions, such as GR, are persistently associated with the DNA methylation machinery and are
thus hypersensitive to global changes in methionine levels. Future experiments will elucidate the mechanisms, which target the
DNA methylation/demethylation machinery to specific genes
such as GR in the hippocampus.
The effects of methionine on binding of NGFI-A to the exon
17 promoter
Differences in methylation of the exon 17 GR promoter are tightly
associated with effects on histone acetylation and NGFI-A binding (Weaver et al., 2004b). We tested the hypothesis that the effect
of methionine on DNA methylation results in (1) decreased histone acetylation at the K9 residue of the H3 histone(s) associated
with the exon 17 GR promoter, (2) decreased interaction between
NGFI-A and the promoter sequence, or (3) changes in both histone acetylation and NGFI-A association with the GR promoter.
We performed a 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 vehicle or methionine (100 g/ml) treated offspring of high and
low LG-ABN mothers were cross-linked in vivo with paraformaldehyde perfusion. We then selectively immunoprecipitated protein–DNA complexes with an antibody against either acetylated
histone H3-K9 or NGFI-A. The protein–DNA complexes were
uncross-linked, and the precipitated genomic DNA was sub-
Weaver et al. • Altering Epigenetic Marking Later in Life
Figure 2. Methionine eliminates maternal effect on NGFI-A binding independently of histone acetylation. 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 methionine-treated (100 g/ml) adult offspring of high and low LGABN mothers (n ⫽ 4 animals per 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 immunoprecipitated (N) hippocampal extracts. a, Representative Southern blot of the amplified exon 17 region from acetyl-histone H3-K9 immunoprecipitated hippocampal tissue (194 bp band). DNA loading was controlled using primers
specific for the ubiquitously expressed -actin promoter-␣ region (171 bp band). b, Representative Southern blot of the amplified exon 17 region of the GR from NGFI-A immunoprecipitated
hippocampal tissue (194 bp band). Exon 1b ER-␣ 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, ROD (mean ⫾
SEM) of exon 17 sequence amplified from acetyl-histone H3-K9 or NGFI-A immunoprecipitated
hippocampal tissue from vehicle- and methionine-treated (100 g/ml) adult high and low
LG-ABN offspring (n ⫽ 4 animals per group; *p ⬍ 0.01; **p ⬍ 0.001).
jected to PCR amplification with primers specific for the exon 17
GR promoter sequence (Fig. 2a– c). ANOVA revealed a significant group effect (F(1,16) ⫽ 10.0; p ⫽ 0.01) but no significant
group ⫻ treatment interaction effect for histone H3-K9 acetylation (F(1,16) ⫽ 0.8; p ⬎ 0.0). However, there was a significant
group ⫻ treatment interaction effect for NGFI-A (F(1,16) ⫽ 7.9;
p ⫽ 0.01). Post hoc analysis showed that vehicle- and methioninetreated offspring of high LG-ABN mothers showed significantly
(*p ⬍ 0.01) greater H3-K9 association with the exon 17 sequence
than any other group (Fig. 2a,c), suggesting no effect of methionine treatment on H3-K9 acetylation. In contrast, vehicle-treated
offspring of high LG-ABN mothers showed significantly (**p ⬍
0.001) greater NGFI-A association with the exon 17 sequence
than any other group (Fig. 2b,c). These results indicate decreased
binding of NGFI-A protein to the hippocampal exon 17 GR promoter in the adult offspring of methionine-treated high LG-ABN
Weaver et al. • Altering Epigenetic Marking Later in Life
mothers compared with the vehicle-treated offspring of high LGABN mothers (Fig. 2b,c); there were no significant differences
between methionine-treated offspring of high LG-ABN mothers
and either vehicle- or methionine-treated offspring of low LGABN dams. The absence of any methionine effect on the offspring
of low LG-ABN mothers was expected because, in these animals,
the exon 17 GR promoter region shows little NGFI-A binding. In
summary, methionine treatment decreased binding of NGFI-A
to the hippocampal exon 17 GR promoter but did not affect levels
of histone acetylation. DNA methylation is known to silence gene
expression through two different mechanisms. The first is sitespecific DNA methylation, such as methylation of the 5⬘ CpG
dinucleotide within the NGFI-A binding site, which directly
blocks binding of the transcription factor to its cognate sequence
and thus inhibits transcription without necessarily altering chromatin structure. The second mechanism is indirect. Methylated
DNA binding proteins, such as methyl-CpG binding protein-2
(MeCP-2), are recruited to a region of dense methylation and
target HDACs to the chromatin, resulting in histone deacetylation and subsequent chromatin inactivation (Jones et al., 1998;
Nan et al., 1998). Although we cannot preclude the involvement
of other acetylation sites within histone H3 or the involvement of
acetylation sites within other histones (e.g., histone H4), our data
are consistent with the hypothesis that the methionine-induced
remethylation of the NGFI-A binding site inhibits transcription
by the direct mechanism through blocking NGFI-A binding
without altering histone acetylation. This mechanism is also consistent with previous data that revealed an absence of MeCP-2
binding within the hypermethylated GR exon 17 promoter region
of the adult low LG-ABN offspring (I. C. G. Weaver, M. J.
Meaney, and M. Szyf, unpublished observation). Thus, methylation in this case does not result in recruitment of methylated
DNA binding proteins and histone deacetylation. We showed
previously that, although site-specific methylation is sufficient to
block NGFI-A binding to the GR exon 17 promoter later in adulthood (as shown here), the GR promoter is both methylated and
associated with deacetylated histones at postnatal day 1 (Weaver
et al., 2004b). We propose that induction of histone acetylation
between postnatal days 1 and 6 enables demethylation of the 5⬘
CpG dinucleotide and binding of NGFI-A, resulting in transcription activation. Thus, there is a unidirectional causal relationship
between the state of histone acetylation and the state of methylation of GR exon 17 promoter, whereby the state of methylation is
determined by the state of histone acetylation and either developmental or pharmacological change in histone acetylation results in demethylation, but methylation does not necessarily alter
histone acetylation. Thus, the increased methylation of the 5⬘
CpG site on the exon 17 promoter with methionine treatment
may be sufficient to inhibit NGFI-A binding, even in the absence of
changes in histone acetylation, through site-specific exclusion of
transcription factor association.
Effects of methionine on GR expression
The effect of maternal care on HPA responses to stress is associated with differences in hippocampal GR gene expression and
glucocorticoid feedback sensitivity (Meaney, 2001). GR gene expression in the hippocampus is increased in the adult offspring of
high compared with low LG-ABN mothers (Francis et al., 1999).
This difference is 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 subsequent alteration of
histone acetylation and NGFI-A binding to the exon 17 sequence
(Weaver et al., 2004b). If the differential epigenetic marking reg-
J. Neurosci., November 23, 2005 • 25(47):11045–11054 • 11051
ulates the expression of the exon 17 GR promoter in high versus
low LG-ABN offspring, then reversal of the epigenetic marking
should accompany a decrease in hippocampal GR expression. To
examine whether maternal LG-ABN behavior or methionine
treatment affected hippocampal exon 17 GR mRNA levels, RTPCR analysis was performed using purified hippocampal mRNA
from the vehicle- or methionine-treated adult offspring of high
and low LG-ABN mothers (Fig. 3a). As a positive control, hippocampal mRNA from TSA-treated adult offspring of low LGABN mothers was also analyzed because this manipulation has
been shown previously to increase GR protein expression.
ANOVA revealed a significant effect group (F(1,16) ⫽ 9.0; p ⫽
0.01) and treatment (F(1,16) ⫽ 8.0; p ⫽ 0.01) and significant
group ⫻ treatment interaction (F(1,16) ⫽ 11.0; p ⬎ 0.05). Post hoc
analysis showed that TSA-treated offspring of low LG-ABN
mothers and vehicle-treated offspring of high LG-ABN dams
showed significantly (*p ⬍ 0.001 and **p ⬍ 0.002, respectively)
greater exon 17 GR mRNA levels than any other group (Fig. 3b),
suggesting that methionine treatment significantly reduces hippocampal exon 17 GR mRNA levels in the offspring of high LGABN dams. This is further supported by the results (Fig. 3c,d)
showing that hippocampal GR protein expression was also significantly decreased in methionine-treated offspring of high LGABN mothers to levels that were comparable with those of either
the vehicle- or methionine-treated offspring of low LG-ABN
mothers. ANOVA revealed highly significant main effects of
group (F(1,16) ⫽ 8.2; p ⫽ 0.01) and treatment (F(1,16) ⫽ 16.9; p ⬍
0.0001), as well as a significant group ⫻ treatment interaction
effect (F(1,16) ⫽ 4.2; p ⫽ 0.05). Post hoc analysis indicated that
methionine treatment significantly decreased hippocampal GR
expression in the offspring of high LG-ABN mothers (vehicletreated high LG-ABN offspring vs methionine-treated high LGABN offspring, *p ⬍ 0.001), eliminating the difference in hippocampal GR expression between the offspring of low or high
LG-ABN mothers (methionine-treated high LG-ABN offspring
vs methionine-treated low LG-ABN offspring, p ⬎ 0.90). Although methionine treatment significantly reduced GR expression in high LG-ABN adult offspring, global abundance of protein in the hippocampus was not apparently decreased, as indicated
by the equal ␣-tubulin immunoreactivity (Fig. 3c).
Effects of methionine on HPA responses to stress
As adults, the offspring of high LG-ABN mothers show increased
hippocampal GR expression, enhanced glucocorticoid feedback
sensitivity, and more modest HPA responses to stress than the
offspring of low LG-ABN mothers (Liu et al., 1997). Given that
methionine treatment reversed the group difference in hippocampal GR expression, we examined the adrenocortical responses to stress in a separate cohort of vehicle- and methioninetreated animals (Fig. 3e). Statistical analysis of the plasma
corticosterone data revealed significant effects of group (F(1,34) ⫽
4.3; p ⫽ 0.05), treatment (F(1,34) ⫽ 5.2; p ⫽ 0.05), and time (F(1,34)
⫽ 22.4; p ⬍ 0.0001), as well as a significant group ⫻ treatment
interaction effect (F(1,34) ⫽ 7.8; p ⫽ 0.001). Post hoc analysis
revealed that plasma corticosterone responses to restraint stress
in the vehicle-treated adult offspring of high LG-ABN mothers
were significantly (*p ⬍ 0.01) lower than those of methionineand vehicle-treated adult offspring of low LG-ABN mothers or
methionine-treated offspring of high LG-ABN mothers. The
HPA response to stress in the offspring of low LG-ABN mothers
was unaffected by methionine treatment.
Weaver et al. • Altering Epigenetic Marking Later in Life
11052 • J. Neurosci., November 23, 2005 • 25(47):11045–11054
The effects of GR on behavioral
responses to stress
Rodents forced to swim in a restricted
space from which they cannot escape
eventually cease apparent attempts to escape and become immobile (Porsolt et al.,
1977). Such learned-helplessness-induced
immobility is selectively sensitive to antidepressant treatments (Porsolt et al.,
1978). Although adrenalectomized rats acquire the immobile response normally,
they are unable to retain the response in
subsequent testing, and the effects are reversed by glucocorticoid administration
(Funder, 1989). Because maternal behavior affects the level of circulating glucocorticoids and hippocampal GR expression in
the offspring, we used the forced-swim test
as a model to assess the effects of methionine treatment on behavioral responses to
stress in the adult offspring of high and low
LG-ABN mothers (Fig. 4). Statistical analysis of the length of time the animals spent
immobile revealed significant effects of
group (F(1,34) ⫽ 4.3; p ⫽ 0.048) and treatment (F(1,34) ⫽ 4.3; p ⫽ 0.046), as well as a
significant group ⫻ treatment interaction
(F(1,34) ⫽ 7.7; p ⫽ 0.009). Post hoc analysis
revealed that methionine treatment significantly increased the length of time the offspring of high LG-ABN mothers spent immobile in the forced-swim test,
comparable with the low LG-ABN animals. Thus, the length of time the vehicletreated adult offspring of high LG-ABN
mothers spent immobile was significantly
( p ⬍ 0.01) shorter compared with the
methionine- and vehicle-treated adult offspring of low LG-ABN mothers or
methionine-treated offspring of high LGABN mothers. Forced-swim test performance by the low LG-ABN offspring was
unaffected by methionine treatment. In
summary, methionine treatment reversed
endocrine and behavioral responses to
stress in the adult offspring of high LGABN mothers as well as epigenetic programming of the exon 17 GR promoter.
Discussion
Figure 3. Methionine eliminates the maternal effect on hippocampal GR expression and HPA responses to stress. a, Representative RT-PCR illustrating absolute levels of hippocampal GR exon 17 mRNA transcript levels from vehicle-treated (Veh), TSAtreated (100 ng/ml), and methionine-treated (Met) (100 g/ml) adult offspring of high and low LG-ABN mothers (n ⫽ 5 animals
per group). Molecular weight markers (MBI Fermentas Life Sciences) correspond to a single major band at 514 bp. The bottom
panel shows the RT-PCR for -actin, illustrating absolute hippocampal mRNA transcript levels. Molecular weight markers correspond to a single major band at 470 bp, and the intensity of the signal was similar in all lanes. b, ROD (mean ⫾ SEM) of
hippocampal GR exon 17 mRNA transcript levels from vehicle-, TSA (100 ng/ml)-, and methionine (100 g/ml)-treated adult
offspring of high and low LG-ABN mothers (n ⫽ 5 animals per group; *p ⬍ 0.002; **p ⬍ 0.001). c, Representative Western blot
illustrating absolute levels of electrophoresed hippocampal GR immunoreactivity from vehicle- and methionine-treated (100
g/ml) adult offspring of high and low LG-ABN mothers (n ⫽ 5 animals per group). Molecular weight markers (SeeBlue; Santa
Cruz Biotechnology) correspond to a single major band at 92 kDa. The bottom panel shows the membrane reprobed for ␣-tubulin
immunoreactivity, 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. d, ROD
(mean ⫾ SEM) of hippocampal GR immunoreactivity levels from vehicle- and methionine-treated (100 g/ml) adult offspring of
high and low LG-ABN mothers (n ⫽ 5 animals per group; *p ⬍ 0.001). e, Mean ⫾ SEM plasma corticosterone responses to a 20
min period of restraint stress (solid bar) in vehicle- and methionine-treated (100 g/ml) adult offspring of high and low LG-ABN
mothers (n ⫽ 10 animals per group; *p ⬍ 0.01).
These studies reveal that methionine, a
well established dietary modulator of macromolecule methylation, reverses both the
DNA methylation pattern of GR promoter
and stress responses, thus demonstrating a
causal relationship between the programming of responses to stress by maternal behavior and the epigenomic status of the
adult offspring. The current data demonstrate that, although
epigenomic marks involving stable covalent modifications of the
DNA triggered by maternal behavior are stable through life, these
marks are potentially reversible even in terminally differentiated
tissues such as the hippocampus. Methylation patterns are altered
by inhibition of DNMTs or during a change in the supply of
methyl donors in cell division during synthesis of the unmethylated nascent strand. However, it is generally believed that methylation patterns remain stable in postmitotic tissues. Our findings
provide novel support for a model that suggests a more dynamic
Weaver et al. • Altering Epigenetic Marking Later in Life
Figure 4. Methionine eliminates the maternal effect on behavioral responses to stress.
Mean ⫾ SEM time spent immobile during a 5 min period of forced-swim stress in vehicle- and
methionine-treated (100 g/ml) adult offspring of high and low LG-ABN mothers (n ⫽ 10
animals per group; *p ⬍ 0.01).
process in which methylation status is modifiable even in postmitotic cells. These studies, together with our previous paper
(Weaver et al., 2004b) showing demethylation after chronic histone deacetylase blockade, demonstrate that the enzymatic machinery necessary for both demethylation and remethylation are
operative in differentiated cells such as neurons.
Our previous studies (Weaver et al., 2004b) demonstrate that
maternal behavior alters the histone conformation and methylation status of the 5⬘ CpG (site 16) dinucleotide within the
NGFI-A consensus sequence and the binding of NGFI-A to its
cognate sequence in vivo. These data provide an epigenetic mechanism for long-term programming of stress responsivity by experience early in life. Moreover, pharmacological inhibition of
histone deacetylation by TSA results in demethylation of the exon
17 GR promoter in the adult hippocampus and reversal of the
effects of low maternal LG-ABN on stress responsivity (Weaver et
al., 2004b). The data presented here show that methionine infusion causes remethylation of the exon 17 GR promoter and reversal of high maternal LG-ABN on stress responsivity, by a mechanism independent of histone H3-K9 acetylation. Considering
that both TSA and methionine alter DNA methylation indirectly
by challenging the accessibility and activity of DNMTs and demethylases, our findings demonstrate that the DNA methylation
and demethylation machinery is present in the adult hippocampus and suggest that the balance of these activities plays a pivotal
role in the stability of the DNA methylation pattern.
Although the experiments presented here involved infusion of
methionine into the left lateral ventricles, they raise the possibility that diet can affect the phenotypes being studied. Because
intracellular levels of methionine seem to be affected by both
dietary intake and polymorphisms of enzymes involved in methionine metabolism, such as methylenetetrahydrofolate-reductase
(Friso et al., 2002), it is tempting to consider, together with our
data, the possibility that diet could modify epigenetic programming in the brain not only during early development but also in
adult life. Human epidemiological and animal model data indicate that susceptibility to adult-onset chronic disease is influenced by persistent adaptations to prenatal and early postnatal
nutrition (Lucas, 1998). Dietary L-methionine is crucial for normal brain development, brain aging, and the pathogenesis of
neurodegenerative disorders, playing an essential role in gene
expression, protein synthesis, cell signaling, lipid transport/metabolism, and neuron survival (Slyshenkov et al., 2002; Van den
J. Neurosci., November 23, 2005 • 25(47):11045–11054 • 11053
Veyver, 2002). DNA methyltransferase requires SAM to establish
or maintain 5-mC patterns. Synthesis of SAM is dependent on
the availability of dietary folates, vitamin B12, methionine, choline, and betaine (Cooney, 1993). Maternal methyl supplements
affect epigenetic variation and DNA methylation and positively
affect health and longevity of the offspring (Wolff et al., 1998;
Cooney et al., 2002; Waterland and Jirtle, 2003; Wylie et al.,
2003). This could also have important therapeutic implications,
because aberrant DNA methylation is involved in neurological
disease, such as fragile X syndrome, and is potentially associated
with multiple psychiatric and behavioral conditions, including
schizophrenia (Grayson et al., 2005). We hypothesize that reversal of epigenetic states in the brain, such as the remethylation of
the exon 17 GR promoter illustrated here, could be triggered not
only by pharmacological agents but also by stable variations in
environmental conditions.
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