early stress evokes dysregulation of histone modifiers in

Early Stress EvokesDysregulation of HistoneModifiers in the MedialPrefrontal Cortex Across theLife Span

ABSTRACT: Early stress has been hypothesized to recruit epigenetic mecha-nisms to mediate persistent molecular, cellular, and behavioral changes. Here,we have examined the consequence of the early life stress of maternal separation(ES) on the gene expression of several histone modifiers that regulate histoneacetylation and methylation within the medial prefrontal cortex (mPFC), a keylimbic brain region that regulates stress responses and mood-related behavior.ES animals exhibit gene regulation of both writer (histone acetyltransferasesand histone methyltransferases) and eraser (histone deacetylases and histonelysine demethylases) classes of histone modifiers. While specific histone modifiers(Kat2a, Smyd3, and Suv420h1) and the sirtuin, Sirt4 were downregulated acrosslife within the mPFC of ES animals, namely at postnatal Day 21, 2 months, and15 months of age, we also observed gene regulation restricted to these specifictime points. Despite the decline noted in expression of several histone modifierswithin the mPFC following ES, this was not accompanied by any change inglobal or residue-specific H3 acetylation and methylation. Our findings indicatethat ES results in the regulation of several histone modifiers within the mPFCacross life, and suggest that such perturbations may contribute to the alteredprefrontal structural and functional plasticity observed following early adversity.� 2015 Wiley Periodicals, Inc. Dev Psychobiol

Keywords: maternal separation; histone deacetylase; histone acetyltransferase;histone methyltransferase; histone demethylase; sirtuin

INTRODUCTION

Early adversity is a risk factor for the development of

adult psychopathology (Felitti et al., 1998; Nemeroff,

2004), with clinical evidence highlighting the role of

adverse early history in the predisposition to anxiety,

depression, bipolar disorder, and schizophrenia (Larsson

et al., 2013; Nanni, Uher, & Danese, 2012; Rosenberg,

Lu, Mueser, Jankowski, & Cournos, 2007). Preclinical

studies demonstrate that diverse early stressors, includ-

ing maternal separation (ES), evoke lasting increases in

anxiety and depressive behavior, and a disruption of

stress responses (Daniels, Pieterson, Carstens, & Stein,

2004; Levine, 1967; Meaney, 2001; Rinc�on-Cort�es &

Sullivan, 2014; Weiss, Franklin, Wizi, & Mansuy,

Manuscript Received: 1 April 2015Manuscript Accepted: 11 September 2015Conflicts of interest: The authors have no conflicts of interest to

declare.Correspondence to: Vidita A. Vaidya, Department of Biological

Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai400005, Maharashtra, India

Contract grant sponsor: TIFR intramural grant (VAV)Contract grant sponsor: Department of Biotechnology, Centre of

Excellence in Epigenetics, IISER (VAV)Contract grant number: BT/01/COE/09/07Article first published online in Wiley Online Library

(wileyonlinelibrary.com).DOI 10.1002/dev.21365 � � 2015 Wiley Periodicals, Inc.

Developmental Psychobiology

Madhavi Pusalkar1

Deepika Suri1

Ashwin Kelkar2

Amrita Bhattacharya1

Sanjeev Galande2

Vidita A. Vaidya1

1Department of Biological SciencesTata Institute of Fundamental Research

Mumbai, Maharashtra, IndiaE-mail: [email protected]

2Centre of Excellence in EpigeneticsIndian Institute of Science Education and

ResearchPune, Maharashtra, India

2011). These behavioral changes observed are accom-

panied by age-dependent structural and functional

changes within multiple limbic brain regions (Chocyk

et al., 2013; Lippmann, Bress, Nemeroff, Plotsky, &

Monteggia, 2007; Muhammad, Carrol, & Kolb, 2012;

Suri et al., 2013), including the medial prefrontal cortex

(mPFC), which plays a key role in top-down control

of anxiety and depressive behavior (Kumar et al.,

2013). Though the mechanistic underpinnings of

these changes are currently unclear, epigenetic mech-

anisms that drive a lasting perturbation of gene

expression have been hypothesized to alter the

function of specific neurocircuits, such as the mPFC,

following early adversity (McClelland, Korosi, Cope,

Ivy, & Baram, 2011).

Epigenetic machinery is thought to be recruited by

environmental cues to influence transcriptional changes

through histone modifications, DNA methylation, or

through microRNA-mediated regulation (Sweatt, 2013).

Histone-modifying enzymes include histone acetyl-

transferases (HATs), histone deacetylases (HDACs),

histone methyltransferases (HMTs), and histone deme-

thylases (HDMs), which catalyze multiple modifica-

tions at specific lysine or arginine residues of histone

tails (Bagot, Labont�e, Pe~na, & Nestler, 2014; Khare

et al., 2011). However, it remains poorly understood

whether epigenetic enzymatic machinery, including

histone modifying enzymes, serves as targets for

regulation by early stress experience.

Clinical reports indicate that mood disorder patients

exhibit disrupted expression of specific HDACs within

peripheral blood mononuclear cells (Hobara et al.,

2010). Studies on rodent models of early life adversity

indicate altered expression of specific histone modifica-

tions and regulation of specific histone and DNA-

modifying enzymes within limbic brain regions (Blaze

& Roth, 2013; Dong et al., 2015). Early stress is also

correlated with altered epigenetic signatures at the

promoters of trophic factors (Roth, Lubin, Funk, &

Sweatt, 2009; Suri et al., 2013), glucocorticoid recep-

tors (Zhang, Labont�e, Wen, Turecki, & Meaney, 2013),

and plasticity-associated genes (Suri, Bhattacharya,

& Vaidya, 2014). Further, antidepressant-mediated

reversal of stress-evoked behavioral and neurogenic

changes is correlated with the rescue of specific

epigenetic modifications (Suri et al., 2013; Tsankova

et al., 2006), and HDAC inhibitors also ameliorate

stress-induced depressive behaviors (Covington et al.,

2009). These studies support a role for histone

modifiers in contributing to the effects of stress and

provide impetus to consider histone modifiers as

putative therapeutic targets. We hypothesized that

ES history may influence the expression of HATs,

HDACs, HMTs, and HDMs, as well as the sirtuin

family, within the mPFC, thus targeting the gene

regulation of epigenetic enzymes.

In the present study, we examined whether the early

stress of maternal separation (ES) influences the tran-

scriptional regulation of histone modifiers, namely

HATs, HDACs, including the sirtuin family, HMTs, and

histone (lysine) demethylases (KDMs) within the

mPFC of ES animals across the life span. We focused

on the mPFC as several previous studies indicate

disruption of transcriptional regulation, neuronal

cytoarchitecture and functional responses within the

mPFC of ES animals (Benekareddy, Goodfellow,

Lambe, & Vaidya, 2010; Soztutar, Colak, & Ulupinar,

2015; Stevenson, Halliday, Marsden, & Mason, 2008).

Our results demonstrate that the expression of several

histone modifiers is regulated in ES animals across life,

with the predominant pattern revealing (1) a significant

downregulation of multiple histone modifiers, (2) a

persistent pattern of a subset of changes that is

observed across the life span, and (3) gene expression

changes that are unique to specific time points and

indicative of an age-dependent temporal regulation of

histone modifiers in animals with an ES history.

METHODS

Animals

Male Sprague–Dawley rats bred in the Tata Institute of

Fundamental Research (TIFR) animal facility were used for

all experiments. Animals were group housed and maintained

on 12-hr light dark cycle with access to food and water ad

libitum. All experimental procedures were conducted as per

the national guidelines of the Committee for Supervision and

Care of Experimental Animals (CPCSEA) and were approved

by the TIFR Institutional Animal Ethics committee.

Early Life Stress Paradigm

Animals were subjected to the early life stress of maternal

separation (ES). Prior to mating, dams were maintained in

groups of three per cage, and mating involved individual

housing of dams with a single male rat for 2–3 days.

Following observation of a vaginal plug, dams were single

housed during gestation and maintained with their litter

until weaning. In brief, litters were assigned to control and

ES groups at random, with animals from at least three

litters included per experimental group to minimize any

litter-specific effects. All experimental litters were chosen to

ensure for relative consistency of size of litter (8–10) and

sex ratios (�50%). The maternal separation paradigm

involved separation of the entire litter from the dam, once

daily for 3 hr per day (11 am–2 pm) from postnatal Day 2

(P2) to postnatal Day 14 (P14). Daily separation involved

the removal of the dam from the home cage, followed by

the removal of the entire litter from the home cage into a

2 Pusalkar et al. Developmental Psychobiology

separate container lined with nesting material, which was

placed on a heating pad to maintain euthermic conditions.

The litter was returned to the home cage at the end of the

daily 3-hr separation, followed by replacement of the dam.

Control dams and their litters were subjected to routine

animal facility handling.

ES animals were sacrificed at three specific time points

for qPCR analysis namely, postnatal Day 21 (n¼ 8 per

group, derived from three litters with two to three animals

from each litter), at 2 months of age in adulthood (n¼ 8

per group, derived from three litters with two to three

animals from each litter), and at 15 months of age in

middle-aged life (n¼ 6 for control, derived from three

litters with two animals from each litter and n¼ 10 for ES

groups, derived from four litters with two to three animals

from each litter). A separate cohort of animals was

sacrificed following ES at P21 (n¼ 4 and five control and

ES groups respectively, derived from three litters with one

to two animals from each litter) and 2 months (n¼ 5 per

group, derived from three litters with one to two animals

from each litter) of age for Western blotting analysis.

Animals from the same litters were used for both qPCR and

Western blotting analysis at P21, 2 and 15 months; however

separate litters contributed to distinct time points. For the

purpose of this study we used only males to minimize

effects due to variation resulting from the estrous cycle.

Animals were sacrificed by decapitation and the brain was

rapidly removed to dissect out the medial prefrontal cortex

(mPFC). The tissue was flash frozen in liquid nitrogen and

stored at �80˚C till further use.

Quantitative PCR (qPCR)

Quantitative PCR (qPCR, Bio-Rad, Hercules, CA) was

performed on RNA extracted from the mPFC of ES animals

and age-matched controls at P21, 2 months and 15 months.

RNA was extracted using Trizol reagent (Trizol, Sigma,

Carlsbad, CA), and spectroscopic analysis of RNA quality

was determined on the NanoDrop spectrophotometer (Eppen-

dorf, Hamburg, Germany). RNA was reverse transcribed to

synthesize cDNA (High capacity cDNA Reverse Transcrip-

tion Kit, Applied Biosystems, Foster City, CA), which was

further amplified using primers specific for genes of interest

(Supplementary Table S1). We profiled the expression of the

following histone modifiers; HATs: Cyclic-AMP response

element binding (CREB) binding protein (Crebbp), Histone

acetyltransferase 1 (Hat1), K (lysine) acetyltransferase 2a

(Kat2a), K (lysine) acetyltransferase 5 (Kat5), K (lysine)

acetyltransferase 6a (Kat6a), Nuclear receptor coactivator 1

(Ncoa1), Nuclear receptor coactivator 2 (Ncoa2), E1A bind-

ing protein 300 (Ep300); HDACs: Histone deacetylase 1

(Hdac1), Histone deacetylase 2 (Hdac2), Histone deacetylase

3 (Hdac3), Histone deacetylase 4 (Hdac4), Histone deacety-

lase 5 (Hdac5), Histone deacetylase 6 (Hdac6), Histone

deacetylase 7 (Hdac7), Histone deacetylase 8 (Hdac8),

Histone deacetylase 9 (Hdac9), Histone deacetylase 10

(Hdac10), Histone deacetylase 11 (Hdac11); Sirtuins: Sirtuin

1 (Sirt1), Sirtuin 2 (Sirt2), Sirtuin 3 (Sirt3), Sirtuin 4

(Sirt4), Sirtuin 5 (Sirt5), Sirtuin 6 (Sirt6), Sirtuin 7 (Sirt7);

HMTs: ASH1 (Absent, small, or homeotic)- like (Drosophila)

(Ash1l), ASH2 (Absent, small, or homeotic)- like (Drosophila)

(Ash2l), Euchromatic histone-lysine N-methyltransferase 1

(Ehmt1), Euchromatic histone-lysine N-methyltransferase 2

(Ehmt2), Enhancer of Zeste homologue 1 (Ezh1), Enhancer of

Zeste homologue 2 (Ezh2), Mixed lineage leukemia 1 (Mll1),

Mixed lineage leukemia 2 (Mll2), Mixed lineage leukemia 3

(Mll3), PR domain containing 2 (Prdm2), SET domain,

bifurcated 1 (Setdb1), SET and MYND domain containing 3

(Smyd3), Suppressor of variegation 3-9 homolog 1 (Suv39h1),

Suppressor of variegation 4-20 homolog 1 (Suv420h1);

KDMs: Jumonji domain containing 3 (Jmjd3), Lysine (K)-

specific demethylase 1A (Kdm1a), Lysine (K)-specific deme-

thylase 2A (Kdm2a), PHD finger protein 8 (Phf8). qPCR data

analysis was performed using the DDCt method as described

previously (Bookout & Mangelsdorf, 2003). Data for all

groups were normalized to the endogenous housekeeping

gene, hypoxanthine-guanine phosphoribosyltransferase (Hprt),

which was found to be unchanged across treatment conditions.

The results are represented as fold change� SEM.

Western Blot Analysis

Protein lysates from the mPFC derived from control and ES

groups (P21 and 2 months) were subjected to Western

blotting analysis to monitor the levels of specific histone

modifications. Protein lysates were separated using SDS–

polyacrylamide gel (12.5%) electrophoresis and were then

transferred to polyvinylidene difluoride membranes (GE

Healthcare, Buckinghamshire, UK). Following blocking,

membrane blots were incubated with specific primary anti-

bodies for histone modifications namely rabbit anti H3

acetylation (panH3Ac, 1:1,000; Abcam, Cambridge, UK),

rabbit anti H3K9 acetylation (H3K9Ac, 1:1,000, Millipore

Bioscience Research Reagents, Darmstadt, Germany), rabbit

anti H3K14 acetylation (H3K14Ac, 1:1,000, Millipore Bio-

science Research Reagents), rabbit H3K4 dimethylation

(H3K4me2, 1:1,000; Millipore Bioscience Research

Reagents), mouse anti H3K9 dimethylation (H3K9me2,

1:1,000; Abcam), rabbit anti H3K9 trimethylation (H3K9me3,

1:1,000; Abcam), or rabbit anti H3 (H3, 1:1,000; Abcam)

along with a loading control of rabbit anti-beta-III-tubulin

(1:5,000; Covance, Princeton, NJ). Following primary anti-

body incubation overnight at 4˚C, blots were washed and then

incubated in a 1:10,000 dilution of horseradish peroxidase-

conjugated donkey anti-rabbit or sheep anti-mouse secondary

antibodies (GE Healthcare). Protein-antibody complexes were

visualized using an ECL substrate (GE Healthcare). Histone

modification westerns were normalized to total amount of

histone H3 and loading was normalized using beta-tubulin.

Densitometric analysis was performed using Image J 1.48

(NIH, Bethesda, Maryland).

Statistical Analysis

qPCR and Western blot data were subjected to statistical

analysis using the unpaired Student’s t-test with significance

determined at p< 0.05 (Prism, Graphpad Software, Inc., La

Jolla, CA).

Developmental Psychobiology Early Stress Evokes Dysregulation of Histone Modifiers 3

RESULTS

Early Stress Evokes Long-Lasting Changes inthe Expression of Specific HistoneAcetyltransferases (HATs) and HistoneDeacetylases (HDACs)

We examined the gene regulation of several HATs and

HDACs, including the sirtuin family members, within the

mPFC at several time points across the life span

following a history of ES (Fig. 1A and B). qPCR analysis

revealed a regulation of several HATs and HDACs within

the mPFC at P21 in ES animals as compared to age-

matched controls (Fig. 1C and D). ES animals predom-

inantly exhibited a significant decline in the expression of

several HATs (Kat2a, Kat6a, and Ncoa2) as well as

HDACs (Hdac2, Hdac10, and Hdac11), with the excep-

tion of Hdac3 that was significantly upregulated

(Fig. 1D). Downregulation of HDACs was observed

across several classes namely class I (Hdac2), class II

(Hdac10), and class IV (Hdac11) histone deacetylases.

Expression of several members of the sirtuin family was

also downregulated. Sirtuins are NADþ-dependent pro-tein deacetylases, with specific sirtuin family members

exhibiting HDAC activity (Dai & Faller, 2008). Strik-

ingly, we noted a significant reduction in the expression

of all three mitochondrial sirtuins, Sirt3, 4, and 5 in the

mPFC of ES animals at P21 (Fig. 1C and D).

ES animals in adulthood (2 months) continued to

exhibit a significant decline in gene expression of the

HAT Kat2a and specific HDACs (Hdac10 and Hdac11)

within the mPFC as compared to age-matched controls

(Fig. 1C and E). The decreased expression of specific

sirtuin family members, namely Sirt3 and Sirt4, noted at

P21 in ES animals was also observed in adult ES animals.

However, certain gene expression changes noted in ES

animals at P21 did not persist into adulthood, namely the

decline in Kat6a, Hdac2, and Sirt5 observed at P21 was

no longer observed in ES adult animals. We did observe

a trend (p¼ 0.09) toward an age-dependent opposing

regulation with reduced Kat6a expression noted at P21

and enhanced levels noted in ES animals in adulthood.

Further, ES animals in adulthood exhibited gene regu-

lation unique to this time point and distinct from that

observed in ES animals at P21, with downregulation of

specific HDACs (Hdac4 and Hdac6) and sirtuins (Sirt1,

Sirt6, Sirt7), as well as upregulation of Hdac7.

Kat2a and Sirt4 downregulation observed in ES

animals at both P21 and 2 months of age was persistent

in middle-aged animals (15 months) (Fig. 1C and F).

Specific gene expression patterns that emerged in

adulthood (2 months) in ES animals continued to

exhibit similar regulation across the life span with

reduced Hdac4, Hdac6, and Sirt6 mRNA levels at

15 months (Fig. 1C and F). While changes observed in

15-month-old ES animals substantially overlapped with

2-month-old ES animals, the pattern of regulation was

more extensive at 2 months of age, and specific gene

regulation (Hdac7, Hdac10, Hdac11, Sirt3, Sirt7)

observed at 2 months in ES animals did not persist into

middle-aged life (Fig 1C and F). In addition, ES

animals in middle-aged life also showed gene expres-

sion changes exclusive to this particular time point,

with a decline noted in Kat5, Ep300, and Hdac8

expression (Fig. 1C and F). Further, similar to the

changes noted in ES animals at P21, middle-aged ES

animals also showed a significant decline in the

expression of Ncoa2 and Hdac2 (Fig. 1C, D, and F).

Taken together, our results indicate that animals

with a history of ES primarily exhibit downregulation

of several histone modifiers within the mPFC, with

specific enzymes (Kat2a and Sirt4) showing a persis-

tent downregulation across the life span.

Early Stress Evokes Changes in the GeneExpression of Histone Lysine Methyltransferases(HMTs) and Histone (Lysine) Demethylases(KDMs) Across the Life Span

Next, we characterized the gene regulation of several

HMTs and KDMs, within the mPFC in ES animals at

P21, 2 months and 15 months of age (Fig. 2A and B).

Quantitative gene expression profile qPCR studies

revealed that while several HMTs (Ash2l, Mll1, Mll3,

Smyd3, Suv39h1, and Suv420h1) were significantly

downregulated in the mPFC of ES animals at P21

(Fig. 2C and D), no change was observed in the

expression pattern of the KDMs profiled. Adult ES

animals showed a similar pattern of gene regulation of

HMTs, with a significant decrease noted in specific

HMTs including an overlap with gene downregulation

at P21 (Smyd3, Suv39h1, and Suv420h1). Further,

unique patterns of changes were observed for Ehmt1,

Ehmt2, Ezh1, Ezh2, Mll2, and Setdb1, all of which

were downregulated in adult ES animals (Fig. 2C and

E). We also observed that gene expression changes in

specific HMTs observed at P21 in ES animals did not

persist into adulthood, with the downregulation of Mll1

and Ash2l no longer observed. Mll3 mRNA levels

showed an opposing pattern of changes at the two ages,

with a decline in expression at P21 and upregulation at

2 months of age. ES animals in adulthood also showed

a decrease in the expression of specific KDMs (Kdm1a

and Kdm2a) within the mPFC (Fig. 2C and E).

The reduced expression of specific HMTs (Smyd3

and Suv420h1) noted in the mPFC in ES animals at

both P21 and 2 months of age was also observed to

persist across the life span with a significant decline


FIGURE 1 Influence of early stress (ES) on the expression of histone acetyltransferases (HATs)

and histone deacetylases (HDACs) in the medial prefrontal cortex across the life span. (A) Shown

is a schematic representation of the early stress (ES) paradigm of maternal separation wherein rat

pups were separated from their dams for 3 hr daily from postnatal (P) Days 2–14 and mRNA

expression of HATs and HDACs was profiled within the medial prefrontal cortex (mPFC) (B) at

P21, 2 and 15 months of age. (C) Shown is the key for the fold changes in gene expression of

HATs and HDACs. qPCR analysis results indicated that a history of ES is associated with the

gene regulation of mRNA levels of several HATs and HDACs at P21 (D), 2 months (E) and

15 months (F) of age. The data are represented as fold change�SEM and significance is

determined at �p< 0.05, Student’s t-test (P21: n¼ 8 per group, 2 months: n¼ 8 per group, and

15 months: n¼ 6 and 10 control and ES groups respectively).


observed in middle-aged animals with a history of ES

(Fig. 2C and F). Further, gene expression changes in

specific HMTs and KDMs that emerged in adult ES

animals at 2 months of age also consisted of similar

trend of downregulation in 15-month-old ES animals

(HMTs: Mll2, Ehmt2, and Setdb1; KDMs: Kdm1a and

Kdm2a) (Fig. 2C and F). However, in contrast to the

decline in the mRNA expression of the HMT Ezh1

FIGURE 2 Influence of early stress (ES) on the expression of histone methyltransferases

(HMTs) and histone (lysine) demethylases (KDMs) in the medial prefrontal cortex across the life

span. (A) Shown is a schematic representation of the early stress (ES) paradigm of maternal

separation wherein rat pups were separated from their dams for three hours daily from postnatal

(P) Day 2–14 and mRNA expression of HMTs and KDMs was profiled within the medial

prefrontal cortex (mPFC) (B) at P21, 2 and 15 months of age. (C) Shown is the key for the fold

changes in gene expression of HMTs and KDMs. qPCR analysis results indicated that a history of

ES is associated with the gene regulation of mRNA levels of several HMTs and KDMs at P21

(D), 2 months (E) and 15 months (F) of age. The data are represented as fold change� SEM and

significance is determined at �p< 0.05, Student’s t-test (P21: n¼ 8 per group, 2 months: n¼ 8 per

group, and 15 months: n¼ 6 and 10 control and ES groups respectively).


noted in 2-month-old ES animals, middle-aged ES

animals showed contrasting pattern of regulation with a

robust twofold upregulation noted in Ezh1 mRNA levels.

Though changes in gene expression noted in ES animals

at 15 months showed a considerable overlap with the

changes noted in ES animals at 2 months of age, we did

observe a more widespread gene regulation of HMTs at

2 months as compared to 15 months in animals with an

ES history, with changes in the expression of specific

HMTs (Ehmt1, Ezh2, Mll3, and Suv39h1) lost in

middle-aged ES animals (Fig. 2C and F).

Collectively, our results highlight a strong pattern of

downregulation of several HMTs and specific KDMs

within the mPFC of animals with an ES history, with

Smyd3 and Suv420h1 exhibiting significant decline in

transcript levels across the life span.

Early Stress Does Not Alter Global Levels ofHistone Acetylation and Histone Residue-Specific Methylation Within the mPFC

Given the widespread gene regulation of histone

modifiers within the mPFC of ES animals, we next

sought to examine whether such changes were accom-

panied by alterations in global histone acetylation and

methylation levels in ES animals. We selected the

postnatal (P21) and adult time points following ES

to profile total and residue-specific histone acetylation

(panH3Ac, H3K9Ac, and H3K14Ac) and residue-

specific histone methylation (H3K4me2, H3K9me2,

and H3K9me3) within the mPFC. Western blot analysis

for pan H3 acetylation (panH3Ac), H3K9Ac, H3K14Ac,

H3K4me2, H3K9me2, and H3K9me3 normalized to H3

levels revealed no change in these histone modifications

within the mPFC of ES animals at P21 (Fig. 3A–G)

and in adulthood (Fig. 3H–N) as compared with their

respective age-matched controls.

DISCUSSION

Our results indicate that ES experience evokes long-

lasting perturbations in the expression of both writer

(HATs and HMTs) and eraser (HDACs and KDMs)

classes of histone modifiers in the mPFC. ES-evoked

gene regulation of histone modifiers within the mPFC

FIGURE 3 Influence of early stress (ES) on residue-specific histone methylation and global

histone acetylation levels in the medial prefrontal cortex. Levels of residue-specific histone

methylation (H3K9me2, H3K9me3, H3K4me2) and global and residue-specific histone acetyla-

tion (panH3Ac, H3K9Ac, H3K14Ac) were assessed by Western blot analysis in the medial

prefrontal cortex of control and ES animals at P21 (A–G) and 2 months (H–N) of age. The levels

of the residue-specific histone methylation, H3K9me2, H3K9me3, H3K4me2, and global and

residue-specific histone acetylation panH3Ac, H3K9Ac, and H3K14Ac were normalized to H3

levels. Shown are representative images from a control and ES animal, as well as densitometric

analysis with results expressed as fold-change of control-C for H3K9me2 (P21: A, B and

2 months: H, I), H3K9me3 (P21: A, C and 2 months: H, J), H3K4me2 (P21: A, D and 2 months:

H, K), panH3Ac (P21: A, E and 2 months: H, L), H3K9Ac (P21: A, F and 2 months: H, M), and

H3K14Ac (P21: A, G and 2 months: H, N). The data are fold change of control� SEM, P21:

n¼ 4 and five control and ES groups respectively) and two months: n¼ 5 per group.


revealed predominantly a decline in gene expression,

with downregulation of specific genes noted across the

entire life span (Kat2a, Smyd3, and Suv420h1) (Fig. 4).

The mitochondrial sirtuin, Sirt4 was also found to

exhibit a consistent decline across life following ES.

Further, we noted gene regulation that emerged in a

temporal manner and was unique to specific time points

(Fig. 4). While robust gene expression changes were

noted in several histone modifiers after ES, this was

not associated with any change in global and residue-

specific levels of H3 acetylation and residue-specific

histone methylation within the mPFC. Our results

provide novel evidence that a history of ES is

associated with persistent gene regulation of several

histone modifiers within the mPFC, raising the intrigu-

ing possibility that such changes could influence

chromatin remodeling and transcriptional regulation,

and thus impinge on the regulation of prefrontal

function and plasticity.

To the best of our knowledge, our study provides the

first extensive profiling of the effects of early adversity

on the expression of several histone modifiers and

the sirtuin family across the life span. Strikingly, ES

animals showed a life-long decline in gene expression

of the HAT, Kat2a, the mitochondrial sirtuin, Sirt4, and

the histone methyltransferases Smyd3 and Suv420h1

that catalyze histone covalent modifications of

H3K4me2/3 and H3K20me2/3, respectively (Fig. 4).

While the role of Kat2a in the PFC is yet unexplored,

it is noteworthy that Kat2a�/�mice exhibit disruption

of plasticity-associated hippocampal gene expression

accompanied by impaired synaptic plasticity and long-

term memory (Stilling et al., 2014). Expression of the

mitochondrial sirtuin, Sirt4, that has been implicated in

regulation of metabolic homeostasis (Chalkiadaki &

Guarente, 2012), was persistently downregulated in the

mPFC after ES. Recent reports also indicate that

decreased Sirt4 expression in neuronal cells results in a

FIGURE 4 Influence of early stress on histone modifiers in the medial prefrontal cortex across

the life span. Shown is a schematic depicting the transcriptional regulation of histone

acetyltransferases (HATs), histone deacetylases (HDACs), sirtuins, histone methyltransferases

(HMTs), and histone (lysine) demethylases (KDMs) in the medial prefrontal cortex across the life

span (postnatal Day 21, 2 and 15 months of age) following the early stress (ES) of maternal

separation. Shown are the histone modifications catalyzed by the HATs, HDACs, sirtuins, HMTs,

and KDMs. Genes found to be consistently regulated across the life span in animals with an ES

history are boxed. Red indicates upregulation and green indicates downregulation of mRNA

levels.


failure to successfully buffer excitotoxic insults (Shih,

Liu, Mason, Higashimori, & Donmez, 2014). In this

regard, it is tempting to speculate that reduced Sirt4

expression may contribute to the inflammatory damage

observed in prefrontal cortical neurons in ES animals

through aberrant glutamatergic function (Wieck, Ander-

sen, & Brenhouse, 2013). The role of the HMTs Smyd3

and Suv420h1 is presently not understood in the context

of neuronal cells. A persistent decline in their expres-

sion within prefrontal circuits following ES raises the

possibility that histone methylation marks catalyzed by

these enzymes may be altered, affecting chromatin

structure and gene expression at specific gene loci.

Though histone modifications are thought to be rela-

tively transient (min to days), recent evidence suggests

that interactions between histone and DNA modifica-

tions may directly impact DNA methylation changes,

thus setting up a template for long-lasting stable

epigenetic marks (Blackledge et al., 2010; Cedar &

Bergman, 2009; Hashimoto, Vertino, & Cheng, 2010;

Huang, Xu, & Zhu, 2013; Wang et al., 2009). While

we did not observe global modifications of residue-

specific histone methylation and acetylation following

ES, a caveat of our studies is that we would miss

histone modifications restricted to specific neuronal

subtypes and those at specific gene loci. Future studies

are required with chromatin immunoprecipitation fol-

lowed by next-generation sequencing to unveil changes

in histone modification marks at specific gene loci.

Further, it is important to note that all our studies

were performed in male rats and involved dissection

of the entire mPFC, hence additional studies will be

required to address whether the results of the present

study can be generalized to both genders and are

applicable to both the prelimbic and infralimbic

subdivisions of the mPFC, that differ functionally

(Chudasama, 2011).

Gene Regulation of HATs in ES Animals

ES animals exhibited significant decreases in several

HATs with the most widespread pattern of down-

regulation noted in middle-aged ES animals (Kat2a,

Kat5, Ncoa1, Ncoa2, and Ep300). A decline in

prefrontal Kat5 and Ncoa1 expression has also been

observed in the social defeat stress model (Kenworthy

et al., 2014), suggesting a possible overlap in the gene

regulation of specific HATs following early and adult

stress exposure. A decline in several HATs noted in ES

animals as they age is of interest given that HATs

impact transcriptional regulation through effects on

gene locus-specific histone acetylation and chromatin

architecture, and may serve as “network hubs” for

diverse protein interactions, including the ability to

influence healthy aging (Bedford & Brindle, 2012).

Previous studies indicate that middle-aged ES animals

exhibit transcriptional changes in several genes associ-

ated with the aging process accompanied by both a

cognitive and neurogenic decline suggestive of “accel-

erated aging” (Suri et al., 2013, 2014). It is tempting to

speculate that perturbed expression of histone modifiers

may also contribute to the transcriptional regulation

noted in ES animals as they age, and could thus

impinge on the aging-associated structural and behav-

ioral alterations noted in middle-aged ES animals (Suri

et al., 2013, 2014).

Gene Regulation of HDACs in ES Animals

Accompanying the decline noted in HATs, we also

observed predominantly a decrease in the expression of

several HDACs in the mPFC of ES animals across life.

The exceptions to this pattern were Hdac3 and Hdac7

that were upregulated at P21 and 2 months, respec-

tively. The mechanisms that evoke such differential

effects on these particular HDACs are at present

unknown. While a decline in Hdac10 and Hdac11 was

noted in common at P21 and 2 months of age, Hdac4

and Hdac6 showed a common pattern of reduced gene

expression at 2 and 15 months of age. The role of

Hdac10 and Hdac11 in the brain is not yet character-

ized. Hdac4 has previously been implicated in experi-

ence-dependent synaptic plasticity via its recruitment

on the promoters of plasticity-associated genes (Kim

et al., 2012; Sando et al., 2012). Hdac4 also has been

reported to influence stress-induced transcriptional

memory (Sailaja, Cohen-Carmon, Zimmerman, Soreq,

& Meshorer, 2012). A previous study (Levine, Worrell,

Zimnisky, & Schmauss, 2012) observed that early stress

evoked biphasic changes in the expression of forebrain

neocortical Hdac1, Hdac3, Hdac8, Hdac10 with an

induction noted in postnatal life (P21 and P28) and a

decline observed in adulthood. Our results are in

agreement with specific aspects of this previous study

and also show a transient induction in mPFC of Hdac3

expression restricted to postnatal life. Interestingly, in

contrast to the upregulation we observed with Hdac3

levels, adult-onset stress evoked a decline in cortical

Hdac3 expression (Tordera et al., 2011), suggesting

that the nature of regulation of Hdac3 by stress is

dependent on the temporal window of stress experi-

ence. The differences noted in the pattern of gene

regulation across these studies could also arise due to

differences in brain regions studied and species-specific

effects. It will be important to uncover whether the

widespread decline in specific HDACs noted in the

mPFC of ES animals is relevant to the adaptive or

maladaptive consequences of ES.


The observations of a decline in both HATs and

HDACs following ES may seem counter intuitive as

HATs and HDACs encode for functionally opposite

histone modifications. However, histone marks are regu-

lated by a complex network of a variety of chromatin

remodelers, therefore the effects of a downregulation of

several HATs and HDACs simultaneously do not neces-

sarily cancel the effect of each other. Our results strongly

point to the possibility that the epigenetic landscape with

reference to histone acetylation marks is likely to be

substantially altered following ES.

Sirtuin Regulation in ES Animals

While originally classified as Class III HDACs, the

sirtuin family that consists of NADþ-dependent proteindeacetylases and/or ADP-ribosyltransferases plays an

important role in metabolic regulation (Choi & Masto-

slavsky, 2014). Specific sirtuin family members have

been implicated in the regulation of neuroinflammation,

neuronal plasticity, cognition, and mood-related behav-

ior (Herskovits & Guarente, 2014; Satoh & Imai,

2014). Strikingly, we observed a significant reduction

in all three mitochondrial sirtuins, Sirt3, Sirt4, and

Sirt5 at P21 in ES animals, with a decline still observed

in Sirt3 and Sirt4 in adulthood, and Sirt4 persistently

downregulated across life. Mitochondrial sirtuins,

which are transcribed by the nuclear genome, are

implicated in regulation of metabolism and energy

homeostasis (Wu, Wu, & Wei, 2014), with reports of a

role for Sirt3 in the regulation of gene expression in

response to cellular stress (Iwahara, Bonasio, Narendra,

& Reinberg, 2012). While a downregulation of Sirt4

across the life span in ES animals was observed, the

exact role of Sirt4 in brain function remains elusive,

with recent reports indicating that Sirt4 may play an

important role in buffering excitotoxicity (Shih et al.,

2014). ES-evoked downregulation of sirtuin family

members was also noted at specific time points, with

several sirtuins decreasing at 2 (Sirt1, Sirt3, Sirt4,

Sirt6, Sirt7) and 15 months (Sirt4, Sirt6) of age. Sirt6

has been strongly implicated in maintaining longevity

in mice and is associated with the aging process (Kanfi

et al., 2012). Our studies provide impetus to further

examine the impact of ES on the activity of sirtuin

family members in the prefrontal cortex, and to address

whether global changes in protein acylation are

observed in ES animals.

Gene Regulation of HMTs and KDMs in ESAnimals

The HMTs Smyd3 and Suv420h1 that catalyze H3K4

and H3K20 di- and tri-methylation, respectively (Khare

et al., 2011) and whose function in the brain is poorly

understood were found to be downregulated across life

following ES. Further, specific members of the methyl-

transferase mixed-lineage leukemia (MLL) family,

which catalyze H3K4 di- and tri-methylation, were

downregulated in ES animals at P21 (Mll1, Mll3), 2

(Mll2, Mll3) and 15 (Mll2) months. Decreased prefron-

tal H3K4 trimethylation has been observed in schizo-

phrenia, and MLL1 has been implicated in rodent

models as a target for antipsychotics (Huang et al.,

2007) and in the regulation of memory through a

modulation of permissive histone methylation (Aguilar-

Valles et al., 2014; Bharadwaj et al., 2014).

Distinct histone modifiers that catalyze H3K9

methylation were decreased at all three ages following

ES. While ES animals at P21 showed reduced levels

of Ash2l and Suv39h1, the maximal extent of down-

regulation (Ehmt1, Ehmt2, Suv39h1, Setdb1) was

noted at 2 months with the decline in Ehmt2 and

Setdb1 persisting up to 15 months of age. Interestingly,

decreased prefrontal expression of ASH2L is reported

in a neurodevelopmental model of schizophrenia,

accompanied by perturbed H3K9 methylation

(Ma�ckowiak, Bator, Latusz, Mordalska, & Wedzony,

2014). Previous reports indicate that ES evokes a

biphasic regulation of hippocampal Suv39h1 expression

with an early decline and an opposing pattern as ES

animals age (Suri et al., 2014). Suv39h1 in the mPFC

did not show such a biphasic pattern of regulation,

rather within this limbic brain region, we observed an

early decline maintained into adulthood that was lost

as ES animals aged. The HMT Ezh1 that is involved

in H3K27 methylation showed ES-evoked biphasic

changes with a downregulation in adulthood and a

robust twofold upregulation in middle aged. Interest-

ingly, RNA-Seq analysis in short-lived fish demon-

strated an age-dependent upregulation of Ezh1

expression (Baumgart et al., 2014). It is tempting to

speculate that the significant induction of Ezh1 in

ES animals may contribute to cognitive decline and

disruption of aging-associated processes. Further,

Setdb1 has been implicated in the regulation of

chromatin conformation changes within the regulatory

regions of the glutamate receptor genes in the PFC

(Bharadwaj et al., 2014), and drug-evoked decreases in

cortical SETDB1, accompanied by reduced H3K9

methylation, are hypothesized to result in a permissive

epigenetic landscape (Chase & Sharma, 2013). Of the

KDMs profiled in ES animals we noted a robust

downregulation of Kdm1a and Kdm2a at 2 and

15 months of age, which was not observed at the early

time-point of P21. While we did not observe any

change in global levels of H3K9me2, H3K9me3, or

H3K4me3, it is certainly possible that altered expres-

sion of HMTs that catalyze repressive and active


histone methylation marks, as well as a change in

levels of KDMs, has the potential to impact specific

gene loci and influence bivalent marks that are

associated with poised genes (Peter & Akbarian, 2011).

It will also be interesting to address the mechanisms

that contribute to the transcriptional regulation of

several histone modifiers in the mPFC of ES animals.

While at present the upstream pathways that mediate

such a gene regulation in ES animals remain unclear,

sustained alterations in neurotransmitter and neuro-

endocrine signaling that arise as a consequence of ES

history may play a potential role (Benekareddy et al.,

2010; Karsten & Baram, 2013).

Overall, our data suggest widespread gene regulation

of several histone modifiers in animals with a history of

ES with both overlapping, as well as unique changes

that emerge in a temporally dependent manner. These

results suggest that the early adverse experience of

maternal separation may result in the disruption of an

epigenetic landscape across the life span, thus laying

down a substratum for persistent patterns of altered

transcription and long-lasting molecular, cellular and

behavioral consequences of early stressful experience.

NOTES

We acknowledge Dr. Shital Suryavanshi and Dr. Sachin Atole

for technical assistance. This research was supported by a

TIFR intramural grant (VAV) and support from the Department

of Biotechnology, Centre of Excellence in Epigenetics, IISER

(VAV; BT/01/COE/09/07).

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