Early Environmental Regulation of Gene Expression: How ...Sept_2013... · Early Environmental Regulation of Gene Expression: How Early Experience Exerts a Sustained Influence on Neuronal

Early Environmental Regulation of Gene

Expression: How Early Experience Exerts a

Sustained Influence on Neuronal Function

[email protected]

Michael J Meaney

Douglas Mental Health University Institute

McGill University

and

Singapore Institute for Clinical Sciences

Summary

The experience of the child is “biologically

embedded” and serves to influence health and

capacity over the lifespan.

Summary

The experience of the child is “biologically embedded”

and serves to influence health and capacity over the

lifespan.

This effect is apparent even at the level of the

DNA of the individual; the activity of genes implicated

in brain development and function

are directly regulated by the social environment.

Summary

The experience of the child is “biologically embedded”

and serves to influence health and capacity over the

lifespan.

This effect is apparent even at the level of the

DNA of the individual; the activity of genes implicated

in brain development and function

are directly regulated by the social environment.

This effect is potentially stable over time; the

imprint of of childhood adversity on the genome

is apparent at later ages, providing a biological basis

for an enduring effect on health and capacity.

The development of an individual is an active

process of adaptation that occurs within a

social and economic context:

• To resource (food, shelter, safety)

availability.

• To social interactions (e.g., parental

signals).

The development of an individual is an active

process of adaptation that occurs within a

social and economic context:

• To resource (food, shelter, safety)

availability.

• To social interactions.

This influence is apparent in the epigenetic mechanisms

that regulate genomic structure and function

Early experience

Abuse

Family strife

Emotional neglect

Harsh discipline

Health Risks

Depression

Drug abuse

Anxiety

Diabetes

Heart disease

Obesity

Developmental Origins of Adult Disease

Early experience

Abuse

Family strife

Emotional neglect

Harsh discipline

Health Risks

Depression

Drug abuse

Anxiety

Diabetes

Heart disease

Obesity

Individual differences

in neural and endocrine

responses to stress (defensive responses)

How does family life influence health over the life span?

Early experience

Abuse

Family strife

Emotional neglect

Harsh discipline

Health Risks

Depression

Drug abuse

Anxiety

Diabetes

Heart disease

Obesity



responses to stress

Poverty

Nutrient Supply

Mate Quality

Violence

Infection

Population density

Parental

investment

Defensive Strategies

Foraging/Metabolism

Reproductive Strategies

Environmental Parental Developmental

condition signal outcome

Robert Hinde: Evolution has shaped the young to use parental

signals as a ‘forecast’ of the quality of the environment

into which they have been born. For most species, there is

no single, optimal phenotype.

Evolutionary biology - Maternal effects

Health and

Human Capacity

Early experience

Abuse

Family strife

Emotional neglect

Harsh discipline



responses to stress

Adversity

Environmental regulation of phenotypic variation

What is the biological basis for ‘programming’ effects

whereby environmental signals acting over perinatal

development associate with stable changes in transcription

and complex phenotypes (physiology, behaviour)?

Summary

• Parental care affects the activity of

genes in the brain that regulate stress

responses, neural development and

reproduction.

• This parental effect involves a form

a “plasticity” at the level of the DNA.

Epigenetics: Any functional change in the genome that

does not involve an alteration of DNA sequence.

If they ask you anything you don’t know,

just say its due to epigenetics.

Nucleosome core particle: ribbon traces for the 146-bp DNA phosphodiester backbones

(brown and turquoise) and eight histone protein chains (Luger et al. Nature 1997).

+ -

Prevents TF

binding to DNA

TF binding involves

alteration of

chromatin structure



+ -

Prevents TF

binding to DNA

TF binding involves

alteration of

chromatin structure

Acetyl group

B. Turner. Chromatin structure and gene regulation. 2001.



+ -

Prevents TF

binding to DNA

TF binding involves

alteration of

chromatin structure

C T A C G T A C T C G G A A T C T C G CH

3 CH

3 CH

3

C T A C G T A C T C G G A A T C T C G

Genetic code is defined by the sequence of four

nucleotides that produce proteins.

RNAs, proteins

Epigenetic effects refer to modifications of the DNA

that alter the activity of the gene, but not its function.

SIN

SIN3a

CH 3

CH 3

MeCP2/

MBD2

HDAC MeCP2/

MBD2

DNA Methylation can inhibit gene expression

by blocking transcription factor binding

HDAC HDAC: Histone deacetylase

Methylated DNA

binding protein CH 3

CH 3

CH 3

CH 3

HDAC




+ -

Prevents TF

binding to DNA

TF binding involves

alteration of

chromatin structure

Every cell in your body has the same nuclear genes,

but…?

Multiple phenotypes from a common genotype

Maternal licking/grooming

Environmental epigenetics hypothesis: Environmental

events activate intracellular signals that remodel

the epigenome, leading to sustained alterations in

the structure and function of the genome, and thus

stable effects on gene transcription.

Parental care Phenotype

Gene

expression

Epigenetic

marks

Parental signals as a source of phenotypic plasticity?


Gene

expression Epigenetic

marks


Maternal licking/grooming

Source of tactile stimulation/nurturance: Enhances

activity of endocrine systems (e.g., GH/IGF) that

promote somatic growth, suppresses those

(glucocorticoids) that inhibit growth

Variations in maternal care

6 11 1 6 0

50

100

150

200

Fre

qu

en

cy c

ou

nt

% Licking/grooming

Stress responses

Neural development

Learning & memory

Metabolism

Reproduction (females)

Broad range of parental effects

High LG Low LG


Gene

expression Epigenetic

marks


Stress responses

Neural development

Learning & memory

Metabolism

Reproduction (females)

Broad range of parental effects

Glucocorticoid Receptor

Hypothalamus

Pituitary

Adrenals

Hippocampus

Glucocorticoids

Stress

(-)

CRF/AVP

ACTH

CRF: corticotropin releasing factor. ACTH: adrenocorticotropin

Hypothalamus

Pituitary

Adrenals

High LG offspring

Glucocorticoids

(-)

CRF

ACTH

(-)

Hypothalamus

Pituitary

Adrenals

Low LG offspring

Glucocorticoids

(-)

CRF

ACTH

(-)

Individual differences in glucocorticoid receptor

levels lead to altered pituitary-adrenal responses

to stress

High LG offspring show more modest HPA stress responses

Pre S10 S20 P20 P40 P60 P120 0

200

400

600

800

1000 High LG Low LG

Pla

sm

a A

CT

H (

pg

/ml)

Pre 10 20 40 60 80 100 0

10

20

30

40

50

60

70

80 High LG Low LG

Co

rtic

oste

ron

e (

µg

/dl)

Disruption of hippocampal GR signaling eliminates this

maternal effect

Hypothalamus

Pituitary

Adrenals

High LG offspring

Glucocorticoids

(-)

CRF

ACTH

(-)

Hypothalamus

Pituitary

Adrenals

Low LG offspring

Glucocorticoids

(-)

CRF

ACTH

(-)

Cross-fostering reveals evidence for direct, postnatal

effects of maternal care

Hippocampal GR(17) mRNA

1 2 3 4 5 6 7 8 9 1011 12 2 3 4 5 67 8 9

5’ 3’

DG CA1 CA2 CA3 0.00

0.05

0.10

0.15

High LG

Low LG

Re

lati

ve

O

DU

Hippocampal GR(17) mRNA

*

* * *

1 2 3 4 5 6 7 8 9 1011 12 2 3 4 5 67 8 9

5’ 3’

1681 ccc

1741 ctctgctagt gtgacacact t1cg2cgcaact c3cgcagttgg 4cggg5cg6cgga ccacccctg7c

1801 ggctctgc8cg gctggctgtc accct9cgggg gctctggctg c10cgaccca11cg ggg12cgggct

1861 c13cgag14cggtt ccaagcct15cg gagtggg16cg gggg17cgggag ggagcctggg agaa

DNA sites that regulate glucocorticoid receptor gene

NGFI-A

GR Promoter 17 Sequence

1 2 3 4 5 6 7 8 910 1112 2 3 4 5 6 78 9

Variable exon 1 region Constant region

5’ 3’

NGFI-A GR gene

GR mRNA

NGFI-A regulates GR gene expression

5’ 3’ 0.0

0.2

0.4

0.6

0.8

1.0

Low LG

High LG

c-M

eth

yla

tio

n

*

NGFI-A site

5’…tgcgggggcgggg…3’

NGFI-A

CH 3 CH

3

Low/Low High/High Low/High High/Low

0.0

0.2

0.4

0.6

0.8

c-m

eth

yla

tio

n

5' CpG region of NGFI-A/RE

5’…tgcgggggcgggg…3’

NGFI-A

CH 3

CH 3

CH 3

SIN

SIN

CH 3

CH 3

CH 3

CH 3

MeCP2/

MBD2

HDAC CH 3

CH 3

MeCP2/

MBD2

DNA Methylation can inhibit gene expression

by blocking transcription factors binding

HDAC HDAC: Histone deacetylase

Methylated DNA

binding protein

HDAC


High Low

0.0

0.2

0.4

0.6

0.8

1.0

*

0.0

0.2

0.4

0.6

0.8

1.0

An

tib

od

y/I

np

ut

*

Histone

Acetylation

NGFI-A

Association

Increased methylation of the exon 17 GR promoter associates with

decreased H3K9ac, reduced NGFI-A binding and GR expression

0

0.1

0.2

0.3

0.4

GR

IR

(R

OD

-IO

D)

*

GR

Expression

High Low High Low

Effects are reversed with intra-hippocampal infusion of HDAC inhibitor

Low/Veh Low/TSA High/Veh High/TSA

Basal Stress Stress + 40 Stress + 60 Stress + 90 0

10

20

30

40

50

Co

rtic

oste

ron

e (

µ

g/d

l)

Time (min)

Low/Veh

Low/TSA

High/Veh

High/TSA

Glucocorticoid stress response

GGAGCCTGGGAGAACCAAGCCTCGGAGTGGGCGGGGGCGGGAG CH

3 CH

3

GGAGCCTGGGAGAACCAAGCCTCGGAGTGGGCGGGGGCGGGAG CH

3

Offspring of Low LG mothers

Offspring of High LG mothers

Lower levels of GR in hippocampus -

Increased stress response

High levels of GR in hippocampus -

Modest stress response

cAMP

5-HT7 receptor

5-HT Tactile stimulation

(maternal LG)

PKA CBP

Summary of in vivo and in vitro studies

NGFI-A (egr-1, zif-268, krox-24)

(T4 - T3)

SP1

CBP NGFI-A SP1

CpGme Exon 17 promoter

C T A C G T A C T C G G A A T C T C G

CH 3

CH 3

CH 3

cAMP

NGFI-A

5-HT7 rec

5-HT

GR gene

PKA CBP

SP1

CBP SP1

Do comparable processes occur in humans?

• Post-mortem studies of hippocampus.

• Samples from suicide victims/controls.

• QSBB (Gustavo Turecki) – forensic phenotyping.

• Human exon 1F promoter (Turner & Muller,

2005)

Human glucocorticoid receptor gene

A B CD E F G H I J 2 3 4 5 67 8 9


5’ 3’

Exon 1F is the human ortholog of the rat exon 17 GR promoter

(70% homology) and contains an NGFI-A consensus sequence.

Human glucocorticoid receptor gene

A B C DE F G H I J 2 3 4 5 67 8 9


5’ 3’

Control Suicide 0.0

0.5

1.0

1.5 GRtotal

GR

mR

NA

*

Control Suicide 0.0

0.3

0.6

0.9

1.2 GR1F

GR

1-F

vari

an

ts

*

Suicide vs abuse - GR expression

GRtotal

GR

mR

NA

/GA

PD

H (

log

co

nc.)

Control Suicide Suicide

- Abuse + Abuse

0.0

0.4

0.8

1.2

*

GR

-1F

mR

NA

/GA

PD

H (

log

co

nc.)


- Abuse + Abuse

0.0

0.5

1.0

1.5

*

GR1F

GR

1-F

CpG

Meth

yla

tion (

%)


- Abuse + Abuse

0

20

40

60 *

Suicide vs abuse - CpG methylation

Hypothalamus

Pituitary

Adrenals

Controls

Glucocorticoids

(-)

CRF

ACTH

(-)

Hypothalamus

Pituitary

Adrenals

Maltreatment

Glucocorticoids

(-)

CRF

ACTH

(-)

Childhood maltreatment associates with increased central

CRF levels and greater HPA and autonomic responses to stress

(DeBellis et al 1994; Heim et al 2000; Lee et al 2005)

Childhood adversity and NR3C1 promoter methylation in DNA

from periperhal samples

Early experience

Abuse

Family strife

Emotional neglect

Harsh discipline

Health Risks

Depression

Drug abuse

Anxiety

Diabetes

Heart disease

Obesity



responses to stress (defensive responses)

How does family life influence health over the life span?

Childhood adversity and NR3C1 promoter methylation in DNA

from periperhal samples

345 genes

Secure:

TBCD (23.89%: Body)

CEBPE (19.70%: TSS200)

Attachment at 18 months (Secure Vs Insecure)

associates with epigenetic variation (6-8 yrs)

612 probes

≥. 5% (p<0.05)

Insecure:

ABR (21.35%: Body)

SHANK2 (9.4%: 1st Exon)

DRD4 (5.76%: Body)

Microtubule

formation LTP

Synaptic

density

Reference 1: ADHDgene Database (I)

64

213 Genes in the ADHDgene database (24

Genes are hot genes)

36 Probes (25 Genes) of ADHD database

genes are found in the 2907 DMPs list.

Reference 1: ADHDgene Database (II)

ADRA1A FGF10

ANK3 GNAO1

ARRB2 HK1

ATXN1 HLA-DRB1

BAIAP2 ITGA11

BAIAP2 MEIS2

BAIAP2 MEIS2

BAIAP2 NCKAP5

BAIAP2 PTPRG

BDNF PTPRG

CACNA1C SH3BP5

CDH13 SH3BP5

CHRNA4 SLC6A3

CHRNA4 UNC5B

COMT UNC5B

DBH ZNF423

DRD4 ZNF423

EMP2 ZNF423

Child attention problems and co-methylation

Child attention problems (SDQ) associated with

differential methylation of 1747 probes (957

genes).

30 probes (22 genes) associated with child

attention problems (-0.3 < r >0.3) show co-

methylation (-0.6 < r >0.6).

rs= -0.36, p=0.02 rs= 0.36, p=0.02 rs= -0.64, p<0.001

7 Genes(GO) + 94 Genes(Pathway)

--123 Probes--

Hierarchical clustering of pair-wise correlation

DMPs

DM

Ps

Pair-wise Correlation

20 40 60 80 100 120

20

40

60

80

100

120-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

The Most

highly

correlated

cluster (30

probes, Avg

Corr=0.8)

67 Co-methylation Clusters

The ‘partial’ mediating role of maternal

psychopathology

Maternal

depression

(36 months)

Maternal

emotional

neglect

Maternal

depression

(36 months)

Child negative

emotionality (36

moths) p = 0.01

p = 0.008 p = 0.001

The ‘partial’ mediating role of maternal

psychopathology

Maternal

depression

(36 months)

Maternal

emotional

neglect

Maternal

depression

(36 months)

Child negative

emotionality (36

moths) p = 0.01

p = 0.008 p = 0.001

(Parenting)

Do effective treatment programs that target

mothers affect DNA methylation profiles

in the children?

90 Unique Samples

Infinium 450K Array

Blood Samples

27 years old

Variables: methy_grp (0= control/no treatment ;1=intervention)

ETXGROUP: 1 and 2 = different forms of no treatment versus 3 and 4 which represent prenatal intervention and combined pre and postnatal intervention, respectively

CHILDGENDER 1=male 2=female

Child Abuse at age 4 and age 15

Child abuse is physical, sexual or emotional mistreatment or neglect

Principal Component Analysis

Component Fstat p

Gender (3.0%) 7.32 .003 x 10-8

Abuse/4 yrs (3%) 5.80 .002 x 10-2

Abuse/15yrs 1.67 ns

Principal Component Analysis

Component Fstat p

Gender (3%) 7.32 .003 x 10-8

Abuse/4 yrs (3%) 5.80 .002 x 10-2

Abuse/15yrs 1.67 ns

Treatment (8%) 3.07 .002 x 10-4

Conclusions

• The function of the genome is regulated by

epigenetic signals that are subject to

environmental regulation.

• These epigenetic signals reflect the quality of the

dearly environment, and guide the development

and function of the brain.

• These effects are potentially stable, but are

subject to modification, potentially over the

lifespan.

Family

Parent-

child

Family

Parent-

child

Transgenerational

influences

Family

Parent-

child

Family

Parent-

child

Transgenerational

influences

Family

Parent-

child

Family

Parent-

child

Transgenerational

influences

Control Perfume Snake 0

1

2

3

4

Response to snake odours

To

ng

ue-F

lick

Offspring

are significantly

larger and with

longer tails

Defense to snake predation in skink lizards

Most frequent prey

• smaller

• shorter tails

• less reactive to

snake cues

If mother has been exposed to the scent of a

predatory snake then offspring are larger, with

longer tails and ….

Nutrient Supply

Mate Quality

Violence

Infection

Population density

Parental

investment

Defensive Strategies

Foraging/Metabolism

Reproductive Strategies

Environmental Parental Developmental

condition signal outcome

Evolutionary biology - Maternal effects

Documents

Early Environmental Regulation of Gene Expression: How ...Sept_2013... · Early Environmental Regulation of Gene Expression: How Early Experience Exerts a Sustained Influence on Neuronal