Eric J. Nestler

Mount Sinai School of Medicine

New York, NY

Transcriptional and Epigenetic

Mechanisms of Addiction

Dr. Ray Fuller

“There is every reason to

be optimistic that in the

future we will find even

better ways of modifying

the function of specific

parts of the brain which will

be useful in treating a

variety of psychiatric and

neurological diseases.”

Drugs mimic neurotransmitters

by activating receptors:

• Morphine

• Nicotine• Marijuana

Drugs block the

dopamine pump:

• Cocaine

• Amphetamine

Drugs activate or

inhibit channels:

• Alcohol

• PCP, ketamine

Drugs of Abuse Act

at the Synapse

Second messengers

& protein

phosphorylation

Regulation of many

cellular processes

Transcription factors

Stable adaptations

in neural function

Target genes

Drugs Addiction: Drug-Induced

Neural Plasticity Mediated Via

Altered Gene Expression

Drugs

Transporters

Channels

Receptors

Chromatin Studies

Offer Major Advances

• Help identify drug-

regulated genes.

• First ever look at

transcriptional

mechanisms in vivo.

• Unique mechanisms

of long-lasting

adaptations.

Regulation of Gene Expression is

Reflected at the Chromatin Level

Active (open) Inactive (condensed)

HDAC

HMT

HAT

SWI-SNF

Basal

transcription

complex

Histone

N-termini

Nucleosome

Transcription factors

Repressors

DNMT

Distinct Temporal Properties of

Drug-Induced Transcription Factors in NAc

Induction

Inductio

n

Time (hr) Time (hr)

Time (days) Time (days)

2 6 12 2 6 12

2 4 6 8 2 4 6 8

∆FosB CREB

Mediated by CREB

phosphorylation

and ATF induction

c-Fos

Other Fos family

proteins

Stable ∆FosB

isoforms

Accumulating,

persisting ∆FosB Rapid

normalization

Unique Induction of ∆FosB in the NAc

by Chronic Drug Administration

52-58 kD (c-Fos)

46-50 kD (FosB)

40 kD (?Fra1, Fra2)

35-37 kD (modified ∆FosB)

33 kD (unmodified ∆FosB)

High levels of ∆FosB

are induced uniquely

by chronic drug

exposure, creating

a molecular switch.

Behavioral Plasticity Mediated by

Drug-Induced Transcription Factors

Transcription factors mediate distinct aspects of the drug

addiction phenotype.

∆FosB mediates drug sensitization:

• Increases sensitivity to drug and natural rewards.

• Mediates a positive emotional and motivational state.

• Drives drug craving and relapse (positive reinforcement).

CREB mediates drug tolerance and dependence:

• Reduces sensitivity to drug and natural rewards.

• Mediates a negative emotional state during drug withdrawal.

• Drives drug craving and relapse (negative reinforcement).

Bill Carlezon, David Self

mRNA

Effect of chronic cocaine

Identify mRNA’s and non-coding RNA’s (e.g., miRNA’s)

regulated in NAc by chronic drug exposure.

Drug Regulation of Gene Expression:

DNA Microarrays or RNA-seq

mRNA

Repressive H3

methylation (K9, K27)

DNA methylation

Acetylated H3 or H4

Activational H3

methylation (K4)

Overlay chromatin modifications on regulated mRNA’s to improve

accuracy of detection and reveal underlying mechanisms.

Drug Regulation of Chromatin:

ChIP-chip Arrays or ChIP-seq

• Theoretical results:

mRNA

Repressive H3

methylation (K9, K27)

DNA methylation

Acetylated H3 or H4

Activational H3

methylation (K4)

Overlay transcription factor binding to further reveal underlying

mechanisms.

Other∆FosB

CREB

Role of Transcription Factors in Drug

Regulation of Gene Expression

783 221 471

80 3 120

178 31 1444

889 9 194

acH3 acH4

acH3 acH4

me2K9H3 acH3/H4

me2K9H3 acH3/H4

Largely non-overlapping mechanisms of histone modifications

associated with chronic cocaine regulation of gene expression

Histone Modifications Induced in the

NAc by Chronic Cocaine

Genes up-

or downregulated

by cocaine

∆FosB accounts for >25% of all genes regulated in the NAc by

chronic cocaine administration:

Regulation of Gene Expression in

the NAc by ∆FosB and CREB

∆FosB

8%

26%

CREB

Complementary use of ChIP-

chip and gene expression

arrays identifies genes

activated or repressed by

chronic cocaine via ∆FosB.

1355

198 129

769

acH3/4me2K9H3

∆FosB

Chromatin Regulation Helps Identify Genes

↑‘d or ↓‘d by Cocaine and ∆FosB

∆FosB

Enrichment

∆FosB over-

expression

∆cJun over-

expression

∆FosB CREB

Glutamatergic, GABAergic, GluR2, Arc GluR1, NR1

& synaptic plasticity GABAA 2 Piccolo

Neuronal excitability Nav, Kv, Cav Nav, Kv, Cav

CDK5, WASPs Tropomodulin,

Actin BPs, MAP2

Dynorphin Dynorphin, CRFR1

NK1 CART, CCK,

BDNF, TrkB

NF B, c-Fos MEF2, JmjC

G9a

Examples of Target Genes for CREB

and ∆FosB in the NAc

Structural plasticity

Transcriptional regulators

Neuromodulators

& growth factor pathways

∆FosB CREB

Glutamatergic, GABAergic, GluR2, Arc GluR1, NR1

& synaptic plasticity GABAA 2 Piccolo

Neuronal excitability Nav, Kv, Cav Nav, Kv, Cav

CDK5, WASPs Tropomodulin,

Actin BPs, MAP2

Dynorphin Dynorphin, CRFR1

NK1 CART, CCK,

BDNF, TrkB

NF B, c-Fos MEF2, JmjC

G9a

Examples of Target Genes for CREB

and ∆FosB in the NAc

Structural plasticity

Transcriptional regulators

Neuromodulators

& growth factor pathways

ChIP Reveals the In Vivo Mechanism

of Cdk5 Gene Activation in the NAc

1

0.5

0

2

3

Chronic cocaine induces H3 acetylation, SWI-SNF

binding, and ∆FosB binding to the Cdk5 promoter

∆FosB alone induces

marks of activation

acH3 acH4 Brg1 Brg2 ∆FosB FosB acH3 Brg1

Saline

Cocaine*

**

*

∆FosB off

∆FosB on*

Bin

din

g to

Cdk5

pro

mo

ter

(fo

ld c

ha

ng

e o

ve

r co

ntr

ol)

Histone

acetylation

SWI-SNF

factors

Cdk5 promoter

ChIP reveals the mechanism of Cdk5 gene induction in the NAc

in vivo:

Cdk5 promoter

(basal state)

∆FosB Induction of Cdk5 Expression

HDAC

∆FosB Induction of Cdk5 Expression

ChIP reveals that the Cdk5 gene is induced in the NAc via:

• Direct binding of ∆FosB to the Cdk5 promoter.

Cdk5 promoter

∆FosB

HDAC

∆FosB Induction of Cdk5 Expression

ChIP reveals that the Cdk5 gene is activated in the NAc via:

• Direct binding of ∆FosB to the Cdk5 promoter.

• Recruitment of HATs & coactivators, and exclusion of

HDACs, causing increased H3 acetylation.

∆FosB

HAT

SWI-SNF

Cdk5 promoter

(activated state)

HDAC

∆FosB Repression of c-Fos Expression

c-Fos promoter

(basal permissive state)

ChIP reveals the mechanism of c-Fos gene repression in the NAc in

vivo:

∆FosB Regulation of c-Fos Expression

ChIP reveals that the c-Fos gene is repressed in the NAc via:

• Direct binding of ∆FosB to the c-Fos promoter.

c-Fos promoter

∆FosB

∆FosB Regulation of c-Fos Expression

ChIP reveals that the c-Fos gene is repressed in the NAc via:

• Direct binding of ∆FosB to the c-Fos promoter.

• ∆FosB recruitment of HDAC1, causing H4 deacetylation.

• Increased methylation of K9H3 and induction of HMTs

independent of ∆FosB.

c-Fos promoter

(repressed state)

HDAC1

∆FosBHMT

Chronic cocaine induces several global changes in chromatin

modifications in the NAc that promote gene expression:

• Increased histone acetylation (acH3) in NAc.

- Mediated by downregulation of HDAC5.

• Decreased repressive histone methylation (meK9H3) in NAc.

- Mediated by downregulation of G9a, a meK9H3 HMT.

- No persistent changes in K4 or K27 methylation.

• Decreased DNA methylation in NAc.

- Mediated by downregulation of DNMT3a.

In each case, these permissive changes promote cocaine’s

behavioral effects.

Global Changes in Chromatin

Modifications After Chronic Cocaine

Chronic Cocaine Induces a

Permissive State of Gene Regulation

More genes are induced after chronic than acute cocaine:

Acute

Chronic + acute

Chronic + 1-wk-wd + acute

Acute

Chronic + acute

Chronic +

1-wk-wd + acute

0 50 100 150 200 250 300

# Significantly upregulated genes

Acute

Chronic + acute

Chronic + 1-wk-wd + acute

Chronic Cocaine, via ∆FosB,

Represses meK9H3 and G9a in NAc

me2K9H3 G9a

1

-2Fo

ld c

hange

**

Cocaine decreases

levels of me2K9H3

and G9a expression

Acute

Chronic

me2K9H3 G9a

1

-2Fo

ld c

han

ge

**

∆FosB off

∆FosB onThese effects are

mediated by ∆FosB

me3K9H3

Control

Chronic cocaine

me3K9H3 is a marker

of heterochromatin:

chronic cocaine alters

the amount of hetero-

chromatin in NAc

neuronal nuclei.

Repression of meK9H3 Enhances

Behavioral Responses to Cocaine

G9a inhibition in NAc enhances cocaine’s behavioral effects:

Veh BIX

01294

100

0

Dru

g s

ide -

salin

e s

ide (

sec)

*

200

300

400

GFP Cre

100

0

Dru

g s

ide -

salin

e s

ide (

sec) *

200

300

400

AAV vectors

in floxed G9a mice

GFP G9a Merge

AA

V-G

FP

AA

V-C

re

me2K9H3

-tubulin

me2K9H3

-tubulin

GFP Cre

Veh BIX

floxed G9a mice

Induction of meK9H3 Suppresses

Behavioral Responses to Cocaine

G9a overexpression in NAc reduces cocaine’s behavioral effects:

GFP G9a mutG9a

*100

0

200

300

400

Dru

g s

ide -

salin

e s

ide (

sec)

HSV vectors

in wildtype mice

*

1

0

2

3

400

Fo

ld m

e2K

9H

3 4

me2K9H3

-tubulin

me2K9H3

-tubulin

G9a - +

mutG9a - +

ac

NAc

∆FosB CREB

Glutamatergic, GABAergic, GluR2, Arc GluR1, NR1

& synaptic plasticity GABAA 2 Piccolo

Intrinsic excitability Nav, Kv, Cav Nav, Kv, Cav

CDK5, WASPs Tropomodulin,

Actin BPs, MAP2

Dynorphin Dynorphin, CRFR1

NK1 CART, CCK,

BDNF, TrkB

NF B, c-Fos MEF2, JmjC

G9a

Examples of Target Genes for CREB

and ∆FosB in the NAc

Structural plasticity

Neuromodulators

& growth factor pathways

Transcriptional regulators

Numerous ∆FosB

targets are implicated

in dendritic growth

Mining ChIP-chip and Expression Data

to Understand Structural Plasticity

Cocaine

∆FosB

↓

Actin-binding

proteins

Wasps,

WavesNF B CDK5

Ubiquitin

signalingArc

Many

others

Regulation of the actin cytoskeleton and

induction and stabilization of dendritic spines

↓

CREB, others

MEF2

∆FosB Mediates Cocaine-Induced

Structural Changes in NAc Neurons

Viral expression of ∆FosB

in NAc mimics cocaine-

induced increases in spine

density, while ∆JunD blocks

cocaine action

# s

pin

es/1

0

m

0

5

10

15

20

25

30

GFP ∆FosB ∆JunD

Saline

Cocaine

*

*

*

GFP

∆FosB

Numerous ∆FosB

targets are implicated

in dendritic growth

Mining ChIP-chip and Expression Data

to Understand Structural Plasticity

Cocaine

∆FosB

↓

Actin-binding

proteins

Wasps,

WavesNF B CDK5

Ubiquitin

signalingArc

Many

others

Regulation of the actin cytoskeleton and

induction and stabilization of dendritic spines

↓

CREB, others

MEF2

Role for ∆FosB in Cocaine Induced

Structural Changes in NAc Neurons

Repeated drug

exposure

(e.g., via ∆FosB &

numerous target genes

Use-dependent plasticity causing

sensitized responses to drugs and

environmental cues

Normal responses to drugs

• Gene and chromatin arrays provide an unprecedented view of

the transcriptional mechanisms underlying chronic drug action

in the NAc.

• This work is defining complex biochemical pathways underlying

drug action.

• It is crucial to carry out equivalent studies of other brain regions

to define circuit level links between cells and behavior.

• Look at multiple adaptations in concert, not individually.

• Eventually use this information to develop fundamentally novel

diagnostic and treatment approaches for drug addiction.

Summary and Future Directions

My lab