How do transcriptional networks rewire neuronal circuits?

Jesse GrayNeurobiology departmentHarvard Medical School

Animals store life-long memories

Neuronal circuits are rewired by experience according to genomic instructions

Experience…

rewires circuits…

via cell biological modifications…

encoded by the genome.

The genome responds to neuronal activity with bursts of new gene expression

Npas4 NeuN DAPI

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Enrichedenvironment

c/o Alan Mardinly

Activity-dependent bursts of new gene expression are required for circuit rewiring

Hong et al., Neuron (2008)

The Bdnf locus

Promoter IV:control

Promoter IV: CRE mKI

How does the genome respond to neuronal activity? L-type

Voltage-SensitiveCalciumChannels

NMDAreceptor

Ca2+Ca2+

CBP

CaMKII CamKIV

RasRafMEKERKRSK

Transcription ofPlasticityeffector genesCREB

CBP P62/ELK

SRF

A complex network of transcription factors drives activity-regulated transcription

PlasticityNeuronalactivity(calcium)

Post-translationallyModified TFsCrebSrfMef2

AP-1 (Fos/Jun) familyEgr familyNr4a familyNpas4

EffectorgenesArcBdnfHomer1a

Inducible (IEG) TFs

What are the cis-actingand trans-acting elements?

Seminar outline

(1) Identification of thousands of new cis-acting elements.

(2) Investigation of the mechanisms of cis-acting element function.

(3) Future directions: How does this transcriptional network rewire circuits?

Where do TFs bind? (Chromatin immunoprecipitation sequencing, ChIP-Seq)

An experimental system for genome-wide analysis of activity-regulated gene expression

neuronal activation via KCl depolarization

mouse cortical neurons

- KCl

ChIP-SeqRNA-Seq

+ KCl

ChIP-SeqRNA-Seq

promoter

Extragenic CBP and transcription factor binding at the fos locus

20 kb

conservationfos gene

KCl

conservation

KCl

Conclusions from the Fos locus

(1) TF binding is both genic and extragenic.

(2) TF binding can be inducible or constitutive.

(3) CBP binding is predominantly extragenic

(4) CBP binding is overwhelmingly activity-dependent

conservation

Questions about activity-regulated CBP-bound loci

(1) Is extragenic, inducible CBP-binding a general phenomenon?

(2) What is the nature of these extragenic CBP-bound loci?

Where in the genome does CBP bind?

CBP binds predominantly outside promoter regions

To what extent is CBP-binding to the genome activity-regulated?

CBP binding (unstimulated)

CBP

bind

ing

(KCl

)

Properties of activity-regulated CBP-bound loci

(1) Extragenic activity-regulated CBP binding is a general phenomenon. 25,000 non-promoter sites.

(2) What is the nature of these CBP-bound loci?

CREB

Do extragenic CBP-bound sites function as transcriptional enhancers, promoters, or neither?

PromoterH3K4me3

EnhancerH3K4me1

RNAPIISRF

CBPCBP

RNAPII

CREB

RNAPIICBP

SRFCBP

ENCODE, 2007Heintzman et al, 2007Roh et al, 2005Visel et al, 2009

ChIP-Seq:

H3K4me3H3K4me1

CBP

CBP

CBP

CBPH3K4me1

H3K4me1 is present at extragenic CBP sites

12,000 enhancersDefined by CBP and H3K4me1!

Luciferasecoding sequence

7kb Arc upstream region

Arc (or other) enhancerArc proximal promoter

Do CBP and H3K4Me1-marked loci function as enhancers?

Kawashima et al, 2008Pintchovsky et al, 2009

0

5

10

15

20

25

noenhancer

Arcenhancer

E1 E2 E3 E4 E5 E6 E7

KCl induction (luciferase activity)

without promoterwith promoter

CBP and H3K4Me1-marked loci function as activity-dependent transcriptional enhancers

Are CBP-bound loci evolutionarily conserved?

Conserved non-coding “islands” are mostly regulatory factor binding sites, not non-coding RNAs

Properties of activity-regulated CBP-bound loci

(1) Extragenic activity-regulated CBP binding is a general phenomenon. 25,000 non-promoter sites.

(2) An estimated 12,000 of these sites are enhancers.

Seminar outline

(1) Identification of thousands of new cis-acting elements.

(2) Investigation of the mechanisms of cis-acting element function.

(3) Future directions: How does this transcriptional network rewire circuits?

Questions about activity-regulated enhancersBefore neuronal activation

Enhancer(H3K4me1)

Promoter(H3K4me3)

SRFCREB

After neuronal activation

EnhancerH3K4me1

PromoterH3K4me3

NPAS4

SRFCBP

CREB

RNAPII

RNA Polymerase II at enhancers:Masternak et al., Nature Immunology 2003Tuan et al, PNAS 1992Heintzmann et al, Nature Genetics 2007

Do activity-regulated enhancers bind RNA Polymerase II (RNAPII)?

?

fos enhancers bind RNA Polymerase II

ChIP:

fos promoter

Questions about activity-regulated enhancers

CBPH3K4me1

H3K4me3

RNAPII

RNAPII

Do enhancers bind RNA Polymerase II (RNAPII)? YES

Transcription at enhancers:Tuan et al, PNAS 1992Masternak et al., Nature Immunology 2003Wang et al, Nature Genetics 2008

Does RNAPII at enhancers synthesize RNA?

What genomic loci are transcribed before and after neuronal activation (RNA-Seq)?

Little extragenic transcription observed in polyA+ RNA

total RNA

mRNA

RNA-Seq for detection of non-polyadenylated RNA

Enhancers at the fos locus produce enhancer RNAs

total RNA

mRNA

0 hr

1 hr

6 hr

senseantisense

senseantisense

senseantisense

Enhancer RNAs are transcribed bidirectionally from CBP-bound enhancer centers

Enhancer transcription is correlated globally with promoter transcription

Induction = (KCl - unstim) / (KCl + unstim)

R2 = 0.8

Induction index:(KCl - unstimulated) / (KCl + unstimulated)

Questions about activity-regulated enhancers

Enhancer

Promoter

RNAPII

RNAPII

Do enhancers bind RNA Polymerase II (RNAPII)? YES

Does RNAPII at enhancers transcribe DNA into RNA? YES

Can enhancers independently recruit RNAPII?eRNAs in other cell types:Natoli laboratory (Milan)Wysocka laboratory (Stanford)

The Arc gene and enhancer locus

The Arc enhancer can recruit RNAPII without the presence of the Arc promoter

Arc+/+, unstimArc+/+, KCl+Arc-/-, unstimArc-/-, KCl+

WT; KCl-WT; KCl+Arc promoter-/-; KCl-Arc promoter-/-; KCl+

Questions about activity-regulated enhancers

Do enhancers bind RNA Polymerase II (RNAPII)? YES

Does RNAPII at enhancers transcribe DNA into RNA? YES

Can enhancers independently recruit RNAPII? YES

Can enhancers independently transcribe eRNAs?

Enhancer

Promoter

RNAPII

RNAPII

Arc eRNA induction depends on the Arc promoter

WT Arc promoter -/-

Questions about activity-regulated enhancers

Do enhancers bind RNA Polymerase II (RNAPII)? YES

Does RNAPII at enhancers transcribe DNA into RNA? YES

Can enhancers independently recruit RNAPII? YES

Can enhancers independently transcribe eRNAs? NO

Enhancer

Promoter

RNAPII

RNAPII

Possible functions for eRNA transcription

(1) eRNA transcription is required to modify enhancer chromatin.

(2) eRNA transcription is an epiphenomenon with no function.

(3) eRNA transcripts function in trans to regulate gene expression.

Enhancer

Promoter

RNAPII

RNAPII

Enhancer RNAs coincide with the H3K4me1 modification

+ strandRNA

- strand RNA

H3K4me1

H3K

4me1

bin

ding

Possible functions for eRNA transcription

(1) eRNA transcription is required to modify enhancer chromatin.

(2) eRNA transcription is an epiphenomenon with no function.

(3) eRNA transcripts function in trans to regulate gene expression.

Enhancer

Promoter

RNAPII

RNAPII

Seminar outline

(1) Identification of thousands of new cis-acting elements.

(2) Investigation of the mechanisms of cis-acting element function.

(3) Future directions: How does this transcriptional network rewire circuits?

Genomics is currently in a great descriptive wave

How do transcriptional networks rewire neuronal circuits? Big questions.

(1) What are the trans- and cis-acting components?

(2) What is the wiring diagram?

(3) How do different factors cooperate to induce effector genes?

(4) Are there plasticity rules encoded in transcriptional logic?

(5) How does the network make decisions or implement circuit rewiring?

Future directions

(1) How does the activity-regulated transcriptional network process information?

Control activity (usinglight-gated ion channels)

Assay gene expression(using RNA-Seq andhigh-throughput qPCR)

Future directions

(1) How does the activity-regulated transcriptional network process information?

(2) How does each inducible transcription factor contribute to effector gene induction?

Future directions

(1) How does the activity-regulated transcriptional network process information?

(2) How does each inducible transcription factor contribute to effector gene induction?

(3) How does the activity-regulated network contribute to homeostatic scaling?

Ibata et al., Neuron 2008

Neuronalactivityblock(TTX)

0 hr 2 hr1 hr 3 hr 4 hr

Surface glutamate receptor (GluA2-YFP) levels increase upon activity blockade

How do transcriptional networks rewire circuits to store memories?

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Acknowledgements

Life TechnologiesScott Kuersten

Gina CostaKevin McKernan

Molecular Genetics CoreChildren’s Hospital Boston

Kellie HaleyHal Schneider

Harvard Medical SchoolBiopolymer facility

Kristin WaraskaRobert Steen

The Helen Hay Whitney foundationMartin Hemberg

Paul Worley labJohns Hopkins

Jing Wu

Tae-Kyung Kim Michael Greenberg

Gabriel KreimanChildren’s Hospital Boston

Athar MalikBrenda BloodgoodAllen CostaJoseph LingEirene Markenscoff-PapadimitriouDan BearMike LaptewiczShannon RobichaudJanine ZiegEric Griffith

Mike Springer

Greenberg laboratoryHarvard Medical School

David Harmin

mRNA dynamics from a steady-state RNA-Seq snapshot

Neuronal activity-regulated enhancers