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
Standardhousing
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?
Npas4 NeuN DAPI
Standardhousing
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