How do transcriptional networks rewire
neuronal circuits?
Jesse Gray
Neurobiology department
Harvard 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
Standard
housing
Npas4
NeuN
DAPI
Enriched
environment
c/o Alan Mardinly
Activity-dependent bursts of new gene expression are
required for circuit rewiring
The Bdnf locus
Promoter IV:
CRE mKI
Promoter IV:
control
Hong et al., Neuron (2008)
How does the genome respond to neuronal activity?
L-type
VoltageSensitive
Calcium
Channels
NMDA
receptor
Ras
Ca2+
MEK
ERK
CaMKII
CBP
Raf
Ca2+
CamKIV
RSK
P62/ELK
CBP
SRF
CREB
Transcription of
Plasticity
effector genes
A complex network of transcription factors drives
activity-regulated transcription
Inducible
(IEG) TFs
What are the cis-acting
and trans-acting elements?
Neuronal
activity
(calcium)
AP-1 (Fos/Jun) family
Egr family
Nr4a family
Npas4
Post-translationally
Modified TFs
Effector
genes
Creb
Srf
Mef2
Arc
Bdnf
Homer1a
Plasticity
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
+ KCl
ChIP-Seq
RNA-Seq
ChIP-Seq
RNA-Seq
Extragenic CBP and transcription factor binding at the
fos locus
KCl
fos gene
conservation
promoter
20 kb
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 activitydependent
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
CBP binding (KCl)
To what extent is CBP-binding to the genome activityregulated?
CBP binding (unstimulated)
Properties of activity-regulated CBP-bound loci
(1) Extragenic activityregulated CBP binding is a
general phenomenon.
25,000 non-promoter
sites.
(2) What is the nature of these
CBP-bound loci?
Do extragenic CBP-bound sites function as
transcriptional enhancers, promoters, or neither?
CBP
CBP
CREB
SRF
Enhancer
H3K4me1
Promoter
H3K4me3
ChIP-Seq:
CBP
SRF
CBP
RNAPII
H3K4me3
H3K4me1
RNAPII
RNAPII
CREB
ENCODE, 2007
Heintzman et al, 2007
Roh et al, 2005
Visel et al, 2009
H3K4me1 is present at extragenic CBP sites
H3K4me1
CBP
CBP
CBP
CBP
12,000 enhancers
Defined by CBP and
H3K4me1!
Do CBP and H3K4Me1-marked loci function as
enhancers?
Arc (or other) enhancer
7kb Arc upstream region
Arc proximal
promoter
Luciferase
coding
sequence
Kawashima et al, 2008
Pintchovsky et al, 2009
CBP and H3K4Me1-marked loci function as activitydependent transcriptional enhancers
25
K C l in d u c tio n (lu c ife ra se a c tiv ity )
without promoter
with promoter
20
15
10
5
0
no
enhancer
Arc
enhancer
E1
E2
E3
E4
E5
E6
E7
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 activityregulated 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 enhancers
Before neuronal activation
Enhancer
(H3K4me1)
CREB
SRF
After neuronal activation
Enhancer
H3K4me1
NPAS4
CREB
CBP
SRF
?
RNAPII
Promoter
(H3K4me3)
Promoter
H3K4me3
Do activity-regulated enhancers bind RNA Polymerase II (RNAPII)?
RNA Polymerase II at enhancers:
Masternak et al., Nature Immunology 2003
Tuan et al, PNAS 1992
Heintzmann et al, Nature Genetics 2007
fos enhancers bind RNA Polymerase II
ChIP:
fos promoter
Questions about activity-regulated enhancers
CBP
H3K4me1
RNAPII
RNAPII
H3K4me3
Do enhancers bind RNA Polymerase II (RNAPII)? YES
Does RNAPII at enhancers synthesize RNA?
Transcription at enhancers:
Tuan et al, PNAS 1992
Masternak et al., Nature Immunology 2003
Wang et al, Nature Genetics 2008
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
0 hr
sense
antisense
total RNA
1 hr
sense
antisense
mRNA
6 hr
sense
antisense
Enhancer RNAs are transcribed bidirectionally from
CBP-bound enhancer centers
Enhancer transcription is correlated globally with
promoter transcription
Induction =
index:
Induction
(KCl - unstim) / (KCl + unstim)
(KCl - unstimulated) / (KCl + unstimulated)
R2 = 0.8
Questions about activity-regulated enhancers
Enhancer
RNAPII
RNAPII
Promoter
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+/+, unstim
WT; KClArc+/+, KCl+
WT; KCl+
Arc-/-, unstim
Arc promoter-/-;
KClArc promoter-/-;
KCl+
Arc-/-, KCl+
Questions about activity-regulated enhancers
Enhancer
RNAPII
RNAPII
Promoter
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?
Arc eRNA induction depends on the Arc promoter
WT
Arc promoter -/-
Questions about activity-regulated enhancers
Enhancer
RNAPII
RNAPII
Promoter
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
Possible functions for eRNA transcription
Enhancer
RNAPII
RNAPII
Promoter
(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 RNAs coincide with the H3K4me1 modification
+ strand
RNA
H3K4me1
H3K4me1 binding
- strand
RNA
Possible functions for eRNA transcription
Enhancer
RNAPII
RNAPII
Promoter
(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.
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 cisacting 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 (using
light-gated ion channels)
Assay gene expression
(using RNA-Seq and
high-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?
Surface glutamate receptor (GluA2-YFP) levels
increase upon activity blockade
0 hr
1 hr
2 hr
3 hr
4 hr
Neuronal
activity
block
(TTX)
Ibata et al., Neuron 2008
How do transcriptional networks rewire circuits to
store memories?
Standard
housing
Npas4
NeuN
DAPI
Neuronal
activityregulated
enhancers
Acknowledgements
Greenberg laboratory
Harvard Medical
School
Athar Malik
Tae-Kyung Kim
Brenda Bloodgood
Allen Costa
Joseph Ling
Eirene Markenscoff-Papadimitriou
Dan Bear
Mike Laptewicz
Shannon Robichaud
Janine Zieg
Michael Greenberg Eric Griffith
mRNA dynamics from a steadystate RNA-Seq snapshot
Gabriel Kreiman
Children’s Hospital Boston
Paul Worley lab
Johns Hopkins
Jing Wu
Molecular Genetics Core
Children’s Hospital Boston
Kellie Haley
Hal Schneider
Harvard Medical School
Biopolymer facility
Kristin Waraska
Robert Steen
Life Technologies
Scott Kuersten
Gina Costa
Kevin McKernan
Martin Hemberg
Mike Springer
David Harmin
The Helen Hay Whitney foundation