How do transcriptional networks rewire neuronal circuits?

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 encoded by the genome. Experience… rewires circuits… via cell biological modifications…

The genome responds to neuronal activity with bursts of new gene expression Standard housing Enriched environment c/o Alan Mardinly Npas4 NeuN DAPI

Activity-dependent bursts of new gene expression are required for circuit rewiring • Promoter IV: • CRE mKI Promoter IV: control The Bdnf locus Hong et al., Neuron (2008)

How does the genome respond to neuronal activity? Ras Raf MEK ERK RSK CREB SRF L-type Voltage- Sensitive Calcium Channels NMDA receptor Ca2+ Ca2+ CamKIV CaMKII CBP P62/ELK Transcription of Plasticity effector genes CBP

A complex network of transcription factors drives activity-regulated transcription Inducible (IEG) TFs AP-1 (Fos/Jun) family Egr family Nr4a family Npas4 What are the cis-acting and trans-acting elements? Neuronal activity (calcium) Post-translationally Modified TFs Effector genes Plasticity Creb Srf Mef2 Arc Bdnf Homer1a

Seminar outline Identification of thousands of new cis-acting elements. Investigation of the mechanisms of cis-acting element function. 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 • KCl • ChIP-Seq • RNA-Seq + KCl ChIP-Seq RNA-Seq neuronal activation via KCl depolarization mouse cortical neurons

Extragenic CBP and transcription factor binding at the fos locus KCl conservation KCl fos gene conservation promoter 20 kb

Conclusions from the Fos locus TF binding is both genic and extragenic. TF binding can be inducible or constitutive. CBP binding is predominantly extragenic CBP binding is overwhelmingly activity-dependent conservation

Questions about activity-regulated CBP-bound loci Is extragenic, inducible CBP-binding a general phenomenon? 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 (KCl) CBP binding (unstimulated)

Properties of activity-regulated CBP-bound loci Extragenic activity-regulated CBP binding is a general phenomenon. 25,000 non-promoter sites. What is the nature of these CBP-bound loci?

Do extragenic CBP-bound sites function as transcriptional enhancers, promoters, or neither? CBP CBP CBP CBP SRF RNAPII RNAPII CREB CREB SRF RNAPII Enhancer H3K4me1 Promoter H3K4me3 ChIP-Seq: H3K4me3 H3K4me1 ENCODE, 2007 Heintzman et al, 2007 Roh et al, 2005 Visel et al, 2009

H3K4me1 is present at extragenic CBP sites H3K4me1 CBP 12,000 enhancers Defined by CBP and H3K4me1! CBP CBP CBP

Do CBP and H3K4Me1-marked loci function as enhancers? Arc proximal promoter Arc (or other) enhancer 7kb Arc upstream region Luciferase coding sequence Kawashima et al, 2008 Pintchovsky et al, 2009

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 Extragenic activity-regulated CBP binding is a general phenomenon. 25,000 non-promoter sites. An estimated 12,000 of these sites are enhancers.

Questions about activity-regulated enhancers Before neuronal activation After neuronal activation NPAS4 Enhancer (H3K4me1) Enhancer H3K4me1 CBP CREB SRF CREB 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 RNAPII 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 CBP H3K4me1 RNAPII H3K4me3 Do enhancers bind RNA Polymerase II (RNAPII)? YES

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 1 hr sense antisense 6 hr sense antisense total RNA mRNA

Enhancer RNAs are transcribed bidirectionally from CBP-bound enhancer centers

Enhancer transcription is correlated globally with promoter transcription Induction index: (KCl - unstimulated) / (KCl + unstimulated) R2 = 0.8 Induction = (KCl - unstim) / (KCl + unstim)

Questions about activity-regulated enhancers RNAPII Enhancer 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; KCl- WT; KCl+ Arc promoter-/-; KCl- Arc promoter-/-; KCl+ Arc+/+, KCl+ Arc-/-, unstim Arc-/-,KCl+

Questions about activity-regulated enhancers RNAPII Enhancer 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 RNAPII Enhancer 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 RNAPII Enhancer RNAPII Promoter eRNA transcription is required to modify enhancer chromatin. eRNA transcription is an epiphenomenon with no function. eRNA transcripts function in trans to regulate gene expression.

Enhancer RNAs coincide with the H3K4me1 modification - strand RNA + strand RNA H3K4me1 H3K4me1 binding

Possible functions for eRNA transcription RNAPII Enhancer RNAPII Promoter eRNA transcription is required to modify enhancer chromatin. eRNA transcription is an epiphenomenon with no function. eRNA transcripts function in trans to regulate gene expression.

Genomics is currently in a great descriptive wave

How do transcriptional networks rewire neuronal circuits? Big questions. What are the trans- and cis-acting components? What is the wiring diagram? How do different factors cooperate to induce effector genes? Are there plasticity rules encoded in transcriptional logic? How does the network make decisions or implement circuit rewiring?

Future directions Assay gene expression (using RNA-Seq and high-throughput qPCR) • How does the activity-regulated transcriptional network process information? Control activity (using light-gated ion channels)

Future directions • How does the activity-regulated transcriptional network process information? • How does each inducible transcription factor contribute to effector gene induction?

Future directions 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 does the activity-regulated transcriptional network process information? • How does each inducible transcription factor contribute to effector gene induction? • How does the activity-regulated network contribute to homeostatic scaling?

How do transcriptional networks rewire circuits to store memories? Standard housing Npas4 NeuN DAPI

How do transcriptional networks rewire neuronal circuits?