0

50

100

0

50

100

Instance 1Instance 2

WT Mut 1 Mut 2 Mut 3 Mut 4 Mut 5

Rat

io to

pG

L4.2

3LIC

G_NEUROD1_01

AG

A

CGCAGC

ATGAG

GC

G_NF1_03

GCT

C

G

A

T

CAT

TCTGC

GCTA

AGC

J_NFIC

C

A

TGT

CT

G

A

CC

AC

T

GA

J_TLX1_NFIC

CATTGGC

C

G

T

A

A

CT

T

A

C

GA

T

CG

GATGCCA

Nfi Nfi dimer Neurod/g Nfi

0

2000

4000

6000

0 5 10 15 20

Number of Repeat Instances Overlapped

Fre

qu

en

cy

Observed

Overlap

Expected Overlap

** ** ** *

** ** **

The Genomics of Neurodevelopment: Transcriptional Networks Underlying the Developing Neocortex

James H. Notwell1, Aaron M. Wenger1, Shoa L. Clarke2, Tisha Chung3, Geetu Tuteja3, Harendra Guturu4, Whitney Heavner3,5, Bruce T. Schaar3, Gill Bejerano1,3

Departments of 1Computer Science, 2Genetics, 3Developmental Biology, 4Electrical Engineering, and 5Biology, Stanford University

shuffled p300 x 10,000

genome

p300

repeat family

O

OOO

O

O

O

O

O

O

OOO

O

OOOOO

O

O

O

O

OO

OO

O

O

O O O O

O

0

20

40

60

E11.5

fore

bra

in

E11.5

heart

E11.5

lim

b

E11.5

midbra

in

E14.5

neoco

rtex

Ob

se

rve

d /

Exp

ecte

d

MER130

UCON11

UCON31

MER124

MER121

MER131

B

A

HumanDog

OpossumChicken

FrogZebrafish

SINELINELTRDNA

20 kb mm913,150,000 13,170,000 13,190,000 13,210,000

BAC End Pairs

UCSC Genes

Enhancer Candidates Based on Conservation

Placental Mammal Conservation by PhastCons

Multiz Alignments of 30 Vertebrates

E14.5 Dorsal Cerebral Wall p300 ChIP-seq Signal

E14.5 Dorsal Cerebral Wall Input ChIP-seq Signal

E14.5 Dorsal Cerebral Wall p300 ChIP-seq peaks

Repeating Elements by RepeatMasker

RP23-141E17

Fezf2 Cadps

E4 E3E2

E1

e14dcw_p300.1848

HumanDog

OpossumChicken

X_tropicalisZebrafish

SINELINELTRDNA

20 kb mm961,620,000 61,640,000 61,660,000 61,680,000

UCSC Genes

Placental Mammal Conservation by PhastCons

Multiz Alignments of 30 Vertebrates

E14.5 Dorsal Cerebral Wall p300 ChIP-seq Signal

E14.5 Dorsal Cerebral Wall Input ChIP-seq Signal

E14.5 Dorsal Cerebral Wall p300 ChIP-seq peaks

Repeating Elements by RepeatMasker

Psmd14Tbr1

e14dcw_p300.3376

AmnSine1

Mouse July 2007 (NCBI37/mm9)

Mouse July 2007 (NCBI37/mm9)

Key Transcription Factors Control Neocortical Development

Auts2, we investigated whether the loss of Tbr1 expression inupper layer neurons in Satb2LacZ/LacZ mutants coincides withchanges in the expression of Auts2. We observed a striking lossof Auts2 expression in the upper layers of Satb2 mutants (Fig. 7M and N), similar to the loss of Tbr1 in Satb2 mutants (Fig. 5B);there was no change in Auts2 expression in layers 5 or 6 (Fig. 7Mand N) relative to controls (Fig. 7 K and L). These data areconsistent with the possibility that Satb2 regulates the expressionof Tbr1, which in turn is required for Auts2 expression in callosalprojection neurons. These results may have implications for theetiology of autism.

Expression of EphA4 and Unc5H3 Restores Callosal Projections inSatb2 Mutants. Previously we identified several genes that showaltered expression in Satb2LacZ/LacZ mutants (9). In particular,three axonal guidance molecules (EphA4, PlxnA4, and Unc5H3)are down-regulated in upper layers of mice lacking Satb2. Priorstudies have implicated Ephs and ephrins in callosal develop-ment (13, 28, 29). EphA4 is normally expressed in upper layercallosal neurons and the glial wedge (28). In Satb2 mutants,EphA4 expression is lost in cortical neurons, but expression inthe glial wedge is maintained (9). Unc5H3 mutants have noreported callosal deficiencies (13), but mice lacking Netrin, a li-gand for Unc5H3, lack both the CC and the anterior commissure(29, 30). To test the hypothesis that one or more of these genesis required for the proper guidance of callosal axons to theirdestinations, we attempted to rescue the formation of callosalprojections in Satb2 mutants by reintroducing the expression ofindividual axon guidance molecules into upper layer neurons. Inutero electroporation of PlxnA4 failed to rescue callosal pro-jections (Fig. 7 I and J) in Satb2LacZ/LacZ mutants, but electro-poration of EphA4 (Fig. 7 E and F) or Unc5H3 (Fig. 7 G and H)resulted in the extension of β-gal+ axons across the CC. Theseresults suggest that EphA4 and Unc5H3 are critical downstreamtargets of Satb2 in callosal fate specification.

DiscussionOur data suggest that cortical projection neurons actively repressalternate fates to promote appropriate fate choices during de-velopment (Fig. 7O). When specific repressive interactions areremoved, alternative fates are executed (Fig. S1). Whereas Tbr1,Ctip2, and Satb2 are expressed in postmitotic neurons, Fezf2 isexpressed in cycling cortical progenitors from very early stages ofcorticogenesis (11). Loss of Fezf2 is critical for the specificationof the subcerebral projections of layer 5 neurons, as evidenced by

the loss of corticospinal projections in Fezf2 mutants (11, 14).During the earliest stages of corticogenesis, Sox5 expression insubplate and layer 6 neurons represses the expression of Fezf2(and consequently that of Ctip2) (15, 16). This likely promotesthe expression of Tbr1 in layer 6 neurons. Tbr1 binds to andrepresses the Fezf2 genomic locus (5, 7), thereby suppressing asubcerebral fate and promoting the formation of corticothalamicprojections from layer 6. Sox5 expression is down-regulated earlyin layer 5 neurons (15, 16), leading to a derepression of Fezf2,which consequently leads to a repression of Satb2. The effect ofthis is threefold. First, there are no intracellular triggers topromote a callosal fate. Second, the absence of Satb2-mediatedrepression of Ctip2 and Bhlhb5 leads to the continued expressionof these genes and the extension of subcortical axons. (Inter-estingly, although Bhlhb5 expression in Satb2 mutants at E18does not differ from that in controls, expression at P4 is signifi-cantly increased in Satb2 mutants. This suggests that Satb2 isrequired for the normal down-regulation of Bhlhb5 expressionduring early prenatal development, and that the sustained ex-pression of Bhlhb5 in Satb2 mutants might be important in ex-ecuting or maintaining the new subcerebral fate of theseneurons.) Third, our data indicate that the absence of Satb2-mediated activation of Tbr1 suppresses corticothalamic projec-tions. Thus, Fezf2-expressing layer 5 neurons extend their axonssubcerebrally.We hypothesize that during production of the upper layers,

the absence of Fezf2 in cortical progenitors allows their daugh-ters to express Satb2, which in turn promotes a callosal identity(in part, surprisingly, by activating Tbr1 in upper layer neurons).Simultaneously, activation of Satb2 results in the repressionof Ctip2 and Bhlhb5, ensuring that executors of subcorticalidentity remain inactive in callosal neurons. Thus, each phase ofcorticogenesis and neuronal fate specification deploys an activerepression of previous fates and a promotion of the appropriatelayer-specific projection fate (see SI Discussion for further details).Tbr1 seems to play distinct roles at different stages of cortical

development. At early stages, Tbr1 promotes a frontal identitywhile suppressing caudal identity (4). During the formation oflayer 6, Tbr1 plays an essential role in specifying the fates andprojection patterns of corticothalamic neurons (4, 5, 7, 17). In-terestingly, although corticothalamic projections are decreasedin Tbr1 mutants and increased in Fezf2 mutants (which show anup-regulation of Tbr1), these alterations in projections are in-complete: there are still some corticothalamic axons in Tbr1mutants, and Fezf2 mutant neurons do not completely convert to

LacZ

Satb2 Lacz/LacZ (GFP) Satb2 Lacz/LacZ (TBR1) Satb2 Lacz/LacZ (EphA4) Satb2 Lacz/+ (UNC5H3) Satb2 Lacz/LacZ (PlxnA4)

A C E G I

CCCC

CC CC CC

B D F H J

AUTS

2Ct

ip2

Satb2 LacZ/+ Satb2 LacZ/LacZ

AUTS2 AUTS2

2-4

5

6

1M NK L

Fezf2

Satb2 Tbr1

Ctip2

callosal subcerebral corticothalamic Layer 5 Layer 6

Tbr1

wildtype

Layers 2-5

O

Fig. 7. Tbr1, EphA4, and Unc5h3 are downstreamtargets of Satb2 and can direct callosal projections.β-Gal+ axons fail to cross the CC in Satb2LacZ/LacZ

mutants electroporated with either a GFP controlconstruct (A and B) or a PlxnA4 expression constructtogether with GFP (I and J). β-Gal+ axons cross theCC in Satb2 mutants electroporated with TBR1-IRES-GFP (C and D), EphA4 and GFP (E and F), or Unc5H3and GFP (G and H). E and F are composites of tiledimages to form the full figure. (Scale bar, 100 μm.)(K–N) Auts2 expression is lost in the upper layers ofSatb2 mutants. (K and L) Auts2 protein (green in K,white in L) is expressed in both the upper and deeplayers of control Satb2LacZ/+ brains at P0. Ctip2 ex-pression is shown in red. (M and N) Auts2 expressionis maintained in layer 6 of Satb2mutants but is down-regulated in the upper layers. (Scale bar, 50 μm.) (O)Model of genetic interactions between Fezf2, Ctip2,Satb2, and Tbr1 in wild type cortex. Fezf2 expressionin layer 5 neurons represses Tbr1 and Satb2, whichrepress corticothalamic and callosal fates, respec-tively. The repression of Satb2 enables expression ofCtip2 and Bhlhb5, which are required for the spec-ification and execution of a subcerebral identity. InSatb2 expressing neurons, Ctip2 is repressed, leading to a repression of subcerebral identity and acquisition of callosal and corticocortical axonal projections.

Srinivasan et al. PNAS | November 20, 2012 | vol. 109 | no. 47 | 19077

NEU

ROSC

IENCE

INAUG

URALART

ICLE

•Decades of careful neurodevelopmental studies1-3 have revealed some of the key transcription factors that control neocortical development.•The identity of other key genes, the targets of these factors, and the enhancers that mediate their regulation remain largely unknown.

Candidate Enhancers Drive Laminar Expression in the Developing Mouse Neocortex

p300 Peaks Next to Key Neocortical Developmental Genes

•A single E14.5 p300 peak next to Fezf2 contains the E4 developmental enhancer whose genomic deletion leads to aberrant cortico-spinal projection fates, similar to those found in its Fezf2 target gene conditional knock-out7.

MER130 is Strongly and Distinctly Enriched Among Neocortex Enhancers

•For each combination of repeat family and p300 set, we shuffled the p300 set across the genome 10,000 times and compared the expected number of p300 elements overlapping each repeat family to the observed number.•22 of 90 (24%) MER130 instances identified in the mouse genome overlap our E14.5 set.

MER130 Contains a Preserved Core of Transcription Factor Binding Sites

Mutating MER130 Core Modulates Enhancer Activity

•Mutating each of the preserved binding sites resulted in a two-fold induction or reduction of reporter activity, relative to the un-mutated construct, when transfected into dissociated cortical neurons.

Summary•We performed ChIP-seq on E14.5 dorsal cerebral wall to identify active enhancers and found 6,629 p300 bound sites.•When tested in vivo, these putative enhancers drive laminar expression patterns throughout the developing neocortex.•The regulatory domains of several key neocortical developmental genes contain a single proximal p300 peak, and some of these peaks appear to be derived from transposable elements.•We identified the transposable repeat family MER130 to be surprisingly enriched in our set of putative developing cortex enhancers, and validated that the putative enhancers function in vitro.•MER130 elements contain a preserved code of transcription factor regulatory logic that modulates enhancer activity when mutated.

References:1. Molyneaux, B. J., Arlotta, P., Menezes, J. R. L., & Macklis, J. D. (2007). Neuronal subtype specification in the cerebral cortex. Nature reviews. Neuroscience.2. Rubenstein, J. L., & Rakic, P. (1999). Genetic control of cortical development. Cerebral cortex (1991).3. Leone, D. P., Srinivasan, K., Chen, B., Alcamo, E., & McConnell, S. K. (2008). The determination of projection neuron identity in the developing cerebral cortex. Current opinion in neurobiology.4. Wenger, A. M., Clarke, S. L., Notwell, J. H., Chung, T., Tuteja, G., Guturu, H., & Bejerano, G. (2013). The enhancer landscape of early neocortical development reveals patterns of dense regulation, pleiotropy and co-option. PLoS Genetics.5. McLean, C. Y., Bristor, D., Hiller, M., Clarke, S. L., Schaar, B. T., Lowe, C. B., et al. (2010). GREAT improves functional interpretation of cis-regulatory regions. Nature Biotechnology.6. Ayoub, A. E., Oh, S., Xie, Y., Leng, J., Cotney, J., Dominguez, M. H., et al. (2011). Transcriptional programs in transient embryonic zones of the cerebral cortex defined by high-resolution mRNA sequencing. Proceedings of the National Academy of Sciences.7. Shim, S., Kwan, K. Y., Li, M., Lefebvre, V., & Šestan, N. (2012). Cis-regulatory control of corticospinal system development and evolution. Nature.

O

O

O

O

O

O

O

O

O

O

O

OO

OO

O

O

O

O

O

OO

O

O

O

O

O

O

O

O

O

O

O

O

O

O

OO O

O

O

O

O

O

O

O

OO

O

O

O

O

O

O

O

O

O

OO

O

O O

O

O

O

O

O

O

O

OOO

O O

O

O

O

O

O

O

O

OOO0

50

100

p300

Enrich

ed

p300

Dep

leted

Aver

age

DN

ase

inte

nsity

O

O

Not TestedTested

1

10

100

Elements with decreasing p300 signal

Rat

io to

pG

L4.2

3LIC

p300 depletedp300 enriched

MER130 Instances Function as Cortical Enhancers in vitro

•21/22 (95.5%) of the MER130 candidates hypothesized to function as cortex enhancers produced greater than 2-fold expression relative to the empty vector when transfected into dissociated cortical neurons (p300 enriched: black bars).•Surprisingly, 6/7 (85.7%) of the MER130 candidates hypothesized not to function as cortex enhancers based on p300 ChIP-seq measurements drove greater than 2-fold expression relative to the empty vector (p300 depleted: gray bars). •The MER130 instances that were not not marked by p300, yet functioned as enhancers in vitro, were depleted for DNaseI cleavage when compared to the other MER130 instances, suggesting they are inactive in vivo.

B

A

HumanDog

OpossumChicken

FrogZebrafish

SINELINELTRDNA

20 kb mm913,150,000 13,170,000 13,190,000 13,210,000

BAC End Pairs

UCSC Genes

Enhancer Candidates Based on Conservation

Placental Mammal Conservation by PhastCons

Multiz Alignments of 30 Vertebrates

E14.5 Dorsal Cerebral Wall p300 ChIP-seq Signal

E14.5 Dorsal Cerebral Wall Input ChIP-seq Signal

E14.5 Dorsal Cerebral Wall p300 ChIP-seq peaks

Repeating Elements by RepeatMasker

RP23-141E17

Fezf2 Cadps

E4 E3E2

E1

e14dcw_p300.1848

HumanDog

OpossumChicken

X_tropicalisZebrafish

SINELINELTRDNA

20 kb mm961,620,000 61,640,000 61,660,000 61,680,000

UCSC Genes

Placental Mammal Conservation by PhastCons

Multiz Alignments of 30 Vertebrates

E14.5 Dorsal Cerebral Wall p300 ChIP-seq Signal

E14.5 Dorsal Cerebral Wall Input ChIP-seq Signal

E14.5 Dorsal Cerebral Wall p300 ChIP-seq peaks

Repeating Elements by RepeatMasker

Psmd14Tbr1

e14dcw_p300.3376

AmnSine1

Mouse July 2007 (NCBI37/mm9)

Mouse July 2007 (NCBI37/mm9)

•A single E14.5 p300 pan-mammalian conserved peak proximal to the Tbr1 gene has likely been seeded by the co-option of an AmnSine1 instance at its center.

Measure the Active Enhancer Landscape During Early Neocortical Development

•In mammals, the dorsal portion of the telencephalon gives rise to the neocortex.•We dissected the dorsal cerebral wall, which includes the developing neocortex and its progenitor populations, from embryonic day 14.5 (E14.5) mouse embryos.•We performed chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) with an antibody against the enhancer-associated p300 co-activator complex4.•Using GREAT5 (great.stanford.edu), we found that the 6,629 p300 bound sites are enriched for cortical development terms.

Telencephalon

abnorm al neuron different iat ioncom plete perinatal lethality

abnorm al nervous system t ractabnorm al brain com m issure m orphology

abnorm al forebrain developm entabnorm al brain white m at ter m orphology

abnorm al dorsal telencephalic com m issure m orphologyabnorm al corpus callosum m orphology

abnorm al brain vent ricle m orphologyabnorm al telencephalon developm ent

abnorm al neuronal precursor proliferat ionabsent dentate gyrus

abnorm al axon guidancem icrophthalm ia

abnorm al dentate gyrus m orphologyabnorm al eye size

m icrognathiaabnorm al secondary palate developm ent

abnorm al palate developm entabnorm al cerebellar foliat ion

0 5 10 15 20 25 30 35 40 45 50 55 60 6565.25

63.9957.5056.76

55.7255.13

42.6042.18

35.7332.08

30.5229.88

28.0927.9027.7327.5027.3327.2926.78

25.59

M ouse Phenot ype-log10(Binom ial p value)

elt13/3

Mammalia

elt27/7

Mammalia

elt38/8

Eutheria

elt46/7

Gnathostomata

elt56/10

Theria

elt66/6

Amniota

elt76/7

Theria

elt88/9

Theria

ElementNeocortex / Transgenics

Evolutionary conservation

Whole mount

Coronal section

Coronal section(zoom)

Candidate targetgene in situ

from Eurexpressor Allen Brain Atlas

Candidate targetgene E14.5 expression

by laser micro dissection and

RNA-seq

SVZ-IZ and VZ CP and SVZ-IZ CP and SVZ-IZ CP and SVZ-IZ CP, SVZ-IZ, and VZ SVZ-IZ and VZ CP and SVZ-IZ CP, SVZ-IZ, and VZ

Element

Ratio

to pG

L4.23

1

10

100

elt1 elt2 elt3 elt4 elt5 elt6 elt7 elt8 elt9 elt10

pGL4.23

AG

Eomes Satb2 Neurod2 Tbr1 Auts2 Id4 Bhlhb5 Auts2Adjacent gene

A B C D E F G H

I J K L M N O P

Q R S T U V W X

Y Z AA AB AC AD AE AF

RNA-seq6

p300 Peaks Derived From Transposable Elements Next to Key Neocortical

Developmental Genes

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470

MER130.0/1-448

MER130.1/1-165

MER130.2/1-310

MER130.3/1-123

MER130.4/1-191

MER130.5/1-254

MER130.6/1-268

MER130.7/1-215

MER130.8/1-302

MER130.9/1-212

MER130.10/1-263

MER130.11/1-306

MER130.12/1-95

MER130.13/1-168

MER130.14/1-242

MER130.15/1-437

MER130.16/1-232

MER130.17/1-309

MER130.18/1-255

MER130.19/1-338

MER130.20/1-344

MER130.21/1-291

MER130.22/1-117

- - - T T T G G C A C A G C C T T G T C A A C A G G T G T G A T C A A C A C T A A G G A A G C A T G C A G T G C C A A C A C C C C T G T T C T G G T C C A G G A T G A - - - - - - G A A C C C A C C A C T G A A A A G A T G G C A T T C A C A G C C G C A G G C A G A C T G C C A - - - T G A A A T C G T T G T T C C - T T G G C A C T G G T G C C C A T G G A G - - - - - A G T G T C T A G G C A A C T T T T G A - - T T G G G C A C C A T G C C C T C C A A G - C A G A T G G G C A T G A A T G T A - G G C A G G C A A G A G A - - - - - - - - - - C C A T T T A T T T A T A T C A A C T G T A T T T G C T G A G G G - C A G G G T T C C C A G C T G G G A A C A T C A A A A C A G C T C T T T G T C C T G G C T G T C T G A A T C T C T G A T G G A G G T G A T G C A G C C T T G C A A A T T C A G C T A G A T T A A T T T T G A A A A G T A A A A A C A A T G T T T C T A T G G A C A C C G G G G G A C A T T T T T G G C T C C C A T C A A T G G T T C A G A G C A C A G A G C A G G C T

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - G A G C A A A G A T G G C A T T C A G A G T G A A T C T C A G G C T G C C T G T T A G A - - - - - - - - - - - G C C T A C C A T T C A T G T C C C G G G G C - - - - - T G G G G C C A A C C A A C T T T C T G - - - - G G G C A C C G T G C C A T C A T G A - C A G A T G G G C A T G G G G - - - - G G C T G C C A G G A T C - - - - - - - - - - - C T C G A G C C T G G G A C C A T T G T G T G T G C T C T G A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

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- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A G A G C T G T C T T T G G G C - - - - - - - A T C T G T A T G - - - - - - - - - - - - - - - - - - - - A G T T T T G T A A G G G C T T C T T G T C T A A - - - A G C C T T T T C - T T G G C C C C T G G G T T T T G G C A A - - - - A G C C A G T C G A C T C T C C T A C A A - - G C T G G C A C A A G G C C A T G G T A A - C A G A T G G C C A G G A G C G T A - G G C A G C A A G G A A A G C A A A G G A A A G C A T G T A G G A G C G T C T G T A G C G A G G G C T G A G G G - C G A G T G C T T C A G A C - - - - A C A G C C T A G A G T C T G T - - - - - C T G T C C - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

C T G C T T T T G G C A T T A G C A A G T C A A C A G G T G T G A C A A A G T T T A A A T G G A T G T A A G A T G C C A C C T G C T T T T C T A A C T C T G G C A T G A G T A A C G G C C T C C T G T A A G A A G A G C T G C C T C C T G G A A C A T T T T G T T A G C T G T C A - - - - - - - - - - - - - - - T T G G C T G G G A C T C A T G T C A T A G T A T - - - - A A G A G A C C T G C C G G C T T C T A A - - G C A G G C A A C C C G C C A T C T A A G - C A G A T G G G C A T G G A T T C T - - - C A G C C A A G A G G - - - - - - - - - - C C A A G T G T A T A C A G C A A A C A T A G G T A T C G G G G C - C T C G T T T C C C A C - - - A G A G C T T G C T A A C G T C T G T T T G T C T T G A T A T - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T G G C A C - - - - - - - - - - - - - - - - - - - - A G G T C T G A C C A A C T C A G A A - - G G A G G C A T T C T G C C A A G C A C A - C A G A T G G - C A T G G G G G G A - A A G T G C C A A A A A A - - - - - - - - - - - - - - A C A T G A G A A T C A A C T G T G G G T A C T G G T G G - C A T G A T T C C C A G A C A T G G A A G T C C A A A C A C T T G T C C A T A C C A A G C T T A T A G C A C A T A C T T A C A G A T A T G C A G A T T T G C C A A C A G T A A T C T A G C A C T G T T A G G T T A A T T T T T A A A A A T A T T T T T A A A G C T A C C G T T G G C T C C T G A A G C T A G - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A A A A T T G T C A C T T G T A G T A T T A T G C A G G C T G C C C A T C T A A A G C C A T C A T T G C - C T G G C A C C T A C A C A C T A A G G G - - - C A A G G A T C C A G C C A A C T T G T G A - - G T G G G C A C C A G G C C A T C T G A G - C A G A T G G G C A T G A A A G T C - G G C A G C C A G T A G A - - - - - - - - - - C C A T G T A T G T G C A T C A A T G G A G G G T G T T G A G G G - C A C G A T T C C C A G A G G A G A A T G T C C T A A C A G C T G T - - - - - C T G G C C T G A G C C A A C A T A C T T A C A A C C A A G G A A A A G T G C C A T C C T T C A A T C C T G C T G A G T C G G A T T A A C C T T C A A A A C A A A A T C T T C T T T T T T T C A G C A G C A G A A G G T G A G T T T G G G T G T C T C T - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - G C A G C A G A G C T G T C A C C T G C T T T A C T A T G C G G G G T G C C C A G C T T A A C C C G T A T T T C T - T T G G C A A C T G T G C C C C A G T G A G A A A A G C T A G C G G T C C A T A T G T T G A - - G T A G G C A C C A T G C C A T T - - - - - T T G A C A G G C A T G A G T G T C - G G C A A G A A A G G G A - - - - - - - - - - C C A T G T A T G A G C A T C A A A T G T G T T T G T T G T G G G - A A G G A T T C T A G G C C A G G A A T A T T A A A A T A A C T G T - - - - - C T G T C C T T C G C T C T A G C A C A T A T G G T T A A G G A G A T A T G C T A T G A T G A C T C A C A T T A A C A T T G G A A A A A A T A T A T A T A G A A T T T G C A G C C A T G C T A A G T G A G T T T C T G G T G T T G A T A A A T T G G G C T T - - - - - - - - - - - - - -

- - - - - - T T T T G G C T G A A A A A T A A T G A A C A G G T G G T G T G T C G G T A A G A A G C C G T G T G G G T G T T A A T G T G T C A T C - - - - - - - - - - - - - - - - - G T T T T C T A C A G A A C A C G A G A G C A T T T T C A G A A T T T T - - A G G T T G C C A A T C T A C - - - - - - - - - T T C G C T G G C A C C T G C A G T C T A - - - - - - - - A A C T G G A A C A C C A T T T T G A G A - - T C A G G C A T G T T G C C A T T G T A A - C A G A T G G T T A C G G C T A C T - C A C A G C C A A G T G A - - - - - - - - - - T C A T G T A T G T A C A T G G G T T G T G G G T G T T G A A A G - C A A G C T G C T C A T T T A G G A A G G T C A A A T A G C A T - - - - - - - T T A T C C A G C T C T T C - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A - - C C A G G C A T G G T G C C A T A C T G A - C A G A T G G C C G C G T G A G T A - G A T G G C C A A C A G A - - - - - - - - - - T A A T G T A T G A G C A A C A A A T G T G T T T G C T G A G G G - C A G C T T T C C C A G G T A A G A G C T T T A A A G C A A C - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Consensus

- - - - T T T + - A + A + T A + + + A C A A + A G + T G T G + + C C A A T G T A A + G A A G C A T G T G G C C C T G A C + T G G C + + T G C T + + C + C A C G G + A G - - - - - C G + T C T C C T + G C A G A A A A G A T G G C A C T T + C A G T A T T + T G C A G G C T G C C C A T C T A A A C C T G T C T T T G C G T T G G C A C C T A T G C C C T A G T G A G - A A A A C T G G C A T G C C A A C T T G T G A - - G T G G G C A C C A T G C C A T C C T A A - C A G A T G G G C A T G A G T G T A A G G C A G C C A A G A A A G - A - - - - - - A C C A T G T A T G T G C A T C A A + T G T G + G T G T T G A G G G C C A G G A T T C C C A G C + A G G A A + G T C A A A A C A G C T G T T T G T C C T G T C C T + A G C T T T C A C A C T T + T + G + T A A G G + G A T T T G C C A A + T T T A + + C A + + T T + A + T T T G G A A A A A A T T T A + A A A G A + T A T + + A G + C T T C C T G + G + C A C T + T A G G + T A G T + T T A A T + T T T C - G T G - A - - - - - - - - - - -

MER130

G

T

G

G

C

A

C

A

T

T

C

G

G

A

C

T

T

C

AA

T

A

G

T

C

G

AT

G

A

C

A

T

GG

A

C

G

C

T

AT

G

A

C

T

T

A

GG

C

T

G

CT

T

C

G

A

C

TA

G

A

G

T

CT

C

G

A

T

G

T

C

G

C

A

TA

C

G

T

A

T

C

A

G

T

C

A

G

C

T

G

A

C

A

T

GT

A

G

C

A

T

A

G

T

C

C

G

T

G

A

T

C

G

T

A

C

G

A

CTT

G

A

G

T

G

A

CT

C

A

T

C

AGT

G

C

AG

T

CAT

C

GA

T

G

C

AC

T

AGT

C

A

G

C

A

TT

C

AGT

G

G

A

TC

G

C

TA

G

A

TC

G

A

CT

A

G

CTT

G

A

A

G

C

G

C

TAC

A

TG

G

C

A

TT

C

GA

G

A

CT

A

G

C

TG

A

CTT

C

A

GG

A

TCC

T

GAC

T

AGT

CAG

A

G

TC

G

A

CTC

T

AG

A

G

TC

A

GTC

G

A

T

CC

A

C

G

AT

G

A

T

CG

CAT

C

T

GA

G

T

CATG

CAG

A

C

G

TA

CG

A

CTT

C

AG

G

A

CT

C

G

C

A

T

G

A

CT

G

A

CT

G

G

C

G

A

CT

G

C

A

TT

C

AG

A

TCGT

A

G

CT

GCAG

TAC

G

A

TC

A

GCTT

C

GAG

A

C

TT

C

AG

G

A

TC

G

A

T

CG

A

TC

G

A

CTC

T

GAC

T

G

C

GTC

T

AGT

G

C

A

G

C

TAC

T

GA

G

A

C

C

A

G

T

T

C

AG

A

TG

ATCT

C

AT

T

C

AG

TGA

CG

TACT

GCAC

T

A

G

TAC

C

GAT

GCTT

A

C

GG

CATC

T

AG

C

T

GA

C

ATG

G

A

CTTAG

A

GGAC

CA

A

TCT

G

A

CG

CA

A

GCT

AT

GCT

CCA

A

G

CTG

A

T

CA

C

G

C

AT

CGA

GA

T

CTAG

GACT

A

GGT

C

AG

G

A

TCT

C

GA

A

G

CT

C

T

AGT

GACAG

C

A

G

TC

T

AG

G

A

CT

G

TCA

T

C

A

GT

A

C

GTG

A

CG

TAT

C

AG

GACTACT

G

C

AGAC

T

AGT

C

GAC

T

GAT

C

GA

A

T

G

C

G

A

TC

G

T

CA

C

G

ATC

TA

GG

C

ATTGA

A

GCTT

C

A

GG

C

ATT

AG

G

TA

CT

GCAG

C

AT

A

GTC

G

TC

AT

GA

G

A

A

GCT

C

A

T

GG

ACTT

AG

C

A

TG

C

A

TG

C

GATT

C

AG

G

A

CT

A

G

CTT

C

A

GT

GAC

ATGC

T

AGTC

A

GT

G

AC

G

C

TA

C

TA

GG

T

C

A

A

G

CT

A

GCT

G

A

TC

A

GTC

G

ATC

C

G

TA

A

C

TGT

A

C

G

TC

T

C

GA

C

T

AGT

C

AGT

C

GAT

C

GA

G

A

T

C

T

A

G

A

GCTG

A

TCT

G

CA

C

G

TAT

G

CAC

T

GAG

T

ACT

C

G

AT

G

G

TAC

G

A

CTC

A

T

GG

C

AT

G

A

TC

G

A

CT

C

T

A

G

G

T

C

G

T

A

CC

G

A

TG

C

A

C

A

T

G

G

T

A

CC

T

C

A

T

A

C

T

A

T

C

T

A

T

CACT

G

C

A

TG

A

A

T

G

G

C

A

TG

A

C

T

AA

GG

T

C

A

G

T

AT

C

A

G

TC

G

G

A

CC

T

G

C

AA A C

C

A

G

TA

T

A

T

A

TA

TT

AT

AT

A

T

A

A

TTCC

C

A

T

GT

G

C

A

T

GA

G_NEUROD1_01

AG

A

CGCAGC

ATGAG

GC

G_NF1_03

GCT

C

G

A

T

CAT

TCTGC

GCTA

AGC

J_NFIC

C

A

TGT

CT

G

A

CC

AC

T

GA

J_TLX1_NFIC

CATTGGC

C

G

T

A

A

CT

T

A

C

GA

T

CG

GATGCCA

Transcription Factor Binding Preferences:

MER130 Consensus Motif:

Multiple Alignment:

Nfi* Nfi* dimer Neurod/g Nfi*

Instance 1 Mutations:

Instance 2 Mutations:

•The multiple alignment of MER130 cortex enhancer instances shows a well-conserved core containing 5 binding sites resembling known motifs: a Neurod/Neurog motif, an Nfi dimer, and two additional Nfi motifs.