The Genomics of Neurodevelopment: Transcriptional

Mouse July 2007 (NCBI37/mm9) 20 kb mm9 A 13,150,000 13,170,000 13,190,000 13,210,000 BAC End Pairs Networks UnderlyingRP23-141E17 the Developing UCSC Fezf2 Cadps Fig. 7. Tbr1, EphA4, and Unc5h3 are downstream Satb2 Lacz/LacZ (GFP) Satb2 Lacz/LacZ (TBR1) Satb2 Lacz/LacZ (EphA4) Satb2 Lacz/+ (UNC5H3) Satb2 Lacz/LacZ (PlxnA4) 1 Enhancer1 Candidates Based on Conservation 2 3 3 E4 E3 E1 targets of Satb2 and can direct callosal projections. AC EJames GI H. Notwell , AaronINAUGURAL ARTICLE M. Wenger , Shoa L. Clarke , Tisha Chung , Geetu Tuteja , LacZ/LacZ E2 β-Gal+ fail to cross the CC in Satb2 4 Placental Mammal Conservation3,5 by PhastCons 3 1,3 mutants electroporated with either a GFP control Harendra Guturu , Whitney Heavner , Bruce T. Schaar , Gill Bejerano Multiz Alignments of 30 Vertebrates construct (A and B)oraPlxnA4 expression construct Human together with GFP (I and J). β-Gal+ axons cross the 1Dog 2 3 Departments ofOpossum Computer Science, Genetics, Developmental Biology, CC in Satb2 mutants electroporated with TBR1-IRES- Chicken LacZ BD F HJ Frog GFP (C and D), EphA4 and GFP (E and F), or Unc5H3 4 5 and GFP (G and H). E and F are composites of tiled Electrical Engineering,E14.5 Dorsal and Cerebral WallBiology, p300 ChIP-seq Signal Stanford University fi images to form the full gure. (Scale bar, 100 μm.) CC E14.5 Dorsal Cerebral Wall Input ChIP-seq Signal (K–N) Auts2 expression is lost in the upper layers of CC Key TranscriptionCC FactorsCC Control CC p300 Peaks Derived From Transposable MER130 Instances Function as Cortical Satb2 mutants. (K and L) Auts2 (green in K, E14.5 Dorsal Cerebral Wall p300 ChIP-seq peaks e14dcw_p300.1848 white in L) is expressed in both the upper and deep Satb2 LacZ/+ Satb2 LacZ/LacZ Neocortical Development Elements NextRepeating Elementsto Key by RepeatMasker Neocortical Enhancers in vitro layers of control Satb2LacZ/+ brains at P0. Ctip2 ex- O wildtype SINE KL MN LINE LTR pression is shown in red. (M and N) Auts2 expression 1 Developmental Genes Not Tested Fezf2 p300 depleted Tested DNA p300 enriched is maintained in layer 6 of Satb2 mutants but is down- 2-4 Mouse July 2007 (NCBI37/mm9) regulated in the upper layers. (Scale bar, 50 m.) (O) 100 μ Satb2 Tbr1 20 kb mm9 100 Model of genetic interactions between Fezf2, Ctip2, B 61,620,000 61,640,000 61,660,000 61,680,000 Ctip2 5 UCSC Genes Satb2,andTbr1 in wild type cortex. Fezf2 expression Psmd14 Ctip2 Tbr1 Tbr1 in layer 5 represses Tbr1 and Satb2, which 6 Placental Mammal Conservation by PhastCons AUTS2 50

10 repress corticothalamic and callosal fates, respec- age DNase intensity r Multiz Alignments of 30 Vertebrates tively. The repression of Satb2 enables expression of Layers 2-5 Layer 5 Layer 6 e

v Human Ratio to pGL4.23LIC A callosal subcerebral corticothalamic Ctip2 and Bhlhb5, which are required for the spec- AUTS2 AUTS2 Dog 1-3 Opossum Decades of careful neurodevelopmental studies have revealed some of the key fi • i cation and execution of a subcerebral identity. In Chicken 1 0 Satb2 expressing neurons, Ctip2 is repressed, leading to a repression of subcerebraltranscription identity factors and acquisition that control of neocortical callosal and development. corticocortical axonal projections. X_tropicalis Zebrafish •The identity of other key genes, the targets of these factors, and the enhancers E14.5 Dorsal Cerebral Wall p300 ChIP-seq Signal p300 Enriched p300 Depleted that mediate their regulation remain largely unknown. Elements with decreasing p300 signal Auts2, we investigated whether the loss of Tbr1 expression in the loss of corticospinal projections in Fezf2 mutants (11, 14). E14.5 Dorsal Cerebral Wall Input ChIP-seq Signal •21/22 (95.5%) of the MER130 candidates hypothesized to function as cortex LacZ/LacZ enhancers produced greater than 2-fold expression relative to the empty vector upper layer neurons in Satb2 mutants coincides with DuringMeasure the earliest the stagesActive of Enhancer corticogenesis, LandscapeSox5 expression in E14.5 Dorsal Cerebral Wall p300 ChIP-seq peaks e14dcw_p300.3376 when transfected into dissociated cortical neurons (p300 enriched: black bars). changes in the expression of Auts2. We observed a striking loss subplate and layer 6 neurons represses the expression of Fezf2 Repeating Elements by RepeatMasker During Early Neocortical Development SINE Surprisingly, 6/7 (85.7%) of the MER130 candidates hypothesized not to function of Auts2 expression in the upper layers of Satb2 mutants (Fig. 7 (and consequently that of Ctip2) (15, 16). This likely promotes LINE AmnSine1 • LTR as cortex enhancers based on p300 ChIP-seq measurements drove greater than 2- M and N), similar to the loss of Tbr1 in Satb2 mutants (Fig. 5B); the expression of Tbr1 in layer 6 neurons. Tbr1 binds to and DNA there was no change in Auts2 expression in layers 5 or 6 (Fig. 7 M represses the Fezf2 genomic locus (5, 7), thereby suppressing a •A single E14.5 p300 pan-mammalian conserved peak proximal to the Tbr1 fold expression relative to the empty vector (p300 depleted: gray bars). and N) relative to controls (Fig. 7 K and L). These data are subcerebral fate and promoting the formation of corticothalamic has likely been seeded by the co-option of an AmnSine1 instance at its center. •The MER130 instances that were not not marked by p300, yet functioned as consistent with the possibility that Satb2 regulates the expression projections from layer 6. Sox5 expression is down-regulated early enhancers in vitro, were depleted for DNaseI cleavage when compared to the other of Tbr1, which in turn is required for Auts2 expression in callosal in layer 5 neurons (15, 16), leading to a derepression of Fezf2, MER130 is Strongly and Distinctly Enriched MER130 instances, suggesting they are inactive in vivo. projection neurons. These results may have implications for the which consequently leads to a repression of Satb2. The effect of etiology of . this is threefold. First, there are no intracellular triggers to Among Neocortex Enhancers promote a callosal fate. Second, the absence of Satb2-mediated NEUROSCIENCE Mutating MER130 Core Expression of EphA4 and Unc5H3 Restores Callosal Projections in repression of Ctip2 and Bhlhb5 leads to the continued expression Telencephalon genome Modulates Enhancer Activity Satb2 Mutants. Previously we identified several genes that show of these genes and the extension of subcortical axons. (Inter- LacZ/LacZ M ouse Phenot ype altered expression in Satb2 mutants (9). In particular, estingly, although Bhlhb5 expression-log in10(BSatb2inom ial p vamutantslue) at E18 0 5 10 15 20 25 30 35 40 45 50 55 60 65 p300 abnorm al differentiation 65.25 fi three axonal guidance molecules (EphA4, PlxnA4, and Unc5H3) does not differ fromcom plet thate perinat inal let controls,hality expression at P4 is63.9 signi9 - abnorm al nervous system tract 57.50 are down-regulated in upper layers of mice lacking Satb2. Prior cantly increasedabnorm al brai inn commSatb2issure m omutants.rphology This suggests that56.76 Satb2 is abnorm al forebrain developm ent 55.72 shuffled p300 x 10,000 100 studies have implicated Ephs and ephrins in callosal develop- abnorm al brain white m atter m orphology 55.13

required for the normal down-regulation of Bhlhb5 expression Instance 1 abnorm al dorsal telencephalic commissure m orphology 42.60 ment (13, 28, 29). EphA4 is normally expressed in upper layer during earlyabno prenatalrm al corpus callo development,sum m orphology and that the42.18 sustained ex- ** ** ** * abnorm al brain ventricle m orphology 35.73 repeat family callosal neurons and the glial wedge (28). In Satb2 mutants, abnorm al telencephaSatb2lon developm ent 32.08 pression ofabno Bhlhb5rm al neuronal pr inecursor prolifermutantsation might30 be.52 important in ex- Expected Overlap absent dentate gyrus 29.88 50 EphA4 expression is lost in cortical neurons, but expression in Inecuting mammals, or the maintainingdorsal portionabnorm al ofaxo then the gu idtelencephalonanc newe subcerebral gives28 .rise09 to the fate neocortex. of these • m icrophthalm ia 27.90 the glial wedge is maintained (9). Unc5H3 mutants have no neurons.) Third,abnorm al d ourentate g datayrus m orp indicatehology that the27.73 absence of Satb2- 6000 •We dissected the dorsal cerebralabn owall,rm al e ywhiche size includes the27 developing.50 neocortex reported callosal deficiencies (13), but mice lacking Netrin, a li- mediated activation of Tbr1m icrognasuppressesthia corticothalamic27.33 projec- and its progenitorabno rpopulations,m al secondary pa lfromate dev embryonicelopm ent day 14.5 (E14.5)27.29 mouse . 0 gand for Unc5H3, lack both the CC and the anterior commissure tions. Thus, Fezf2abn-expressingorm al palate developm layerent 5 neurons26.78 extend their axons 4000 We performed chromatinabnor mimmunoprecipitational cerebellar foliation followed25 .5by9 high-throughput Observed (29, 30). To test the hypothesis that one or more of these genes • subcerebrally. Frequency Overlap is required for the proper guidance of callosal axons to their sequencingWe hypothesize (ChIP-seq) with that an antibody during against production the enhancer-associated of the upper p300 layers,co- 2000 activator complex4. 100 destinations, we attempted to rescue the formation of callosal the absence of Fezf2 in cortical progenitors allows their daugh- Instance 2

0 Ratio to pGL4.23LIC •Using GREAT5 (great.stanford.edu), we found that the 6,629 p300 ** ** ** projections in Satb2 mutants by reintroducing the expression of ters to express Satb2, which in turn promotes a callosal identity 0 5 10 15 20 bound sites are enriched for cortical development terms. Number of Repeat Instances Overlapped individual guidance molecules into upper layer neurons. In (in part, surprisingly, by activating Tbr1 in upper layer neurons). 50 utero electroporation of PlxnA4 failed to rescue callosal pro- Simultaneously, activation of Satb2 results in the repression MER130 jections (Fig. 7 I and J) in Satb2LacZ/LacZ mutants, but electro- Candidateof Ctip2 and EnhancersBhlhb5, ensuring Drive that Laminar executors Expression of subcortical poration of EphA4 (Fig. 7 E and F) or Unc5H3 (Fig. 7 G and H) identity remain inactive in callosal neurons. Thus, each phase of 60 0 + in the Developing Mouse Neocortex resulted in the extension of β-gal axons across the CC. These corticogenesis and neuronal fate specification deploys an active WT Mut 1 Mut 2 Mut 3 Mut 4 Mut 5 Nfi Nfi dimer Neurod/g Nfi results suggest that EphA4 and Unc5H3 are critical downstream Element elt1 elt2 elt3 elt4 elt5 elt6 elt7 elt8 G_NF1_03 J_TLX1_NFIC G_NEUROD1_01 J_NFIC Neocortexrepression / Transgenics 3/3 of previous7/7 fates8/8 and6/7 a promotion6/10 6/6 of the6/7 appropriate8/9 fi Evolutionary conservation Mammaliafi Mammalia Eutheria Gnathostomata Theria Amniota Theria Theria C G G C G T T A C G A T G G Satb2 A T T targets of in callosal fate speci cation. C G A T G A A A C C SI Discussion CCC A A C G T T layer-speci c projection fate (see for further details). TAC A T G G T A G A G C C G C C G GGC GCC G G T C G C C C C A C TT T A T T A AA A T A A BCDEFGH 40 UCON11 Tbr1 seems to play distinct roles at different stages of cortical UCON31 •Mutating each of the preserved binding sites resulted in a two-fold induction or Discussion Whole mount development. At early stages, Tbr1 promotes a frontal identity ed / Expected

v MER121 reduction of reporter activity, relative to the un-mutated construct, when

r Our data suggest that cortical projection neurons actively repress while suppressing caudal identity (4). During the formation of MER131 transfected into dissociated cortical neurons. alternate fates to promote appropriate fate choices during de- layer 6, IJKLMNOPTbr1 plays an essential role in specifying the fates and 20 Obse velopment (Fig. 7O). When specific repressive interactions are projection patterns of corticothalamic neurons (4, 5, 7, 17). In- MER124 Coronal section Summary removed, alternative fates are executed (Fig. S1). Whereas Tbr1, terestingly, although corticothalamic projections are decreased Tbr1 Fezf2 Ctip2, and Satb2 are expressed in postmitotic neurons, Fezf2 is in QRSTUVmutants and increased in mutantsW (whichX show an 0 We performed ChIP-seq on E14.5 dorsal cerebral wall to identify active enhancers Coronal section • expressed in cycling cortical progenitors from very early stages of up-regulation(zoom) of Tbr1), these alterations in projections are in- x t te and found 6,629 p300 bound sites. fi rain r rain r corticogenesis (11). Loss of Fezf2 is critical for the speci cation complete:Adjacent gene Eomes there Satb2 are still Neurod2 some Tbr1 corticothalamic Auts2 Id4 axons Bhlhb5 in Auts2Tbr1 Y Z AA AB AC AD AE AF oreb •When tested in vivo, these putative enhancers drive laminar expression patterns of the subcerebral projections of layer 5 neurons, as evidenced by mutants, and Fezf2 mutant neurons do not completely convert to f Candidate target E11.5 hea E11.5 limb throughout the developing neocortex. gene in situ E11.5 E11.5 midb from Eurexpress E14.5 neoco or Allen Brain Atlas •For each combination of repeat family and p300 set, we shuffled the p300 set •The regulatory domains of several key neocortical developmental genes contain a Srinivasan et al. PNAS | November 20, 2012 | vol. 109 | no. 47 | 19077 single proximal p300 peak, and some of these peaks appear to be derived from Candidate target across the genome 10,000 times and compared the expected number of p300 gene E14.5 expression by laser micro 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 transposable elements. dissection and 6 elements overlapping each repeat family to the observed number. RNA-seqRNA-seq •22 of 90 (24%) MER130 instances identified in the mouse genome overlap our •We identified the transposable repeat family MER130 to be surprisingly enriched in our set of putative developing cortex enhancers, and validated that the putative AG E14.5 set. p300 Peaks Next to Key Neocortical enhancers function in vitro. 100 Developmental Genes MER130 Contains a Preserved Core of •MER130 elements contain a preserved code of regulatory logic that modulates enhancer activity when mutated. Mouse July 2007 (NCBI37/mm9) Transcription Factor Binding Sites 10 20 kb mm9 A 13,150,000 13,170,000 13,190,000 13,210,000 References: BAC End Pairs Transcription Factor Binding Preferences: Ratio to pGL4.23 to Ratio Nfi* Nfi* dimer Neurod/g Nfi* RP23-141E17 G_NF1_03 J_TLX1_NFIC G_NEUROD1_01 J_NFIC 1. Molyneaux, B. J., Arlotta, P., Menezes, J. R. L., & Macklis, J. D. (2007). Neuronal 1 UCSC Genes C G C GG T T A C G A T G G A T T C G A T A A A Nature reviews. Neuroscience G C C CCC A A C G T subtype specification in the . . T TAC A T G G T A G A G C C G C C G GGC GCC G G T C G C C C C A C Fezf2 Cadps TT T A T T A AA A T A

Enhancer Candidates Based on Conservation MER130 Consensus Motif: MER130 E4 E3 E1 2. Rubenstein, J. L., & Rakic, P. (1999). Genetic control of cortical development. elt1 elt2 elt3 elt4 elt5 elt6 elt7 elt8 elt9 A T G TG A AG G G G T C G G C T A C G A C A A C A G GT E2 G C A G T C G C C T C T G G T CA T G C CCC G A CT C T A C G G T G C T G A T A C A C A T C A T T A C A A T G T A G C elt10 A A G A T T C A G T C G T CT T C A G A G G A C T T G T A A A G G C C T T C A A A T C C T A G C A A G T G G T T C G A G T C GA A T A TT T G A T C G CA C T A A A C AA GAG C A C T T T G A A A C AC G G A A C CA A C AAC A GA T A G C CC C C A T C TC A A CA C C T T T T A C G T C G A C C A C C C A T T A TTTT C AC T T A T A G T T T C C TC T A C A A G G TC AG AT T AC G T C G T T T G T A GA A T T G CT C A C A T A CG A T T G G G T T A C G A C G A T T C G C C G C A T C A G G G A T A G A C A T T G T C A G G A T G T A T T T C GC A C G A C G A A G C C T A G G C G G G G C T G A A T A C C A A C A C T C A A A A C A G C T G T A G G A C A G C T A C A T G G T T G G A C C G T C C C G G C A A C C G A G C AAA C G A C G C C A G A G C G A T A T T T C A G A T T T G A A C A A G G CC T GT G G A C A C C A A A G C G G A A A T T C GC C G A T T G T T T AA C A A A T T G TG G G TAG A C C A G A A C T T G T C T A G A A C C A C T A T A C CA G T T T C A A A T T T A C ACT G A T C A A G A G A C C T A A C A G A A G A C A G A A T C A T A T C C A T T T T T T C TTT C T T TCC A A T T C C A A C T A A C C A GA C CA A C G A A C C G A T G A T C T GA A C A A A G C A C G A G G T G G A T T A C C T T G G A A A AT A A A C A C T A A T G T T T T C G G TG C T T T T T C C G A A A G G C A C C C A C A T G C G G A C C T A T C A T T C AT A C C T A G G G A C C G T A T T T T G G T T G A C G T A G C T G T T A T A C C A T G A C G T T G T T G G C A G G T T A AT G G A C C G G GG C G C C C G G G G GG C GC C C G GC G CTC G G G G G G C C C C C G G GGC G G A G T A T A G C G G T C T C G G G C T T CG T G T T T A C G G G T T G G G C C G G G T G A T G A G T G T T T A T C G C G CA G T C G T T TC GT GG G GTGA TG CTA AA GGC T G GT TT ATG G C C T T T TGC CG G AC G G GT A CC TTT C G C AT A C AT C T T A AG A CTT A T TC T G G G G C T C C TA A T TT A Cerebral cortex (1991). Placental Mammal Conservation by PhastCons pGL4.23 Element Multiple Alignment: 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 2703. Leone,280 D. P.,290 Srinivasan,300 K.,310 Chen,320 B., Alcamo,330 E.,340 & McConnell,350 360 S. K.370 (2008).380 390 400 410 420 430 440 450 460 470 MER130.0/1-448 ---TTTGGCACAGCCTTGTCAACAGGTGTGATCAACACTAAGGAAGCATGCAGTGCCAACACCCCTGTTCTGGTCCAGGATGA------GAACCCACCACTGAAAAGATGGCATTCACAGCCGCAGG CAGACTGCCA---TGAAATCGTTGTTCC-TTGGCACTGGTGCCCATGGAG-----AGTGTCTAGGCAACTTTTGA--TTGGGCACCATGCCCTCCAAG-CAGATGGGCATGAATGTA-GGCAGGCAA GAGA------CCATTTATTTATATCAACTGTATTTGCTGAGGG-CAGGGTTCCCAGCTGGGAACATCAAAACAGCTCTTTGTCCTGGCTGTCTGAATCTCTGATGGAGGTGATGCAGCCTTGC AAATTCAGCTAGATTAATTTTGAAAAGTAAAAACAATGTTTCTATGGACACCGGGGGACATTTTTGGCTCCCATCAATGGTTCAGAGCACAGAGCAGGCT MER130.1/1-165 ------GAGCAAAGATGGCATTCAGAGTGAATCMultiz Alignments of 30 Vertebrates T CAGGCTGCCTGTTAGA------GCCTACCATTCATGTCCCGGGGC-----TGGGGCCAACCAACTTTCTG----GGGCACCGTGCCATCATGA-CAGATGGGCATGGGG----GGCTGCCAG GATC------CTCGAGCCTGGGACCATTGTGTGTGCTCTGA------MER130.2/1-310 ------ATGGTG TATCCCATCCAAACAA---TTCATCTTTGTTGGCTCCTGCGCCTCGCTGC----TACTGGCATGCCACCATGATA--GCGGGCATGATGCCACAGTAA-CAGATGGGCATCAACACACACATGCCAA GCAG------TCAGTTGGTGCATCAAGGCTGGGTATTAGTATTCACTCTTCCT--CCAAGAAATTCATAAACTACATGAGGGCAAATCTAAGTTTCTACAACTGTGTTAGTGAGATATGCCA ACTCAAAGCTAGATTAATTTTAAAACTCTTTTTTTAAAAGCAAACATGTTTCCAGGGCCACAGAAGGTAAGTT------MER130.3/1-123 ------Human ------TTATGA--TTGGGCAAAATGCCACCTGCAACAGATGGATATGAATACA-GGCAACCAG AGAG------GCTGTGTTATACCCCAAGTCCTGGCATCTGTGGCC--GACTCCCTCACCAGGCTGTCAAAACAGCT------The determination of projection neuron identity in the developing cerebral cortex.------MER130.4/1-191 ------CACCGTCATGACTGCC CAGGTTGCTGGAGTAAA-CTGTTACTCTTCTGGCACCTACACACTGAGGC------AGGAGTCCAAACTTGTAG--ATGGGCAGCCAGCCATGTCAG-CAGATGGGCACAGGGAAA--GCGGCCAA GGAA------GCATGTGTGTGCATCCAGCATAGGAGTGCAGAA-CAGGAGCTCCAGCTGAGAGCATCA------MER130.5/1-254 ------ACAAGTGCCAACAGATGTGACCAATTTAATGAAGCATTCGTCTCCGACTTGGCACTCCTTCCAGAGGGAAGCCCTCCTCCCTCCTTTCAGCAAGAGCAGCAC------ATTGTDog A CAAGCTAGCCCTCTTC-ACCACCCTCTGCCTGGCA-----GCTCTGCGGAGCGAAACTGGCACATCATGGTGTGT--GTGGGAACCGGGCCATCACGA-CAGGTGGTCACAGCTCTGTAGCTGCCAG AGCT------ACACAATGAGCATCAGACTCGTGT------MER130.6/1-268 ------CTACAGATGTAAGGAGGCATGTGGCCTTGGACTAAAGCTGTTATGGAACAGCAG------GATTGCCTGGCCCAAGAAATGGCACTCGCTGTGTGATOpossum G AAAGCCGCCCCTCTAAAACCGTGTTTGG-TTAGCACCTATGCCCCACTGC----AGTTGGCAGGGCACATCCTCT--GTGGGCATGCTGCCAGCCTAA-CAGATGGGCCTGAATGTAACCCTGCCAG AAAA------TCAGTGTGTGCAATGAGTTCACAACTGTGG---CTAGATTCCTTTCCAAGGATGTT------Current opinion in neurobiology. ------MER130.7/1-215 ------CTGCTTGTCAGCTGTCACGTGTCAC------AGCTTTAGTCAACTCGAGG--GCAGGAATCCCTCCATCCTAA-CAGATGGATGGGAATTTA-ACCAGACAA TAAA------CCATGTACGTGCCATAAATATGTTTGCCAAGGG-CATGGTTCCCAGTGCGGGGC-TCTGCACAGCTGT-----CTTCCCGTGAACTCCCCGATTCTTGGTGAGATGATTTGTT AACTCTGAAACAG------MER130.8/1-302 ------AATGTGTGGCACCAGGGCCTGTGGAGTCTTAATGG------AGAGGATTTGGCAGTAAAACGGTCCCTGACTAAACCCChicken C CAGGTTGCCTATTAACACCTGCCATTAC-CTGCCATCTAAATGTAAGTTA----AATAATCTTCCCAATTTGTAT--TTTGGCAAAACGCCACCACAG-CAGATGGAAAAGACTGTA-TGTAACTAG GAAA------TTATGCATATACATTTAATGTGCGGACTGTGTG-CATTAGTCCTTTCGAGGAACATCCGAGCATCCTT-----CTGCTCTCGGCTTTTTTTATACTGTTTACACCATATGCCA ------MER130.9/1-212 ------AGGAGCTGCAGCAGCTGCGAAGCTCTGTAATGAAGCATTTGCCCCTCATTTGGCATCGCTTGCACTTGAGAG------GATTTTCTTACAAAAGATCAACTATTTGATGAATTCCA CAGGCTATCGCTCTGAAATTCCACATA--TTGGCACCTACATGCTGTTGA-----AATTGACTACCAGCTTCTGC---ATGGCACAGTGCCCTCATCA-CAGATGGCCAGGTG------4. Wenger, A. M., Clarke, S. L., Notwell, J. H., Chung, T., Tuteja, G., Guturu, H.,------& - MER130.10/1-263 ------GCCAGGATGACACTCCTGCCACGAGGGAG------GGATTTCCTAC-GAACAGATGTCACTTAGAGTATTATFrog G TGGACTATCTATCTCAACAAATAATTGC-CTGGCACTTCTGCCCTATGGC-----AAGGGTCTGAAAACGTGTTATGGTGAGCAC-ACACTATACAAG-CAGATGGGCACAGAAAAC------ACAG AATGGAAGGAAGCACAAGGAGCCTGCAGCATGTGTGGGAAAG------CCAGGTGGGAGTACTGAAGCAGCTGT-----CTCTGCTCAGCCTCCA------MER130.11/1-306 ------GTAGTACTGTCACAGAAT------TTTCCCCCTGCTGAAGAGCAGGCTCTCACAGTGCTGTG CAAGCTGCCCATTTAAAACTATCATTGC-TTGGCACCTATGCCCTAGTGA------CAGTTTGACCAACATGTGA--GCAGGCATACTGCCATCCTGA-CAGATGGGTGTGAGTACA-GGCAGCCAA GAAA------CCATGTATCTGCATCTAGTGTATTTGTTAAGGG-CAGGATTCCCAGTTGGG-ATGTTCAAACGTCTGT-----CCATCCTGAGCTTTCACGCCTGTGGATAAAGGGTTGTCAT CATTAATCCCAGCTCTGACTGTGA------MER130.12/1-95 ------Zebrafish ------CTTCTAAAGCTATCTT----TTGGCGCCTTTGTCCCTGGGA----AGCCAGCACACCATCTGGCAG--GTAGGCACAGTGCCACTAAG--CAGATGGG------ACCAA GCGG------MER130.13/1-168 ------TGAAAAACATCTGGCTGGCTTGGCACTTAAGC-----TGAGTAAAAATAGCAAGCCATCTTGTGA--GTGGGCACCACGCCACTGTGG-CAGATGGAGAATAATGCT-GGCAGCCAA GCGA------CGGTAGAATGGTACCCAATGAATTTGCAGAGGG-CAGACCTCGCAGCTGGAAGGGTC------Bejerano, G. (2013). The enhancer landscape of early neocortical development ------MER130.14/1-242 ------CACAGATGTTGCCCATTGCAGAGGAGCACACTGCCCTGTCCTGCTGCAATTGCCAAACAGAAG------GTTCTCTTGGCAGGCATGTTGATGCATACTGTGCTCAE14.5 Dorsal Cerebral Wall p300 ChIP-seq Signal G CGGGCTGTGCCCCTGAACCTGCTTTTCTATTGGCAAATGGGCCTCCTCAG----AACTGGCTTGCCTCATTCTCC--ACAGGCCTCCTGCCACCCAAA-CAGATGGGCACAAGGGTAATTGTGCCAG GGGC------TAAGAATGCACATCAGC------MER130.15/1-437 ------AGAAGCATCTACAGATGCAGTGTGATCCCAGGCAGTGAAGCATGTGGCCCTTCATTGGCATTAGACGATAACAGGAG------GATCTCCTGGCAGAAGAACTGACTTTCAGTGTGTTCTG CATGCTGCCCCTCCAAACCCATCTTTGA-TTGGCACCTATGCCCTATTGA----AACTGGCATCACACCTTCTGA--ATGGGCACTGTGCCAGACTAG-CAGATGGGTATGGGCAAAGGCATGCCAG AAAC------CCAGTATGTGAATCAAGTTTAAGTATCAGGGGCCCAAACTCCCATCCAGGGATCTAAAAACTGCAGT-----CTGTTAAGAGCTTTTTGTGCTTAAGGTTAAAGGACATGCC ATTTCTACTCTAGCTTAGAGGTTGATTAATTTTGAAAGTCAAACAGGAACTCCTGTGGTGGTGTCAGGCAAAATTCTGTCTTTGGTGAATGA------MER130.16/1-232 ------AGAGCTGTCTTTGGGC------ATCTGTATG------AGTTTTGTA AGGGCTTCTTGTCTAA---AGCCTTTTC-TTGGCCCCTGGGTTTTGGCAA----AGCCAGTCGACTCTCCTACAA--GCTGGCACAAGGCCATGGTAA-CAGATGGCCAGGAGCGTA-GGCAGCAAG GAAAGCAAAGGAAAGCATGTAGGAGCGTCTGTAGCGAGGGCTGAGGG-CGAGTGCTTCAGAC----ACAGCCTAGAGTCTGT-----CTGTCC------reveals patterns of dense regulation, pleiotropy and co-option. PLoS Genetics. ------MER130.17/1-309 CTGCTTTTGGCATTAGCAAGTCAACAGGTGTGACAAAGTTTAAATGGATGTAAGATGCCACCTGCTTTTCTAACTCTGGCATGAGTAACGGCCTCCTGTAAGAAGAGCTGCCTCCTGGAACATTTTG TTAGCTGTCA------TTGGCTGGGACTCATGTCATAGTAT----AAGAGACCTGCCGGCTTCTAA--GCAGGCAACCCGCCATCTAAG-CAGATGGGCATGGATTCT---CAGCCAA GAGG------CCAAGTGTATACAGCAAACATAGGTATCGGGGC-CTCGTTTCCCAC---AGAGCTTGCTAACGTCTGTTTGTCTTGATAT------MER130.18/1-255 ------E14.5 Dorsal Cerebral Wall Input ChIP-seq Signal ------TGGCAC------AGGTCTGACCAACTCAGAA--GGAGGCATTCTGCCAAGCACA-CAGATGG-CATGGGGGGA-AAGTGCCAA AAAA------ACATGAGAATCAACTGTGGGTACTGGTGG-CATGATTCCCAGACATGGAAGTCCAAACACTTGTCCATACCAAGCTTATAGCACATACTTACAGATATGCAGATTTGCC AACAGTAATCTAGCACTGTTAGGTTAATTTTTAAAAATATTTTTAAAGCTACCGTTGGCTCCTGAAGCTAG------MER130.19/1-338 ------AAAATTGTCACTTGTAGTATTATG CAGGCTGCCCATCTAAAGCCATCATTGC-CTGGCACCTACACACTAAGGG---CAAGGATCCAGCCAACTTGTGA--GTGGGCACCAGGCCATCTGAG-CAGATGGGCATGAAAGTC-GGCAGCCAG TAGA------CCATGTATGTGCATCAATGGAGGGTGTTGAGGG-CACGATTCCCAGAGGAGAATGTCCTAACAGCTGT-----CTGGCCTGAGCCAACATACTTACAACCAAGGAAAAGTGCC ATCCTTCAATCCTGCTGAGTCGGATTAACCTTCAAAACAAAATCTTCTTTTTTTCAGCAGCAGAAGGTGAGTTTGGGTGTCTCT------MER130.20/1-344 ------GCAGCAGAGCTGTCACCTGCTTTACTATG CGGGGTGCCCAGCTTAACCCGTATTTCT-TTGGCAACTGTGCCCCAGTGAGAAAAGCTAGCGGTCCATATGTTGA--GTAGGCACCATGCCATT-----TTGACAGGCATGAGTGTC-GGCAAGAAA GGGA------CCATGTATGAGCATCAAATGTGTTTGTTGTGGG-AAGGATTCTAGGCCAGGAATATTAAAATAACTGT-----CTGTCCTTCGCTCTAGCACATATGGTTAAGGAGATATGC5. McLean, C. Y., Bristor, D., Hiller, M., Clarke, S. L., Schaar, B. T., Lowe, C. B.,T ATGATGACTCACATTAACATTGGAAAAAATATATATAGAATTTGCAGCCATGCTAAGTGAGTTTCTGGTGTTGATAAATTGGGCTT------et - MER130.21/1-291 ------TTTTGGCTGAAAAATAATGAACAGGTGGTGTGTCGGTAAGAAGCCGTGTGGGTGTTAATGTGTCATC------GTTTTCTACAGAACACGAGAGCATTTTCAGAATTTT- -AGGTTGCCAATCTAC------TTCGCTGGCACCTGCAGTCTA------AACTGGAACACCATTTTGAGA--TCAGGCATGTTGCCATTGTAA-CAGATGGTTACGGCTACT-CACAGCCAA GTGA------TCATGTATGTACATGGGTTGTGGGTGTTGAAAG-CAAGCTGCTCATTTAGGAAGGTCAAATAGCAT------TTATCCAGCTCTTC------MER130.22/1-117 ------E14.5 Dorsal Cerebral Wall p300 ChIP-seq peaks ------A--CCAGGCATGGTGCCATACTGA-CAGATGGCCGCGTGAGTA-GATGGCCAA CAGA------TAATGTATGAGCAACAAATGTGTTTGCTGAGGG-CAGCTTTCCCAGGTAAGAGCTTTAAAGCAAC------al. (2010). GREAT improves functional interpretation of cis-regulatory regions.------Consensus e14dcw_p300.1848 ----TTT + - A + A + T A +++ ACAA + A G + TGTG + + CCAATGTAA + GAAGCATGTGGCCCTGAC + TGGCRepeating+ + TGCT + + C +ElementsCACGG + AG-----C by RepeatMaskerG + TCTCCT + GCAGAAAAGATGGCACTT + CAGTATT + T G CAGGCTGCCCATCTAAACCTGTCTTTGCGTTGGCACCTATGCCCTAGTGAG-AAAACTGGCATGCCAACTTGTGA--GTGGGCACCATGCCATCCTAA-CAGATGGGCATGAGTGTAAGGCAGCCAA GAAAG-A------ACCATGTATGTGCATCANature BiotechnologyA + TGTG + GTGTTGAGGGCCAGGATTCCCAG. C + AGGAA + GTCAAAACAGCTGTTTGTCCTGTCCT + AGCTTTCACACTT + T + G + TAAGG + GATTTGCC A A + TTTA + + C A + + T T + A + TTTGGAAAAAATTTA + AAAGA + TAT + + A G + CTTCCTG + G + CACT + TAGG + TAGT + TTAAT + TTTC-GTG-A------SINE Instance 1 Mutations: LINE 6. Ayoub, A. E., Oh, S., Xie, Y., Leng, J., Cotney, J., Dominguez, M. H., et al.

LTR Instance 2 Mutations: DNA (2011). Transcriptional programs in transient embryonic zones of the cerebral

•A single E14.5 p300 peak next Mouseto Fez Julyf2 2007contains (NCBI37/mm9) the E4 developmental enhancer cortex defined by high-resolution mRNA sequencing. Proceedings of the National whose genomic deletion leads to20 kbaberrant cortico-spinalmm9 projection fates, similar to •The multiple alignment of MER130 cortex enhancer instances shows a well- Academy of Sciences. B 61,620,000 61,640,000 61,660,000 61,680,000 those found in its Fezf2 target gene conditionalUCSC Genes knock-out7. conserved core containing 5 binding sites resembling known motifs: a Neurod/ 7. Shim, S., Kwan, K. Y., Li, M., Lefebvre, V., & Šestan, N. (2012). Cis-regulatory Psmd14 Tbr1 Neurog motif, an Nfi dimer, and two additional Nfi motifs. control of corticospinal system development and . Nature. Placental Mammal Conservation by PhastCons

Multiz Alignments of 30 Vertebrates Human Dog Opossum Chicken X_tropicalis Zebrafish 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 e14dcw_p300.3376 Repeating Elements by RepeatMasker SINE LINE AmnSine1 LTR DNA