Symmons et al.

SUPPLEMENTARY INFORMATION

Functional and topological characteristics of mammalian regulatory domains

Orsolya Symmons 1, Veli Vural Uslu 1, Taro Tsujimura 1, Sandra Ruf 1, Sonya Nassari 1,

Wibke Schwarzer 1, Laurence Ettwiller 2,# and François Spitz 1,*

1 Developmental Biology Unit – European Molecular Biology Laboratory - Meyerhofstrasse 1 - 69117 Heidelberg – Germany 2 Centre for Organismal Studies – University of Heidelberg – Germany # Present address : New England Biolabs - Ipswich – MA - United States

Supplementary Figures 1-8

Supplementary Table 6

Supplementary Note - Methods

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Supplementary Figure 1

A Enrichment of insertions relative to heart EP300 sites B Enrichment of insertions relative to forebrain EP300 sites

9 9 8

8 7 7 6 6 5 5 4

4 Enrichment 3

3 Enrichment 2 2 1 1

0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 distance to EP300 binding site (kb) distance to EP300 binding site (kb)

C Enrichment of insertions relative to midbrain EP300 sites

12 limb 10 heartmidbrain 8 forebrain

6 midbrain negative

Enrichment 4

2

0 0 200 400 600 800 1000 distance to EP300 binding site (kb)

Enrichment of insertions in the proximity of EP300 sites bound in the same tissue. Enrichment of insertions with tissue-specific LacZ activity (compared to random insertions), at increasing distance (x-axis) from EP300 sites detected in heart (A), forebrain (B) and midbrain (C). Error bars represent one standard deviation from the mean. Colours indicate the tissue in which insertions were expressed (limb: green; heart: purple; forebrain: blue; midbrain: red; no LacZ activity: grey). EP300 data is taken from (Blow et al. 2010) and (Visel et al. 2009). The overlap and proximity between EP300 sites (Visel et al. 2009) may account for the enrichment of forebrain activity around midbrain EP300 sites, and vice versa.

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Supplementary Figure 2

B Hpse2 A 2 bp ex4 13 kb ex3 chr15:63,550,550 chr19:43,106,401

192857 179039 183610 194246

C 178338a remob256

Brinp3 100 kb 135 kb 183 kb

chr1:148,846,484 +/- 1kb remob100 remob12 remob58 remob191

e

pl

178338a remob100 remob256 remob58

remob12 remob191

Orientation-independent activity of the regulatory sensor. Precisely overlapping lacZ expression patterns obtained in E11.5 mouse embryos with adjacent insertions, indicating that the orientation of the insertion relative to the activating enhancer does not influence enhancer-sensor interactions. (A) Two insertions separated by 2 bp in a gene desert on mouse chromosome 15. 192857 was produced by remobilization from 179039, and inserted at the same position, into the duplicated TA scar. (B) Two independent insertions into an intron of Hspe2, on mouse chromosome 19. (C) Multiple insertions in a 300 kb region downstream of Brinp3, on mouse chromosome 1, inserted in different orientations and separated by distances from 6 bp to 183 kb. In each case, LacZ staining was detected in the developing ear (e) and proximal limb bud (pl). Further examples of orientation-independent activation are also shown in Supplementary Figures 4 and 8.

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Supplementary Figure 3.

Hi-C TAD Shh ZRS Rbm33 Rnf32 Nom1 Ube3c 9530036O11Rik Mnx1 Lmbr1

203881e12

ShhSB1-8c ShhSB2-9a regulatory domain

ZPA ZPA ZPA ZPA

203881e12 Shh::lacZ ShhSB1-8c ShhSB2-9a

Long-range detection of a single limb enhancer at the Shh locus. Expression of Shh (indicated with a Shh::lacZ allele, kindly provided by Andreas Kottmann (Gonzalez-Reyes et al. 2012)) in the zone of polarizing activity (ZPA, orange arrow) in the limb bud has been shown to arise from a single enhancer, the ZRS (orange oval), as indicated by loss-of- function experiments and enhancer-scanning of the entire gene desert (Jeong et al. 2006; Sagai et al. 2005). Insertions into this gene desert, as well as an insertion 30kb downstream of the gene recapitulate multiple aspects of the Shh expression pattern, including expression in the limb bud (orange arrow). This indicates that the ZRS – the only enhancer with limb activity in this region – can activate the regulatory sensor at long-distance. The large regulatory domain defined by these insertions largely overlaps with a topological domain defined by Hi-C, although the insertion downstream of Shh (203881e12) falls within the TAD boundary. A schematized version of the locus is shown, with genes indicated by arrows (Shh in orange, protein-coding genes in black, non-coding gene in grey). Hi-C data (Dixon et al. 2012) is represented by two-dimensional heat maps, and TADs are shown by white, brown and green bars, with the unstructured region indicated by a dashed line.

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Supplementary Figure 4.

a b d e f Evx2 Hoxd13

HoxD centromeric regulatory domain

TADs * b d a c e f Hoxd13-11 cohesin

Atf2 CTCF (invariant)

Lnp CTCF (limb) Atp5g3 Evx2

Hoxd limb regulatory archipelago

a b c d e Hoxd13

The regulatory sensor integrates the input from several non-redundant enhancers at multiple positions of the Hoxd regulatory domain. Multiple enhancers with diverse spatially restricted activities (blue ovals, patterns of activity represented on a limb outline) have been identified in a large gene-poor region centromeric to the Hoxd cluster (Spitz et al. 2003; Gonzalez et al. 2007; Montavon et al. 2011), forming a regulatory archipelago. Multiple insertions have been obtain within this interval (a-e: SB sensor, this work and (Ruf et al. 2011); f: Hoxd9lac transgene, rel5 (Montavon et al. 2011)). Expression patterns in E11.5 (E11 for b) embryos are shown, including details of the forelimb (anterior: left; posterior: right). At all insertions (except e, which showed only extremely weak expression in the posterior limb, arrow), the reporter gene showed the typical expression pattern resulting from the integrated ouput of the archipelago, and not only the specific activity of the nearby enhancer element. CTCF (red lollipops = cell-invariant sites detected by ChIP, red lollipops with white center = ChIP-chip data from embryonic limbs (Soshnikova et al. 2010)) and cohesin-complex (purple rings) (including embryonic limb data from (DeMare et al. 2013)) are interspersed within the relatively homogeneous regulatory domain. The regulatory domain (extended here to include the endogenous genes) and the corresponding TAD are shown as colored bars.

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Supplementary Figure 5

B A

Hi-C TADs

1260 kb

Foxg1 hs566hs1168hs1523 Prkd1 hs342 hs433 Hi-C chr8:91M-92M TADs

hs342 hs848 hs71 * Snx20 Cyld hs152 280kb 140kb hs566

Nkd1 Nod2 Sall1 195041 216512 182529a

202250e1 202250 177175-emb7

C chr19:42.5M-43.6M Hi-C 195041 216512 Sall1 182529a TADs

Loxl4 405 kb

Hpse2 211532 194246 Hps1 Cnnm1 Pyroxd2 183610

fm fm al fm al D mnp mnp pl pl pl sc sc sc

211532 183610 194246

chr3:15.8M-18.8M

Hi-C TADs E Hi-C chr8:58.9M-60.0M 694 kb 404 kb 514 kb TADs

Cyp7b1 526 kb Cep44 Scrg1 Ythdf3 hs1239 Mir124a-2 di rh Fbxo8 limb Hand2 EP300

te

md sc md sc hs1239 196680-5 Mir124 (Makeyev et al, 2007) lb lb

198191 Hand2

Overlap between the functional and topological subdivision of the genome Extended regions of co-expression, outlined by insertions of the regulatory sensor (defining a regulatory domain, A-C) or by an endogenous gene and flanking insertions (D-E) are contained within a single topological domain. The different loci (coordinates given are based on NCBIM37/mm9 mouse genome assembly) are represented schematically, with the extent of the RD and distances between genes (black boxes) and insertions indicated. The constitutive topological domains (Hi-C TADs as defined by (Dixon et al. 2012) (data shown from mouse ES cells) are represented as two- dimensional heat maps and as brown and white coloured bars, with unstructured regions shown as dashed lines. CTCF and cohesin binding sites are represented by red lollipops and purple circles, respectively, with the intensity indicating degree of tissue-invariance. Enhancers (ovals) and EP300- bound regions (diamonds) are indicated. Outlines of embryos show corresponding colors in the regions where enhancer were found active (data from (Visel et al. 2007)), or from which the EP300-

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ChIP data was generated (Visel et al. 2009; Blow et al. 2010). (A). Multiple insertions in the gene desert surrounding Sall1 define a RD based on expression at the midbrain-hindbrain boundary, in the posterior limb bud and the tail. The RD is contained within a single TAD. (B). Insertions in the large gene-desert flanking Foxg1 (as shown in Figure 4B-adapted from Chen et al. 2013) define a large RD that is included in a TAD containing several forebrain EP300 sites and confirmed enhancers (Visel et al. 2013), the activities of which correspond to the ones detected by the sensor (dark green=active broadly in the forebrain; light green=restricted activity to sub-regions of the forebrain; brown=cranio- facial) (C). Expression in the facial mesenchyme (fm), proximal limb bud (pl) and sclerotome of three insertions on chromosome 19 outlines a RD, which is included in a single TAD. Within the RD, the two insertions in an intron of Hpse2 (183610 and 194247) show additional expression in the anterior limb bud (al) and the medial nasal process (mnp), indicating the possible presence of substructures with the RD. (D). The miRNA gene Mir124-2 is located at the boundary of a gene desert. The entire gene desert is part of a single TAD, and a distant insertion into this gene desert captured the expression domain of the miRNA gene (in situ panel reprinted from Molecular Cell, Makeyev et al. 2007, with permission from Elsevier) in the spinal cord (sc), rhombencephalon (rc), diencephalon (dl) and telencephalon (te). One putative enhancer with expression in the spinal cord, also located in the gene desert, has been documented in the VISTA enhancer browser. (E). The 198191 insertion half a megabase away from the Hand2 gene shares expression with the gene in the posterior limb bud (lb) and the mandibulary process (md), indicating a possible co-regulation. The interval is contained within a single TAD.

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Supplementary Figure 6.

A B

Hi-C Hi-C chr8:94.48M-96.04M TADs TADs chr7:142.4M-143.9M

Lpcat2 Mmp2 Ces1 cluster Irx5 Irx6 Slc6a2 100 kb Ptpre Mki67 100 kb

197116-9 193083 193883 179860a

Overlap between functional transition zones and topological boundaries Insertions bounding a transition zone are separated by a topological boundary. The different loci (coordinates given are based on NCBIM37/mm9 mouse genome assembly) are represented schematically. The constitutive topological domains (Hi-C TADs as defined by (Dixon et al. 2012), data shown from mouse ES cells) are represented as two-dimensional heat maps and as brown and white coloured bars, with unstructured regions shown as dashed lines. CTCF and cohesin binding sites are represented by red lollipops and purple circles, respectively (A). Two insertions downstream of Irx6 show strikingly different expression, outlining a TZ. The TZ overlaps a TAD boundary. (B) A TZ defined by two insertions in a gene desert upstream of Mki67 overlaps the boundary between two adjacent TADs, as well as the presence of multiple tissue-invariant CTCF and cohesin binding sites.

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Supplementary Figure 7

Distribution of putative boundary elements, genome-wide

B D E A CTCF sites bound in 15+ tissues C all SINE B2 elements SINE B1 (X35S) elements CTCFPlot sites RDs vs CTCF4mbound using in mm9 4 asor sampling less space tissues Plot RDs vs CTCF15p using mm9 as sampling space Plot RDs vs TSS usingTSS mm9 as sampling space Plot RD vs SINEB2 all using mm9 as sampling space Plot RDs vs X35S using mm9 as sampling space 0.10 0.6 ** *

** * ** * 0.8 ** * ** * 0.08 0.5 0.15 0.08 0.07

0.6

0.4 0.06 0.06 0.10 0.05 0.3 0.4 Frequency (per 10 Kb) Frequency (per 10 Kb) Frequency (per 10 Kb) Frequency (per 10 Kb) Frequency (per 10 Kb)

0.04 0.04 Frequency per 10kb 0.2 0.05 0.2 0.02 0.03

A B C D A B C D A B C D A B C D A B C D A B RD TZ A B RD TZ A B RD TZ A B RD TZ A B RD TZ Ranks (ASC) : A=916 ; B=761 ; C=1 ; D=49 (rdm set num=1000) Ranks (ASC) : A=839 ; B=780 ; C=1 ; D=46 (rdm set num=1000) Ranks (ASC) : A=925 ; B=758 ; C=1 ; D=17 (rdm set num=1000) Ranks (ASC) : A=328 ; B=567 ; C=1 ; D=20 (rdm set num=1000) Ranks (ASC) : A=255 ; B=761 ; C=2 ; D=29 (rdm set num=1000)

F CTCF sites bound in 9+ tissues, G SINE B2 elements, H Plot RDs vsnot CTCF9OhneTSS overlapping using mm9 TSS as sampling space notPlot overlapping RDs vs SINEB2ohneCTCF CTCF using mm9 binding as sampling sitesspace Plot truncatedintraRDs vs SINEB2truncated SINE usingB2 mm9elements as sampling space 0.14 0.14 ** 0.6 ** * ** * 0.12 0.12 0.5 0.10 0.10

0.4

0.08 0.08

0.3 Frequency (per 10 Kb) Frequency (per 10 Kb) Frequency (per 10 Kb)

0.06 0.06 Frequency per 10kb 0.2 0.04 0.04

0.1

A B C D A B C D A B C D A B RD TZ A B RD TZ A B RD TZ Ranks (ASC) : A=667 ; B=845 ; C=1 ; D=147 (rdm set num=1000) Ranks (ASC) : A=238 ; B=468 ; C=1 ; D=34 (rdm set num=1000) Ranks (ASC) : A=320 ; B=674 ; C=1 ; D=13 (rdm set num=1000)

Distribution of putative boundary elements, within topological domains

J L M I K all SINE B2 elements SINE B1 (X35S) elements CTCFPlot intraRD sites vs CTCF4miboundnus usingin 4 TADs or as less sampling tissues space CTCFPlot intraRDs sites vs CTCF15 bound using T ADsin as15+ sampling tissues space Plot RDs vs TSS usingTSS TADs as sampling space Plot intraRDs vs SINEB2full using TADs as sampling space Plot intraRDs vs X35S using TADs as sampling space 0.8 0.10

0.15 ** ** 0.7 1.0 0.15 0.6 0.08 0.8 0.10 0.5 0.06 0.10 0.6 0.4 Frequency (per 10 Kb) Frequency (per 10 Kb) Frequency (per 10 Kb) Frequency (per 10 Kb) Frequency (per 10 Kb) 0.4 0.3 0.05 0.04 Frequency per 10kb

0.05 0.2 0.2 0.02 0.1 0.00

A B C D A B C D A B C D A B C D A B C D A B RD TZ A B RD TZ A B RD TZ A B RD TZ A B RD TZ Ranks (ASC) : A=301 ; B=708 ; C=217 ; D=404 (rdm set num=1000) Ranks (ASC) : A=387 ; B=717 ; C=86 ; D=376 (rdm set num=1000) Ranks (ASC) : A=536 ; B=549 ; C=3 ; D=137 (rdm set num=1000) Ranks (ASC) : A=22 ; B=678 ; C=1 ; D=169 (rdm set num=1000) Ranks (ASC) : A=29 ; B=751 ; C=133 ; D=66 (rdm set num=1000)

Distribution of boundary elements relative to operationally defined domains. Tissue-specific (bound in 4 or less tissues; (A)) and highly tissue-invariant (bound in 15 or more tissues; (B)) CTCF sites, as well as transcriptional start sites (TSS; (C)), SINE B2 (D) and a subclass of SINEB1 elements (X35S; (E)) are depleted within regulatory domains (“RD”) when compared to a randomized dataset. The same features are less or not depleted in transition zones (“TZ”) and control regions (class “A” and “B”).. This specific depletion was also observed when we considered only CTCF sites not overlapping TSS (F), or SINE B2 elements not overlapping CTCF binding sites (G) or truncated SINE B2 elements, which contain neither CTCF, nor PolII-PolIII binding sites, which have been suggested to contribute to SINE B2 boundary activity (Lunyak et al. 2007) (H). When considering only intra- TAD regions TSS (K) and SINE B2 elements (L) maintained depletion. Tissue-specific CTCF sites (I), and X35S elements were also no longer depleted when comparing only intraTAD regions, whereas highly tissue-invariant CTCF sites show lower density, albeit with barely statistical significance (p=0.086), in RDs (J), (M). For all panels the frequency distribution of the randomized dataset is shown by a grey box-plot, while the true observed measurement is represented by a red dot. Cases where the difference between the real observation and the randomization is statistically significant (P<0.05) are indicated by a single blue asterisk, and cases where it is highly statistically significant (P<0.01) is indicated by two asterisks.

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Supplementary Figure 8

A C

Scale 500 kb mm9 chr7: 122,000,000 122,500,000 123,000,000 Calcb Insc AK136021 Sox6 1700003G18Rik Insc AK043303 Sox6 1110004F10Rik Sox6 1110004F10Rik Sox6 Sox6 Sox6 Sox6 Sox6 Hi-C 177182b-144 178338a-150 007921 GSM800624 TADs GSM876518 cohesin GSM876521 GSM912911 GSM1036127 ChIP-seq GSM1036128 TAD TAD GSM1036133 GSM1184316 GSM883646 boundary ? insulator? ??? boundary ? peaks GSM1184315_Nipbl

}

} } } 5 - Cb 8w CTCF 0.2 _ 5 - cohesin Kidney 8w CTCF 0.2 _ 5 - cell-invariant Cortex 8w CTCF 0.2 _ CTCF 5 - CTCF Heart 8w CTCF Insc 0.2 _ Sox6 Plekha7 5 - 100 kb ChIP-seq BM 8w CTCF 0.2 _ 5 - MEF 8w CTCF 0.2 _ 5 - Spleen 8w CTCF 0.2 _ 5 - Thymus 8w CTCF 0.2 _

177182b-144 178338a-150 007921

B D

Scale 100 kb mm9 chr5: 28,400,000 28,450,000 28,500,000 28,550,000 28,600,000 Insig1 En2 Cnpy1 Cnpy1 207770e1 198124e6 ShhSB2-330kb

GSM800624 Hi-C GSM876518 cohesin GSM876521 TADs GSM912911 GSM1036127 GSM1036128 ChIP-seq GSM1036133 GSM1184316 En2 GSM883646 Shh peaks GSM1184315_Nipbl

5 - Cb 8w CTCF 0.2 _ 5 - insulator? ??? insulator? Kidney 8w CTCF 0.2 _ 5 - cohesin Cortex 8w CTCF 0.2 _ En2 Cnpy1 5 - cell-invariant CTCF Heart 8w CTCF 0.2 _ CTCF 5 - ChIP-seq BM 8w CTCF 0.2 _ 5 - Insig1 MEF 8w CTCF 0.2 _ 5 - Spleen 8w CTCF 0.2 _ 5 - Thymus 8w CTCF 0.2 _

207770e1 197626 198124e6

Constitutive CTCF sites within extended regulatory domains. (A). Schematic representation of a large TAD extending from Insc to Plekha7 and encompassing the Sox6 gene. Insertions showed both shared expression in the developing limb and facial chondrocytes (black arrowheads and arrows, respectively), and specific ones (e.g. forebrain, red arrow). Constitutive CTCF sites (dark red lollipops) or more cell-type variable ones (light red lollipops) are dispersed throughout the region. Constitutive CTCF sites are largely co-bound by cohesin (purple circles). Whereas some of these CTCF-cohesin sites may correlate with topological or regulatory boundaries, the role and function of others is more elusive. (B). An extended regulatory domain encompasses the En2-Cnpy1 genes, with multiple insertions showing LacZ staining at the midbrain-hindbrain boundary in E13 embryos (expression is also seen in E11.5 embryos – see Figure 3). This expression matches the activity of a documented enhancer (green oval), and several additional EP300 binding sites in the midbrain have been identified in this region (green diamonds). Three constitutive CTCF sites (red lollipops) that overlap tissue-invariant cohesin binding sites are found interspersed in this region. (C- D) Screenshots of the Insc-Plekha7 and En2-Cnpy1 regions in the UCSC browser, showing extensive overlaps between cell-invariant cohesin and CTCF peaks. CTCF and cohesin data assembled from published resources (Supplementary Table 6). Heat maps for Hi-C data taken from (Dixon et al. 2012).

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Supplementary Tables

Table S1. List of all transposon insertions used for large-scale comparative analysis.

Positions are based on the NCBI37/mm9 mouse genome assembly. Details available on the

TRACER database (Chen et al. 2013).

Table S2. List of insertions (after clustering of neighboring ones) for expression analysis, annotated for activity in the limb, midbrain, forebrain and heart (Y=expression detected) in

E11-12 embryos.

Table S3. Neighbouring enhancer-transposon insertions pairs (within 200kb), with comparison of their activity/expression patterns. Enhancer names and references (PMID) are given. Two additional tables list the results of the randomisations performed on the data with the corresponding annotations of the pairs

Table S4. List of the enhancer-gene-transposon insertions triplets. Relative positions were calculated by taking the enhancer as a reference (position 1 = middle of the enhancer fragment), and adapting orientations to have the target gene TSS located at a “plus” position.

Loci where the enhancers (can) regulate multiple target genes are indicated in comments.

Table S5. List of regulatory domains and transitions zones, and other intervals with regulatory information. The IDs and position of the insertions determining the ends of the intervals are given. Their type is annotated as described in the Experimental Methods. Their position with respect to overlapping topological structures was defined using data from

(Dixon et al. 2012): “intra-TADs” labels interval that are contained within a single TAD;

“interrupted” is for intervals which span two or more adjacent TADs; intervals that are extend from a TAD into an adjacent unstructured domain are labelled as “unstructured”.

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Table S6. – Source of the datasets used for comparative analysis.

Type of Data Reference Web / details SB sensor (Chen et al. 2013) http://tracerdatabase.embl.de expression data in or mouse embryos http://www.ebi.ac.uk/panda-srv/tracer/index.php Hi-C, mouse (Dixon et al. 2012) http://chromosome.sdsc.edu/mouse/hi- (ES, cortex) c/download.html p300 (Blow et al. 2010) ChIP peaks 1 CTCF (Shen et al. 2012) http://hgdownload.cse.ucsc.edu/goldenPath/mm ChIP peaks 2 9/encodeDCC/wgEncodeLicrTfbs/ Cohesin 3 (Remeseiro et al. 2012) GSM800624: SA1; MEFs ChIP peaks GSM876521: SMC3; MEFs GSM876518: SMC1; MEFs (Seitan et al. 2013) GSM1184316: Rad21; thymocytes GSM1184315: Nipbl; thymocytes (DeMare et al. 2013) GSM1036133: SMC1_cortex_e14.5 GSM1036128: SMC1_limb_e11.5 GSM1036127: SMC1_limb_e11.5 (Phillips-Cremins et al. GSM883646: SMC1; NPC 2013) (The ENCODE Project GSM912935: Rad21; MEL Consortium et al. 2012) GSM912911: Rad21; CH12 TSS UCSC genome browser http://genome.ucsc.edu/index.html Mm9; from RefSeq Track SINEB2 4 UCSC genome browser http://genome.ucsc.edu/index.html Mm9; from Repeat Masker Track (repClass=SINE; repFamily=B2) SINEB1 (Roman et al. 2008) provided by Angel Carlos Roman. X35S Enhancers (Visel et al. 2007) http://enhancer.lbl.gov/ In situ gene Emage http://www.emouseatlas.org/emage/ expression data (Richardson et al. 2010) EMBRYS http://embrys.jp/embrys/html/MainMenu.html (Yokoyama et al. 2009) GXD http://www.informatics.jax.org/expression.shtml (Finger et al. 2011) MAMEP http://mamep.molgen.mpg.de/ (Geffers et al. 2012)

Notes 1 For EP300 sites the position considered was the centre of the peak. 2 For sites bound by CTCF in different tissues we merged sites (taking the position of their median as new position) when they overlapped by at least 1 bp. 3 We define as a cohesin binding sites any region identified as a peak in ChIP-seq experiments targeting any protein of the cohesin complex. For the analyses carried out, we restricted to “invariant” regions, occupied by the different proteins in the different tissues and cell-types assessed. Importantly, we found a very large overlap between the different datasets. 4 From the SINE B2 list, we further distinguished the elements that were truncated to only the last 90 bases (or less) of the consensus sequence, and therefore did not contain the PolII, PolIII and CTCF binding site.

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Supplementary Note 1.

Enrichment analysis for EP300-SB comparison Enrichment was calculated as the mean of

Nspecific,x/Nrandom,x, with Nspecific,x being the number of true insertions and Nrandom,x being the number of random insertions located within x bp of the nearest EP300 site. Nrandom,x was set to 1 if no random insertions were found within x. Only x with Nspecific,x of 5 or more were considered. We tested enrichment at a distance from 0 to 1 Mb with window sizes increasing by 500bp increments. Randomisation was repeated 200 times for each enrichment analysis. Since EP300 sites from different tissues often cluster together, particularly for midbrain and forebrain (Visel et al. 2009) ,we removed EP300 sites, which were less than 10kb away from a EP300 site in the second tissue considered, but the effect is still appreciable for the brain tissues.

Calculating the (p-value) of the frequency of certain features (eg. topological boundaries, CTCF sites, SINE B2 elements) for a given sequence set RSA (eg “regulatory domains”, class C). 1000 sequence sets were randomly generated, each of which contained non-overlapping sequences mimicking sequences of RSA: for each original region of RSA, a region of the same size was randomly sampled from the chromosome of the original RSA region restricted to the allowed genome space. The allowed genome space considered for sampling random regions was matched to RSA properties and was either the whole genome or restricted to the TAD domains. In the later case, only the subset of the region set that was intra-TAD was used as

RSA. For each random sequence set, we determined the feature frequency in random regions.

The observed feature frequency in RSA (Fobs) is ranked within the 1000 random feature frequencies and the p-value is estimated given the formula: rankobs/(Nrdm+1), where rankobs is the rank of Fobs and Nrdm is the number of random dataset used in the simulation (always 1000 in this study). Note that with 1000 random sets, 0.000999001 is the best p-value one can obtain.

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