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ABSTRACT CpG islands (CGIs) represent a widespread feature of vertebrate genomes, being associated with ~70% of all gene promoters. CGIs control transcription initiation by conferring nearby

promoters with unique chromatin properties. In addition, there are thousands of distal or orphan CGIs (oCGIs) whose functional relevance is barely known. Here we show that oCGIs are an

essential component of poised enhancers that augment their long-range regulatory activity and control the responsiveness of their target genes. Using a knock-in strategy in mouse embryonic

stem cells, we introduced poised enhancers with or without oCGIs within topologically associating domains harboring genes with different types of promoters. Analysis of the resulting cell

lines revealed that oCGIs act as tethering elements that promote the physical and functional communication between poised enhancers and distally located genes, particularly those with large

CGI clusters in their promoters. Therefore, by acting as genetic determinants of gene–enhancer compatibility, CGIs can contribute to gene expression control under both physiological and

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support SIMILAR CONTENT BEING VIEWED BY OTHERS PROMOTER-PROXIMAL CTCF BINDING PROMOTES DISTAL ENHANCER-DEPENDENT GENE ACTIVATION Article 04 January 2021 THE CHROMATIN, TOPOLOGICAL AND

REGULATORY PROPERTIES OF PLURIPOTENCY-ASSOCIATED POISED ENHANCERS ARE CONSERVED IN VIVO Article Open access 16 July 2021 INCREASED ENHANCER–PROMOTER INTERACTIONS DURING DEVELOPMENTAL

ENHANCER ACTIVATION IN MAMMALS Article 20 March 2024 DATA AVAILABILITY All the 4C–seq data generated in this study are available through the GEO (GSE156465). All the generated transgenic ESC

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Central  Google Scholar  Download references ACKNOWLEDGEMENTS We thank the Rada-Iglesias laboratory members for insightful comments and critical reading of the manuscript. T.P. is supported

by a doctoral fellowship from the DAAD (Germany). V.S.-G. is supported by a doctoral fellowship from the University of Cantabria (Spain). Work in the Rada-Iglesias laboratory was supported

by the EMBO Young Investigator Programme; CMMC intramural funding (Germany); the German Research Foundation (DFG) (Research Grant no. RA 2547/2-1); ‘Programa STAR-Santander Universidades,

Campus Cantabria Internacional de la convocatoria CEI 2015 de Campus de Excelencia Internacional’ (Spain); the Spanish Ministry of Science, Innovation and Universities (Research Grant nos.

PGC2018-095301-B-I00 and RED2018-102553-T REDEVNEURAL 3.0); and the European Research Council (ERC CoG ‘_PoisedLogic_’; grant no. 862022). The Landeira laboratory is funded by grants from

the Spanish Ministry of Science and Innovation (grant nos. BFU2016-75233-P and PID2019-108108GB-I00) and the Andalusian Regional Government (grant no. PC-0246-2017). AUTHOR INFORMATION

AUTHORS AND AFFILIATIONS * Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany Tomas Pachano, Tore Bleckwehl & Alvaro Rada-Iglesias * Institute of

Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/Universidad de Cantabria/SODERCAN, Santander, Spain Tomas Pachano, Víctor Sánchez-Gaya, Thais Ealo, Maria Mariner-Faulí, Patricia

Respuela, María Muñoz-San Martín, Endika Haro & Alvaro Rada-Iglesias * Centre for Genomics and Oncological Research (GENYO), Granada, Spain Helena G. Asenjo & David Landeira *

Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, University of Granada, Granada, Spain Helena G. Asenjo & David Landeira * Instituto de Investigación

Biosanitaria ibs.GRANADA, Hospital Virgen de las Nieves, Granada, Spain Helena G. Asenjo & David Landeira * Max Planck Institute for Molecular Biomedicine, Muenster, Germany Sara

Cruz-Molina * Center for Biomics, Erasmus University Medical Center, Rotterdam, the Netherlands Wilfred F. J. van IJcken * Cologne Excellence Cluster for Cellular Stress Responses in

Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Alvaro Rada-Iglesias Authors * Tomas Pachano View author publications You can also search for this author inPubMed 

Google Scholar * Víctor Sánchez-Gaya View author publications You can also search for this author inPubMed Google Scholar * Thais Ealo View author publications You can also search for this

author inPubMed Google Scholar * Maria Mariner-Faulí View author publications You can also search for this author inPubMed Google Scholar * Tore Bleckwehl View author publications You can

also search for this author inPubMed Google Scholar * Helena G. Asenjo View author publications You can also search for this author inPubMed Google Scholar * Patricia Respuela View author

publications You can also search for this author inPubMed Google Scholar * Sara Cruz-Molina View author publications You can also search for this author inPubMed Google Scholar * María

Muñoz-San Martín View author publications You can also search for this author inPubMed Google Scholar * Endika Haro View author publications You can also search for this author inPubMed 

Google Scholar * Wilfred F. J. van IJcken View author publications You can also search for this author inPubMed Google Scholar * David Landeira View author publications You can also search

for this author inPubMed Google Scholar * Alvaro Rada-Iglesias View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS T.P. and A.R.-I.

conceptualized the project. Experimental investigations were performed by T.P., T.E., M.M.-F., H.G.A., P.R., M.M.-S. and E.H. T.P., V.S.-G. and T.B. performed data analyses. T.P. and A.R.-I.

wrote, reviewed and edited the manuscript. S.C.-M., W.F.J.v.I., D.L. and A.R.-I. were responsible for obtaining resources. A.R.-I. was responsible for supervision and funding acquisition.

CORRESPONDING AUTHOR Correspondence to Alvaro Rada-Iglesias. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW

INFORMATION _Nature Genetics_ thanks Darío Lupiáñez, Robin Andersson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are

available. PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 GENETIC

AND EPIGENETIC FEATURES OF THE OCGIS ASSOCIATED WITH PES. A, Comparison of CpG%, observed/expected CpG ratio, GC% and sequence length between random regions (n = 436000), NMIs associated to

_PE-distal_ (_PE-NMIs_; n = 345) and NMIs associated to the _devTSS_ (_devTSS-NMIs_; n = 1476) (Methods). The p-values were calculated using two-sided unpaired Wilcoxon tests with Bonferroni

correction for multiple testing; black numbers indicate median fold-changes; green numbers indicate non-negligible Cliff Delta effect sizes. The coloured area of the violin plot represents

the expression values distribution and the center line represents the median. B, H3K27me3 ChIP-seq levels14,24 around: _PE-distal_ with overlapping TFBS/p300 peaks and CAP-CGIs (n = 135),

_PE-distal_ with TFBS/p300 peaks separated by 1bp-1kb from CAP-CGIs (n = 65), _PE-distal_ with TFBS/p300 peaks separated by 1-3kb from CAP-CGIs (n = 53), _PE-distal_ without CAP-CGIs within

3kb (n = 254) and AEs without CAP-CGI within 3kb (n = 8115). C, % of CpG methylation at CAP-CGI associated with _PE-distal_ (PE-CAP-CGI; n = 276) and CAP-CGI associated with the TSS of

developmental genes (devTSS-CAP-CGI; n = 1926) in the indicated cell types (Methods). D, For the identification of the _PE Sox1(+35)CGI_ deletion, primer pairs flanking each of the deletion

breakpoints (1 + 3 and 4 + 2), located within the deleted region (5 + 6) or amplifying a large or small fragment depending on the absence or presence of the deletion (1 + 2) were used. E,

H3K27me3 levels at _PE Sox1(+35)_ were measured by ChIP-qPCR in WT ESCs and in n = 2 independent _PE Sox1(+35)CGI_−_/_− ESCs clones using primers adjacent to the deleted region. The bars

display the mean of n = 3 technical replicates (black dots). F, Independent biological replicate for the data presented in Fig. 1d. _Sox1_ expression was investigated by RT-qPCR in ESCs and

AntNPC with the indicated genotypes. n = 2 independent _PE Sox1 CGI_−_/_− ESC clones (circles and diamonds) and n = 1 _PE Sox1_−_/_− clone were studied. For each cell line, n = 2 replicates

of the AntNPC differentiation were performed. Expression values were normalized to two housekeeping genes (_Eef1a_ and _Hprt_) and are presented as fold-changes with respect to WT ESCs. The

coloured area of the violin plot represents the expression values distribution and the center line represents the median. EXTENDED DATA FIG. 2 ENGINEERING OF PES MODULES WITHIN THE GATA6-TAD

AND FOXA2-TAD. A, Epigenomic and genomic features of two previously characterized PEs14 (_PE Six3(_−_133)_; _PE Lmx1b(+59)_) in which the oCGIs overlap with conserved sequences bound by

p300 and, thus, likely to contain relevant TFBS. B, The different _PE Sox1(+35)_ insertions were identified using primer pairs flanking the insertion borders (1 + 3 and 4 + 2; 1 + 5 and 6 + 

2; 1 + 3 and 6 + 2), amplifying potential duplications (4 + 3, 3 + 2 and 4 + 1; 6 + 5, 5 + 2 and 6 + 1) and amplifying a large or small fragment depending on the absence or presence of the

insertion (1 + 2), respectively. The PCR results obtained for WT ESCs and for two ESC clonal lines with homozygous insertions of the _PE Sox1(+35)_ modules in the _Gata6_-TAD are shown. C,

Independent biological replicate for the data presented in Fig. 2b. D-E, Strategy used to insert the _PE Wnt8b(+21)_ (d) or the _PE Sox1(+35)_ (e) components into the _Gata6_-TAD (d) or

_Foxa2_-TAD (e), respectively. The right panels shows the TADs in which _Gata6_ (d) or _Foxa2_ (e) are included according to publically available Hi-C data80,81, with the red triangle

indicating the integration site of the PE modules, approximately 100 Kb downstream of _Gata6_ (d) or _Foxa2_ (e). F-G, For identifying the successful insertion of the different _PE

Sox1(+35)_ (f) or _PE Wnt8b__(+21)_ (g) modules, primer pairs flanking the insertion borders (1 + 3 and 4 + 2; 1 + 5 and 6 + 2; 1 + 3 and 6 + 2), amplifying potential duplications (4 + 3, 3 

+ 2 and 4 + 1; 6 + 5, 5 + 2 and 6 + 1) and amplifying a large or small fragment depending on the absence or presence of the insertion (1 + 2), respectively, were used. The PCR results

obtained for two ESC clonal lines with homozygous insertions of the indicated PE modules in the _Foxa2_-TAD (f) or _Gata6_-TAD (g), respectively, are shown. H-I, Independent biological

replicates for the data shown in Fig. 2c (h) and Fig. 2d (i). In (c), (h) and (i), the expression differences between AntNPCs with the TFBS + CGI module and AntNPCs with the other PE modules

were calculated using two-sided non-paired t-tests (**: foldchange>2 & p<0.001; *: foldchange> 2 & p<0.05; ns: not significant; fold-change<2 or p>0.05). EXTENDED

DATA FIG. 3 PES ARE ENRICHED IN CPG-RICH MOTIFS AND ARE BOUND BY CXXC-DOMAIN CONTAINING PROTEINS. A, Comparison of the TF motifs enriched in either PEs with a CAP-CGI in <3kb and active

enhancers without CAP-CGIs in <3kb. Motif enrichment analyses were performed with _Homer_82 (left) and _AME_107 (right). B, ChIP-seq signals for KDM2B31 (upper panel) and TET132 (lower

panel) are shown around: _PE-distal_ with overlapping TFBS/p300 peaks and CAP-CGIs (n = 135), _PE-distal_ with TFBS/p300 peaks separated by 1bp-1kb from CAP-CGIs (n = 65), _PE-distal_ with

TFBS/p300 peaks separated by 1-3kb from CAP-CGIs (n = 53) and _PE-distal_ without CAP-CGIs within 3kb (n = 254). ChIP-seq profile plots were generated using either the p300 peaks (left) or

the CAP-CGIs (right) associated with the PEs as midpoints. EXTENDED DATA FIG. 4 ENGINEERING OF ESC LINES CONTAINING THE _PE SOX1(+35)_ TFBS AND AN ARTIFICIAL CGI WITHIN THE _GATA6_-TAD. A,

Strategy used to insert the _PE Sox1(+35)TFBS_ alone or together with an aCGI into the _Gata6_-TAD. The upper left panel shows the epigenomic and genetic features of the _PE Sox1(+35)_. The

lower left panel shows the _PE Sox1(+35)_ modules inserted into the _Gata6_-TAD. The right panel shows the _Gata6_-TAD according to publically available Hi-C data80,81. The red triangle

indicates the integration site of the _PE Sox1(+35)_ modules approximately 100 Kb downstream of _Gata6_. B, For the identification of the _PE Sox1(+35)TFBS+aCGI_ insertion, primer pairs

flanking the insertion borders (1+3 and 4+2), amplifying potential duplications (4 + 3 and 4 + 4) and amplifying a large or small fragment depending on the absence or presence of the

insertion (1 + 2), respectively, were used. The PCR results obtained for two ESC clonal lines with homozygous insertions of _PE Sox1(+35)TFBS+aCGI_ in the _Gata6_-TAD are shown. C,

Independent biological replicate for the data presented in Fig. 2f. The expression differences between AntNPCs with the TFBS+CGI module and AntNPCs with the other PE modules were calculated

using two-sided non-paired t-tests (*: foldchange> 2 & p<0.05; ns: not significant; fold-change<2 or p>0.05). D, For the identification of the aCGI insertion alone, primer

pairs flanking the insertion borders (1 + 3 and 4 + 2), amplifying potential duplications (4 + 3 and 4 + 4) and amplifying a large or small fragment depending on the absence or presence of

the insertion (1 + 2), respectively, were used. The PCR results obtained from two ESC clonal lines with heterozygous insertions of aCGI in the _Gata6_-TAD are shown. E, The expression of

_Gata6_ and _Sox1_ was measured by RT-qPCR in cells that were either WT or heterozygous for the aCGI insertion in the _Gata6_-TAD (two different clones; circles and diamonds). For each cell

line, n = 2 replicates of the AntNPC differentiation were performed. The results obtained in n = 2 independent biological replicates are presented in each panel (Rep1 and Rep2). EXTENDED

DATA FIG. 5 _GATA6_ EXPRESSION PATTERNS IN CELL LINES WITH THE _PE SOX1(+35)_ MODULES INSERTED WITHIN THE _GATA6_-TAD. A, _Gata6_ and _Sox1_ expression was measured by RT-qPCR in ESCs and at

intermediate stages of AntNPC differentiation (Day 3 and Day 4). The analysed cells were either WT or homozygous for the insertions of the different _PE Sox1(+35)_ modules within the

_Gata6_-TAD. For the cells with the PE module insertions, n = 1 clonal cell line was studied. For each cell line, n = 2 replicates of the AntNPC differentiation were performed. Expression

values were normalized to two housekeeping genes (_Eef1a_ and _Hprt_) and are presented as fold-changes with respect to WT ESCs. _B_, Quantification of cells expressing GATA6 or SOX1

according to immunofluorescence assays as the ones shown in Fig. 2g. The analysed cells were either WT of homozygous for the insertions of the different _PE Sox1(+35)_ modules within the

_Gata6_-TAD. C, The expression patterns of GATA6 (upper panel) and SOX1 (lower panel) were investigated by immunofluorescence in WT ESCs or AntNPCs that were either WT, homozygous for the

insertion of the _PE Sox1(+35)TFBS_ + _aCGI_ in the _Gata6_-TAD or heterozygous for the insertion of the aCGI alone in the _Gata6_-TAD. Nuclei were stained with DAPI. Scale bar = 100µm. D,

Quantification of cells expressing GATA6 or SOX1 according to the immunofluorescence assays described in (c). In (b) and (d), the bars display the mean of n = 3 technical replicates (black

dots). EXTENDED DATA FIG. 6 EPIGENETIC AND TOPOLOGICAL CHARACTERIZATION OF THE _GATA6_-TAD CELL LINES. A, Bisulfite sequencing data presented in Fig. 3a for the indicated _Gata6_-TAD cell

lines. The circles correspond to individual CpG dinucleotides located within the TFBS module. Unmethylated CpGs are shown in white, methylated CpGs in black and not-covered CpGs in gray. B,

Chromatin accessibility at the endogenous _PE Sox1(+35)_ and the _Gata6_-TAD insertion site (P1 and P2) were measured by FAIRE-qPCR in cells with the indicated genotypes. C, DNA methylation

and nucleosome occupancy at the TFBS were simultaneously analyzed by NOMe-PCR in the indicated _Gata6_-TAD ESC lines. In the upper panels, the black and white circles represent methylated or

unmethylated CpG sites, respectively. In the lower panels, the blue or white circles represent accessible or inaccessible GpC sites for the GpC methyltransferase, respectively. Red bars

represent inaccessible regions large enough to accommodate a nucleosome. The dotted line indicates where the TFBS starts. The grey shaded area represents a nucleosome-depleted region. D,

Scatter plots showing population-averaged nucleosome occupancy (red) and DNA methylation (black) levels within the TFBS in the indicated _Gata6_-TAD ESC lines. The grey shaded area

represents a nucleosome depleted region. E-F, H3K4me1, H3K4me3, H2AK119ub, CBX7 and PHC1 levels at the endogenous _PE Sox1(+35)_ and the _Gata6_-TAD insertion site (P1 and P2) were measured

by ChIP-qPCR in cells with the indicated genoytpes. ChIP-qPCR signals were calculated as described in Fig. 3. G, 4C-seq experiments were performed using the _Gata6_ promoter as a viewpoint

in AntNPC with the indicated genotypes. H, Pile-up plots showing average Hi-C7,52 signals in ESC between two groups of PE-gene pairs: PEs and developmental genes with CGI-rich promoters; PEs

and genes with CGI-poor promoters. For each PE-gene pair, both the PE and the gene were located within the same TAD. Left panels include all the considered PE-gene pairs (n = 401 pairs for

developmental genes; n = 900 for CGI-poor promoters; middle panels includes PE-gene pairs with the same genomic size in the two groups (n = 401 pairs); right panels consist of PE-gene pairs

with the same genomic size and genes with expression levels <1 FPKM9 (n = 290 pairs) (Methods). EXTENDED DATA FIG. 7 GENERATION OF CELL LINES WITH ENGINEERED _PE SOX1(+35)_ MODULES WITHIN

THE _GRIA1-TAD_ AND GLOBAL CHARACTERIZATION OF H3K27AC AND ERNA LEVELS AT ACTIVE ENHANCERS. A, ESC clonal lines with insertions of the different _PE Sox1(+35)_ modules were identified using

primer pairs flanking the insertion borders (1 + 3 and 4 + 2; 1 + 5 and 6 + 2; 1 + 3 and 6 + 2), amplifying potential duplications (4 + 3, 3 + 2 and 4 + 1; 6 + 5, 5 + 2 and 6 + 1) and

amplifying a large or small fragment depending on the absence or presence of the insertion (1 + 2), respectively. The PCR results obtained for WT ESCs or two ESC clonal lines with homozygous

insertions of the different _PE Sox1(+35)_ modules in the _Gria1_-TAD are shown. B, Independent biological replicate for the data presented in Fig. 4b. The expression differences between

AntNPCs with the TFBS + CGI module and AntNPCs with the other PE modules were calculated using two-sided non-paired t-tests (ns: not significant; fold-change<2 or p>0.05). C, Bisulfite

sequencing analyses of ESC lines with the indicated _PE Sox1(+35)_ modules inserted in the _Gria1_-TAD. The circles correspond to individual CpG dinucleotides located within the TFBS:

unmethylated CpGs (white), methylated CpGs (black) and not-covered CpGs (gray) are shown. The plot on the right summarizes the DNA methylation levels measured within the TFBS in the

indicated ESC lines. D, Active enhancers (AEs) identified in ESCs based on the presence of distal H3K27ac peaks were classified into three categories (Methods): Class I (AEs in TADs

containing only poorly expressed genes; n = 271(left); n = 340 (middle, right); Class II (AEs in TADs with at least one highly expressed gene; n = 271(left); n = 2353(middle); n = 

340(right)); Class III (AEs whose closest genes in the same TAD is highly expressed; n = 271(left); n = 1262(middle); n = 340(right)). The violin plots show the H3K27ac and eRNA levels in

ESC for each AE category. P-values were calculated using unpaired Wilcoxon tests with Bonferroni correction for multiple testing; the numbers in black indicate the median fold-changes

between the indicated groups; the coloured numbers correspond to Cliff Delta effect sizes: negligible (red) and non-negligible (green). In the left and right panels, eRNA levels for the

three enhancers classes are compared after correcting for H3K27ac differences (Methods). EXTENDED DATA FIG. 8 GENERATION AND CHARACTERIZATION OF CELL LINES WITH PE INSERTIONS AT THE _GRIA1_

AND _SOX7/RP1L1_ TADS. A, H2AK229ub and SUZ12 levels at the endogenous _PE Sox1(+35)_, the _Gria1_ promoter and the _Gria1_-TAD insertion site (P1 and P2; Fig. 4d) were measured by ChIP-qPCR

in ESCs with the indicated genotypes. ChIP-qPCR signals were calculated as in Fig. 3. B, ESC clonal lines in which a pCGI was inserted 380bp upstream of the _Gria1_-TSS in cells with the

indicated _PE Sox1(+35)_ modules 100Kb upstream from _Gria1_ were identified using the indicated primer pairs. PCR results for clonal ESC lines with the indicated double homozygous

insertions are shown. C, eRNA levels at the endogenous _PE Sox1(+35)_ and the _Gria1_-TAD insertion site (P1 and P2) were measured by RT-qPCR in cells with the indicated genotypes.

Expression values were calculated as in Fig. 3. D, Strategy to insert the indicated _PE Sox1(+35)_ modules 380bp upstream (red triangle) of the _Gria1_-TSS. E, ESC clonal lines with the _PE

Sox1(+35)_ modules 380bp upstream of the _Gria1-_TSS were identified using the indicated primer pairs. PCR for ESC clonal lines with homozygous insertions of the indicated _PE Sox1(+35)_

modules are shown. F, Independent biological replicate for the data presented in Fig. 5e. G, ESC clonal lines with the _PE Sox1(+35)_ modules within the _Sox7/Rp1l1_-TAD were identified

using primers flanking the insertion borders (1 + 3 and 4 + 2; 1 + 3 and 6 + 2), amplifying potential duplications (4 + 3, 3 + 2 and 4 + 1) and amplifying a large or small fragment depending

on the absence or presence of the insertion (1 + 2), respectively. PCR results for ESC clonal lines with homozygous insertions of the indicated _PE Sox1(+35)_ modules are shown. H,

Independent biological replicate for the data presented in Fig. 5g. In (a) and (c), the bars display the mean of n = 3 technical replicates (black dots). In (f) and (h), the expression

differences between AntNPCs with the TFBS + CGI module or the other PE modules were calculated using two-sided non-paired t-tests (***: foldchange> 2 & p<0.0001; ns: not

significant; fold-change<2 or p>0.05). EXTENDED DATA FIG. 9 GENERATION OF ESC LINES WITH STRUCTURAL VARIANTS. A, ESC lines with the _Six3/Six2_ TAD boundary deletion were identified

using primers flanking the deleted region (1 + 3 and 4 + 2), amplifying the deleted fragment (5 + 6) and amplifying a large or small fragment depending on the absence or presence of the

deletion (1 + 2), respectively. The PCR results for two ESC clonal lines with 36Kb homozygous deletions (_del36_) are shown. B, ESC lines with the _Six3/Six2_ inversion were identified using

primer pairs flanking the inverted region (1 + 3, 4 + 2, 1 + 4 and 3 + 2) and amplifying potential duplications (4 + 3, 3 + 3 and 4 + 4). The PCR results for two ESC clonal lines with 110Kb

homozygous inversions (_inv110_) are shown. C, Epigenomic and genetic features of a CTCF binding site112 (CBS; highlighted in grey) located upstream of the _PE Six1(_−_133)_ (highlighted in

yellow). D, ESC lines with the CBS deletion were identified using primers flanking the deleted region (1 + 2) or located in the CBS (3 + 4). The PCR results for two ESC clonal lines with

homozygous CBS deletions are shown. E, The expression of _Six3_ and _Six2_ was measured by RT-qPCR in cells with the indicated genotypes. For each of the engineered structural variants, n = 

2 independent clonal cell lines were generated (circles and diamonds). In each plot, the number of circles and/or diamonds corresponds to the number of AntNPC differentiations performed. The

results obtained in n = 2 independent biological replicates are presented in each panel (Rep1 and Rep2). Expression values are presented as fold-changes with respect to WT ESCs. F, ESC

lines with the _Lmx1a_-TAD boundary inversion were identified using primers flanking the inverted region (1 + 3, 4 + 2, 1 + 4 and 3 + 2) and amplifying potential deletions (1 + 4) or

duplications (4 + 3, 3 + 3 and 4 + 4). The PCR results for three ESC clonal lines with 260 Kb homozygous inversions (_inv260_) are shown. EXTENDED DATA FIG. 10 EXAMPLES OF HUMAN CONGENITAL

DISEASES CAUSED BY STRUCTURAL VARIANTS THAT DISRUPT DEVELOPMENTAL LOCI WITH PE-ASSOCIATED OCGIS. A, Upper panel: heterozygous inversion in a patient with Branchio-oculo-facial syndrome

(BOFS)5. Lower panel: epigenomic and genetic features of _TFAP2A_ neural crest (NC) cognate enhancers (left), 6q16.2 genes (middle) and _TFAP2A_ (right). In the lower left panel, enhancer

reporter assays in chicken embryos are shown for two representative _TFAP2A_ enhancers5. Computational CGI and NMIs are represented as green rectangles. The inversion places one _TFAP2A_

allele into a novel TAD and impairs its normal expression in NC cells due to the physical disconnection from its enhancers. _TFAP2A_ has a promoter with a large CGI cluster and marked with a

broad H3K27me3 domain in ESCs. Some _TFAP2A_ NC enhancers are associated with oCGIs and marked with H3K27me3 in ESCs. Moreover, this inversion places genes originally found within the

6q16.2 locus in proximity of the _TFAP2A_ NC enhancers within a shuffled domain. The promoters of these 6q16.2 genes (i.e _GPR63_ and _NDUFAF4_) contain a short CGI centered on their TSSs.

In agreement with our findings, none of the 6q16.2 genes is responsive to the _TFAP2A_ NC enhancers5. B, Upper panel: deletion found in families with brachydactyly involving a TAD boundary

located between the _EPHA4_ and the _PAX3_ loci63. Lower panel: epigenomic and genetic features of the _Epha4_ cognate enhancers in the mouse E11.5 limb (left) and in human ESCs (right).

Representative reporter assay in E11.5 mouse embryos for the hs1507 element is shown in the middle63. The deletion includes _EPHA4_, a gene highly expressed in the developing limb, and the

TAD boundary separating the _EPHA4_ and _PAX3_ TADs. As a result, enhancers that control _EPHA4_ expression in the limb establish ectopic interactions with _PAX3_ (that is enhancer adoption)

and strongly induce its expression in the limb. The _PAX3_ promoter contains a large CGI cluster and is marked with H3K27me3 in ESCs, while one of the major _EPHA4_ enhancers (hs1507) is

associated with an oCGI and is marked with H3K27me3 in ESCs. The high responsiveness of _PAX3_ to the _EPHA4_ enhancers is in agreement with our findings. SUPPLEMENTARY INFORMATION REPORTING

SUMMARY PEER REVIEW INFORMATION SUPPLEMENTARY DATA 1 List of oligonucleotides and antibodies. SUPPLEMENTARY DATA 2 List of knock-in donor sequences. RIGHTS AND PERMISSIONS Reprints and

permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Pachano, T., Sánchez-Gaya, V., Ealo, T. _et al._ Orphan CpG islands amplify poised enhancer regulatory activity and determine target gene

responsiveness. _Nat Genet_ 53, 1036–1049 (2021). https://doi.org/10.1038/s41588-021-00888-x Download citation * Received: 05 August 2020 * Accepted: 17 May 2021 * Published: 28 June 2021 *

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