Large-Scale Discovery of Enhancers from Human Heart Tissue · Large-Scale Discovery of Enhancers...

Large-Scale Discovery of Enhancers from Human Heart Tissue

Dalit May, Matthew J. Blow, Tommy Kaplan, David J. McCulley, Brian C. Jensen, Jennifer A. Akiyama, Amy Holt, Ingrid Plajzer-Frick, Malak Shoukry, Crystal Wright, Veena Afzal,

Paul C. Simpson, Edward M. Rubin, Brian L. Black, James Bristow, Len A. Pennacchio, Axel Visel

- Supplementary Information -

Nature Genetics: doi:10.1038/ng.1006

May et al: ChIP-seq Discovery of Human Heart Enhancers - 2

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Supplementary Note

Enrichment of p300/CBP peaks near PolII binding sites

To further investigate the association between p300/CBP binding sites in the human heart and transcription of nearby genes, we used ChIP-seq to map genome-wide binding of RNA polymerase II (PolII) in human fetal heart (Methods). Enrichment analysis of this data-set identified 5,540 proximal peaks (less than 2.5kb of transcriptional start sites). There is a significant enrichment of human heart candidate enhancers near proximal PolII peaks, extending up to 150kb away (Supplementary Fig. 4). These results show that p300/CBP peaks are correlated with RNA polymerase II peaks, supporting our findings that the identified p300/CBP peaks are linked to transcription in the human heart.

Functional conservation of human candidate enhancers in mice

Limited sequence conservation of candidate enhancers is potentially indicative of functional changes in regulatory architecture that have occurred across species 1-3. To further evaluate the functional conservation of human candidate heart enhancers across mammalian species, we performed p300/CBP ChIP-seq on mouse heart tissue at postnatal day 2, a stage at which the developmental progression and gene expression profile of the mouse heart is broadly similar to that of human fetal heart at gestational week 16 4,5. Using identical methods as for human heart tissue, we identified 6,564 candidate enhancers (distal ChIP-seq peaks, Supplementary Table 1) and compared them to the human fetal candidate enhancer sets. We catalogued the human heart candidate enhancers by their p300/CBP binding conservation to mouse into 4 classes: 1) Significant p300/CBP binding at the orthologous genomic site in the mouse heart (21%); 2) Human peaks whose mouse homologous regions were not called as p300/CBP peaks but exceeded the genome-wide background (25%); 3) p300/CBP binding at the orthologous genomic site in the mouse genome does not exceed background levels (41%); 4) Human candidate enhancers that could not be aligned to a unique position in the mouse genome (13%) 6 (Supplementary Fig. 7a, Methods). For each class of peaks,



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we further characterized the degree of sequence constraint across multiple mammalian species using their phastCons score 7 (Supplementary Fig. 7b). These results suggest that while a fraction of the human candidate heart enhancers are functionally conserved between the two species, more than half (54%) of all candidate enhancers identified from human heart tissue did not show any evidence of an orthologous p300/CBP binding site in the mouse heart data set. A considerable proportion (1,296; 26%) of fetal human heart candidate enhancers show neither substantial sequence constraint nor binding conservation to the mouse genome and therefore could not have been identified using sequence-based or binding conservation (asterisks in Supplementary Fig. 7b).



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Supplementary Figures

Supplementary Figure 1: Reproducibility of p300/CBP ChIP-seq. The enrichment of reads from the primary human fetal heart sample analyzed in this study was compared to that from a second human fetal heart sample sequenced at lower coverage (see Methods), using the 2,233 candidate adult human heart enhancers identified in this study as anchoring points. Read coverage was cumulated across all enhancers, and corrected for the different number of reads from each sample. Both the original and validation fetal heart data sets exhibit highly significant enrichment at candidate heart enhancers (*, p < 1e-90, Fisher’s Exact test). In contrast, no enrichment was observed near heart enhancers in human input DNA (see Methods). The quantitatively similar degree of enrichment in the two fetal heart data sets supports the reproducibility of p300/CBP ChIP-seq from human heart tissue.



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Supplementary Figure 2: p300/CBP binding conservation in fetal and adult human heart. p300/CBP binding sites in the human genome were compared between fetal and adult human heart. a) Heat map of human ChIP-seq read density in 100kb windows surrounding each of the human adult heart candidate enhancers in the adult human heart (left), compared to ChIP-seq read density at the same sites in the fetal data set (right). b) Heat map of human ChIP-seq read density in 100kb windows surrounding each of the human fetal heart candidate enhancers in the fetal human heart (left), compared to ChIP-seq read density at the same sites in the adult data set (right). Peaks fall into three categories, based on ChIP-seq signal in the respective other data set (from top): 1. significant binding in both data sets, 2. read density in the respective other data set is above background, but below peak significance threshold, 3. read density in the respective other data set not exceeding genome-wide background (stage-specific). c) Overlap of candidate enhancers identified in both fetal and adult heart tissues, with stage-specific and suggestively enriched regions shown in separate colors. Minor differences between total peaks and counts in categories of overlap are due to cases of peaks overlapping more than one peak in the respective other dataset.



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Supplementary Figure 3: p300/CBP distal peaks near heart-expressed and non-heart-expressed genes. Frequency of adult human heart candidate enhancers (red) compared to matched random regions (black) near genes highly expressed in adult heart (a) and near genes highly expressed in 10 other tissues (b). The graphs display the frequency of the different elements up to 200kb away from the transcription start site and show significant enrichment of heart p300/CBP candidate enhancers in proximity to heart-expressed genes (starting at 2.5kb away from TSS). Fold enrichment of the most proximal peaks (2.5-10kb from TSS) is 3.6 (p = 1e-11, binomial distribution). The significant enrichment over random peaks and over non-heart genes is maintained up to 100kb (binomial distribution p < 0.002). Errors bars indicate 95% confidence intervals.

Supplementary Figure 4: Enrichment of human heart p300/CBP ChIP-seq peaks near human heart RNA polymerase II peaks. Frequency of fetal human heart candidate enhancers near genes occupied by RNA polymerase II in the fetal human heart (red) compared to random sequences (black) showing significant enrichment up to 130kb away from the nearest PolII peak (5,540 peaks). Error bars indicate 95% confidence intervals.



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Supplementary Figure 5: p300/CBP distal peaks are enriched near heart disease-associated genes. Cumulative plot of frequency of fetal (a-b) and adult (c-d) human heart candidate enhancers (red) compared to matched random regions (black) near genes associated with heart diseases (a,c) and control genes not associated with heart diseases (b,d). The graphs display the cumulative frequency of the different elements up to 200kb away from the transcription start and show significant enrichment of heart p300/CBP candidate enhancers in proximity to heart disease-associated genes (starting at 2.5kb away from TSS). The significant enrichment over random peaks and over non-heart genes is maintained up to 200kb (for all bins, binomial distribution p < 0.05). Errors bars indicate 95% confidence intervals.



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Supplementary Figure 6: Sequence constraint properties of human heart candidate enhancers. Direct comparison between the constraint properties of human heart candidate enhancers and previously identified embryonic mouse candidate enhancers. To enable a direct comparison between data sets, 1kb windows centered on the peak maxima of the 500 most significant peaks in each data set were assigned the score of the most highly constrained overlapping vertebrate phastCons 7 element in the relevant genome 8, resulting in a larger proportion of peaks with no evidence of constraint compared to Figure 3. a) Fraction of candidate enhancers that were under extreme evolutionary constraint (defined as a phastCons score of > 600). b) Fraction of candidate enhancers that were under no detectable constraint (no overlapping vertebrate constrained element).



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Supplementary Figure 7: Cross-species functional conservation. Heart tissue p300/CBP binding sites in the human genome (left) were compared to p300/CBP binding at orthologous positions in the mouse genome (right). a) Heat map of human ChIP-seq read density in 100kb windows surrounding each of the human fetal heart candidate enhancers in the human genome (left), compared to ChIP-seq read density at orthologous sites in the mouse genome (right). Human peaks fall into four categories based on ChIP-seq signal in the mouse (from top): 1. significant binding at orthologous site in the mouse genome, 2. read density in the mouse genome above background, but below peak significance threshold, 3. read density in mouse genome not exceeding genome-wide background, 4. human peak sequence not alignable to mouse genome. b) Proportion of peaks in the different binding conservation categories and their sequence constraint properties (as determined by maximum score of overlapping phastCons elements). Asterisks indicate enhancer populations that are both poorly constrained and absent in the mouse genome, precluding their discovery by comparative genomics or from mouse-derived ChIP-seq data. Highly constrained, phastCons > 450; Moderately constrained, phastCons 350-450; Weakly constrained, phastCons ≤350; No detectable constraint, no overlapping phastCons element.



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Supplementary Figure 8: Intersections with ENCODE datasets. a-c) Percentage of p300/CBP fetal human heart candidate enhancers overlapping ENCODE datasets (red) compared to expected overlap for random distribution (black). a) Histone mark H3K4me1 in 8 human cell lines (fold enrichment 9.5-19.8, hypergeometric distribution p < 1e-15). b) Histone mark H3K27ac in 8 human cell lines (fold enrichment 7.8-13.9, hypergeometric distribution p < 1e-15). c) Clusters of DNaseI hypersensitivity regions (DHS) in different cell lines (fold enrichment 12.2, hypergeometric distribution p < 1e-15). d-f) Number of positive in vivo enhancers tested previously 9,10 overlapping ENCODE datasets: d) Histone mark H3K4me1 in 8 human cell lines. e) Histone mark H3K27ac in 8 human cell lines. f) Clusters of DNaseI hypersensitivity regions (DHS) in different cell lines. The vast majority of genome regions identified by the cell line-derived marks considered here do not act as in vivo heart enhancers. The ENCODE datasets used are: clusters of DNaseI hypersensitivity sites (DHS), the top 100,000 H3K4me1 peaks in 8 human cell lines and the top 100,000 H3K27ac peaks in 8 human cell lines.



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Supplementary Figure 9: In vivo testing of heart enhancer predictions. Comparison of the frequency of in vivo heart enhancers among tested sequences in different data sets. Human fetal heart p300/CBP ChIP-seq predictions (n=65): the elements tested in this study. Mouse e11.5 heart p300-ChIP-seq (n=130): mouse data sets 8 showing similar success rate in predicting active heart enhancers. Conserved elements near heart-expressed genes (n=33): a subset of 33 tested elements from previously described data sets 9,10 located within 100kb from the transcript start sites of highly heart-expressed genes. Conserved elements near heart genes (n=17): the testing of 17 moderately conserved elements located near genes implicated in embryonic heart development through genetic or developmental biology studies (Supplementary Table 8). In addition to moderate evolutionary conservation, these sequences contained sequence motifs hypothesized to be cardiac-associated 11. * p < 0.001, chi-square test.



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Supplementary Figure 10: Transcription factor binding site analysis of candidate heart enhancers. The top 500 p300/CBP peaks from each tissue were divided into equally sized bins containing the 250 most conserved and 250 least conserved candidate enhancers, respectively, and searched using the JASPAR database12 of non-redundant vertebrate transcription factor binding motifs (see Methods). Results are expressed as fold enrichment / depletion of transcription factor binding sites compared to random genomic DNA, and sorted by average fold enrichment across most conserved and least conserved candidate enhancers from P2 mouse heart. Notably, human and mouse p300/CBP peaks, including those with weak evolutionary conservation, tend to be enriched in the same types of transcription factor binding sites. This suggests that while many heart enhancers are poorly conserved between human and mouse, they nonetheless share a similar repertoire of binding sites, providing a possible explanation for the activity of even poorly conserved human heart enhancers in the mouse assay.



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Supplementary Figure 11: Reproducible enhancer activity at e11.5 of adult vs. fetal human candidate heart enhancers. 41 of the 65 tested elements are candidate enhancers in the adult heart, and the proportion of reproducible enhancers is similar to the annotated activity of the complete set of tested sequences (n=65).



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Supplementary Figure 12: Transgenic results for cardiac enhancers tested in mice at e11.5 and at postnatal day 28. See Fig. 4 for description of panels. The four elements shown in Fig. 4 are also reproduced here for comparison. a) hs1750 – The activity of the enhancer is seen throughout the right and left ventricle and the outflow tract with minimal activity in the atria, both at e11.5 and in the P28 heart. b) hs1963 - The enhancer is active throughout the chambers (left and right atria and ventricles) with an atria predominance in the P28 heart. c) hs1751 – The enhancer is active primarily in the outflow tract in both the embryonic and P28 heart. At e11.5, there is also activity in the right ventricle and minimal activity in the left ventricle. At 4 weeks of age, the pattern is more restricted to the outflow tract, with sporadic activity in the chambers. d) hs1766 - At e11.5, the enhancer shows activity in the heart, with the main activity located in the outflow tract myocardium and with sporadic activity in the myocardium of the ventricles. This pattern is maintained at P28. e)/f) hs1760 and hs1763 – The enhancers are active throughout the embryonic and P28 heart. g) hs1959 – The enhancer is active primarily in the left ventricle with minimal activity in the right ventricle both at e11.5 and in the P28 heart. h) hs1753 – The tested element is active mainly in the left and right ventricle both at e11.5 and in the P28 heart. LV-left ventricle; RV-right ventricle; LA-left atrium; RA-right atrium; OFT-outflow tract; PA-pulmonary artery; Ao-Aorta; DA-dorsal aorta.



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Supplementary Tables

Human fetal heart p300/CBP

Human adult heart p300/CBP

Mouse P2 heart p300/CBP

Total Reads 27,073,513 100.0% 26,246,243 100.0% 27,181,826 100.0% Reads mapping to reference genome 13,966,403 51.6% 16,444,832 62.7% 12,720,771 46.8%

Reads mapping to unique location in the reference genome

11,463,876 42.3% 13,261,649 50.5% 9,596,917 35.3%

Non-clonal reads mapping to unique location in the reference genome

10,090,784 37.3% 9,021,512 34.4% 8,641,854 31.8%

Total peaks 12,055 100.0% 5,659 100.0% 17,146 100.0% Peaks passing filtering 10,064 83.4% 4,488 79.3% 12,686 74.0% Proximal peaks (<=2.5kb from nearest TSS) 5,017 49.9% 2,255 50.3% 6,121 48.3%

Distal peaks (>2.5kb from nearest TSS) 5,047 50.1% 2,233 49.7% 6,564 51.7%

Supplementary Table 1: Properties of ChIP-seq data sets. Human fetal heart, 16 week gestational age; Human adult heart, adult failing heart; Mouse P2 heart, CD1 strain hearts from postnatal day 2.



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Phenotypes Binom FDR Q-value Binom fold enrichment abnormal cardiac muscle morphology 3.32E-44 2.1

abnormal vitelline vasculature 1.13E-31 2.2

abnormal epicardium morphology 5.23E-27 5.7

thin ventricular wall 3.43E-22 2.6

abnormal myocardial trabeculae morphology 7.74E-22 2.3

decreased cardiac muscle contractility 2.79E-21 2.3

abnormal renal glomerulus morphology 3.34E-21 2.1

anemia 1.14E-20 2.0

pericardial effusion 7.44E-20 2.9

abnormal cardiac muscle contractility 4.35E-19 2.1

abnormal erythrocyte cell number 2.83E-18 2.2

abnormal placenta vasculature 4.35E-18 2.4

impaired muscle contractility 6.80E-18 2.0

abnormal vascular branching morphogenesis 9.51E-18 3.3

abnormal erythrocyte morphology 1.66E-17 2.0

abnormal looping morphogenesis 4.90E-17 2.2

abnormal myocardial fiber morphology 6.41E-17 2.0

poorly developed ventricular trabeculae 7.91E-17 2.9

abnormal dorsal aorta morphology 8.31E-17 2.3

abnormal liver development 1.33E-16 2.6

abnormal liver/biliary system development 3.66E-16 2.5

abnormal capillary morphology 1.02E-15 3.6

disorganized myocardium 1.32E-15 3.5

thin myocardial wall 1.72E-15 2.6

abnormal tail development 4.23E-15 3.8

abnormal ventricular trabeculae morphology 1.48E-14 2.3

cortical renal glomerulopathies 1.49E-14 2.4

branchial arch hypoplasia 1.79E-14 4.3

abnormal mitochondrial physiology 3.86E-14 2.5

abnormal placenta labyrinth morphology 6.09E-14 2.1

abnormal outflow tract septation 7.91E-14 2.1

decreased heart rate 2.91E-13 2.1

dilated left ventricle 7.53E-13 2.4

abnormal wound healing 2.03E-12 2.6

dilated heart ventricles 2.78E-12 2.1

congestive heart failure 6.52E-12 2.2

pale yolk sac 6.48E-12 2.2

cardiac hypertrophy 7.02E-12 2.1

abnormal exercise endurance 7.49E-12 4.2

abnormal heart tube morphology 7.45E-12 2.4

decreased ventricle muscle contractility 3.60E-11 2.2

abnormal trophoblast layer morphology 7.03E-11 2.2



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abnormal vascular endothelial cell morphology 7.13E-11 2.4

abnormal cell differentiation 1.06E-10 2.9

hydrops fetalis 1.87E-10 2.2

increased stomach pH 3.86E-10 5.4

abnormal muscle weight 7.92E-10 3.8

failure of looping morphogenesis 8.63E-10 2.6

abnormal QRS complex 1.43E-09 2.6

abnormal placental labyrinth vasculature morphology 5.25E-09 2.2

Supplementary Table 2: Top enriched annotations of putative target genes near candidate human heart enhancers.



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Chr Peak

start Peak end Gene

symbol SNPs

chr1 154339500 154343200 LMNA rs12760757, rs34732197, rs6661281, rs35567642, rs11264438, rs4020356, rs72201856, rs11268414, rs67356867, rs12760770

chr1 199545525 199548175 TNNT2 rs1779284, rs2268163, rs763208, rs73074616, rs1779304, rs17576470, rs1772848, rs2268164, rs1779283, rs10920170, rs832153, rs17190, rs1615071, rs1772849, rs1772852, rs1772850, rs1772851, rs1772853, rs832152, rs832151, rs1772855, rs1772856, rs832150, rs1772857

chr1 199564875 199566325 TNNT2 rs1361904, rs1572969, rs1572968, rs6697983, rs6698269, rs6685490, rs1361903, rs6660897, rs1361902, rs6700827, rs1361901, rs61819984, rs947376, rs947375, rs947374, rs7541805

chr1 228952525 228957875 AGT rs7542539, rs12058807, rs1316445, rs1316446, rs74144823, rs2493153, rs2478546, rs74144824, rs1887208, rs66917618, rs74144827, rs74144825, rs2493154, rs4847010, rs74144826, rs59907258, rs67651579, rs61828378, rs66833529, rs72171772, rs66649095, rs66833528, rs66730980, rs71891768, rs11590877, rs74144828, rs12060898, rs12728983, rs3000060, rs2986386, rs2986387, rs3000061, rs11577947, rs12118743, rs2986384, rs3000062, rs7543422, rs2354846, rs3000063, rs1887209, rs7542919, rs11811761, rs71563450, rs868143, rs28359591, rs28359594, rs9969999, rs2493148, rs28359595, rs72101134, rs61035893, rs71929931, rs72181029, rs71835020, rs72225934, rs2986385, rs58791351, rs28359596, rs2493155, rs28359597, rs12045784, rs55833939, rs55733925, rs2493145

chr1 228962275 228963725 AGT rs28359613, rs16852586, rs11579420, rs28359614, rs7515210, rs28359615, rs28359617, rs12023382, rs28359616, rs12026211, rs28359618, rs72448283, rs28359619, rs28359620, rs28359621, rs59135397

chr1 234889225 234890675 ACTN2 rs661219, rs663798, rs507130, rs507841, rs662513, rs535411, rs676377, rs537306

chr1 234919400 234921025 ACTN2 rs1768048, rs1773439, rs697660, rs4659701, rs71178340, rs34214344

chr1 234922375 234924525 ACTN2 rs6688531, rs9943223, rs59360301, rs891337, rs891338, rs67531528, rs71937704, rs35891713, rs12410300, rs72167259, rs4006425, rs707201, rs12755962, rs12730785

chr1 234929775 234931225 ACTN2 rs10925200, rs12736189, rs72760272, rs3905576, rs10465610, rs3905125, rs58455499

chr1 235223950 235228725 RYR2 rs1252152, rs6666878, rs59323044, rs1252148, rs12139583, rs6704137, rs16834731, rs1252149, rs1252150, rs1252151, rs12093589, rs7524778, rs12737188, rs73120019, rs10715956, rs72047939, rs10546053, rs10577856, rs16834733, rs57011109, rs1252153, rs1252154, rs12036945, rs6698554, rs12047281, rs1252155

chr1 235241350 235242800 RYR2 rs28578664, rs6698022

chr1 235372575 235374025 RYR2 rs73119307, rs16834891, rs2225886, rs12564070, rs73119310

chr10 75395000 75396450 VCL rs12264611, rs74146308, rs66804664

chr10 88397900 88401550 LDB3 rs2675688, rs17333786, rs57257372, rs36059654, rs3033309, rs2675687, rs10788520, rs55952431, rs2250515, rs2803547, rs12269312, rs7069508, rs17333800, rs10887636, rs10887637, rs12773473, rs12779508, rs12779522

chr10 88413375 88422000 LDB3 rs45578532, rs11202111, rs2675697, rs34219180, rs12262894, rs33936589, rs35723943, rs2675696, rs35387448, rs36050631, rs35422767, rs2675695, rs2675694, rs36064638, rs34410271, rs34549734, rs2803553, rs36117636, rs35264027, rs34088875, rs11202113, rs35357978, rs35891072, rs2803554, rs34880769, rs3740340, rs2736687, rs35282464, rs17425463, rs34157063, rs3740341, rs17106907, rs11812601, rs34106786, rs35511066, rs11594957, rs35882322, rs41291400, rs17425484, rs61857109, rs35305953, rs7088351, rs34346910, rs4934240, rs34339902, rs34499525, rs2675693, rs34959905, rs4256897, rs34073498, rs35777670, rs2675692, rs2803555, rs2803558, rs2803557, rs45584436, rs2803556, rs45491495, rs34972863, rs45453092, rs11816975, rs45501499, rs12259201, rs3740342, rs10887639, rs2803559, rs35305714, rs10887640, rs67549220, rs12569813,



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rs4492716, rs12570315, rs2354363, rs2803560, rs2244769, rs12248422, rs10887641, rs57702354, rs11202117, rs11202118, rs11202119, rs11202120

chr10 88434000 88436925 LDB3 rs3802662, rs4934249, rs4934247, rs4934248, rs7908743, rs56339490, rs4934250, rs61246957, rs61662518, rs59195415, rs7893160, rs11593608, rs7923558, rs11595553, rs35422936, rs3740345, rs73344165, rs11202126, rs2803571, rs3802663, rs1986382, rs1986383, rs1578895, rs12570147, rs73344172, rs1140170, rs1140171, rs3204236, rs1140172

chr10 88482800 88484250 LDB3 rs45462791, rs57203586, rs45601731, rs45493398, rs45494496, rs45513796, rs45541534, rs45591436, rs45446600, rs45537636, rs11277810, rs3802664, rs45567939, rs45516591, rs45595736, rs45602332

chr10 92673950 92675400 ANKRD1 rs34336222, rs11594233

chr10 92678500 92681150 ANKRD1 rs7895858, rs10458744, rs35231118, rs59529525, rs7896058, rs7896199

chr11 2510675 2513450 KCNQ1 rs179436, rs2074247, rs74048676, rs179437, rs72847668, rs757086, rs2074248, rs11023217

chr11 2753275 2760050 KCNQ1 rs41282928, rs55647309, rs35511783, rs1057128, rs81205, rs163149, rs163150, rs12720453, rs28730759, rs56021860, rs12417409, rs55812754, rs81204, rs3852523, rs163148, rs163147, rs2411343, rs71302039, rs2741950, rs11826215, rs17221854, rs189261, rs2237884, rs55771042, rs56305460, rs55882753, rs163146, rs233434, rs58972425, rs34309112, rs12794000, rs74049757, rs163145, rs11607946, rs35937910, rs234886, rs56166304

chr11 2765275 2769600 KCNQ1 rs3892488, rs149145, rs33915190, rs11024084, rs3899907, rs2521915, rs12271773, rs2521916, rs233439, rs151317, rs34664040, rs149373, rs2237885, rs234875, rs2237886, rs56094341, rs2237887, rs3852524, rs5789273, rs12790738

chr11 2785150 2786600 KCNQ1 rs11601574, rs59809880, rs12275085

chr11 2788075 2790375 KCNQ1 rs35891657, rs9971505, rs8181588, rs4930008, rs60702106, rs515774, rs2237888, rs59497747, rs11024184

chr11 2866650 2870700 KCNQ1 rs371972, rs10832869, rs441613, rs400643, rs72850019, rs73421376, rs439926, rs445108, rs444956, rs446396, rs384037, rs10832870, rs57417691, rs73421378, rs2237901, rs367003, rs429573, rs2583437, rs376984

chr11 19150250 19153375 CSRP3 rs11025042, rs72156532, rs10833067, rs7111850, rs4757764, rs2278148, rs4757763, rs9645622, rs4757765, rs34811457, rs61876752, rs11608112, rs61449427

chr11 19176600 19179550 CSRP3 rs12276811, rs66540504, rs66762612, rs11025054

chr11 19184400 19188175 CSRP3 rs57725010, rs11025055, rs73429792, rs11025056, rs793280, rs7104244, rs72904181, rs35530213, rs793279, rs7108302, rs11025057, rs55714085, rs11604807, rs7112377

chr11 19195725 19198075 CSRP3 rs4757774, rs4757773, rs924692, rs924691, rs998263, rs58560650, rs60270944, rs56851677, rs2114117

chr12 109839525 109847725 MYL2 rs2857729, rs10849916, rs35227242, rs2857730, rs71458382, rs11065774, rs35724597, rs2857726, rs34242515, rs3782888, rs2857728, rs34280033, rs35022714, rs35631280, rs35720387, rs34695960, rs61943011, rs2520205, rs36077898, rs2520204, rs28932774, rs3932753, rs2106403, rs7488411, rs61943012, rs12301951, rs10849917, rs34684305, rs17550549, rs2520203, rs2857727, rs2520202, rs2520201, rs34290874, rs34718537, rs2040571, rs34945811, rs2857724, rs2857725, rs34297524, rs35977393, rs34608943, rs2857723, rs2520200, rs5800907, rs2857722, rs2857720, rs3216816, rs34204180, rs2233258, rs2857721, rs2857719, rs2520199, rs35427495, rs11538904, rs2857718, rs2233257, rs35206048, rs2233256, rs35011057, rs2301359, rs34545235, rs34482519, rs2301358, rs35968763, rs34995642, rs34991693, rs2857731, rs58464605, rs61943013, rs12427105, rs4766517, rs55897251, rs12321621, rs2028005, rs12302980, rs61943014, rs11830879, rs12828640, rs61943015, rs11065776, rs4766518, rs71442743, rs34293369, rs73421379, rs7312138, rs6489834

chr12 113318250 113319700 TBX5 rs2114328, rs56267344, rs58269686, rs2555007, rs2555006



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chr12 113335950 113339025 TBX5 rs1248053, rs71853167, rs12809488, rs71769019, rs61931005, rs12426272, rs1248052, rs1106023, rs61931006, rs73394826, rs11067103, rs7303733, rs56172437, rs61931008, rs58079209, rs11067104, rs4766730

chr12 113363900 113365350 TBX5 rs5801057, rs1568595, rs7132160, rs2686573, rs2859670, rs73394857, rs4572187, rs1248032, rs57671151, rs72426552

chr15 61073250 61074700 TPM1 rs11852785, rs8024248, rs8025434, rs74020952, rs2035714, rs16946216, rs16946217, rs74020953, rs2035713

chr15 61076450 61080100 TPM1 rs35981648, rs8026456, rs73436139, rs11634528, rs4775592, rs56146346, rs12913459, rs11071708, rs74020954, rs73436143, rs35817592, rs16946228, rs35071721, rs10851720, rs12050680, rs10775183, rs34642222, rs12595433, rs59637534, rs73436154, rs12440897, rs73436150, rs73436151, rs58389234, rs12903994, rs11071709, rs62011026, rs1489315

chr17 35082150 35090825 TCAP rs1976292, rs2934955, rs907087, rs2941503, rs2247862, rs4344777, rs35969488, rs903502, rs62074882, rs903504, rs35734596, rs903503, rs2934956, rs2941504, rs1565922, rs2934954, rs72089406, rs55681053, rs1976293, rs1565921, rs66689310, rs59462337, rs907088, rs10558975, rs1976291, rs62929565, rs2941505, rs1565920, rs1495102, rs1495101, rs1495100, rs2934953, rs2934952, rs2941506, rs2952152, rs11656146, rs2934951, rs12150603, rs732083, rs2313171, rs9675194, rs907089, rs907090, rs732084, rs12150298, rs62074883, rs72832915, rs8078228, rs8077172, rs1018246, rs62074884, rs11078919, rs1476278, rs9303274, rs73985211, rs12940986, rs2952153

chr17 35114075 35116500 TCAP rs4252605, rs34006795, rs11078920, rs4252601, rs35527921, rs2952154, rs2952155, rs35517009, rs35244499, rs4252603, rs4252604, rs4252608, rs4252606, rs4252607

chr17 37147550 37149000 JUP rs71373448, rs55727144, rs58413662, rs7224601

chr17 37197900 37204375 JUP rs1030827, rs67631308, rs11869167, rs2353023, rs2353021, rs2353022, rs28590627, rs68109828, rs72075217, rs73983661, rs71373454, rs72402792, rs67705634, rs56039779, rs58002761, rs12939653, rs2883071, rs753663, rs12939597, rs12939614, rs12939617, rs12939654, rs1811138, rs1865840, rs873150, rs2883072, rs71373455, rs9903086, rs34761055, rs34768220, rs4796608, rs11079013, rs72359914, rs4796609, rs35394132, rs8080677, rs12453367, rs9910080, rs1030826, rs4796703, rs9909529, rs34548959

chr17 45608600 45610950 SGCA rs2696287, rs2696286, rs62062993, rs2696285, rs12945599, rs67824140, rs2586480, rs2696284

chr17 45641950 45643400 SGCA rs73340532, rs2696275, rs9900013, rs62063000, rs12325763, rs11652362, rs62063001, rs2246839

chr19 60314375 60320925 TNNI3 rs73617642, rs604216, rs10425386, rs10573756, rs682884, rs61688601, rs73935111, rs56367991, rs34521018, rs73617639, rs56350931, rs73935112, rs45620341, rs73932601, rs575144, rs73932600, rs73935306, rs12981737, rs667451, rs73060940, rs73935305, rs73932602, rs73935303, rs73935304, rs66707428

chr2 71582800 71587350 DYSF rs4346411, rs13389635, rs12713753, rs12713752, rs4995934, rs4852262, rs7571056, rs34836478, rs11888263, rs4502445, rs4535069, rs73940806, rs57013453, rs4606960, rs55901624, rs71416711, rs72827517, rs4493287, rs10181951, rs11681784, rs10205355, rs12469262, rs10181971, rs9309455

chr2 71694150 71697300 DYSF rs11896727, rs1863810, rs13409105, rs56163347, rs6724003, rs62145899, rs58344325, rs72902695, rs13416470, rs6546705

chr2 71779825 71786900 DYSF rs934068, rs13428177, rs35888135, rs57904051, rs5832066, rs13428172, rs5832067, rs35589033, rs13428188, rs934066, rs67241256, rs62147860, rs57465669, rs55955032, rs4852821, rs10166424, rs934067, rs750355, rs34557670, rs34658789, rs5832068, rs751473, rs750356, rs751474, rs2114811, rs10170252, rs35596062, rs72259872, rs751475, rs34657914, rs58901933, rs13388078, rs55817837, rs66527974, rs55785582, rs55920073, rs62147861, rs34751818, rs12620909, rs13394724, rs4309602, rs1427689, rs6736164

chr2 71793500 71800450 DYSF rs66888842, rs35660836, rs7598653, rs10196615, rs2162626, rs59333450, rs67131910, rs1559980, rs34460551, rs10192575, rs72906619, rs35121695, rs57121650, rs2589513, rs62147873,



- 21 -

rs71416717, rs35970254, rs62147874, rs73942384, rs934069, rs749314, rs2004421

chr2 219976575 219984325 DES rs16859882, rs4672929, rs7572996, rs12466021, rs4674390, rs7572681, rs16859887, rs11306834, rs11306835, rs61117405, rs35262707, rs58148431, rs4672928, rs4674391, rs72963577, rs67324509, rs56206641, rs12988290, rs72963579, rs62191870, rs1877473, rs73085247, rs73085248, rs7571880, rs2175325, rs12466871, rs7571806, rs4674392, rs55881109, rs71352187, rs72613758, rs13396695, rs12475092, rs12467950, rs4341919, rs4519510, rs4470334, rs4365453

chr21 34631025 34632475 KCNE2 rs9983110, rs2834455, rs2834456, rs9983253

chr21 34639025 34640475 KCNE2 rs2834462, rs7283269, rs2834463, rs28458780, rs2834464, rs73205256, rs55787537, rs1004851

chr21 34830125 34831575 KCNE1 rs724386, rs11278136, rs56655934, rs36026948

chr21 34838650 34840100 KCNE1 rs2409523, rs9636617, rs9636882, rs2834518, rs9636618, rs2834519, rs9636619, rs57216941, rs2251280, rs73208707

chr3 38548000 38551650 SCN5A rs1543762, rs7642814, rs11332245, rs7650025, rs62239351, rs7647657, rs17037830, rs6802071, rs60465424, rs6792762, rs12638647, rs6810361, rs17037837, rs12638664, rs10049479, rs4679070, rs1473804, rs12497866

chr3 38557525 38563075 SCN5A rs56283404, rs12634016, rs9932, rs6767797, rs13320543, rs71323667, rs55880069, rs73825588, rs55787218, rs13078236, rs56254602, rs13078265, rs13078756, rs6781009, rs34342189, rs9832296, rs4073198, rs12490047, rs73067126, rs62239356, rs12490200, rs57298738

chr3 38592200 38600200 SCN5A rs41312409, rs41310771, rs41258454, rs71323669, rs41313653, rs41312403, rs41310769, rs41313655, rs45626031, rs55755759, rs41312405, rs57339204, rs71288084, rs56103436, rs41312407, rs7428882, rs7373819, rs34691493, rs4073103, rs9858585, rs41311131, rs41311133, rs41310773, rs34986547, rs7626962, rs1805125, rs41313657, rs41313659, rs67925888, rs41310775, rs45443294, rs41312411, rs71085329, rs73825598, rs72257817, rs67508199, rs41262044, rs67933130, rs60484872, rs59817472, rs41261544, rs58918127, rs41312413, rs9825527, rs41313663, rs61501808, rs9863293, rs45518333, rs57360866, rs71085330, rs62245085, rs56887724, rs41313661, rs4072710, rs41313665, rs72549412, rs41312415, rs55834287, rs7430407, rs41313667, rs45480800, rs45491996, rs41311135, rs61737825, rs41260344, rs6772421, rs3922844, rs6790619, rs9870213, rs6773055, rs6772989, rs6773076, rs73070963, rs58988678, rs3922843, rs41312417, rs34519218, rs71323670, rs41313669

chr3 38631650 38634000 SCN5A rs7431920, rs41314314, rs41311079, rs41312945, rs73054549, rs7430361, rs7433203, rs9867047, rs35349722, rs34767956, rs7433206, rs11710077, rs11926428, rs73054554, rs41314316

chr3 52443650 52445100 TNNC1 rs613519, rs61729091, rs34058757

chr5 172553575 172557200 NKX2-5 rs10516096, rs9313618, rs3131918, rs2544583, rs7701353, rs55816030, rs73333060, rs10039302, rs35969514, rs58410632, rs10516097

chr5 172588600 172591325 NKX2-5 rs3132141, rs12514371, rs3132140, rs61087188, rs3095872, rs73806240, rs55892953, rs13361978, rs13362000, rs3131916, rs3131917, rs2339832, rs10072156, rs72818026, rs3132142, rs5873349, rs12658373

chr5 172601400 172607750 NKX2-5 rs58142804, rs9313620, rs73317193, rs10054760, rs10071514, rs73317200, rs73317197, rs7715857, rs61576269, rs869236, rs1055336, rs10045147, rs72818029, rs6891790, rs72818030, rs10476068, rs10035505, rs10035534, rs11134781, rs62385111, rs61589835, rs6884881, rs34952680, rs34220532, rs5873351, rs34249218, rs6873714, rs6890510, rs62385112, rs62378333, rs6895287, rs72818031, rs6873521, rs6895304, rs6873973, rs6874428, rs62378332

chr5 172610075 172613650 NKX2-5 rs59532306, rs72818036, rs72818035, rs6890391, rs6891373, rs73318913, rs60785242, rs73318910, rs4868245, rs4867697, rs10476069, rs57043327, rs10476070, rs59574502, rs72818041, rs10072402



- 22 -

chr5 172616725 172618175 NKX2-5 rs9313621, rs10064148, rs60220184, rs876580, rs9313622, rs876579, rs3028715, rs73318935

chr5 172626400 172631775 NKX2-5 rs73318956, rs12518519, rs6556066, rs72421982, rs34978944, rs11953681, rs6880241, rs13158689, rs73318953, rs13158679, rs73318955, rs6897902, rs71611173, rs67739418, rs60307290, rs3132146, rs3095874, rs28579041, rs3132147, rs72818047, rs73804115, rs72818049

chr5 172638700 172640150 NKX2-5 rs11134784, rs34521880, rs10079619, rs12518366, rs11134786, rs11134785, rs11134787

chr5 172641925 172643375 NKX2-5 rs59611961, rs10037238, rs60097659, rs13361570, rs7714904, rs4077555, rs4077556, rs71611174, rs13357140, rs71912465, rs56201849, rs67696839, rs13361556, rs4505973

chr6 7483175 7484775 DSP rs1986222, rs35495774, rs72825054, rs1570283, rs34881022, rs5874099, rs1986223

chr7 150956300 150957750 PRKAG2 rs10229321, rs73478092, rs73478085, rs73478086, rs73478088, rs73478091

chr7 150965400 150967900 PRKAG2 rs73480022, rs4725421, rs4725420, rs4726075, rs4726076, rs73479892, rs73479887, rs59152884, rs73479896

chr7 151000100 151001700 PRKAG2 rs73158145, rs2727534, rs10261373, rs10260723, rs10231614, rs13228496, rs10260883, rs10247362, rs10247579, rs2727535, rs73481967, rs10250833, rs10235453, rs56039541, rs60355157

chr7 151005600 151007050 PRKAG2 rs9690322, rs2727539, rs35636043, rs2536094, rs2536093, rs2727540, rs2727541

chr7 151020500 151026975 PRKAG2 rs10274750, rs10271654, rs6971188, rs10452851, rs6953809, rs1860745, rs73728270, rs12673429, rs58291553, rs35159455, rs1860744, rs62478181, rs2536084, rs35907177, rs10275386, rs9769358, rs10232066, rs12673432, rs28550316, rs10278860, rs9771283, rs2727551, rs35004814, rs10046458, rs61023859, rs2536082, rs2536083, rs7778356, rs10225256, rs6949482, rs73484108, rs10269789, rs6950066, rs2727552, rs7792467, rs10269404, rs73728272, rs56765232, rs34248709, rs56145758, rs9648723, rs7780804, rs9648664, rs73484113

chr7 151046325 151053950 PRKAG2 rs10279256, rs10254101, rs10952316, rs73484147, rs10952317, rs12670430, rs35755070, rs35143182, rs67905656, rs73158190, rs62478182, rs6945529, rs6965359, rs7792937, rs7793421, rs13229708, rs13229709, rs73484149, rs7808855, rs2079988, rs73484151, rs2536075, rs1860743, rs62478183, rs73158191, rs2536073, rs59938864, rs61584957, rs10278669, rs4726086, rs7777428, rs2536072, rs2536071, rs55651005, rs2536070, rs7782461, rs58412371, rs7782653, rs2536069

chr7 151063150 151064600 PRKAG2 rs60176015, rs60791946, rs1104897, rs34632609, rs58772035, rs34078741, rs1547470, rs11767633, rs72199014, rs11366779, rs72350282, rs72025776, rs57903206, rs56797283, rs35986515, rs35019754, rs71657488

chr7 151071200 151072650 PRKAG2 rs60415078, rs1001116, rs35381838, rs5888449, rs59875794, rs4546566

chr7 151082100 151084775 PRKAG2 rs57892148, rs13224958, rs62478188, rs13224874, rs13224962, rs12537154, rs7778077, rs62478208, rs73158202, rs62478209, rs7801616, rs35937356

chr7 151195825 151198500 PRKAG2 rs58051053, rs73160065, rs56118280, rs60144398, rs13240743, rs12703165, rs12669153, rs60995722, rs59082716, rs71534419, rs66515231, rs56921306, rs55969839, rs61213961, rs34656135, rs72076103, rs71784381, rs71792523, rs73728437

chr8 11570375 11579750 GATA4 rs71205012, rs4510815, rs34199071, rs6987901, rs10102524, rs13250571, rs62489310, rs10102512, rs35101655, rs5889380, rs3030023, rs36217163, rs71539738, rs34226447, rs58467083, rs56399324, rs7386288, rs7824996, rs2409803, rs28468438, rs71205013, rs4840575, rs10095214, rs17808884, rs4376467, rs55994985, rs10086192, rs4368939, rs11780085, rs11783999, rs34336487, rs5029613, rs4367493, rs2409802, rs60668163, rs3030025, rs13251684, rs55869537, rs5889381, rs56406719, rs2409804, rs57547181, rs2898291, rs62489312, rs34065675, rs4284012, rs55758210, rs11784455, rs11784691, rs73535993

chr8 11636100 11639050 GATA4 rs73666003, rs10090884, rs2132714, rs2898296, rs11989007, rs2132713, rs10103483, rs73539813, rs71205019, rs66494852,



- 23 -

rs7819212, rs62489352, rs67120440, rs67120441, rs62489353, rs71521636, rs12681769, rs11785912, rs9644751, rs7832826, rs4841584, rs13275657

chr8 11641025 11642900 GATA4 rs73666005, rs2029969, rs28626371, rs4841586, rs17153707, rs17153710, rs904006, rs56294240, rs28709984, rs28628715, rs2173117

chr9 138582875 138588150 NOTCH1 rs59012671, rs71384089, rs4880103, rs7034271, rs10564413, rs56011832, rs7859652, rs59456072, rs4880104, rs73564874, rs7042742, rs17065115, rs6560637, rs56077004, rs6560636, rs7874114, rs58131031, rs10870085, rs67976492, rs56000923, rs55748806, rs34485221

chrX 153249600 153256325 FLNA rs5904390, rs2070817, rs12843193, rs12837195, rs5904389, rs2239470, rs5987248, rs12846495, rs28935169, rs5904391, rs5986975, rs5904392, rs5987249, rs5945422, rs12846320, rs12846370, rs35392838, rs59074156, rs34163302, rs6655285, rs5987250, rs5904390, rs2070817, rs12843193, rs12837195, rs5904389, rs2239470, rs5987248, rs12846495, rs28935169, rs5904391, rs5986975, rs5904392, rs5987249, rs5945422, rs12846320, rs12846370, rs35392838, rs59074156, rs34163302, rs6655285, rs5987250

chrX 153301450 153302900 TAZ rs35902788, rs41311704, rs73638278, rs35902788, rs41311704, rs73638278

Supplementary Table 3: Candidate enhancers near genes implicated in heart diseases and their corresponding SNPs. Coordinates are in human genome version hg18.

SNP Candidate enhancer location (hg18) Gene

rs1476413 chr1:11769325-11775875 MTHFR

rs4979848 chr10:80612475-80616575 ZMIZ1

rs1015089 chr14:22676100-22682825 DHRS4

rs4725925 chr7:149955500-149960500 GIMAP4

rs2260187 chr19:5750050-5752350 DUS3L

rs1628955 chr15:62973250-62975650 ANKDD1A

rs17726002 chr10:73538675-73540125 ASCC1

rs7940871 chr11:45183725-45185175 PRDM11

rs8140884 chr22:41662950-41664400 PACSIN2

rs7036030 chr9:34075075-34076525 NUDT2

rs10876864 chr12:54686550-54689225 RPS26

Supplementary Table 4: SNPs within candidate heart enhancers that are eQTLs for gene expression in primary human cells 13.



- 24 -

Maximum scoring phastCons element overlapping core 1kb centered on peak

summit

Maximum scoring phastCons element overlapping region

Fetal human heart candidate enhancers

(n=5,047)

Tested elements (n=65)

Fetal human heart candidate

enhancers (n=5,047)

Tested peaks (n=65)

Tested element (cloned region)

(n=65)

No detectable constraint 1931 (38%) 22 (34%) 793 (16%) 5 (8%) 1 (2%)

Weakly constrained 1284 (26%) 20 (31%) 1204 (24%) 18 (28%) 20 (31%)

Moderately constrained 752 (15%) 11 (17%) 1004 (20%) 16 (25%) 18 (28%)

Highly constrained 1080 (21%) 12 (18%) 2046 (40%) 26 (40%) 26 (40%)

Supplementary Table 5: Sequence constraint properties of human heart candidate enhancers and tested elements. The distribution of tested elements is overall similar to the genome-wide set of ChIP-seq peaks. A moderate bias towards constrained elements among the tested regions is in part due to the frequent inclusion of additional weakly or moderately constrained sequences flanking the peak region in the tested constructs.



- 25 -

Candidate enhancers Tested peaks (n=65) Top 100 Top 500 Top 1000 All (n=5,047)

Significant in mouse 52 (52%) 217 (43%) 361 (36%) 1047 (21%) 24 (37%)

Sub-significant in mouse 31 (31%) 183 (37%) 369 (37%) 1285 (25%) 25 (38%)

Human-specific 12 (12%) 64 (13%) 185 (18.5%) 2079 (41%) 9 (14%)

Non-alignable 5 (5%) 36 (7%) 85 (8.5%) 635 (13%) 7 (11%)

Supplementary Table 6: Functional conservation properties of human heart candidate enhancers and in vivo tested elements. Candidate human fetal heart enhancers were ranked by the statistical significance of ChIP-seq enrichment at the respective peaks, and the top 100, 500, and 1000 peaks were examined for functional conservation in the mouse genome. See Supplementary Fig. 7 for definitions of functional conservation classes. Overall, more significant binding is positively correlated with increased likelihood of functional conservation of peaks between human and mouse. All categories of functional conservation are represented among the in vivo tested candidate enhancers.



- 26 -

ID Annotation Whole-mount 1747 Tested peak: chr3 50611100 50616900

Reproducibility: 7/12 Flanking genes: HEMK1-CISH (intergenic)

1667 Tested peak: chr18 18300275 18307650

Reproducibility: 5/6 Flanking genes: CTAGE1-RBBP8 (intergenic)

1748 Tested peak: chr11 74941850 74953100

Reproducibility: 3/4 Flanking genes: GDPD5-SERPINH1 (intergenic)

1750 Tested peak: chr2 240843300 240849300

Reproducibility: 7/7 Flanking genes: OTOS-GPC1 (intergenic)

1751 Tested peak: chr18 12267650 12271125

Reproducibility: 8/8 Flanking genes: CIDEA-TUBB6 (intergenic)

1752 Tested peak: chr5 148776325 148786625

Reproducibility: 5/6 Flanking genes: IL17B-CSNK1A1 (intergenic)

1753 Tested peak: chr7 158579925 158585700

Reproducibility: 14/18 Flanking genes: VIPR2 (intronic)

1754 Tested peak: chr19 11108900 11115900

Reproducibility: 9/11 Flanking genes: LDLR-SPC24 (intergenic)

1881 Tested peak: chr15 41958350 41961550

Reproducibility: 6/14 Flanking genes: LOC728758-CASC4 (intergenic)

1759 Tested peak: chr11 65011450 65018025

Reproducibility: 8/13 Flanking genes: NCRNA00084-MALAT1 (intergenic)



- 27 -

1760 Tested peak: chr16 65490225 65501200 Reproducibility: 14/14 Flanking genes: PDP2-CDH16 (intergenic)

1763 Tested peak: chr10 134288500 134298550

Reproducibility: 4/5 Flanking genes: INPP5A (intronic)

1764 Tested peak: chr2 218507550 218516100

Reproducibility: 7/9 Flanking genes: TNS1 (intronic)

1766 Tested peak: chr1 229049800 229054950

Reproducibility: 12/12 Flanking genes: C1orf198 (intronic)

1767 Tested peak: chr10 80649625 80653600

Reproducibility: 7/12 Flanking genes: ZMIZ1 (intronic)

1769 Tested peak: chr17 35082150 35090825

Reproducibility: 6/7 Flanking genes: PGAP3 (intronic)

1882 Tested peak: chr8 142488500 142497275

Reproducibility: 8/8 Flanking genes: GPR20-PTP4A3 (intergenic)

1886 Tested peak: chr2 232234175 232241350

Reproducibility: 5/8 Flanking genes: C2orf57-PTMA (intergenic)

1887 Tested peak: chr6 35564700 35569550

Reproducibility: 4/5 Flanking genes: TEAD3 (intronic)

1891 Tested peak: chr1 154896350 154906400

Reproducibility: 7/7 Flanking genes: BCAN-NES (intergenic)

1909 Tested peak: chr2 43297800 43301025

Reproducibility: 9/9 Flanking genes: HAAO-ZFP36L2 (intergenic)



- 28 -

1912 Tested peak: chr1 3252200 3260250 Reproducibility: 4/4 Flanking genes: PRDM16 (intronic)

1915 Tested peak: chr11 119261250 119270500

Reproducibility: 4/9 Flanking genes: PVRL1-TRIM29 (intergenic)

1919 Tested peak: chr2 96287250 96291800

Reproducibility: 8/13 Flanking genes: TMEM127 (intronic)

1920 Tested peak: chr11 69017550 69021725

Reproducibility: 9/10 Flanking genes: MYEOV-CCND1 (intergenic)

1927 Tested peak: chr8 22884625 22889875

Reproducibility: 5/6 Flanking genes: PEBP4-RHOBTB2 (intergenic)

1931 Tested peak: chr14 99109300 99114775

Reproducibility: 11/11 Flanking genes: CCDC85C (intronic)

1932 Tested peak: chr15 97073025 97079950

Reproducibility: 6/14 Flanking genes: IGF1R (intronic)

1933 Tested peak: chr2 101092600 101096250

Reproducibility: 5/9 Flanking genes: TBC1D8 (intronic)

1937 Tested peak: chr2 240956075 240963875

Reproducibility: 8/9 Flanking genes: OTOS-GPC1 (intergenic)

1945 Tested peak: chr2 238185675 238191550

Reproducibility: 4/9 Flanking genes: RAB17-LRRFIP1 (intergenic)

1948 Tested peak: chr19 46623876 46631900

Reproducibility: 7/10 Flanking genes: B3GNT8-C19orf69 (intergenic)



- 29 -

1950 Tested peak: chr10 93338125 93345100 Reproducibility: 4/4 Flanking genes: HECTD2-PPP1R3C (intergenic)

1951 Tested peak: chr2 237885675 237889550

Reproducibility: 11/18 Flanking genes: COPS8-COL6A3 (intergenic)

1955 Tested peak: chr6 164299525 164306475

Reproducibility: 5/8 Flanking genes: QKI-C6orf118 (intergenic)

1958 Tested peak: chr10 3078000 3080675

Reproducibility: 4/4 Flanking genes: ADARB2-PFKP (intergenic)

1959 Tested peak: chr16 81325114 81327548

Reproducibility: 7/7 Flanking genes: CDH13 (intronic)

1962 Tested peak: chr8 30254050 30260575

Reproducibility: 15/17 Flanking genes: DCTN6-RBPMS (intergenic)

1963 Tested peak: chr9 133097950 133106275

Reproducibility: 12/12 Flanking genes: NUP214-FAM78A (intergenic)

1967 Tested peak: chr2 47148125 47152750

Reproducibility: 8/9 Flanking genes: TTC7A (intronic)

1971 Tested peak: chr20 55447725 55454000

Reproducibility: 10/10 Flanking genes: RBM38-HMGB1L1 (intergenic)

1974 Tested peak: chr10 79641225 79648100

Reproducibility: 4/5 Flanking genes: RPS24-LOC283050 (intergenic)

1466 Tested peak: chr14 76452675 76458100

Reproducibility: 4/8 Flanking genes: C14orf166B-C14orf4 (intergenic)



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Supplementary Table 7: Summary of in vivo heart enhancers. Representative images of whole embryos of all 43 positive in vivo heart enhancers identified in this study. ID: identifier of the tested element in the Vista enhancer browser (http://enhancer.lbl.gov/). Tested peak: coordinates of the injected DNA sequence in human genome hg18; Reproducibility: fraction of stained embryos with reproducible staining in the exact same sub-region shown in the representative whole-mount; Flanking Genes: nearest UCSC Known Genes upstream and downstream the tested element.

ID Region (hg18) phastCons Nearest heart gene

In vivo testing results Distance

494 chr4:174672888-174673445 416 HAND2 negative 14787

495 chr10:92680001-92680654 474 ANKRD1 other (hindbrain, midbrain, nose)

-9315

497 chr22:18121204-18121707 408 TBX1 negative -2769

498 chr12:113144750-113145389 474 TBX5 negative 181016

500 chr4:111797722-111798276 428 PITX2 negative -20042

502 chr18:18027981-18028530 557 GATA6 heart 24842

503 chr5:88584156-88586256 808 MEF2C heart -370426

504 chr2:219979738-219980250 479 DES negative -11349

505 chr5:172565402-172566203 359 NKX2-5 negative 29119

507 chr4:111749623-111750222 505 PITX2 negative 13780

508 chr8:11641591-11642104 464 GATA4 heart 42722

509 chr5:50743324-50744961 599 ISL1 negative 29428

511 chr10:103517183-103518383 699 FGF8 other (tail) 8034

513 chr10:103530170-103531020 509 FGF8 other (dorsal root ganglion, cranial nerve)

-4778

514 chr5:153851820-153852169 576 HAND1 negative -13977

515 chr22:18129356-18130055 470 TBX1 other (limb) 5481

516 chr5:44424923-44426224 538 FGF10 other (limb) -1033

Supplementary Table 8: Summary of the testing of 17 moderately conserved elements located near genes implicated in embryonic heart development through genetic or developmental biology studies. In addition to moderate evolutionary conservation, these sequences contained sequence motifs hypothesized to be cardiac-associated 11. We observed that 3 of the 17 sequences (18%) were reproducible heart enhancers.



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Human fetal heart

p300/CBP-ChIP-seq predictions

e11.5 mouse heart p300 ChIP-seq predictions

Total tested elements 65 130 Elements with reproducible cardiac patterns 43 (66%) 81 (60%) Average positive embryos per element 7.2 5.8 Reproducible LacZ staining in the heart 310 469 Average reproducibility 7.2/9.3 (77%) 5.8/7.7 (75%) Average peak size (STD) 6123.8 (2326.6) 837.2 (292.9) Average element size (STD) 3719 (808) 1648 (868)

Supplementary Table 9: Reproducibility of positive heart enhancers in the present study and comparison with mouse heart enhancer predictions 8. While average peak size in the human data set was substantially larger than in the mouse data set, elements tested in transgenic mice typically included only the most significantly covered subregions of longer peaks (see example in Fig. 1), resulting in a more moderate difference in the average size of tested elements.



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mCS/CS264 Mouse ortholog (mm9) Human ortholog (hg18) ID mm123 hs 1652 Tested region chr6:72,165,180-72,168,822 chr2:85,850,978-85,854,516 Reproducibility - heart - other

heart 5/11 Limb 7, neural tube 9, nose 7

heart 6/8 Other 6

humanmouse lift-over 87.7% of bases, 100.0% of span Flanking genes intronic Atoh8 Whole-mount

P300/CBP coverage (called yes/no) 55 (yes) mCS/CS271 Mouse ortholog (mm9) Human ortholog (hg18) ID mm130 hs1659 Tested region chr9:24,691,516-24,694,512 chr7:35,379,250-35,382,874 Reproducibility - heart - other

Heart 7/10 other 7

heart 9/9

humanmouse lift-over 78.8% of bases, 99.4% of span Flanking genes Tbx20 / Herpud2 Whole mount

P300/CBP coverage (called yes/no) 27 (yes) mCS/CS272 Mouse ortholog (mm9) Human ortholog (hg18) ID mm131 hs1660 Tested region chr7:69,037,006-69,038,618 chr5:132,704,481-132,706,219 Reproducibility - heart - other

Heart 6/7 ear 6

-- Ear 5/8

humanmouse lift-over 86.1% of bases, 99.9% of span Flanking genes Intronic Auts2 Whole-mount

P300/CBP coverage (called yes/no) 5 (no)



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mCS/CS273 Mouse ortholog (mm9) Human ortholog (hg18) ID mm132 hs1661 Tested region chr13:46,666,689-46,669,718 chr5:153,556,100-153,558,157 Reproducibility - heart - other

heart 7/7 branchial arch 5, eye 6, midbrain 6, somite 6

-- --

humanmouse lift-over 93.3% of bases, 100.0% of span Flanking genes Intronic CAP2 Whole mount

P300/CBP coverage (called yes/no) -- mCS/CS277 Mouse ortholog (mm9) Human ortholog (hg18) ID mm136 hs1665 Tested region chr8:28,216,799-28,218,903 chr8:37,802,248-37,804,505 Reproducibility - heart - other

Heart 7/10, blood vessels 4 other 6

Heart 5/7, blood vessels 7

humanmouse lift-over 82.5% of bases, 100.0% of span Flanking genes Intronic GPR124 Whole mount

P300/CBP coverage (called yes/no) 14 (no) mCS/CS279 Mouse ortholog (mm9) Human ortholog (hg18) ID mm138 hs1667 Tested region chr18:11,371,312-11,374,453 chr18:18,303,642-18,306,902 Reproducibility - heart - other

heart 8/8 heart 5/6

humanmouse lift-over 87.3% of bases, 100.0% of span Flanking genes Gata6 – Rbbp8 CTAGE1 - Rbbp8 Whole mount

P300/CBP coverage (called yes/no) 87 (yes)



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mCS/CS284 Mouse ortholog (mm9) Human ortholog (hg18) ID mm143 hs1672 Tested region chr3:104,400,858-104,404,522 chr1:113,341,579-113,343,543 Reproducibility - heart - other

heart 3/3 heart 7/7

humanmouse lift-over 87.0% of bases, 99.9% of span Flanking genes Slc16A1 – LRIG2 Whole mount

P300/CBP coverage (called yes/no) 17 (no) mCS/CS287 Mouse ortholog (mm9) Human ortholog (hg18) ID mm146 hs1675 Tested region chr11:77,235,971-77,237,943 chr17:25,018,828-25,021,000 Reproducibility - heart heart 9/9 heart 8/10 - other humanmouse lift-over 55.2% of bases, 100.0% of span Flanking genes Intron SSH2 Whole mount

P300/CBP coverage (called yes/no) 23 (yes)

Supplementary Table 10: Summary of mouse–human orthologous elements tested in vivo. To establish that the transgenic mouse enhancer assay correctly reports the activity of human heart enhancers, we tested the human orthologous sequences of eight mouse heart enhancers originally identified through ChIP-seq from mouse heart tissue 8. Six of eight (75%) human sequences showed positive heart enhancer activity at e11.5. While the reporter staining in the heart appeared in several cases fainter with the human heart enhancer, we observed overall no significant difference in the reproducibility of the positive enhancers in mouse vs. human (t-test, P=0.71). These results suggest that, with few exceptions, this transgenic assay accurately reports the in vivo activity of human heart enhancers.



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Supplementary Table 11: Properties of eight elements that were tested at e11.5 and in 4-week old mice. TSS-transcript start site. See Supplementary Fig. 7 for definitions of binding conservation classes.

ID Location Start End hs1747 chr3 50611482 50615162 hs1758 chr15 66778003 66780739 hs1667 chr18 18303642 18306902 hs1778 chr10 97163113 97166280 hs1748 chr11 74941714 74944400 hs1750 chr2 240846030 240848828 hs1751 chr18 12267700 12271650 hs1752 chr5 148781886 148785269 hs1753 chr7 158581530 158584957 hs1754 chr19 11112931 11115703 hs1881 chr15 41958540 41961183 hs1759 chr11 65011280 65014970 hs1760 chr16 65494577 65497061 hs1763 chr10 134292019 134296802 hs1764 chr2 218509416 218511808 hs1766 chr1 229051146 229054604 hs1767 chr10 80650322 80654048 hs1769 chr17 35084650 35088052 hs1882 chr8 142490529 142495534 hs1885 chr2 26796157 26799604 hs1886 chr2 232235504 232240353 hs1887 chr6 35565107 35568578 hs1889 chr3 124172992 124177979 hs1890 chr5 148801548 148805056 hs1891 chr1 154896875 154901908

ID Distance

to nearest

TSS (bp)

Chr Peak start Peak end Element size (bp)

# of reads (fetal)

# of reads (adult)

Highest phastCons

Binding conservation to mouse

hs1750 119529 chr2 240846030 240848828 2798 73 21 290 significant

hs1751 25248 chr18 12267700 12271650 3950 66 28 391 sub-significant

hs1753 47060 chr7 158581530 158584957 3427 72 29 350 human-specific

hs1760 14267 chr16 65494577 65497061 2484 104 24 521 significant

hs1763 24292 chr10 134292019 134296802 4783 97 29 261 non-alignable






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hs1903 chr14 76576624 76581102 hs1908 chr7 150296482 150300994 hs1909 chr2 43298225 43300800 hs1912 chr1 3251953 3256221 hs1913 chr22 34312628 34317304 hs1914 chr3 37785705 37789508 hs1915 chr11 119264943 119268868 hs1916 chr16 85456216 85459375 hs1917 chr17 17379914 17384912 hs1919 chr2 96287429 96290813 hs1920 chr11 69017366 69021712 hs1927 chr8 22884661 22889291 hs1931 chr14 99109408 99114079 hs1932 chr15 97075267 97079751 hs1933 chr2 101092532 101095924 hs1934 chr20 55167930 55171001 hs1935 chr4 185542044 185545204 hs1937 chr2 240956654 240961667 hs1941 chr2 119961106 119965510 hs1944 chr15 63164722 63168467 hs1945 chr2 238187121 238191306 hs1947 chr7 2617812 2621342 hs1948 chr19 46628830 46630731 hs1950 chr10 93338469 93341700 hs1951 chr2 237886563 237890012 hs1952 chr3 188147609 188151169 hs1953 chr4 140902316 140905736 hs1955 chr6 164302336 164306379 hs1958 chr10 3077624 3080441 hs1959 chr16 81325114 81327548 hs1961 chr3 37781005 37784084 hs1962 chr8 30254588 30258451 hs1963 chr9 133100117 133105005 hs1967 chr2 47149282 47152936 hs1968 chr2 238468111 238472336 hs1971 chr20 55448120 55452699 hs1972 chr6 21694829 21697501 hs1973 chr12 122112881 122117678 hs1974 chr10 79642400 79647338 hs1466 chr14 76454394 76457450

Supplementary Table 12: hg18 coordinates of 65 tested elements. ID corresponds to the identifier of the tested element in the Vista enhancer browser (http://enhancer.lbl.gov/).



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Supplementary References

1. Kunarso, G. et al. Transposable elements have rewired the core regulatory network of human embryonic stem cells. Nat Genet 42, 631-4 (2010).

2. Odom, D.T. et al. Tissue-specific transcriptional regulation has diverged significantly between human and mouse. Nat Genet 39, 730-2 (2007).

3. Schmidt, D. et al. Five-vertebrate ChIP-seq reveals the evolutionary dynamics of transcription factor binding. Science 328, 1036-40 (2010).

4. Henderson, D.J. & Anderson, R.H. The development and structure of the ventricles in the human heart. Pediatr Cardiol 30, 588-96 (2009).

5. Coppen, S.R. et al. Comparison of connexin expression patterns in the developing mouse heart and human foetal heart. Mol Cell Biochem 242, 121-7 (2003).

6. Kent, W.J. et al. The human genome browser at UCSC. Genome Res 12, 996-1006 (2002).

7. Siepel, A. et al. Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res 15, 1034-50 (2005).

8. Blow, M.J. et al. ChIP-Seq identification of weakly conserved heart enhancers. Nat Genet 42, 806-810 (2010).

9. Pennacchio, L.A. et al. In vivo enhancer analysis of human conserved non-coding sequences. Nature 444, 499-502 (2006).

10. Visel, A. et al. Ultraconservation identifies a small subset of extremely constrained developmental enhancers. Nat Genet 40, 158-60 (2008).

11. Leung, G. & Eisen, M.B. Identifying cis-regulatory sequences by word profile similarity. PLoS One 4, e6901 (2009).

12. Bryne, J.C. et al. JASPAR, the open access database of transcription factor-binding profiles: new content and tools in the 2008 update. Nucleic Acids Res 36, D102-6 (2008).

13. Dimas, A.S. et al. Common regulatory variation impacts gene expression in a cell type-dependent manner. Science 325, 1246-50 (2009).


Large-Scale Discovery of Enhancers from Human Heart Tissue · Large-Scale Discovery of Enhancers...

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