Large-Scale Discovery of Enhancers from Human Heart Tissue

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1 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 -

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

3 May et al: ChIP-seq Discovery of Human Heart Enhancers - 3 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)

4 May et al: ChIP-seq Discovery of Human Heart Enhancers - 4 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

5 May et al: ChIP-seq Discovery of Human Heart Enhancers - 5 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. -5 -

May et al: ChIP-seq Discovery of Human Heart Enhancers - 6 Supplementary Figure 3: p300/cbp distal peaks near heart-expressed and non-heart-expressed genes.

6 May et al: ChIP-seq Discovery of Human Heart Enhancers - 6 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 nonheart 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

May et al: ChIP-seq Discovery of Human Heart Enhancers - 7 Supplementary Figure 5: p300/cbp distal peaks are enriched near heart disease-associated genes.

7 May et al: ChIP-seq Discovery of Human Heart Enhancers - 7 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

8 May et al: ChIP-seq Discovery of Human Heart Enhancers - 8 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)

9 May et al: ChIP-seq Discovery of Human Heart Enhancers - 9 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 ; Weakly constrained, phastcons 350; No detectable constraint, no overlapping phastcons element

10 May et al: ChIP-seq Discovery of Human Heart Enhancers - 10 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 , hypergeometric distribution p < 1e-15). b) Histone mark H3K27ac in 8 human cell lines (fold enrichment , 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 linederived 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

11 May et al: ChIP-seq Discovery of Human Heart Enhancers - 11 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

May et al: ChIP-seq Discovery of Human Heart Enhancers - 12 Supplementary Figure 10: Transcription factor binding site analysis of candidate heart enhancers.

12 May et al: ChIP-seq Discovery of Human Heart Enhancers - 12 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 database 12 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

May et al: ChIP-seq Discovery of Human Heart Enhancers - 13 Supplementary Figure 11: Reproducible enhancer activity at e11.5 of adult vs. fetal human candidate heart enhancers.

13 May et al: ChIP-seq Discovery of Human Heart Enhancers - 13 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)

May et al: ChIP-seq Discovery of Human Heart Enhancers - 14 Supplementary Figure 12: Transgenic results for cardiac enhancers tested in mice at e11.5 and at postnatal day 28. See Fig.

14 May et al: ChIP-seq Discovery of Human Heart Enhancers - 14 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) hs 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) hs 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

15 May et al: ChIP-seq Discovery of Human Heart Enhancers - 15 Supplementary Tables Human fetal heart p300/cbp Human adult heart p300/cbp Mouse P2 heart p300/cbp Total Reads 27,073, % 26,246, % 27,181, % Reads mapping to reference genome Reads mapping to unique location in the reference genome Non-clonal reads mapping to unique location in the reference genome 13,966, % 16,444, % 12,720, % 11,463, % 13,261, % 9,596, % 10,090, % 9,021, % 8,641, % Total peaks 12, % 5, % 17, % Peaks passing filtering 10, % 4, % 12, % Proximal peaks (<=2.5kb from nearest TSS) Distal peaks (>2.5kb from nearest TSS) 5, % 2, % 6, % 5, % 2, % 6, % 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

16 May et al: ChIP-seq Discovery of Human Heart Enhancers - 16 Phenotypes Binom FDR Q-value Binom fold enrichment abnormal cardiac muscle morphology 3.32E abnormal vitelline vasculature 1.13E abnormal epicardium morphology 5.23E thin ventricular wall 3.43E abnormal myocardial trabeculae morphology 7.74E decreased cardiac muscle contractility 2.79E abnormal renal glomerulus morphology 3.34E anemia 1.14E pericardial effusion 7.44E abnormal cardiac muscle contractility 4.35E abnormal erythrocyte cell number 2.83E abnormal placenta vasculature 4.35E impaired muscle contractility 6.80E abnormal vascular branching morphogenesis 9.51E abnormal erythrocyte morphology 1.66E abnormal looping morphogenesis 4.90E abnormal myocardial fiber morphology 6.41E poorly developed ventricular trabeculae 7.91E abnormal dorsal aorta morphology 8.31E abnormal liver development 1.33E abnormal liver/biliary system development 3.66E abnormal capillary morphology 1.02E disorganized myocardium 1.32E thin myocardial wall 1.72E abnormal tail development 4.23E abnormal ventricular trabeculae morphology 1.48E cortical renal glomerulopathies 1.49E branchial arch hypoplasia 1.79E abnormal mitochondrial physiology 3.86E abnormal placenta labyrinth morphology 6.09E abnormal outflow tract septation 7.91E decreased heart rate 2.91E dilated left ventricle 7.53E abnormal wound healing 2.03E dilated heart ventricles 2.78E congestive heart failure 6.52E pale yolk sac 6.48E cardiac hypertrophy 7.02E abnormal exercise endurance 7.49E abnormal heart tube morphology 7.45E decreased ventricle muscle contractility 3.60E abnormal trophoblast layer morphology 7.03E

17 May et al: ChIP-seq Discovery of Human Heart Enhancers - 17 abnormal vascular endothelial cell morphology 7.13E abnormal cell differentiation 1.06E hydrops fetalis 1.87E increased stomach ph 3.86E abnormal muscle weight 7.92E failure of looping morphogenesis 8.63E abnormal QRS complex 1.43E abnormal placental labyrinth vasculature morphology 5.25E Supplementary Table 2: Top enriched annotations of putative target genes near candidate human heart enhancers

18 May et al: ChIP-seq Discovery of Human Heart Enhancers - 18 Chr Peak start Peak end Gene symbol SNPs chr LMNA rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr TNNT2 rs , rs , rs763208, rs , rs , rs , rs , rs , rs , rs , rs832153, rs17190, rs , rs , rs , rs , rs , rs , rs832152, rs832151, rs , rs , rs832150, rs chr TNNT2 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs947376, rs947375, rs947374, rs chr AGT rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs868143, rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr AGT rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr ACTN2 rs661219, rs663798, rs507130, rs507841, rs662513, rs535411, rs676377, rs chr ACTN2 rs , rs , rs697660, rs , rs , rs chr ACTN2 rs , rs , rs , rs891337, rs891338, rs , rs , rs , rs , rs , rs , rs707201, rs , rs chr ACTN2 rs , rs , rs , rs , rs , rs , rs chr RYR2 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr RYR2 rs , rs chr RYR2 rs , rs , rs , rs , rs chr VCL rs , rs , rs chr LDB3 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr LDB3 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs ,

19 May et al: ChIP-seq Discovery of Human Heart Enhancers - 19 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr LDB3 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr LDB3 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr ANKRD1 rs , rs chr ANKRD1 rs , rs , rs , rs , rs , rs chr KCNQ1 rs179436, rs , rs , rs179437, rs , rs757086, rs , rs chr KCNQ1 rs , rs , rs , rs , rs81205, rs163149, rs163150, rs , rs , rs , rs , rs , rs81204, rs , rs163148, rs163147, rs , rs , rs , rs , rs , rs189261, rs , rs , rs , rs , rs163146, rs233434, rs , rs , rs , rs , rs163145, rs , rs , rs234886, rs chr KCNQ1 rs , rs149145, rs , rs , rs , rs , rs , rs , rs233439, rs151317, rs , rs149373, rs , rs234875, rs , rs , rs , rs , rs , rs chr KCNQ1 rs , rs , rs chr KCNQ1 rs , rs , rs , rs , rs , rs515774, rs , rs , rs chr KCNQ1 rs371972, rs , rs441613, rs400643, rs , rs , rs439926, rs445108, rs444956, rs446396, rs384037, rs , rs , rs , rs , rs367003, rs429573, rs , rs chr CSRP3 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr CSRP3 rs , rs , rs , rs chr CSRP3 rs , rs , rs , rs , rs793280, rs , rs , rs , rs793279, rs , rs , rs , rs , rs chr CSRP3 rs , rs , rs924692, rs924691, rs998263, rs , rs , rs , rs chr MYL2 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr TBX5 rs , rs , rs , rs , rs

20 May et al: ChIP-seq Discovery of Human Heart Enhancers - 20 chr TBX5 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr TBX5 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr TPM1 rs , rs , rs , rs , rs , rs , rs , rs , rs chr TPM1 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr TCAP rs , rs , rs907087, rs , rs , rs , rs , rs903502, rs , rs903504, rs , rs903503, rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs907088, rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs732083, rs , rs , rs907089, rs907090, rs732084, rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr TCAP rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr JUP rs , rs , rs , rs chr JUP rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs753663, rs , rs , rs , rs , rs , rs , rs873150, rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr SGCA rs , rs , rs , rs , rs , rs , rs , rs chr SGCA rs , rs , rs , rs , rs , rs , rs , rs chr TNNI3 rs , rs604216, rs , rs , rs682884, rs , rs , rs , rs , rs , rs , rs , rs , rs , rs575144, rs , rs , rs , rs667451, rs , rs , rs , rs , rs , rs chr DYSF rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr DYSF rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr DYSF rs934068, rs , rs , rs , rs , rs , rs , rs , rs , rs934066, rs , rs , rs , rs , rs , rs , rs934067, rs750355, rs , rs , rs , rs751473, rs750356, rs751474, rs , rs , rs , rs , rs751475, rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr DYSF rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs ,

21 May et al: ChIP-seq Discovery of Human Heart Enhancers - 21 rs , rs , rs , rs , rs934069, rs749314, rs chr DES rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr KCNE2 rs , rs , rs , rs chr KCNE2 rs , rs , rs , rs , rs , rs , rs , rs chr KCNE1 rs724386, rs , rs , rs chr KCNE1 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr SCN5A rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr SCN5A rs , rs , rs9932, rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr SCN5A rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr SCN5A rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr TNNC1 rs613519, rs , rs chr NKX2-5 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr NKX2-5 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr NKX2-5 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs869236, rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr NKX2-5 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs

22 May et al: ChIP-seq Discovery of Human Heart Enhancers - 22 chr NKX2-5 rs , rs , rs , rs876580, rs , rs876579, rs , rs chr NKX2-5 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr NKX2-5 rs , rs , rs , rs , rs , rs , rs chr NKX2-5 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr DSP rs , rs , rs , rs , rs , rs , rs chr PRKAG2 rs , rs , rs , rs , rs , rs chr PRKAG2 rs , rs , rs , rs , rs , rs , rs , rs , rs chr PRKAG2 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr PRKAG2 rs , rs , rs , rs , rs , rs , rs chr PRKAG2 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr PRKAG2 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr PRKAG2 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr PRKAG2 rs , rs , rs , rs , rs , rs chr PRKAG2 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr PRKAG2 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr GATA4 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr GATA4 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs ,

23 May et al: ChIP-seq Discovery of Human Heart Enhancers - 23 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chr GATA4 rs , rs , rs , rs , rs , rs , rs904006, rs , rs , rs , rs chr NOTCH1 rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chrx FLNA rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs chrx TAZ rs , rs , rs , rs , rs , rs 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 rs chr1: MTHFR rs chr10: ZMIZ1 rs chr14: DHRS4 rs chr7: GIMAP4 rs chr19: DUS3L rs chr15: ANKDD1A rs chr10: ASCC1 rs chr11: PRDM11 rs chr22: PACSIN2 rs chr9: NUDT2 rs chr12: RPS26 Supplementary Table 4: SNPs within candidate heart enhancers that are eqtls for gene expression in primary human cells

24 May et al: ChIP-seq Discovery of Human Heart Enhancers - 24 No detectable constraint Weakly constrained Moderately constrained Highly constrained Maximum scoring phastcons element overlapping core 1kb centered on peak summit Fetal human heart candidate enhancers (n=5,047) Tested elements (n=65) Maximum scoring phastcons element overlapping region Fetal human heart candidate enhancers (n=5,047) Tested peaks (n=65) Tested element (cloned region) (n=65) 1931 (38%) 22 (34%) 793 (16%) 5 (8%) 1 (2%) 1284 (26%) 20 (31%) 1204 (24%) 18 (28%) 20 (31%) 752 (15%) 11 (17%) 1004 (20%) 16 (25%) 18 (28%) 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 May et al: ChIP-seq Discovery of Human Heart Enhancers - 25 Candidate enhancers Top 100 Top 500 Top 1000 All (n=5,047) Tested peaks (n=65) 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 May et al: ChIP-seq Discovery of Human Heart Enhancers - 26 ID Annotation Whole-mount 1747 Tested peak: chr Reproducibility: 7/12 Flanking genes: HEMK1-CISH (intergenic) 1667 Tested peak: chr Reproducibility: 5/6 Flanking genes: CTAGE1-RBBP8 (intergenic) 1748 Tested peak: chr Reproducibility: 3/4 Flanking genes: GDPD5-SERPINH1 (intergenic) 1750 Tested peak: chr Reproducibility: 7/7 Flanking genes: OTOS-GPC1 (intergenic) 1751 Tested peak: chr Reproducibility: 8/8 Flanking genes: CIDEA-TUBB6 (intergenic) 1752 Tested peak: chr Reproducibility: 5/6 Flanking genes: IL17B-CSNK1A1 (intergenic) 1753 Tested peak: chr Reproducibility: 14/18 Flanking genes: VIPR2 (intronic) 1754 Tested peak: chr Reproducibility: 9/11 Flanking genes: LDLR-SPC24 (intergenic) 1881 Tested peak: chr Reproducibility: 6/14 Flanking genes: LOC CASC4 (intergenic) 1759 Tested peak: chr Reproducibility: 8/13 Flanking genes: NCRNA00084-MALAT1 (intergenic)

27 May et al: ChIP-seq Discovery of Human Heart Enhancers Tested peak: chr Reproducibility: 14/14 Flanking genes: PDP2-CDH16 (intergenic) 1763 Tested peak: chr Reproducibility: 4/5 Flanking genes: INPP5A (intronic) 1764 Tested peak: chr Reproducibility: 7/9 Flanking genes: TNS1 (intronic) 1766 Tested peak: chr Reproducibility: 12/12 Flanking genes: C1orf198 (intronic) 1767 Tested peak: chr Reproducibility: 7/12 Flanking genes: ZMIZ1 (intronic) 1769 Tested peak: chr Reproducibility: 6/7 Flanking genes: PGAP3 (intronic) 1882 Tested peak: chr Reproducibility: 8/8 Flanking genes: GPR20-PTP4A3 (intergenic) 1886 Tested peak: chr Reproducibility: 5/8 Flanking genes: C2orf57-PTMA (intergenic) 1887 Tested peak: chr Reproducibility: 4/5 Flanking genes: TEAD3 (intronic) 1891 Tested peak: chr Reproducibility: 7/7 Flanking genes: BCAN-NES (intergenic) 1909 Tested peak: chr Reproducibility: 9/9 Flanking genes: HAAO-ZFP36L2 (intergenic)

28 May et al: ChIP-seq Discovery of Human Heart Enhancers Tested peak: chr Reproducibility: 4/4 Flanking genes: PRDM16 (intronic) 1915 Tested peak: chr Reproducibility: 4/9 Flanking genes: PVRL1-TRIM29 (intergenic) 1919 Tested peak: chr Reproducibility: 8/13 Flanking genes: TMEM127 (intronic) 1920 Tested peak: chr Reproducibility: 9/10 Flanking genes: MYEOV-CCND1 (intergenic) 1927 Tested peak: chr Reproducibility: 5/6 Flanking genes: PEBP4-RHOBTB2 (intergenic) 1931 Tested peak: chr Reproducibility: 11/11 Flanking genes: CCDC85C (intronic) 1932 Tested peak: chr Reproducibility: 6/14 Flanking genes: IGF1R (intronic) 1933 Tested peak: chr Reproducibility: 5/9 Flanking genes: TBC1D8 (intronic) 1937 Tested peak: chr Reproducibility: 8/9 Flanking genes: OTOS-GPC1 (intergenic) 1945 Tested peak: chr Reproducibility: 4/9 Flanking genes: RAB17-LRRFIP1 (intergenic) 1948 Tested peak: chr Reproducibility: 7/10 Flanking genes: B3GNT8-C19orf69 (intergenic)

29 May et al: ChIP-seq Discovery of Human Heart Enhancers Tested peak: chr Reproducibility: 4/4 Flanking genes: HECTD2-PPP1R3C (intergenic) 1951 Tested peak: chr Reproducibility: 11/18 Flanking genes: COPS8-COL6A3 (intergenic) 1955 Tested peak: chr Reproducibility: 5/8 Flanking genes: QKI-C6orf118 (intergenic) 1958 Tested peak: chr Reproducibility: 4/4 Flanking genes: ADARB2-PFKP (intergenic) 1959 Tested peak: chr Reproducibility: 7/7 Flanking genes: CDH13 (intronic) 1962 Tested peak: chr Reproducibility: 15/17 Flanking genes: DCTN6-RBPMS (intergenic) 1963 Tested peak: chr Reproducibility: 12/12 Flanking genes: NUP214-FAM78A (intergenic) 1967 Tested peak: chr Reproducibility: 8/9 Flanking genes: TTC7A (intronic) 1971 Tested peak: chr Reproducibility: 10/10 Flanking genes: RBM38-HMGB1L1 (intergenic) 1974 Tested peak: chr Reproducibility: 4/5 Flanking genes: RPS24-LOC (intergenic) 1466 Tested peak: chr Reproducibility: 4/8 Flanking genes: C14orf166B-C14orf4 (intergenic)

30 May et al: ChIP-seq Discovery of Human Heart Enhancers - 30 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 ( 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: HAND2 negative chr10: ANKRD1 other (hindbrain, midbrain, nose) 497 chr22: TBX1 negative chr12: TBX5 negative chr4: PITX2 negative chr18: GATA6 heart chr5: MEF2C heart chr2: DES negative chr5: NKX2-5 negative chr4: PITX2 negative chr8: GATA4 heart chr5: ISL1 negative chr10: FGF8 other (tail) chr10: FGF8 other (dorsal root ganglion, cranial nerve) 514 chr5: HAND1 negative chr22: TBX1 other (limb) chr5: FGF10 other (limb) 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

31 May et al: ChIP-seq Discovery of Human Heart Enhancers - 31 Human fetal heart p300/cbp-chip-seq predictions e11.5 mouse heart p300 ChIP-seq predictions Total tested elements Elements with reproducible cardiac patterns 43 (66%) 81 (60%) Average positive embryos per element Reproducible LacZ staining in the heart Average reproducibility 7.2/9.3 (77%) 5.8/7.7 (75%) Average peak size (STD) (2326.6) (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

32 May et al: ChIP-seq Discovery of Human Heart Enhancers - 32 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 human mouse lift-over Flanking genes Whole-mount heart 5/11 Limb 7, neural tube 9, nose % of bases, 100.0% of span intronic Atoh8 heart 6/8 Other 6 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 human mouse lift-over Flanking genes Whole mount Heart 7/10 other % of bases, 99.4% of span Tbx20 / Herpud2 heart 9/9 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, ,706,219 Reproducibility - heart - other human mouse lift-over Flanking genes Whole-mount Heart 6/7 ear % of bases, 99.9% of span Intronic Auts2 -- Ear 5/8 P300/CBP coverage (called yes/no) 5 (no)

33 May et al: ChIP-seq Discovery of Human Heart Enhancers - 33 mcs/cs273 Mouse ortholog (mm9) Human ortholog (hg18) ID mm132 hs1661 Tested region chr13:46,666,689-46,669,718 chr5:153,556, ,558,157 Reproducibility - heart - other human mouse lift-over Flanking genes Whole mount heart 7/7 branchial arch 5, eye 6, midbrain 6, somite % of bases, 100.0% of span Intronic CAP 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 human mouse lift-over Flanking genes Whole mount Heart 7/10, blood vessels 4 other % of bases, 100.0% of span Intronic GPR124 Heart 5/7, blood vessels 7 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 human mouse lift-over heart 8/8 heart 5/6 87.3% of bases, 100.0% of span Flanking genes Gata6 Rbbp8 CTAGE1 - Rbbp8 Whole mount P300/CBP coverage (called yes/no) 87 (yes)

May et al: ChIP-seq Discovery of Human Heart Enhancers - 34 mcs/cs284 Mouse ortholog (mm9) Human ortholog (hg18) ID mm143 hs1672 Tested region chr3:104,400,858-104,404,522

34 May et al: ChIP-seq Discovery of Human Heart Enhancers - 34 mcs/cs284 Mouse ortholog (mm9) Human ortholog (hg18) ID mm143 hs1672 Tested region chr3:104,400, ,404,522 chr1:113,341, ,343,543 Reproducibility - heart - other human mouse lift-over Flanking genes Whole mount heart 3/3 heart 7/7 87.0% of bases, 99.9% of span Slc16A1 LRIG2 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 human mouse 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

ChIP-seq Identification of Weakly Conserved Heart Enhancers

ChIP-seq Identification of Weakly Conserved Heart Enhancers Matthew J. Blow, David J. McCulley, Zirong Li, Tao Zhang, Jennifer A. Akiyama, Amy Holt, Ingrid Plajzer-Frick, Malak Shoukry, Crystal Wright,