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Human-gained heart enhancers are associated with species-specific cardiac attributes

An Author Correction to this article was published on 17 November 2022

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Abstract

Heart development is controlled by a relatively conserved network of transcriptional and chromatin regulators; how the human heart has evolved species-specific features to maintain adequate cardiac output and function remains to be defined. In this study, we performed a comparative epigenomic analysis of mouse and human cardiomyocytes at the earliest stages of cardiogenesis and identified enhancers and promoters that are specifically active in human cardiogenesis. These cis-regulatory elements (CREs) are associated with genes involved in heart development and function and are enriched in genetic variants associated with human cardiac phenotypic and disease traits, particularly those differing between humans and mice. Human-gained CREs are also gained within genomic loci of known transcriptional regulators, potentially expanding their role in human heart development. In particular, we found that a human-gained enhancer in the locus of the early developmental regulator ZIC3 regulates ZIC3 induction at the mesoderm stage as well as cardiomyocyte differentiation. Overall, our results illuminate how human-specific CREs can contribute to human-specific cardiac attributes and can expand the role of conserved transcriptional regulators in human cardiac development.

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Fig. 1: Dynamic epigenomic changes correlate with cardiac developmental gene expression programs.
Fig. 2: Comparative human and mouse epigenomic analysis reveals human-gained CREs that are associated with specific cardiac traits.
Fig. 3: Human-gained enhancers in the ZIC3 locus establish a potential ZIC3 mesoderm-related GRN that directs elevated human ZIC3 expression at the mesoderm stage.
Fig. 4: ZIC3 is required for human CM differentiation.
Fig. 5: A ZIC3 human-gained enhancer regulates mesoderm-specific ZIC3 expression and CM differentiation.

Data availability

Sequencing data are available from the Gene Expression Omnibus database (snATAC: GSE192500; bulk sequencing data: GSE192365). A list of all the used sequencing datasets (new and public) and their accession numbers is available in Supplementary Table 13. Bulk sequencing data can be viewed using the UCSC Genome Browser (https://genome.ucsc.edu/s/Cbenner/Chi-211218-ReviewerTracks). All other data supporting the findings in this study are included in the main article and associated files.

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Acknowledgements

We thank M. Swim for help with analysis of flow cytometry data and E. Ines for technical assistance. We thank K. Jepsen and the UCSD Institute for Genomic Medicine for help with sequencing and the UCSD Human Embryonic Stem Cell Core Facility for help with cell sorting. This work was supported, in part, by grants from the National Institutes of Health (1UM1HL128773) to B.R., S.M.E. and N.C.C.; and the American Heart Association (18CDA34080195) to J.B.

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Authors

Contributions

E.D., B.R. and N.C.C. conceived the project and the overall design of the experimental strategy. C.B. designed and performed bioinformatics analyses. E.D., F.Z., S.P., E.F., X.H., A.Y.L. and J.G. conducted experiments. Y.Z., O.B.P. and R.H. helped with bioinformatics analysis. B.R., S.M.E., J.B. and H.S.K. provided critical intellectual input and data interpretation. E.D. and N.C.C. prepared the manuscript, with input from all authors.

Corresponding authors

Correspondence to Chris Benner or Neil C. Chi.

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The authors declare no competing interests.

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Nature Cardiovascular Research thanks Mirana Ramialison and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Integrated supplementary information

Extended Data Fig. 1 Gene regulatory networks dynamically remodel during human cardiomyocyte differentiation.

a, Flow cytometry analysis shows the percentage of cell types generated at each cardiac developmental stage as assessed by the indicated markers. b, Heatmap shows cell-type cardiac enhancers through comparison with ENCODE-derived cell line enhancer data. c, CREs are enriched in intergenic and intronic regions during cardiomyocyte differentiation. d, Number of CREs for different epigenomic assays are shown across the six stages of human vCM differentiation. e, Bar plot shows the number of CREs that are gained or lost compared to the preceding stage during human ventricular cardiomyocyte differentiation. f, Analyzing the ratio of active enhancers (H3K27ac+/H3K4me1+) to all enhancers (H3K4me1+) reveals increased enhancer utilization during human vCM differentiation. g, Heatmap displays enhancer activity as assessed by H3K27ac signal during in vitro vCM differentiation and in purified human fetal CMs. PSC, human pluripotent stem cells; Mes, mesoderm; CMes, cardiac mesoderm; CP, cardiac progenitor; CM, cardiomyocyte; vCM, ventricular cardiomyocyte; fvCM, fetal ventricular cardiomyocyte; CRE, cis-regulatory region; pos., positive; snRNA, small nuclear RNA; snoRNA, small nucleolar RNA; UTR, untranslated region; pseudo, pseudogene; TTS, transcription termination site; ncRNA; non-coding RNA; miRNA, microRNA; HUVEC, human umbilical vein endothelial cells; NHEK, normal human epidermal keratinocytes; fvCM, fetal ventricular cardiomyocyte.

Extended Data Fig. 2 Integrative transcriptional and chromatin accessibility analysis identifies putative key cardiac regulators.

a, Dot plot shows expression (circle color) and transcription factor motif enrichment (circle size) of known and potential novel regulators of human ventricle cardiomyocyte differentiation. b, Heatmap shows differentially regulated super-enhancers during cardiomyocyte differentiation. c, Distribution of H3K27ac signal for each of the indicated stages shows super-enhancer loci with associated genes of interest based on proximity. PSC, pluripotent stem cells; Mes, mesoderm; Cmes, cardiac mesoderm; CP, cardiac progenitor; CM, cardiomyocyte; vCM, ventricular cardiomyocyte; FPKM, fragments per kilobase of transcript per million mapped reads.

Extended Data Fig. 3 Human gained enhancers consist of traditional and super-enhancers during cardiomyocyte differentiation.

a, HGEs are enriched in intergenic and intronic regions during cardiomyocyte differentiation. b, HGEs consist of both traditional (TE) and super-enhancers (SE). c, Violin plots reveal comparable activity of HGEs compared to all other mouse stages combined. Dashed line in violin plots indicates the mean and dotted lines indicate quartiles. d, Shannon entropy calculations show stage-specific expression of genes near gained or stable enhancers. Lower entropy indicates more stage-specific gene expression. Horizontal line in boxplots represents the median, the box indicates the interquartile range and the dots represent outliers. Data was compared using Wilcoxon rank sum test (****p value < 0.0001). PSC-Stable, n = 7339; PSC-Gained, n = 1240; Mes-Stable, n = 6436; Mes-Gained, n = 1924; CP-Stable, n = 7525; CP-Stable, n = 1531; CM-Gained, n = 7531; CM-Gained, n = 1653. e, Stable and gained super-enhancer domains are primarily associated with transcription factors based on GO-term analysis. f, Representative super-enhancer TADs with HGEs (blue boxes) are shown at various cardiomyocyte developmental stages. HGE, human-gained enhancer; PSC, pluripotent stem cells; Mes, mesoderm; CP, cardiac progenitor; CM, cardiomyocyte; UTR, untranslated region; pseudo, pseudogene; TTS, transcription termination site; enh., enhancers; TAD, topologically associated domain; hs, human; ms, mouse.

Extended Data Fig. 4 HGEs are associated with cardiac developmental specific activity and function.

a, Bar graph shows the percentage and number of VISTA validated enhancers driving cardiac LacZ reporter activity in vivo that overlap with stable and gained enhancers at different cardiomyocyte developmental stages. b, GREAT analysis reveals biological processes associated with HGEs at the examined cardiac developmental stages. c-f, ATAC-seq/ChIP-seq profiles show that SNPs associated with (c) atrial fibrillation and (e) QRS duration overlap with HGEs, and luciferase reporter assays (d, f) confirm that the SNPs affect enhancer activity. Sequence comparison surrounding the SNP is shown on the bottom left of (c) and (e) with nucleotides that are not conserved between mouse and human indicated in red. Gene expression in human (grey) and mouse (black) cardiomyocytes is shown on the bottom right of (c) and (e). n = 9 for the luciferase reporter assays. Data was compared using a two-tailed Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Data represented as the mean ± SEM with individual data points. g, Bar graph shows the percentage and absolute number of noncoding mutations found in CHD patients that overlap with stable and gained enhancers at different cardiomyocyte developmental stages. HGE, human-gained enhancer; PSC, pluripotent stem cells; Mes, mesoderm; CMes, cardiac mesoderm; CP, cardiac progenitor; CM, cardiomyocyte; vCM, ventricular cardiomyocyte; snRNA, small nuclear RNA; mitoch., mitochondrial; transl., translation; carb., carbohydrate; catab., catabolism; cyto., cytoplasmic; FDR, false discovery rate; Hs, human; Ms, mouse; expr, expression; FKPM, fragments per kilobase per million reads; EV, empty vector; WT, wild-type; SNP; single nucleotide polymorphism; CHD, congenital heart disease.

Extended Data Fig. 5 Gene expression and transcription factor motif analyses reveal distinct differences between human and mouse mesoderm.

a, Clustered heatmap of human and mouse mesoderm gene expression profiles shows both conserved and divergent gene expression clusters. b, Table shows top transcription factor motifs for human and mouse mesoderm active enhancers. Mes, mesoderm; val., value. Motif enrichment p-values are derived from a one-sided binomial test.

Extended Data Fig. 6 CREs within mesoderm cell subpopulations are associated with distinct transcription factor binding activity.

a, Clustered heatmap shows differentially (FDR < 0.05) accessible CREs between identified snATAC mesoderm cell subpopulations. b, Heatmap of transcription factor binding motif analysis of cluster-specific CREs from human mesodermal snATAC-seq data reveals that ZIC3 along with other cardiac developmental transcription factor motifs are enriched in the pre-cardiac mesoderm subpopulation. c, ZIC3 motif enrichment is projected onto the UMAP of human PSC-derived mesoderm snATAC-seq data. d, (Left) Mouse scRNA-seq data from E7.25-E8.5 Mesp1-Cre; Rosa26-tdTomato embryos are displayed by tSNE (t-distributed stochastic neighbor embedding) plots for all developmental stages combined. (Right) Zic3 expression is projected onto the tSNE plot. PSC, pluripotent stem cell; N, neural; Epi, epiblast; ExE, extra-embryonic; APS/ME, anterior primitive streak/mesendoderm; LPM, lateral-plate mesoderm; PCM, pre-cardiac mesoderm; PM; paraxial mesoderm, PSM, pre-somitic mesoderm; CRE, cis-regulatory region; adj., adjusted.

Extended Data Fig. 7 Generation of clonal ZIC3 HGE deletion lines.

Human PSC ZIC3 HGE1-3 individual knockout (KO) and triple KO (TKO) lines were generated through CRISPR/Cas9-mediated deletion of ZIC3 HGE1-3 enhancer regions (9.2 kb, 1.8 kb, and 1.5 kb, respectively). PCR genotyping using external (ext., that is, outside deleted regions) and internal (int., that is, inside deleted regions) primers confirmed the individual human PSC ZIC3 HGE KOs (left) and the human PSC ZIC3 HGE TKO (right). HGE TKO was confirmed by individual HGE1-3 genotyping. Wild-type (WT) cells were used as control. Note that the WT HGE1 band is 9.2 kb and thus difficult to PCR amplify. M, marker, ext., external; int., internal.

Extended Data Fig. 8 HGE3 KO and HGE TKO human pluripotent stem cell knockout lines display reduced cardiomyocyte differentiation and mesoderm-specific ZIC3 expression defects.

All individual PCR data for WT and ZIC3 HGE knockout (KO) and HGE triple knockout (TKO) lines are shown with statistical significance indicated (ZIC3, n= 9 for all lines; HOPX, n = 9 for WT/HGE1-3 KO lines, n = 12 for WT/HGE TKO lines; APLNR, n = 6 for WT/HGE1 and HGE2 KO lines, n = 9 for WT/HGE3 KO lines, n = 12 for WT/HGE TKO lines; NODAL, n = 6 for WT/HGE1 and HGE2 KO lines, n = 12 for WT/HGE3 KO and HGE TKO lines; LEFTY2, n = 6 for WT/HGE1 and HGE2 KO lines, n = 9 for WT/HGE3 KO lines, n = 12 for WT/HGE TKO lines. TBP gene expression was used for gene expression normalization. b, qPCR analysis of ZIC3 HGE3 KO and HGE TKO human PSC lines reveals no significant changes in ZIC3 expression at the PSC stage compared to WT cells (n = 9). c, qPCR analysis reveals significant changes in the expression of ZIC3 but not neighboring genes, RBMX and FGF13, in HGE TKO human PSC lines when compared to WT human PSC lines at the mesoderm stage (n = 6). d, Representative examples of flow cytometry analyses for TNNT2 staining (x-axis) show that ZIC3 HGE3 and TKO lines (but not HGE1 nor HGE2) display reduced cardiac differentiation. Data is represented as the mean ± SEM with individual data points. Data was analyzed using a two-tailed Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. HGE, human-gained enhancer; expr., expression; PSC, pluripotent stem cells; Mes, mesoderm.

Supplementary information

Supplementary Fig. 1

Reporting Summary

Supplementary Table 1 HGEs for each cardiac stage

Supplementary Table 2 Genes in the vicinity of HGEs

Supplementary Table 3 MGEs for each cardiac stage

Supplementary Table 4 Validated VISTA enhancers overlapping HGEs

Supplementary Table 5 Genes differentially expressed between human and mouse mesoderm

Supplementary Table 6 snATAC-seq statistics

Supplementary Table 7 snATAC-seq-associated genes with cluster-specific gene scores

Supplementary Table 8 Annotated snATAC-seq peaks for mesoderm subclusters

Supplementary Table 9 Cluster-specific snATAC-seq peaks

Supplementary Table 10 TF binding motifs enriched in snATAC-seq peaks

Supplementary Table 11 ZIC3-regulated mesoderm genes

Supplementary Table 12 Sequences of oligonucleotides used in the study

Supplementary Table 13 Raw sequencing datasets used in the study

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Destici, E., Zhu, F., Tran, S. et al. Human-gained heart enhancers are associated with species-specific cardiac attributes. Nat Cardiovasc Res 1, 830–843 (2022). https://doi.org/10.1038/s44161-022-00124-7

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