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. 2024 May 13;25(1):122.
doi: 10.1186/s13059-024-03268-w.

Single-cell 3D genome structure reveals distinct human pluripotent states

Niannian Li #  1   2 , Kairang Jin #  1   3 , Bin Liu #  1   2   4 , Mingzhu Yang #  5   6 , PanPan Shi  1   3 , Dai Heng  1   3 , Jichang Wang  7   8 , Lin Liu  9   10
Affiliations

Single-cell 3D genome structure reveals distinct human pluripotent states

Niannian Li et al. Genome Biol. .

Abstract

Background: Pluripotent states of embryonic stem cells (ESCs) with distinct transcriptional profiles affect ESC differentiative capacity and therapeutic potential. Although single-cell RNA sequencing has revealed additional subpopulations and specific features of naive and primed human pluripotent stem cells (hPSCs), the underlying mechanisms that regulate their specific transcription and that control their pluripotent states remain elusive.

Results: By single-cell analysis of high-resolution, three-dimensional (3D) genomic structure, we herein demonstrate that remodeling of genomic structure is highly associated with the pluripotent states of human ESCs (hESCs). The naive pluripotent state is featured with specialized 3D genomic structures and clear chromatin compartmentalization that is distinct from the primed state. The naive pluripotent state is achieved by remodeling the active euchromatin compartment and reducing chromatin interactions at the nuclear center. This unique genomic organization is linked to enhanced chromatin accessibility on enhancers and elevated expression levels of naive pluripotent genes localized to this region. In contradistinction, the primed state exhibits intermingled genomic organization. Moreover, active euchromatin and primed pluripotent genes are distributed at the nuclear periphery, while repressive heterochromatin is densely concentrated at the nuclear center, reducing chromatin accessibility and the transcription of naive genes.

Conclusions: Our data provide insights into the chromatin structure of ESCs in their naive and primed states, and we identify specific patterns of modifications in transcription and chromatin structure that might explain the genes that are differentially expressed between naive and primed hESCs. Thus, the inversion or relocation of heterochromatin to euchromatin via compartmentalization is related to the regulation of chromatin accessibility, thereby defining pluripotent states and cellular identity.

Keywords: Chromatin accessibility; Genome structure; Human embryonic stem cells; Naive; Pluripotency; Primed.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Distinct transcriptional expression profiles of extended naive and primed states of hESCs. a Morphology of extended naive and primed hESCs originating from H9 cell lines (scale bar, 100 μm). b RNA-seq analysis of differential gene expression between naive and primed hESCs. Red color indicates upregulated genes in naive hESCs; green designates upregulated genes in primed hESCs (representative genes are shown); gray denotes unchanged. c Heatmap showing the relative expression of representative marker genes for naive and primed hESCs. d GSEA of the target genes of pluripotency-related transcription factors in naive and primed hESCs. e KEGG-enrichment analysis of naive and primed hESCs based on our RNA-seq data
Fig. 2
Fig. 2
Genomic structure and chromosomal organization of naive and primed hESCs as revealed by single-cell Dip-C. a Representative single-cell chromatin contact maps after haplotype imputation showing a scattered distribution of chromatin indicative of more chromatin interactions in the primed state but greater localized chromatin distribution suggestive of fewer chromatin interactions in the naive state (five individual cells are shown). b 3D genomic structure of primed and naive states of hESCs that reveal greater intermingling of chromosomes in the primed state but with a more-localized regional distribution of chromosomes in the naive state. Each particle represents 20 kb of chromatin or a radius of ~ 100 nm (five individual cells are shown: the various colored bars indicate chromosome nos. 1, 2, 3.., X/Y; and higher magnification and resolution can be found in Fig. 2f. c Quantification of chromosomal intermingling (vertical axis: the average fraction of nearby particles that were not from the same chromosome) and chromatin compartmentalization (horizontal axis: Spearman’s correlation between each particle’s own CpG frequency and the average of nearby particles). d The same two clusters can also be distinguished by unsupervised clustering via PCA of single-cell chromatin compartments, without the need for bulk data. The two alleles of each locus were treated as two different loci. e Example cross-sections of two cell types, colored by chromosome. f The contacts in chromosome 9 of two groups of five cells. g The position of genetic information on chromosome 9 in the nucleus from both the paternal and maternal parents. h Example cross-sections of two cell types, colored by the multi-chromosome intermingling index. i Structural differences in homologous chromosomes from the parents between naive and primed hESCs (chromosomes 4, 5, 10, and 11 are shown; and other chromosomes can be found in Fig. S3A)
Fig. 3
Fig. 3
3D structures and features of the maternal and paternal X chromosomes in naive and primed hESCs. a ATAC-seq signal tracks for representative genomic regions in the X chromosome; y-axes = reads per million (rpm). b 3D structures of four single cells in naive and primed states showing compartmentalization of euchromatin (green) and heterochromatin (purple), as visualized using CpG frequency as a proxy. c Representative H3K9me3, H3K27me3, and CTCF landscapes across chrX:74,640,777–80,629,783 of the genome, as generated by CUT&Tag in naive and primed hESCs. d Spatial position and structure of the parental X chromosome in the simulated 3D structures of all chromosomes (four individual cells are shown). e Representative images of Xist RNA-FISH in primed and naive hESCs (nuclei were stained with DAPI [blue]; scale bar, 5 μm). f ATAC-seq signal tracks for the cell as a whole; y-axes = reads per million (rpm). g Representative H3K9me3, H3K27me3, and CTCF landscapes of the genome, as generated by CUT&Tag in naive and primed hESCs
Fig. 4
Fig. 4
Distinct interchromosomal contacts in naive and primed hESCs. a Serial cross-sections of a single cell showing compartmentalization of euchromatin (green) and heterochromatin (magenta), as visualized using CpG frequency as a proxy. Note: all five representative cells yielded a consistent pattern of chromatin compartmentalization in the primed and naive states, with each particle representing 20 kb of chromatin (~ 60 nm in radius). Adjacent serial sections are separated by 7.5 particle radii (~ 450 nm). b Radial distribution of CpGs is defined as the mean CpG frequency in each concentric spherical shell, averaged over all single cells of the same cell type. Radial distances (to the nuclear center of mass) in each cell were normalized by their mean. c Genome-wide correlation between radial positioning was defined as the normalized distance to the nuclear center of mass and the CpG frequency of consecutive 1-Mb bins. d Radial positioning along the genome. Radial preferences across the genome, as measured by average distances to the nuclear center of mass. e Lieberman–Aiden’s method was applied to distinguish A/B compartments. f Genome-wide correlation between radial positioning was defined as the normalized distance to the nuclear center of mass and the CpG frequency of consecutive 1-Mb bins in bulk-cell Hi-C data of naive and primed hESCs. g 2D heatmap of the chromosomal contacts and distributions of naive and primed hESCs. Primed/naive designates that primed reflects specific contacts compared to naive. h, i Interchromosomal contact map and percentage of interchromosomal contacts of the naive state compared with the primed state. Chromosomal intermingling was quantified as the percentage of interchromosomal contacts that were increased in primed hESCs
Fig. 5
Fig. 5
Localization of naive and primed genes vis-a-vis chromatin compartmentalization and their interaction sites. a The interaction sites of naive-state enriched genes (e.g., NANOG) in the chromatin compartment and their relationships with interchromosomal contacts differed between naive and primed hESCs (example genes were chosen for cell-type-specific chromatin compartment values; not all genes showed such trends). b The interaction sites of primed state-enriched genes (e.g., ZIC2) in the chromatin compartment and their relationships with interchromosomal contacts differed between naive and primed hESCs (example genes were chosen for cell-state-specific chromatin compartment values; not all genes showed such trends). c Distinct distributions of representative 3D genomic structures of naive (toward the nuclear center) and primed genes (peripheralized regions) on the 3D chromatin structure in five single naive cells and five single primed cells. d Quantification of chromosomal intermingling (vertical axis: the average fraction of nearby particles that were not from the same chromosome) and chromatin compartmentalization (horizontal axis: Spearman’s correlation between each particle’s own CpG frequency and the average of nearby particles). e, f Radial distribution of CpGs averaged over all single cells of the same cell type. Radial distances to the nuclear center of mass in each cell were normalized by their mean
Fig. 6
Fig. 6
Transcriptional regulation and chromatin accessibility in naive and primed states. a Radial positioning along the genome of primed or naive genes and enhancers in naive or primed hESCs. b Heatmaps of ATAC-seq signal distribution around the transcriptional start site (TSS) ± 3000 bp of expressed genes and average profiles of the enrichment at the TSS in naive or primed hESCs (two replicates are shown for each state). c 3D structural differences between the two alleles of the representative marker-gene loci in primed and naive cells. d Joint analysis by ATAC-seq, CUT&Tag, and RNA-seq of the relationships between gene expression and chromatin accessibility in naive (NANOG) and primed states (ZIC2). Genome browser tracks of RNA-seq, ATAC-seq, and CUT&Tag-seq data of CTCF, H3K9me3, and H3K27me3 at the NANOG and ZIC2 loci in naive and primed hESCs
Fig. 7
Fig. 7
Distinct histone and chromatin distributions of naive and primed hESCs. a, b Histone distribution pattern of naive and primed hESCs, as detected by immunofluorescence. Nuclear distributions of H3K9ac, H3K27ac, H3K9me3, and H3K27me3 show differences in primed and naive hESCs (nuclei were stained by DAPI [blue]; scale bar, 10 μm). The ratios of the distances from H3K9me3 foci to the nuclear center were calculated by discerning approximately 130 signals from approximately 25 cellular nuclei, with a ratio = 0 indicating the center of a nucleus and a ratio = 1 indicating the edge of a nucleus. Average fluorescence intensity was calculated based on approximately 25 cell nuclei. Data shown are representative of three independent experiments with biological triplicates per experiment (data are presented as means ± standard deviation [SD]; P values were determined by a two-sided Student’s t test. t-test; ****P < 0.0001). c Representative images of Alu (green) and LINE1 (red) repeats, as revealed by DNA FISH in primed and naive cells (DNA was labeled by DAPI [blue]; scar bar, 20 μm). d, e Representative images of DNA FISH for the HERVH element, and B3GAT1 and KLF5 genomic regions (nuclei were stained with DAPI [blue]; scale bar, 5 μm). Experiments were repeated three times, with approximately 20 signals for 8–10 cells counted (t test. ****P < 0.0001; P values were determined by two-sided Student’s t test). f Proposed simplified model to illustrate differential genomic structure and compartmentalization and preferential localization of specific genes for naive and primed states in human ESCs. Naive genes in naive hESCs were principally localized to the active euchromatin compartment at the central nucleus, whereas heterochromatin was organized at the nuclear center of the primed state, suppressing naive genes. The active chromatin compartment at the nuclear center shows relaxed chromatin interactions that effectively facilitate enhancer activity with respect to the transcription of naive genes for naive pluripotency. This model provides insights into specific gene expression in association with chromatin structure and histone modifications
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