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. 2021 Dec 2;49(21):12167-12177.
doi: 10.1093/nar/gkab1069.

Relaxed 3D genome conformation facilitates the pluripotent to totipotent-like state transition in embryonic stem cells

Yezhang Zhu  1 , Jiali Yu  1 , Jiahui Gu  1 , Chaoran Xue  1 , Long Zhang  1 , Jiekai Chen  2 , Li Shen  1   3   4
Affiliations

Relaxed 3D genome conformation facilitates the pluripotent to totipotent-like state transition in embryonic stem cells

Yezhang Zhu et al. Nucleic Acids Res. .

Abstract

The 3D genome organization is crucial for gene regulation. Although recent studies have revealed a uniquely relaxed genome conformation in totipotent early blastomeres of both fertilized and cloned embryos, how weakened higher-order chromatin structure is functionally linked to totipotency acquisition remains elusive. Using low-input Hi-C, ATAC-seq and ChIP-seq, we systematically examined the dynamics of 3D genome and epigenome during pluripotent to totipotent-like state transition in mouse embryonic stem cells (ESCs). The spontaneously converted 2-cell-embryo-like cells (2CLCs) exhibited more relaxed chromatin architecture compared to ESCs, including global weakening of both enhancer-promoter interactions and TAD insulation. While the former correlated with inactivation of ESC enhancers and down-regulation of pluripotent genes, the latter might facilitate contacts between the putative new enhancers arising in 2CLCs and neighboring 2C genes. Importantly, disruption of chromatin organization by depleting CTCF or the cohesin complex promoted the ESC to 2CLC transition. Our results thus establish a critical role of 3D genome organization in totipotency acquisition.

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Figures

Figure 1.
Figure 1.
Global weakening of the 3D genome conformation during ESC to 2CLC transition. (A) Hi-C contact maps at 250-kb resolution across the entire chromosome 2. (B) PC1 values at 500-kb resolution across the entire chromosome 2. (C) Box plot showing compartment strength in ESCs and 2CLCs. (D) Scatter plot comparing insulation scores at ESC TAD boundaries (23) between ESCs and 2CLCs. (E) Box plot showing the insulation score at TAD boundaries. Note that higher score denotes higher insulation potential. (F) Genome browser shot showing Hi-C contacts (top) and directionality index (DI) (bottom) at 20-kb resolution. Arrows indicate TAD boundaries. O/E, observed/expected. (G) Average DI in a 0.6 Mb region centered on TAD boundaries. (H) Contact probability as a function of the genomic distance in logarithmic bins. Lines represent means of biological replicates; edges of semi-transparent ribbons represent individual data points of the two biological replicates. (I) Aggregate Hi-C contact maps around TAD boundaries. (J) Box plot comparing TAD strengths in ESCs and 2CLCs. (K) Box plot showing inter-TAD interaction frequencies in ESCs and 2CLCs.
Figure 2.
Figure 2.
Loss of enhancer-promoter interactions and down-regulation of pluripotent genes in 2CLCs. (A) Aggregate Hi-C contact maps between pairs of loop anchors (29). (B) Box plot showing loop strengths in ESCs and 2CLCs. (C) Aggregate Hi-C contact maps between ESC enhancer-promoter pairs (32). (D) Box plot showing enhancer-promoter interaction strengths in ESCs and 2CLCs. (E) Down-regulated genes in 2CLCs tend to locate closer to ESC enhancers. Shown are cumulative frequency curves comparing the distances between transcription start sites (TSS) of non-/up-/down-regulated genes and their nearest ESC enhancers. P value was calculated using two-sided Kolmogorov–Smirnov test. (F) Gene set enrichment analysis indicating that down-regulated genes in 2CLCs were enriched for ESC super-enhancer neighboring genes. Red, up-regulated genes in 2CLCs; blue, down-regulated genes in 2CLCs. (G) Box plot comparing the expression of super-enhancer neighboring genes in ESCs and 2CLCs. (H) Scatter plot comparing expression of pluripotent genes in ESCs and 2CLCs. (I) Relative expression levels of pluripotent genes in 2CLCs versus ESCs by RT-qPCR. Data are normalized to Actin and are presented as mean ± SD, **P < 0.01, ***P < 0.001 (multiple t tests).
Figure 3.
Figure 3.
Inactivation of ESC enhancers and formation of new enhancers in 2CLCs. (A–C) Average chromatin accessibility, H3K27Ac, H3K4me1, H3K4me3, H3K27me3 and H3K9me3 signals around MERVL (A), ESC enhancers/super-enhancers (B), and promoters of the indicated genes (C) in ESCs and 2CLCs. SE, super-enhancer. (D) Average chromatin accessibility, H3K4me1 and H3K4me3 signals around putative 2C enhancers. (E) Up-regulated genes in 2CLCs tend to locate closer to putative 2C enhancers. Shown are cumulative frequency curves comparing the distances between TSS of non-/up-/down-regulated genes and their nearest putative 2C enhancers. P value was generated using two-sided Kolmogorov–Smirnov test. (F) Average DUX ChIP-seq signals around putative 2C enhancers. Equal number of random regions were used as the control. (G) Bar plot showing that up-regulated genes tend to have ESC TAD boundaries separating them from neighboring putative 2C enhancers. Values represent enrichment of boundary presence between the promoters of indicated genes and their nearest putative 2C enhancers. Boundary presence between the same promoters and the symmetrical sites of their nearest putative 2C enhancers serves as the control. (H) Genome browser snapshot showing a TAD boundary between the promoter of Nelfa and a putative 2C enhancer (highlighted). Shown are Hi-C contact maps at 20-kb resolution (top) and genome browser tracks of H3K27Ac ChIP-seq, RNA-seq and DUX ChIP-seq signals (bottom). The thicker black line with an arrowhead indicates the weakened TAD boundary, and the two thinner black lines show the region that was zoomed in.
Figure 4.
Figure 4.
Depletion of CTCF or cohesin facilitates ESC to 2CLC transition. (A) The percentage of 2CLCs upon knockdown of Ctcf, Smc1a, Smc3, Rad21, or Yy1. Data are presented as mean ± SD, n = 3. **P < 0.01, ***P < 0.001 (multiple t tests). (B) Relative expression levels of 2C-specific transcripts after knocking down Ctcf, Smc1a, Smc3, Rad21, or Yy1. Data are presented as mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 (multiple t tests). (C) Box plot showing log2 fold change of indicated groups of genes upon acute depletion of the CTCF protein in ESCs by auxin-inducible degron (AID). Analyses were performed using a published RNA-seq dataset (15). (D) Representative FACS analysis of 2C-CTCF-AID cells treated with or without auxin. Note that endogenous CTCF in this cell line is fused with an eGFP tag to indicate CTCF protein levels. (E) Average DI in a 0.6 Mb region centered at TAD boundaries. (F) Aggregate Hi-C contact maps between pairs of loop anchors. (G) Aggregate Hi-C contact maps between ESC enhancer-promoter pairs. (H) The percentages of 2CLCs at indicated time points after auxin treatment or withdrawal. Data are presented as mean ± SD, n = 3. *P < 0.05, ***P < 0.001 (multiple t tests). (I) Relative expression levels of 2C-specific transcripts upon auxin-induced CTCF degradation. Data are presented as mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 (multiple t tests).
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