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. 2022 Jun 15;41(13):e110600. doi: 10.15252/embj.2022110600

Figure EV4. Open site chromatin state dynamics and differential CTCF binding throughout germ cell differentiation.

Figure EV4

  1. Overlap enrichment analysis of consolidated open site clusters against annotations from the Ensembl Regulatory build. P‐values were computed using Fisher’s exact tests. P‐value symbol brackets: **** = [0, 0.0001); ** = [0.001, 0.01); * = [0.01, 0.05); ns = [0.05, 1].
  2. Select ChIP‐seq coverage tracks around a representative cluster 2 loci.
  3. Western blot against CTCF in the chromatin‐bound fraction (top row) and whole‐cell lysate (middle row) as well as α‐tubulin (bottom row) in each cell type. The signals of CTCF from whole‐cell lysates were normalized by α‐Tubulin, while those of the chromatin‐bound fraction was normalized by the mean across all cell types (top panel).
  4. 2D UMAP embedding based on epigenetic signals in promoters for each cell type, with labels derived from semi‐supervised HDBSCAN.
  5. Enrichment of epigenetic signals in each promoter cluster and expression of the cognate gene.
  6. Association between promoter clusters and cell types. Number of open sites per cell type in each cluster (top axis: bars) and their enrichment as odds ratios (bottom axis: dots). Error bars indicate 95% confidence intervals.
  7. Pile‐up plots of intra‐class promoter‐promoter interactions.
  8. Contributors of differential CTCF binding. The aggregate plot of various ChIP‐seq enrichment signals (left) as well as the insulation score (right) near CTCF‐binding sites found both in cell types (“constitutive”) or only GSCs but not in d4c7 mPGCLCs (“GSC‐high”) appear largely identical in their chromatin state yet distinct from those lost in GSCs. n = 35,692/13,364 for constitutive/GSC‐high peaks.
  9. 3D epigenetic landscape rewiring near Ddx4. Observed/expected contact maps at 10 kb resolution for mESCs, EpiLCs, and d2 mPGCLCs are shown alongside select ChIP‐seq coverage tracks. A strongly insulating CTCF peak (highlighted in red) upstream of Ddx4’s TSS is found in all earlier stages and prevents spurious activation.
  10. Coordinated differential expression and E‐P looping between d4c7 mPGCLCs and GSCs. A strong correlation was observed when applying stratified rank‐rank hypergeometric overlap to genes ranked by differential expression versus differential E‐P interactions straddling sites depleted of CTCF binding in GSCs. While increased E‐P looping is correlated with elevated expression regardless of whether the interaction spans differential CTCF‐bound sites, the degree of coordination is stronger (i.e., more significant/brighter) for those that do straddle GSC‐depleted sites.