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. 2020 Aug 19;117(35):21450–21458. doi: 10.1073/pnas.2008672117

Fig. 4.

Fig. 4.

Gata1 HiChIP confirms increased E-P interactions in EryD. (A) Interaction maps of Gata1 HiChIP in EryP and EryD at 500-kb and 5-kb resolution, normalized by square root of vanilla coverage (VC SQRT). Zoom-in views of highlighted regions are in B. (B) Gata1 HiChIP 3D signal enrichment at the Mgll and Abtb1 loci in EryP and EryD cells. (C) Average profiles of E-P interactions of EryP-/EryD-specific genes. Promoters of EryP-specific or EryD-specific genes were set as anchors, and enhancers were defined by Gata1 peaks ±100 kb away from TSS. The average of reads within E-P interactions was plotted. y axis is the average of normalized reads for E-P interactions per gene. Each enhancer bin in the x axis indicates the rank position of the enhancer based on its distance to promoter. Genomic baseline (gray bars) are E-P interactions of a set of randomly selected genes of matched size in both EryP and EryD cells. (D) Quantification of E-P loops in EryP and EryD cells at 25-kb resolution. P value represents permutation test in 1,000 random genes selection of matched size. (E–G) Validation of EryP-shared and EryD-shared enhancers (Enhancer 1, E1). v4C interaction profiles at the E1 in EryP and EryD cells were aligned in E. Quantitative transcript analysis upon E1 deletion in MEL cells and mESCs-derived CD71+/Ter119+ cells were presented in F and G, respectively. (H–J) Validation of EryD-specific enhancer (Enhancer 2, E2). v4C interaction profiles at the E2 in EryP and EryD cells were aligned in H. Quantitative transcript analysis upon E2 deletion in MEL cells and mESCs-derived CD71+/Ter119+ cells were presented in I and J, respectively. Virtual 4C interaction profiles were generated at 1-kb resolution (E and H). E1 and E2 are highlighted in yellow, and promoters of flanking genes are highlighted in gray (E and H). ChIP-seq and RNA-seq track of EryP (blue highlighted) and those of EryD cells (red highlighted) were aligned to the selected genomic region (B, E, and H). Experiments were replicated at least twice in F, G, I, and J. Error bars indicate the SEM; n = 3 (F, G, I, and J). *P < 0.05, **P < 0.01, ***P < 0.001, unpaired one-tailed Student’s t test (F, G, I, and J) and permutation test (D).