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. 2021 Jul 16;12:4344. doi: 10.1038/s41467-021-24641-4

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — A H3K27me3 HiChIP and ChIP-seq profiles generated in mESC are shown around the Lhx5 locus. Both the Lhx5 TSS and a PE previously shown to control this gene in AntNPC (PE Lhx5 (−109kb)) are marked with green vertical lines. Upper panel: heatmap of log2 interaction intensities based on H3K27me3 HiChIP data generated in mESC. Two medium panels: ChIP-seq and 1D HiChIP signals for H3K27me3 in mESC. Lower two panels: significant (p < 0.01; FitHiChIP¹⁰²) interactions were called using the H3K27me3 HiChIP PETs (paired-end tags) and loops in which one of the anchors overlaps (±10 kb) either the PE Lhx5 (−109kb) or the Lhx5 TSS are shown. b Significant (p < 0.01; FitHiChIP) interactions were called using the mESC H3K27me3 HiChIP PETs (paired-end tags). Interaction anchors were overlapped with PEs and their interaction partners were hierarchically annotated with the categories shown in the pie chart (“Methods”). c mESC ChIP-seq profiles for H3K27me3, H3K27ac, and H3K4me3 are shown around the TSS (n = 1083) interacting with PEs according to the H3K27me3 HiChIP loops identified in mESC (p < 0.01; FitHiChIP). d Pathway (green) and Gene Ontology (red) analysis for the TSS/genes interacting with PEs according to the H3K27me3 HiChIP loops identified in mESC (p < 0.01; FitHiChIP) was performed with ConsensusPathDB. e The interactions (called using mESC H3K27me3 HiChIP data; p < 0.01; FitHiChIP) established by PcG domains (“Methods”) and PcG-bound promoters as well as those involving PE-general promoter or PE-bivalent promoter pairs were classified as either intra-TAD (yellow) or inter-TAD (green) depending on whether the two anchors of each loop occur within the same or different TADs, respectively. f, g) Significant (mESC H3K27me3 HiChIP data; p < 0.01; FitHiChIP) interactions between distal PEs (>10 kb from TSS) and bivalent promoters (n = 526) were visualized as pileup plots using Hi-C data generated in vitro³⁶ (2i ESC, serum+LIF ESC; f) and in vivo³⁷ (E3.5 Inner Cell Mass (ICM), E6.5 epiblast, E7.5 ectoderm; g). Hi-C pairwise interactions are shown 250 kb up- and downstream of each PE and bivalent promoter pair. Hi-C interaction matrices were KR-balanced (balanced). The numbers in the upper left corners correspond to “loopiness” values (center intensity normalized to the intensity in the corners).