Fig. 1: Identification of a class of SEs that coordinate cell fate may be mediated by adjacent TFs.

a Workflows for multi-omics analysis in ESCs and differentiated cells to identify SE patterns, including integrated epigenomics (H3K27ac), transcriptomics (scRNA sequencing) and three-dimensional genomics (Hi-C) analysis. b Analysis of SE regions based on H3K27ac ChIP-seq data. SEs were defined by signal levels above the inflection point of the curve. All H3K27ac ChIP-seq reads were aligned to the mouse genome assembly mm10 using Bowtie2, and ChIP-seq peaks were called by MACS with default parameters. The parameter “12.5 kb” was used as the maximum distance between the two regions to be sutured, and ROSE was used to distinguish SE from TSSs. H3K27ac ChIP-seq data: ESCs (GSM6911328, generated in this study), EBs (GSM1816114), NPC (GSM1603409), Mesoderm cells (GSM1163099) and MEF (GSM2912468). Publicly available ChIP-seq data have been listed in Supplementary Table 10. c Heatmap showing cell type-specific SE distribution. H3K27ac ChIP-seq signals in a ± 5 kb window for the same SE region in different cell types. d Gene ontology-biological process (GO-BP) analysis of cell-specific PSEAGs. Genes with the closest TSS to the SE (within a 50 kb window) were defined as predicted SE-associated genes (PSEAGs). GO-BP analyses were carried out using the online tool: DAVID^103,104. e Gene regulatory network (GRN) analysis of SE-related TFs in ESCs. The red circle represents the master transcription factor (Seed), while the green four-sided diamonds are transcription factors (TFs) that interact with the Seed. GRN analyses were carried out using the online tool: NetworkAnalyst (https://www.networkanalyst.ca/NetworkAnalyst/home.xhtml)^105,131. f UMAP embedding of single-cell RNA profiles (dots) of ESCs (green dashed line) and EBs (red dashed line). Color intensity reflects gene expression levels. g Hi-C heatmap (GSE96107), H3K27ac (GSM6911328, generated in this study) and CTCF (GSM699165) signals indicating co-localization of ESC SEs with adjacent master TFs (Klf5 and Esrrb loci). The thick red line, located at the upper part of the H3K27ac peak, delineates the SE region, whereas the thick orange line, situated at the lower part of the Hi-C heatmap, delineates the TAD region. Hi-C data are analysed and presented using online tools: 3D Genome Browser (https://3dgenome.fsm.northwestern.edu/)¹³⁰. Publicly available ChIP-seq data have been listed in Supplementary Table 10. h Proposed SE function model: SE may regulate ESC properties by promoting the activity of adjacent TFs within the same TAD. Taking Pou5f1, Sox2, Esrrb and Klf5 loci as examples in the illustration. Source data are provided as a Source Data file.