Fig. 5: Chromatin changes at enhancers and TSSs adjacent to new and old CTCF loop anchors.

a Formation of CTCF loops by recruiting CTCF to new anchor sites correlates with an increase in ATAC-TF signal at adjacent enhancers and transcription start sites (TSSs). These enhancers also show increased H3K4me1, H3K27ac, and RNAPIIS2ph. Promoters show similar changes over broader regions. b Dissolution of CTCF loops by discarding previously used anchor sites. When this happens at a specific stage, enhancers and TSSs adjacent to discarded CTCF anchor sites lose ATAC-TF signal, H3K4me1, H3K27ac, and RNAPIIS2ph, suggesting that loss of CTCF anchors correlates with inactivation of adjacent enhancers and promoters. c Changes in transcription measured by RNA-seq at genes located inside stage-specific CTCF loops. d Metaplot analysis of interactions between CTCF loop anchors and between enhancers and promoters located within the loop anchors. The top diagram indicates the specific arrangement of CTCF loop anchors and enhancer-promoter interactions analyzed in this specific case. Changes in interactions between enhancer-promoter pairs in two differentiation stages are analyzed in the context of changes in CTCF loops in which the enhancer-promoter pairs are contained. Below the diagram, the top left panel shows a subtraction heatmap of Hi-C interactions in PP and H9 hESCs for PP-specific CTCF loops. Formation of these loops in PP cells (black arrowheads indicating increased interactions at the CTCF loop anchors) correlates with increased enhancer-promoter interactions inside the loops (green arrowheads). The right panel shows a similar analysis comparing CTCF loops present in SC-β organoids but absent in H9 hESCs. The bottom panels show a parallel analysis using RNAPIIS2ph HiChIP instead of Hi-C. Interactions at CTCF loop anchors cannot be detected (absence of black arrowheads) because CTCF anchors lack RNAPIIS2ph; however, enhancer-promoter interactions can be observed as increased signal in RNAPIIS2ph HiChIP data (green arrowheads) e Subtraction heatmaps of H3K27ac HiChIP between consecutive differentiation stages around new CTCF loop anchors formed by extension of one old anchor. f Subtraction heatmaps of H3K27ac HiChIP between consecutive differentiation stages around new CTCF loop anchors formed by extension of two old anchors. Abbreviations: human embryonic stem cells (hESCs), definitive endoderm (DE), primitive gut tube-like (PGT), pancreatic progenitors (PP), and stem cell-derived β-cell organoids (SC-β organoids).