Extended Data Fig. 7: Elevated Runx1 levels in Phase 1 resulted in additional Runx occupancies in post-commitment preferred sites and closed chromatin regions.
From: Runx factors launch T cell and innate lymphoid programs via direct and gene network-based mechanisms
a, Gating strategy to sort control and Runx1 OE Phase 1 cells for C&R is illustrated. Briefly, bone-marrow progenitor cells were co-cultured with OP9-Dll1 cells for 2 days, and empty control or Runx1 overexpressing vector was retrovirally introduced. After 40–42 hours (total 4 days of culture on OP9-Dll1 cells), infection+ Phase 1 cells were sorted. In this system, for most cells to reach Phase 2 normally, 8–10 days of culture are needed1,71]. b, Scatter plots and Venn diagrams compare differential Runx1 occupancies at promoter (top) and non-promoter regions (bottom) when Runx1 concentration was increased. Numbers indicate differential Runx1 binding sites (fold enrichment > 2, Poisson enrichment P < 0.001). c, Runx1 C&R signal intensities from indicated cells are shown. Note increased occupancy even at Group 1 and Group 3 sites which were already bound in control Phase 1 cells. d, Bar graph represents compartment state profiles within different groups of Runx binding sites. e, Testing hypothesis that Runx1 OE accesses sites conditionally occupied in other pro-T related contexts. Area-proportional Venn diagrams show analysis strategy to identify Runx binding sites appearing specifically in Bcl11b knockout DN2b/DN3 cells (left), and ILC2-specific Runx binding sites (middle; Runx1, right; Runx3). f, Bar graph shows percentages of Group 4 peaks overlapping with indicated Runx binding site types. g, Density plots illustrate motif frequencies for PU.1, TCF1 (Tcf7), bHLH, and GATA factors in different types of Runx binding sites. h, Runx1, Runx3 (blue), PU1 (purple)23, TCF1 (red), E2A and HEB (green) binding profiles in non-promoter regions under unperturbed Phase 1 or Phase 2 conditions are shown. Runx1 binding patterns in empty vector control and Runx1 OE transduced conditions are displayed in orange tracks (left). Stage-preferential dynamic binding groups are indicated as color bars. Group 1, Phase 1-preferential; Group 2a, Phase 2-preferential and precociously occupied by OE; Group 2b, Phase 2-preferential but not occupied by OE; Group 3, Phase 1 & Phase 2 shared; Group 4op, OE-specific and open sites; Group 4cl, OE-specific and closed sites. The numbers on the right side indicate percent of each group of peaks within the same color bar. TCF1, E2A, and HEB binding sites were measured in independent replicates using C&R from thymic DN3 cells. PU.1 occupancy was previously determined using ChIP-seq23.
