Fig 2: Endothelial cells lose haemogenic potential upon cell cycle block. a Immunofluorescence staining showing the expression of CDH5 and RUNX1 over time. At EHT D3, media were supplemented either with 0.1% DMSO (CTRL) or with 0.1 μg/ml Nocodazole (Noc). After 48 h, at EHT D5, cells were washed and media refreshed every other day. After cell cycle block is released, cells reprise proliferation, but RUNX1 expression is gradually lost, with positive cells representing very rare events at EHT D12. Haematopoietic clusters are outlined in CTRL and consist of RUNX1+ cells that are mainly round shaped and grouped in clusters. These clusters can also be recognised in the CDH5-stained panel (green), in which they display a typical round morphology, as opposed to the surrounding elongated endothelial cells. Finally, these clusters can also be recognised in the DAPI (grey) panel, in which they are characterised by the typical dense nuclear morphology and brighter appearance compared to endothelial cells. In Nocodazole-treated cultures, all these features are not present: the few RUNX1+ cells are more scattered and not grouped in clusters; they mark elongated endothelial cells rather than round cells; these elongated cells are CDH5 positive (green panel) and thus clearly endothelial; finally, their nuclei do not appear to be denser or brighter than the surrounding cells, as was the case in haematopoietic clusters found in control. Scale bar is 50 μm. b Forty-eight hours after Nocodazole release, at EHT D7, cells were collected and cultured in a CFU assay to assess their differentiation potential. Data are mean ± SEM of 3 independent experiments, with statistical significance compared to control as *P < 0.5, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-tailed unpaired t test. Degrees of freedom = 4; t values for the distinct colonies are BFU-E = 5.803, CFU-E = 4.02, CFU-GM = 6.2, CFU-G = 3.625, CFU-M = 7.407, and CFU-GEMM = 5.547. c Diagram summarising the role of cell cycle progression during EHT

Fig 3: The CRC model of lineage-determining TFs underling corneal epithelial identity.RUNX1/PAX6/SMAD3 complex governs their own SEs and a broader SE network, establishing the active chromatin environment to maintain expression of the corneal epithelial identity genes.

Fig 4: RPS complex positively regulates SE activity.a Hierarchical clustering of the indicated ChIP-seq-binding profiles using affinity (read count) scores. Pearson correlation coefficients between each group are shown. b Motif enrichment within RPS peaks in LSCs. c Co-immunoprecipitation analysis of the interactions among endogenous RUNX1, PAX6, and pSMAD3 in LSCs. d Heatmaps grouped into two clusters using k-means algorithm for the indicated ChIP-seq and ATAC-seq signals at RUNX1-bound sites. e Metaplots of average RPS ChIP-seq signals across TEs and SEs in LSCs. f GSEA of SE-associated gene set in scrambled shRNA-treated versus shRUNX1-treated LSCs and scrambled shRNA-treated versus shSMAD3-treated LSCs. g Venn diagram showing the overlapping and unique peaks bound by RPS. h Genome browser tracks for the indicated ChIP-seq and ATAC signals across the TGFBI, AQP3, and IGF1R loci. i GO BP and KEGG analysis for the genes closest to the overlapping RPS peaks with pvalueCutoff = 0.01 and qvalueCutoff = 0.05.

Fig 5: RUNX1 depletion impairs H3K27ac deposition across lineage-specific enhancers.a Heatmaps showing the indicated ChIP-seq signals across the peaks with H3K27ac reduction in shRUNX1-treated versus scrambled shRNA-treated LSCs. b Scatterplot of H3K27ac enrichment in shRUNX1-treated versus scrambled shRNA-treated LSCs. Sites identified as significantly differentially bound (fold change ≥ 2, FDR < 0.05) are shown in red. c Pie chart showing the percentages of reduced and unchanged H3K27ac signals close to the loci of down-regulated genes upon RUNX1 loss. d Metaplots of average H3K27ac density across the SEs of wild-type LSCs in shRUNX1-treated and scrambled shRNA-treated LSCs. e Pie chart showing the percentages of reduced and unchanged H3K27ac signals across the SEs of wild-type LSCs upon RUNX1 loss. f Genome browser tracks for the indicated ChIP-seq signals across the PAX6 and TGFBI loci in scrambled shRNA-treated and shRUNX1-treated LSCs. g Metaplots of average H3K27ac density across the RUNX1-binding TEs in scrambled shRNA-treated and shRUNX1-treated LSCs.