ETV6 binds to a chromosomal locus encompassing Tnf with upregulation of TNF production in Etv6^R355X/+ HSPCs after transplantation. (A) Overrepresentation analysis of ETV6 targets within chromatin loops and Hallmark gene sets. (B) Regulatory activity at the Tnf locus showing topologically associated domain on chromosome 17 identified by integrative analysis incorporating Hi-C loops at this locus in HPC5 cells; CUT&RUN of Etv6^+/+ LSK cells, Etv6^R355X/+ LSK cells, and Etv6^+/+ HPC5 cells; and ATAC-seq data generated from the HPC5 cells. (C) ETV6 binding sites from CUT&RUN in Etv6^+/+ and Etv6^R355X/+ LSK cells and HPC5 cells, and ATAC-seq from HPC5 cells at Tnf, Nfkbil1, and Lst1 loci encompassed by the chromatin loop at the Tnf locus. (D) Schematic description of the experimental design to examine TNF expression in HSPCs after competitive transplantation. (E) Representative histogram of mean fluorescence intensity (MFI) of TNF staining in Etv6^+/+ and Etv6^R355X/+ LSK cells harvested from the same recipient animal 6 weeks after transplantation compared with phorbol myristate acetate/ionomycin-stimulated or unstimulated BM as well as BM stained with an IgG control antibody. (F) MFI of intracellular TNF within each HSPC subpopulation (n = 6). Data are representative of 3 experiments and show mean ± SD. ∗P < .05; ∗∗P < .001; and ∗∗∗P < .0001, determined by multiple paired t tests.