TNF-α treatment prevents loss of H3H9me3 at intronic L1Md and HSC gene repression. (A) Experimental design for TNF-α treatment in vivo, molecular analysis, and reconstitution experiments using HSCs sorted from mice 1 mo after TBI and previously treated with TNF-α (IR + TNFα) or not (IR), or left untreated (NIR); BMMC, bone marrow mononuclear cells. (B) Plot profile of H3K9me3 enrichment along the L1Md sequences ± 1 kb flanking regions in NIR (blue), IR (green), and IR + TNF-α (red) conditions. Each line represents the `merged CUT&Tag signal from two (NIR) to three (IR and IR + TNF-α) mice ± SEM. (C) H3K9me3 enrichment at intronic L1Md evaluated by ChIP-qPCR. (D) mRNA expression measured by qRT-PCR. Ct values were normalized to RPL32 and HPRT. (C and D) Results are expressed as fold change from the mean value of the IR condition. Each dot represents a pool of four mice. Means ± SEM from two independent experiments, t test. (E and F) GSEA analysis using gene sets. Enrichment plots for TNF-a signaling via NF-κB hallmark (E) and LT-HSC signature (F). *, P < 0.05; **, P < 0.01.