Fig. 4: ZBTB1 regulates HSC multipotency and drives thymic reconstitution potential.

A Experimental schema for T cell differentiation assay and competitive allogeneic HCT with Zbtb1-deficient Kit^lo HSCs generated with 2-mo young Rosa26Cas9-eGFP KI mice. B Frequency of T cell subsets following 6 weeks of culture in M-ATOs. Refer to Supplementary Fig. 13B for gating strategies to define the above populations. Aggregated data across three independent experiments, each performed in duplicates (Control = 3; Zbtb1-KO = 3). C–E Competitive allogeneic HCT with Zbtb1-deficient Kit^lo HSCs generated with 2-mo young Rosa26Cas9-eGFP KI mice. Sixteen weeks after competitive HCT, frequency of donor-derived chimerism of mature lineages in the peripheral blood as described in Fig. 2E (C), donor-derived chimerism of LSK and MP cell subsets in the BM (D), and donor-derived chimerism of T cell precursor thymocyte and mature T cell subsets in the thymus (E). All data are from n = 6 mice/group, (Control = 6; Zbtb1-KO = 6), across two independent experiments. F Experimental schema for T cell differentiation assay and competitive allogeneic HCT with Zbtb1-OE Kit^hi HSCs from young mice. G Frequency of T cell subsets following 6 weeks of culture in M-ATO of young Kit^hi HSCs. Refer to Supplementary Fig. 13B for gating strategies to define the above populations. Aggregated data across three independent experiments, each performed in duplicates (Control = 3; Zbtb1-OE = 3). H–J Competitive allogeneic HCT with Zbtb1-OE Kit^hi HSCs generated with young mice. Sixteen weeks after competitive HCT, frequency of donor-derived chimerism of mature lineages in the peripheral blood as described in Fig. 2E (H), donor-derived chimerism of LSK and MP cell subsets in the BM (I), and donor-derived chimerism of T cell precursor thymocyte and mature T cell subsets in the thymus (J). All data are from n = 6 mice/group, (Control = 6; Zbtb1-OE = 6), across two independent experiments. Refer to Supplementary Fig. 12 for gating strategies to define the above populations. K Previously identified Zbtb1 targets⁶⁹ and Notch1-interacting proteins⁷¹ enriched in Kit^lo HSCs. L Pathway enrichment analysis for non-Notch1 Zbtb1 targets enriched in Kit^lo HSCs by gseapy()¹⁰⁴ using GO_Biological_Process_2023 libraries. Bubble plot showing representative pathways. Error bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. P-values calculated by nonparametric unpaired two-tailed Mann–Whitney U test. Panels (A) and (F) were created in BioRender. Lab, K. (2025) https://BioRender.com/zy2p156. Source data are provided as a Source Data file, Source Data Fig. 4.