Figure 8.
Effects of RUNX, CTCF, or Med12 deletion on DN2 cell generation before and after Notch stimulation in primary BM progenitors. (A) Experimental scheme. BM progenitors from Cas9;Bcl2 Tg mice were transduced with sgRNA and cultured without stromal cells for 1 day. Then, they were transferred onto OP9 stromal cells and cocultured for 4 days. CD45+hNGFR+ sgRNA-transduced cells were gated and analyzed for Lin markers, CD19, CD44, and CD25 expression. Representative plots show Lin/CD19 profiles in CD45+hNGFR+ sgRNA-transduced cells (upper panel) and CD44/CD25 profiles in CD45+hNGFR+Lin−CD19+ cells (lower panel). (B and C) Percentage (B) and relative number (C) of CD25+ cells among CD45+hNGFR+Lin−CD19+ sgRNA-transduced cells (from A) are shown with SD. (D) Alternative experimental scheme. sgRNA-transduced BM progenitors were cultured without stromal cells for 2 days. Then, they were transferred onto OP9-DLL1 stromal cells and cocultured for 4 days. CD45+hNGFR+ sgRNA-transduced cells were gated and analyzed for Lin markers, CD19, CD44, and CD25 expression. Representative plots show Lin/CD19 profiles in CD45+hNGFR+ sgRNA-transduced cells (upper panel) and CD44/CD25 profiles in CD45+hNGFR+Lin−CD19− cells (lower panel). (E and F) Percentage and relative number of CD25+ cells among hNGFR+CD45+Lin−CD19− sgRNA-transduced cells (from D) are shown with SD. (G) Working model of Notch-dependent functional conversion of RUNX TFs regulating the initiation of the T-lineage program. The RUNX/CTCF complex in the LP stage represses T-signature genes. Notch signaling induces the dissociation of RUNX from CTCF and facilitates the redirection of the RUNX/Mediator/p300 complex, thereby triggering T cell differentiation. Results shown in A and D are representative of three independent experiments, whereas data in B, C, E, and F represent the mean values of three independent biological replicates. The data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons (B, C, E, and F). For Notch-: B, *adjusted P = 0.0224 for sgCbfb; **adjusted P < 0.0001 for sgCTCF. For Notch-: C, *adjusted P = 0.0208 for sgCbfb; **adjusted P = 0.0083 for sgCTCF. For Notch+; E, **adjusted P = 0.0014 for sgCbfb; *adjusted P = 0.0337 for sgMed12. For Notch+: F, **adjusted P = 0.0006 for sgCbfb; **adjusted P = 0.0029 for sgMed12. SD, standard deviation.

Effects of RUNX, CTCF, or Med12 deletion on DN2 cell generation before and after Notch stimulation in primary BM progenitors. (A) Experimental scheme. BM progenitors from Cas9;Bcl2 Tg mice were transduced with sgRNA and cultured without stromal cells for 1 day. Then, they were transferred onto OP9 stromal cells and cocultured for 4 days. CD45+hNGFR+ sgRNA-transduced cells were gated and analyzed for Lin markers, CD19, CD44, and CD25 expression. Representative plots show Lin/CD19 profiles in CD45+hNGFR+ sgRNA-transduced cells (upper panel) and CD44/CD25 profiles in CD45+hNGFR+Lin−CD19+ cells (lower panel). (B and C) Percentage (B) and relative number (C) of CD25+ cells among CD45+hNGFR+Lin−CD19+ sgRNA-transduced cells (from A) are shown with SD. (D) Alternative experimental scheme. sgRNA-transduced BM progenitors were cultured without stromal cells for 2 days. Then, they were transferred onto OP9-DLL1 stromal cells and cocultured for 4 days. CD45+hNGFR+ sgRNA-transduced cells were gated and analyzed for Lin markers, CD19, CD44, and CD25 expression. Representative plots show Lin/CD19 profiles in CD45+hNGFR+ sgRNA-transduced cells (upper panel) and CD44/CD25 profiles in CD45+hNGFR+Lin−CD19− cells (lower panel). (E and F) Percentage and relative number of CD25+ cells among hNGFR+CD45+Lin−CD19− sgRNA-transduced cells (from D) are shown with SD. (G) Working model of Notch-dependent functional conversion of RUNX TFs regulating the initiation of the T-lineage program. The RUNX/CTCF complex in the LP stage represses T-signature genes. Notch signaling induces the dissociation of RUNX from CTCF and facilitates the redirection of the RUNX/Mediator/p300 complex, thereby triggering T cell differentiation. Results shown in A and D are representative of three independent experiments, whereas data in B, C, E, and F represent the mean values of three independent biological replicates. The data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons (B, C, E, and F). For Notch-: B, *adjusted P = 0.0224 for sgCbfb; **adjusted P < 0.0001 for sgCTCF. For Notch-: C, *adjusted P = 0.0208 for sgCbfb; **adjusted P = 0.0083 for sgCTCF. For Notch+; E, **adjusted P = 0.0014 for sgCbfb; *adjusted P = 0.0337 for sgMed12. For Notch+: F, **adjusted P = 0.0006 for sgCbfb; **adjusted P = 0.0029 for sgMed12. SD, standard deviation.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal