Figure 7
TMe enhancer site is essential for T-ALL initiation. (A) ChIP-qPCR analysis for Tcf1 binding in murine T-ALL (mT-ALL, n = 3), wild type LSKs (Neg Ctrl, n = 3) and IgG Controls (n = 2) at NMe (each left bar, red) and TMe (each right bar, green). Data are represented as mean ± standard error of the mean (SEM). (B) Genomic localization of TMe in mm10, depicting the strategy for deletion of genomic element. Integrative genomics viewer profiles for N1IC (top, red, n = 3) and N1IC TMe−/− (bottom, green, n = 3) showing chromatin accessibility from ATAC-seq analysis on sorted LSKs. Tracks were group-scaled, scaling is shown in the top left corner. (C) Schematic representation of BM chimeras R26 N1IClox/+Mx1Cre (N1IC), R26 N1IClox/+ TMe+/−Mx1Cre (N1IC TMe+/−), and R26 N1IClox/+ TMe−/−Mx1Cre (N1IC TMe−/−) mice. (D) Kaplan-Meier survival analysis of transplanted mice after last poly(I:C) injection. N1IC mice (red, n = 8), N1IC TMe+/− (orange, n = 10), and N1IC TMe−/− (green, n = 11) were followed for 341 days post poly(I:C) injection. Log-rank (Mantel-Cox) test, *P value < .05; ***P value < .001; ****P value < .0001. (E) Relative percentages of CD45.2+ transplanted cells in peripheral blood of N1IC mice (red, n = 12), N1IC TMe+/− (orange, n = 11), and N1IC TMe−/− (green, n = 12), post poly(I:C) injection. Timepoints are indicated below the graph. Data are represented as mean ± SEM. (F) Flow cytometric-based phenotypic analysis of transplanted (CD45.2+) and induced (eGFP+) BM cells. Plots depict representative profiles from N1IC mouse T-ALL midstage progression (red), N1IC TMe+/− at the endpoint (orange), and N1IC TMe−/− at the endpoint (green). (G) Total protein analysis by western blot for Myc, Tcf1, and β-actin on FACS purified T cells from BM of experimental groups: N1IC CD8+ (n = 2), N1IC DP (CD4+CD8+, n = 2), N1IC TMe+/− DP (CD4+CD8+, n = 4), and N1IC TMe−/− DP (CD4+CD8+, n = 2), as indicated. (H) Total protein analysis by western blot for Myc and β-actin on FACS purified T cell blasts from BM of experimental groups: N1IC CD8+ (n = 3) and N1IC TMe+/− CD8+ (n = 3), as indicated. (I) 3C-qPCR analysis for interactions between Myc promoter and NMe on FACS purified T cells from BM of experimental groups: N1IC CD8+ (n = 3), N1IC DP (n = 3), N1IC TMe−/− DP (n = 3). Bacterial artificial chromosome (BAC) clones spanning the region of interest were used as negative control. Data are represented as mean ± SEM. Unpaired t-test, *P value < .05.

TMe enhancer site is essential for T-ALL initiation. (A) ChIP-qPCR analysis for Tcf1 binding in murine T-ALL (mT-ALL, n = 3), wild type LSKs (Neg Ctrl, n = 3) and IgG Controls (n = 2) at NMe (each left bar, red) and TMe (each right bar, green). Data are represented as mean ± standard error of the mean (SEM). (B) Genomic localization of TMe in mm10, depicting the strategy for deletion of genomic element. Integrative genomics viewer profiles for N1IC (top, red, n = 3) and N1IC TMe−/− (bottom, green, n = 3) showing chromatin accessibility from ATAC-seq analysis on sorted LSKs. Tracks were group-scaled, scaling is shown in the top left corner. (C) Schematic representation of BM chimeras R26 N1IClox/+Mx1Cre (N1IC), R26 N1IClox/+ TMe+/−Mx1Cre (N1IC TMe+/−), and R26 N1IClox/+ TMe−/−Mx1Cre (N1IC TMe−/−) mice. (D) Kaplan-Meier survival analysis of transplanted mice after last poly(I:C) injection. N1IC mice (red, n = 8), N1IC TMe+/− (orange, n = 10), and N1IC TMe−/− (green, n = 11) were followed for 341 days post poly(I:C) injection. Log-rank (Mantel-Cox) test, *P value < .05; ***P value < .001; ****P value < .0001. (E) Relative percentages of CD45.2+ transplanted cells in peripheral blood of N1IC mice (red, n = 12), N1IC TMe+/− (orange, n = 11), and N1IC TMe−/− (green, n = 12), post poly(I:C) injection. Timepoints are indicated below the graph. Data are represented as mean ± SEM. (F) Flow cytometric-based phenotypic analysis of transplanted (CD45.2+) and induced (eGFP+) BM cells. Plots depict representative profiles from N1IC mouse T-ALL midstage progression (red), N1IC TMe+/− at the endpoint (orange), and N1IC TMe−/− at the endpoint (green). (G) Total protein analysis by western blot for Myc, Tcf1, and β-actin on FACS purified T cells from BM of experimental groups: N1IC CD8+ (n = 2), N1IC DP (CD4+CD8+, n = 2), N1IC TMe+/− DP (CD4+CD8+, n = 4), and N1IC TMe−/− DP (CD4+CD8+, n = 2), as indicated. (H) Total protein analysis by western blot for Myc and β-actin on FACS purified T cell blasts from BM of experimental groups: N1IC CD8+ (n = 3) and N1IC TMe+/− CD8+ (n = 3), as indicated. (I) 3C-qPCR analysis for interactions between Myc promoter and NMe on FACS purified T cells from BM of experimental groups: N1IC CD8+ (n = 3), N1IC DP (n = 3), N1IC TMe−/− DP (n = 3). Bacterial artificial chromosome (BAC) clones spanning the region of interest were used as negative control. Data are represented as mean ± SEM. Unpaired t-test, *P value < .05.

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