Il7r is a transcriptional Notch1 target in murine T-ALL pathogenesis. (A) Schematic representation of the 5′ regulatory region of murine Il7r. Numbers represent base pairs from the translation initiation site (+1, atg, initiation codon). The sequence of a putative RBP-Jκ binding site (bold type) was identified at −1937 bp. Binding sequences for other regulators are also shown. (B) Flow cytometry analysis of IL-7R (right panel) and CD44 and CD25 (left panel) expression on thymocytes from embryonic day 14.5 murine fetal thymus. Numbers indicate percentages of positive cells. Shaded graph shows background staining with irrelevant isotype-matched antibodies. (C) Representative ChIP assay of fetal murine thymocytes in (B), using anti–RBP-Jκ or isotype-matched control antibody (IgG1). PCR amplification of input and immunoprecipitated DNA was performed using primer pairs spanning the RBP-Jκ site identified in Il7r or the reported Il2ra RBP-Jκ site. Size in base pairs (bp) is indicated on the left (n = 3); -, empty lane. (D) Quantitative analysis of ChIP assays in (C). Results are shown as percentage of enrichment compared with input. Bars represent mean ± standard error of the mean (SEM) of 3 independent experiments. Luciferase reporter assays of Jurkat cells cotransfected with the reporter vector pGL3 (empty or containing the Il7r RBP-Jκ binding site) along with a retrovirus encoding ICN1 and GFP (ICN1) or GFP alone (GFP) as control (n = 3) (E) or pGL3 containing the wt or a mutated RBP-Jκ binding site in Il7r, along with retrovirus encoding dnMAML1 fused to GFP (dnMAML1) or GFP alone (GFP) (n = 5) (F). Data are fold induction of relative luciferase activity (RLU) ± SEM. *P < .05, **P < .01. ns, not significant.