Figure 3.
Figure 3. CSL/MAML-mediated transcriptional activation of IL7R by active Notch1. (A) Identification of a conserved CSL-binding site in the 5′ regulatory region of human IL7R and mouse Il7ra. Numbers indicate distances in base pairs from the transcription initiation site. (B) Luciferase reporter assays in 293 T cells cotransfected with a reporter construct containing the 5′ regulatory region of IL7R shown in A, along with either a retroviral vector encoding ICN1 and GFP (ICN1) or a GFP-only control vector (GFP). Data are represented as fold induction over luciferase activity of control cells cotransfected with an empty reporter vector (pGL3B) and the GFP-only vector. (C) MAML-dependent activation of IL7R transcription. Reporter assays were performed in Jurkat cells cotransfected with the IL7R reporter and ICN1-GFP, and with or without dnMAML1-GFP. Data are represented as fold induction over luciferase activity of control cells transfected with the GFP-only vector. (D and E) Notch-induced IL7R promoter activity requires an intact CSL-binding site. Reporter assays were performed in RBP-Jκ+/− and RBP-Jκ−/− MEFs cotransfected with the IL7R reporter along with either ICN1-GFP or GFP-only vectors (D) and 293 T cells cotransfected with ICN1-GFP along with a reporter vector containing either the wild-type sequence of the CSL-binding site in the IL7R promoter or the mutated (mut) CSL sequence shown in A (E). Bar graphs represent means ± SEM of triplicate samples from at least four independent experiments. (F) ICN1 binds to the IL7R promoter in vivo. Formaldehyde cross-linked chromatin from primary DN2 human thymocytes, SupT1 pre-T cells and CUTLL1 T-lineage cells, and REH, NALM-6, and HPB-NULL pre-B cells was subjected to ChIP with specific antibodies against human Notch1 (N1; top), or PU.1 (bottom). Goat or rabbit Igs were used, respectively, as controls. PCR was done on input DNA and on immunoprecipitated DNA with primers pairs spanning the CSL sites of HES1 and IL7R (top) or the Ets site of IL7R (bottom). Results are from one representative out of two to three independent experiments.

CSL/MAML-mediated transcriptional activation of IL7R by active Notch1. (A) Identification of a conserved CSL-binding site in the 5′ regulatory region of human IL7R and mouse Il7ra. Numbers indicate distances in base pairs from the transcription initiation site. (B) Luciferase reporter assays in 293 T cells cotransfected with a reporter construct containing the 5′ regulatory region of IL7R shown in A, along with either a retroviral vector encoding ICN1 and GFP (ICN1) or a GFP-only control vector (GFP). Data are represented as fold induction over luciferase activity of control cells cotransfected with an empty reporter vector (pGL3B) and the GFP-only vector. (C) MAML-dependent activation of IL7R transcription. Reporter assays were performed in Jurkat cells cotransfected with the IL7R reporter and ICN1-GFP, and with or without dnMAML1-GFP. Data are represented as fold induction over luciferase activity of control cells transfected with the GFP-only vector. (D and E) Notch-induced IL7R promoter activity requires an intact CSL-binding site. Reporter assays were performed in RBP-Jκ+/− and RBP-Jκ−/− MEFs cotransfected with the IL7R reporter along with either ICN1-GFP or GFP-only vectors (D) and 293 T cells cotransfected with ICN1-GFP along with a reporter vector containing either the wild-type sequence of the CSL-binding site in the IL7R promoter or the mutated (mut) CSL sequence shown in A (E). Bar graphs represent means ± SEM of triplicate samples from at least four independent experiments. (F) ICN1 binds to the IL7R promoter in vivo. Formaldehyde cross-linked chromatin from primary DN2 human thymocytes, SupT1 pre-T cells and CUTLL1 T-lineage cells, and REH, NALM-6, and HPB-NULL pre-B cells was subjected to ChIP with specific antibodies against human Notch1 (N1; top), or PU.1 (bottom). Goat or rabbit Igs were used, respectively, as controls. PCR was done on input DNA and on immunoprecipitated DNA with primers pairs spanning the CSL sites of HES1 and IL7R (top) or the Ets site of IL7R (bottom). Results are from one representative out of two to three independent experiments.

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