Notch1 autoregulates its transcription via direct binding to CSL sites. (A) Notch1 expression in thymocyte subsets. Each fraction was sorted from C57BL/6 thymocytes. qPCR was performed and Notch1 expression relative to EF1α is shown as a mean of values from triplicate wells ±SD. Notch1 expression in DN1 cells was set at 100, and the expression in other subsets was set relative to this. Data are representative of three independent experiments. (B) Schematic representation of the 5′ region of the mouse Notch1 locus indicating potential CSL-binding sites. The CSL-binding sequences from mouse and human are shown. (C) ChIP analysis to identify Notch1 binding to CSL sites in Notch1. Rag2^−/− DN3 thymocytes were used for ChIP analysis. qPCR was performed with primers flanking each putative CSL site. The Hes1 promoter region was the positive control. Values represent the mean of signal intensity relative to input DNA from triplicate wells ±SD. Data are representative of three independent experiments. (D) Schematic representation of the Notch1 promoter sequences used in the luciferase assays. The CSL sites are shown. (E) ICN1 enhances Notch1 promoter activity. NIH3T3 cells were transiently cotransfected with the Notch1 promoter construct containing the −6K, −5K, −1K CSL-binding sites, and with either empty vector or pcDNA3-ICN1. Luciferase activity relative to Renilla luciferase activity was normalized to the pGL3-basic alone. The data are shown as a mean of values from triplicate wells ±SD. Data are representative of at least three independent experiments.

Expression of Notch1 inversely correlates with Id3. (A) DN3a cells and DN3b cells were sorted from thymocytes of C57BL/6 mice. Notch1, Id1, Id2, and Id3 expression relative to EF1α is shown as the mean of values from triplicate wells ±SD. Data are representative of three independent experiments. (B) Rag2^−/− mice were injected with α-CD3 or PBS. Thy1.2⁺ cells were sorted from the thymocytes 6 h or 24 h post-injection. The transcript quantities relative to EF1α are shown as the mean of values from triplicate wells ±SD. Data are representative of three independent experiments. (C) DN3a cells were sorted from C57BL/6 or Id3^−/− thymocytes and stimulated with DMSO or PMA for 2 h. Notch1 expression relative to EF1α was determined by qPCR. Data are relative to untreated samples (UT) of each mouse and represent the mean of three independent experiments ±SD. (D) Dose-dependent Notch1 expression in E2A/HEB double-deficient thymocytes. Notch1 expression relative to EF1α was determined from DN3a cells of Tcf12^f/fTcfe2a^f/fLckCre⁻ (LckCre⁻), Tcf12^f/+Tcfe2a^f/fLckCre⁺ (H^f/+E^f/f), Tcf12^f/fTcfe2a^f/fLckCre⁺ (H^f/fE^f/f) thymocytes. Data are from triplicates of two independent experiments (n = 3) and relative to the LckCre⁻ sample. (***) P < 0.05, Student's t-test, two-tailed. Graphed results are means with error bars representing the standard error of the mean (SEM).

Id3 down-regulates Notch1 expression. (A) Model for Notch1 and E2A transcriptional regulation of Notch1. Both Notch and E2A are positive regulators of Notch1 transcription. Following β-selection, pre-TCR signaling induces Id3 expression. This inhibits transcriptional activation of Notch1 by E2A leading to lower Notch1 protein, which extinguishes the initial positive autoregulatory loops. (B) Time course of Notch1 and Id3 mRNA down-regulation by PMA stimulation in Scid-adh cells. qPCR was performed on Scid-adh cells that were stimulated with PMA (20 ng/mL) for 0 h, 2 h, and 6 h. Notch1 and Id3 expression relative to EF1α is shown as the mean of values from triplicate wells ±SD. Data are relative to the 0-h sample, which was set at 100, and are representative of three independent experiments. (C,D) E2A binding (C) and Notch1 binding (D) to Notch1 is inhibited by pre-TCR stimulation. Scid-adh cells were treated with DMSO or PMA/ionomycin for 2 h or 4 h and fixed for ChIP. qPCR was performed with primers flanking either the E2A-binding sites or CSL-binding sites on the Notch1 locus. E47 and Notch1 binding were also measured at the TCRβ locus or at the Hes1 locus as a control of E47 or Notch1 signaling activity. Values represent the mean of the ratio of the amount of immunoprecipitated DNA/input from values from triplicate wells ±SD. Data are representative of two independent experiments.

Pre-TCR signals down-regulate Notch1 transcription. (A) Notch1 mRNA was diminished by PMA treatment in DN3 cells. DN3 cells from Rag2^−/− mice were treated with DMSO and/or PMA (20 ng/mL) and/or ionomycin (500 ng/mL) for 6 h, and qPCR was performed. Notch1 expression relative to EF1α is shown as the mean of values from triplicate wells ±SD. Notch1 expression in the PMA/ionomycin sample was set at 1, and the values in the other samples were normalized to this. The data are representative of three independent experiments. (B) Time course of Notch1 mRNA expression following PMA stimulation. DN3 cells from Rag2^−/− mice were treated with PMA (20 ng/mL) for 0 h, 2 h, or 6 h, and qPCR was performed. Notch1 expression relative to EF1α is shown as the mean of values from triplicate wells ±SD. Data are relative to the 0-h sample, whose value was set at 100, and representative of three independent experiments. (C) Time course of Notch1 protein expression following PMA stimulation. Rag2^−/− DN3 thymocytes were sorted and treated with DMSO or PMA (20 ng/mL) for 0 h, 2 h, or 6 h. Notch1 and β-actin expression were determined by Western blotting. Data are representative of three independent experiments. (D) Quantification of Notch1 protein expression after PMA stimulation. Protein amounts were measured by densitometer and quantified by Image Quant. The protein amounts were relative to unstimulated control, and error bars show SD from three independent experiments. (E) PMA-induced Notch1 down-regulation is regulated through transcriptional changes. Primary-transcript RT–PCR analysis was performed with DN3 cells from Rag1^−/− mice. Cells were treated with either DMSO or PMA. Primers were designed to flank between Notch1 exon 27 and exon 28 for mRNA and exon 27 and intron 27 for primary RNA. Notch1 mRNA or primary RNA expression is relative to 18S rRNA and is shown as a mean of values from triplicate wells ±SD. Data are relative to the 0-h sample, whose value was set at 100 and is representative of two independent experiments.

E47 directly up-regulates Notch1 transcription. (A) Schematic representation of the 5′ region of the Notch1 locus with putative E2A-binding sites. The translational start site is labeled “0.” Only the first two Notch1 exons are shown. (B) E47 binds to E2A-binding sites of Notch1. Rag2^−/− DN3 thymocytes were subjected to ChIP analysis. qPCR was performed with primers flanking putative E2A-binding sites at 5.5, 4.4, 3, and 0.3 kb upstream of the transcriptional start site as well as intron 1 of Notch1. Graphs represent the mean of the ratio of the amount of immunoprecipitated DNA/input from values from triplicate wells ±SD. Data are representative of two independent experiments. (C) Differential binding activity of acetylated histone 4 to the Notch1 locus in DN3a and DP cells. ChIP analysis to identify acetylated Histone 4 was performed on DN3a and DP cells from wild-type B6 mice. qPCR was performed to detect E2A-binding sites at −0.3 kb and intron 1 of Notch1. Data are representative of two independent experiments. (D) Schematic of the Notch1 promoter luciferase construct including the E2A-binding sites. (E) E47 and HEB transactivate the Notch1 promoter. NIH3T3 cells were transfected with the Notch1 promoter construct containing the indicated E2A-binding sites and with the empty vector, E47, or HEB. Luciferase activity relative to Renilla luciferase activity is shown as the mean of values from triplicate wells ±SD. Data are representative of two independent experiments. (F) Id3 inhibits E47-induced transactivation of the Notch1 promoter. NIH3T3 cells were transduced with either MigR1 or Id3. At 18 h, cells were transiently cotransfected with the Notch1 promoter construct with empty vector or E47. Luciferase activity relative to Renilla luciferase activity is shown as a mean of values from triplicate wells ±SD. Data are representative of three independent experiments.

Id3 down-regulates Notch1 expression and inhibits the growth of Notch1-dependent T-ALL cells. (A) Id3 inhibits T-ALL cell growth. G4A2, LCR5, LCR144, and LCR434 were transduced with either GFP (MigR1), Mig-DNMAML, or Mig-Id3. GFP⁺ cells were sorted at day 1 post-transduction (day 1) and cultured for the indicated days. Extrapolated GFP⁺ cell numbers are shown as a mean of values from triplicate wells ±SD. Data are representative of three independent experiments. (B) Id3 represses Notch1 mRNA expression. T-ALL cells were transduced with either MigR1 or Mig-Id3. GFP-expressing cells were sorted at day 1 post-transduction and cultured for 1 d (day 2). Notch1 expression relative to EF1α was determined at days 1 and 2, and is shown as a mean of values from triplicate wells ±SD. Data are representative of three independent experiments. (C) ICN1 rescues Id3-mediated growth arrest of T-ALL cells. T-ALL cells were cotransduced with the GFP (MigR1), Mig-ICN1, NGFR, and NGFR-Id3 retroviruses; and GFP⁺NGFR⁺ cells were sorted at day 1 post-transduction. GFP⁺NGFR⁺ cells were cultured for the indicated days. The extrapolated cell numbers of the GFP⁺NGFR⁺ fraction are shown as a mean of values from triplicate wells ±SD. Data are representative of three independent experiments.