TCF-1 and activated β-catenin negatively regulate Notch1 and its target genes in DN3 thymocytes. (A) Elevated expression of Notch1 and its target genes in TCF-1–deficient DN3 and DN4 thymocytes. Tcf7^−/− mice were age 8 weeks or younger and without overt signs of thymic malignancy at the time of analysis. DN subsets were sorted from lineage-negative thymocytes from Tcf7^−/− mice or littermate controls and assessed for gene expression. The relative expression level of individual genes was obtained by normalizing to the Hprt1 housekeeping gene. Data are means ± standard deviation from 1 of 3 experiments with similar results (n ≥ 3 in each experiment). (B) Activated β-catenin represses the expression of Notch1 and its targets in DN3 thymocytes. Lineage-negative DN thymocytes were cultured on OP9-DL1 stromal cells¹⁰  overnight in the presence of interleukin-7 (5 ng/mL) and then infected with empty retroviral vector pMIG or that expressing wild-type (WT) or mutant β-catenin. The mutant β-catenin has internal deletions of its N-terminal Ser/Thr phosphorylation sites and is therefore constitutively active.⁸  Twenty-four hours later, the GFP⁺ DN3 thymocytes were sorted and analyzed for expression of indicated genes. After normalization to Hprt1, the expression of each gene in pMIG-infected cells was arbitrarily set to 1, and its relative expression in the presence of WT or mutant β-catenin was then calculated. Data were pooled from at least 3 independent experiments (n ≥ 7). Similar data were obtained with DN4 cells (not shown). (C) β-catenin–mediated Notch repression depends on TCF-1. DN3 thymocytes were sorted from control mice, Vav1-Cre Lef1^−/−6  or Tcf7^−/−, cultured in the presence of 5 µM MetBIO or BIO for 6 hours, and then harvested for gene expression analysis. After normalizing to Hprt1, the expression of each gene in MetBIO-treated cells was arbitrarily set to 1, and its relative expression in BIO-treated samples was then calculated. Data are pooled from 2 independent experiments (n ≥ 3). *, P < .05; **, P < .01; ***, P < .001 by Student t test. Note that although multiple TCF/LEF binding motifs were found within “–30 kb ∼ +10 kb” of transcription initiation sites of the Notch1, Dtx1, and Ptcra genes, we did not find enriched binding of TCF-1 to these motifs in DN3 thymocytes. Further studies are necessary to determine if repression of Notch1 and its targets by TCF-1 is mediated by direct regulation via more distal TCF/LEF motifs or by indirect mechanisms.