Regulation of Lef1 by ICN1 in primary T-cell progenitors and T-cell lymphoma. (A) QPCR analysis of Lef1 and Deltex1 expression in primary T-cell progenitors cultured in vitro on OP9-DL1 in the presence of DMSO or GSI for 12 hours or after infection with MigR1 or MigR1-ICN1 retrovirus. mRNA expression was standardized to HPRT. Comparison of the mouse and human Lef1 gene sequence surrounding the mouse Lef1p + 11-bp CSL site (B) and the Lef1p −2179-bp CSL site (C). The CSL-binding sites are shaded. (D) EMSA using the Lef1p +11-bp and Lef1p −2179-bp CSL sites. Complexes were competed with self-competitor, a CSL consensus sequence (Jκ3), or nonspecific competitor (Oct). The position of the free oligonucleotides and shifted CSL complex are shown. Vertical lines have been inserted to indicate where a gel was cut. All lanes are from the same gel. (E) ChIP analysis. DNA precipitated with IgG or anti-Notch1 from 0531 cells treated with GSI (squlf]) or DMSO (▩) for 15 hours was analyzed by QPCR using primers specific for sequences near the Lef1p −2-kb and Lef1p +11-bp CSL-binding sites, in Lef1 intron 11, and the Hes1 promoter. Results are representative of 4 precipitations. Hes1 promoter P < .001; Lef1 intron P < .25; and Lef1p −2 kb P < .05 using a paired Student t test. The Lef1p +11-bp site was precipitated specifically with anti-Notch1 in 3 of 4 experiments, resulting in P < .25. (F) QPCR analysis of DNA precipitated with IgG or antiacetylated histone H4 (AcH4) from the same samples as in panel E. Histograms represent the means (± SE) of triplicate QPCR amplifications.