Transcriptional reprogramming of the Notch1 locus after deletion of proximal promoter sequences. (A) Chromatin immunoprecipitation-sequence analysis of histone H3 acetylation in the Notch1 locus of T7 IN1C⁻ and the T2 IN1C⁺ cells and WT and Notch1^f/fCD4-Cre⁺ (N1C⁺) thymocytes. Top 4 histograms have a vertical scale of 150. The enlarged histograms in the inset have a vertical scale of 50. The regions identified as I1 and I2 correspond to islands of enriched tag density in the IN1C⁺ thymocytes, which were predicted by the “statistical model for identification of chip-enriched regions” algorithm in the N1C⁺, but not in the WT or input samples (see also Table 1).²⁷  The gap in the T2 IN1C⁺ sample corresponds to the location of the floxed deletion. (B) Real-time PCR measurement of H3 acetylation in the WT and N1C⁺ samples shown in panel A at several positions along the Notch1 locus. Amplicons in intron 2 and islands I1 and I2 were located, respectively, at 29.3, 36.5, and 41.7 kb downstream of the transcription start site. (C) Northern blot of Notch1 transcripts in WT and N1C⁺ thymocytes and in the T1 IN1C⁺ cell line. An amount of 2 μg of poly(A)⁺ RNA was loaded for each sample, and the blots were hybridized with the E30/31 probe (see Figure 6D). The left panel shows a 42-hour exposure; the right panel shows a 7-day exposure of the WT and N1C⁺ lanes. Arrowheads indicate transcripts likely to have initiated from 3′ promoters in the N1C⁺ sample. A photo of the methylene blue staining of the membrane is shown as a loading control in the bottom panel. (D) RT-PCR of exon 27b–containing transcripts in nontransformed thymocytes from 3- to 4-week-old mice with the indicated genotypes. See Figure 5E for details. Samples were defined as nontransformed according to their CD4/CD8 profile and normal CD25 expression. (E) RT-PCR of exon 27b–containing transcripts in the T7.D5 Cre-ERT2⁺ clone cultured in the presence of 4OHT or vehicle for the indicated times (bottom). Cells were also analyzed for the deletion of floxed Notch1 sequences by PCR (top). T2 corresponds to a IN1C⁺ leukemic cell line. Similar results were obtained in 4 independent experiments. In panels D and E, the arrowhead indicates the specific product from a correctly spliced transcript; the asterisk indicates products that may correspond to splicing intermediates of transcripts initiated at upstream locations.