Journal: The Journal of Experimental Medicine
Article Title: Notch interaction with RUNX factors regulates initiation of the T-lineage program
doi: 10.1084/jem.20250911
Figure Lengend Snippet: CTCF motif is highly enriched in LP-specific RUNX1-binding regions and LP-specific interaction of RUNX1 with CTCF. (A) ChIP-seq analysis of RUNX1 was performed using Cas9-LPs with or without Notch stimulation. The Venn diagram shows the numbers of reproducible RUNX1 nonpromoter ChIP peaks in LPs (Notch-) and Notch-stimulated LPs for 2 days (Phase 1; Notch+). (B) Top three enriched sequence motifs among the 4,832 LP-specific, the 11,014 Phase 1–specific, and 17,550 shared reproducible RUNX1 peaks between LP and Phase 1 are shown. Data are based on ChIP-seq peaks scored as reproducible in two replicate samples. (C) Myc- and FLAG-tagged RUNX1-ERT2 vectors were retrovirally transduced into Cas9-LPs. Total extracts from Myc-FLAG-RUNX1-ERT2–expressing LPs treated with tamoxifen for 6 h were subjected to two-step affinity purification followed by SDS-PAGE and silver staining. All of the visible bands were analyzed using mass spectrometry analysis. Phase 1 and Phase 2 cells were stimulated with Notch ligand on OP9-DLL4 for 2 and 10 days, respectively. (D) Representative RUNX1-binding molecules in LP, Phase 1, and Phase 2 cells are shown with Mascot scores. The full list of RUNX1-binding molecules is provided in . (E) Total extracts from Mock- or Myc-FLAG-RUNX1-ERT2–transduced and tamoxifen-treated LPs, with or without Notch stimulation, were subjected to IP with anti-FLAG and anti-Myc mAbs followed by immunoblotting with anti-CTCF, anti-Notch1-IC, or anti-Med12 antibodies (left panels). Nuclear lysates (input) were also immunoblotted with anti-CTCF, anti-Notch1-IC, anti-Med12, and anti-Myc (RUNX1) antibodies, whereas cytoplasmic lysates (input) were immunoblotted with anti-tubulin-α mAb (right panels). Data are representative of three independent experiments. IP, immunoprecipitation. Source data are available for this figure: .
Article Snippet: ChIP-seq libraries were constructed using NEBNext Ultra II DNA Library Prep Kit (E7645S; NEB) with Sample Purification Beads, and NEBNext Multiplex Oligos for Illumina (E6440S; NEB).
Techniques: Binding Assay, ChIP-sequencing, Sequencing, Expressing, Affinity Purification, SDS Page, Silver Staining, Mass Spectrometry, Western Blot, Immunoprecipitation
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