Efficient deletion of Mbd3 in hematopoietic cells of Mbd3^ΔH/ΔH mice destabilizes the NuRD complex and results in the development of T-ALL. (A) PCR amplification of the Mbd3 locus from genomic DNA isolated from whole bone marrow, thymus, and spleen. Data shown are representative of at least three independent experiments using littermate mice. (B) Expression of Mbd3 mRNA in CD150⁺ CD48^– EPCR⁺ CD45⁺ HSCs. Measured by quantitative RT-PCR, normalized to the expression of Ubc. Mean ± SE is shown, summarizing three independent experiments using littermate mice. ***, P < 0.005, Welch’s t test. (C) Western blots of nuclear extracts, detecting NuRD complex proteins in whole bone marrow, spleen, and thymus. The antibodies used for protein detection are shown to the right of each blot, and protein size markers (kD) are shown on the left. Data shown are representative of at least three independent experiments using littermate mice. (D) Coimmunoprecipitation of Chd4 and Hdac1 from whole–bone marrow nuclear extracts. Antibodies used for immunoblotting are shown to the right of each blot, and protein size markers (kD) are shown on the left. All the images in this panel are from the same blot, which was first immunoblotted for Hdac1, then stripped and reprobed for Chd4. Several irrelevant lanes between those shown have been omitted. Data shown are representative of two independent experiments using littermate mice. (E) Kaplan-Meier survival curve, showing the age at which mice became moribund because of thymoma. (F) Enlarged thymus representative of Mbd3^ΔH/ΔH tumors, compared with thymus from sex-matched littermate controls (left; bar, 1 cm). Representative hematoxylin and eosin–stained sections were photographed at 400× magnification (right). Data are representative of four independent experiments using littermate mice. (G) FACS analysis of representative Mbd3^ΔH/ΔH thymus tumor, compared with sex-matched littermate control. The percentage of cells in each plot that lie within the each gate is shown. Data are representative of three independent experiments analyzing six tumor samples and six age- and sex-matched mice from the same mouse colony. (H) Analysis of Dβ₁-Jβ₁ and Dβ₂-Jβ₂ rearrangements at the Tcrb locus by PCR of genomic DNA from the indicated tissues. Data shown summarize six independent DNA extractions using age- and sex-matched mice from the same mouse colony. (I) Schematic diagram of the Notch1 protein, showing the location of mutations detected in Mbd3^ΔH/ΔH T-ALL. The color inside each arrowhead represents a single T-ALL. Arrowheads with red outlines indicate frameshift and nonsense mutations that disrupt the C terminus of the protein.