Gene dose-dependent control of hematopoiesis and hematologic tumor suppression by CBP

Abstract

Mice with monoallelic inactivation of the CBP gene develop highly penetrant, multilineage defects in hematopoietic differentiation and, with advancing age, an increased incidence of hematologic malignancies. The latter are characterized, at least in some cases, by loss of heterozygosity (LOH) at the CBP locus. No such pathology was observed in wild-type or p300 heterozygous null mice of the same age and genetic background. Thus, a full complement of CBP, but not p300, is required for normal hematopoietic differentiation. These results also provide the first experimental evidence for the hypothesis that CBP has tumor-suppressing activity.

Figure 1

Generation of CBP knockout mice. (A) Schematic representation of the CBP WT allele, targeting vector, and the predicted mutant CBP allele resulting from homologous recombination. (A) Asp718; (Bg) BglII; (M) MunI; (Nc) NcoI; (S) SalI. (B) Southern blot analysis of genomic DNA derived from WT (+/+),CBP^+/− and CBP^−/− embryos. The sizes (kb) of the expected fragments corresponding to the WT and mutant (MUT) alleles are indicated. (C) CBP amino-terminal-specific immunoblot analysis of standardized quantities of cell extracts derived from embryos of the indicated genotypes. Immunoblots for GAPDH and p300 are shown for loading normalization.

Figure 1

Generation of CBP knockout mice. (A) Schematic representation of the CBP WT allele, targeting vector, and the predicted mutant CBP allele resulting from homologous recombination. (A) Asp718; (Bg) BglII; (M) MunI; (Nc) NcoI; (S) SalI. (B) Southern blot analysis of genomic DNA derived from WT (+/+),CBP^+/− and CBP^−/− embryos. The sizes (kb) of the expected fragments corresponding to the WT and mutant (MUT) alleles are indicated. (C) CBP amino-terminal-specific immunoblot analysis of standardized quantities of cell extracts derived from embryos of the indicated genotypes. Immunoblots for GAPDH and p300 are shown for loading normalization.

Figure 1

Generation of CBP knockout mice. (A) Schematic representation of the CBP WT allele, targeting vector, and the predicted mutant CBP allele resulting from homologous recombination. (A) Asp718; (Bg) BglII; (M) MunI; (Nc) NcoI; (S) SalI. (B) Southern blot analysis of genomic DNA derived from WT (+/+),CBP^+/− and CBP^−/− embryos. The sizes (kb) of the expected fragments corresponding to the WT and mutant (MUT) alleles are indicated. (C) CBP amino-terminal-specific immunoblot analysis of standardized quantities of cell extracts derived from embryos of the indicated genotypes. Immunoblots for GAPDH and p300 are shown for loading normalization.

Figure 2

Defective hematopoiesis in CBP^+/− mice. Analysis of hematopoietic subpopulations in PB and bone marrow from WT (open bars), p300^+/− (hatched bars), CBP^+/− mice without splenomegaly (shaded bars), and CBP^+/− mice with splenomegaly (solid bars). (A) Total mononuclear cell counts; (C) phenotypic analysis; (D) analysis of the number of CFCs from bone marrow cell suspensions. The actual numbers of myeloid (M) colonies are 10 times the values presented in D. (B) Quantification of B and T lymphocytes, myeloid, and erythroid (E) cells in PB. Values shown represent the mean ± s.d. of three or more separate experiments. Significant differences (P<0.05) are indicated with an asterisk.

Figure 3

Histology of primary and transplanted tumors . (A) Primary plasma-cell infiltrated lymph node from a CBP^+/− mouse (Table 2, mouse 5). Transplantation of bone marrow from this animal into WT recipient mice resulted in diffuse plasmocytomas (arrows) in recipients (B, C). (D) Primary HS in a CBP^+/− mouse. (E) HS (arrow) infiltrating hepatic vasculature, arising in a WT recipient transplanted with bone marrow from a CBP^+/− donor without overt tumor. (F) Infiltration of a lymph node with primary lymphocytic leukemia in a CBP^+/− mouse.

Figure 4

LOH at the CBP locus in tumors. Southern blot analysis of tumors isolated from a mediastinal mass in an animal with myelogenous leukemia (A) (Table 2, mouse 18); HS arising in a WT recipient transplanted with bone marrow from a CBP^+/− mouse (B); and plasmacytomas arising in multiple recipients (T2, T5, T6) of bone marrow from a CBP^+/− mouse (C). Comparisons are made to WT, and CBP^+/− controls. Expected WT and targeted mutant (MUT) alleles are indicated by arrows. (D) p300 and CBP Western blot analysis of protein extracts from plasmacytomas arising in multiple transplant recipients (T2, T5, T6), compared with equivalent amounts of protein extract from spleens of WT and CBP^+/− controls.

Figure 4

LOH at the CBP locus in tumors. Southern blot analysis of tumors isolated from a mediastinal mass in an animal with myelogenous leukemia (A) (Table 2, mouse 18); HS arising in a WT recipient transplanted with bone marrow from a CBP^+/− mouse (B); and plasmacytomas arising in multiple recipients (T2, T5, T6) of bone marrow from a CBP^+/− mouse (C). Comparisons are made to WT, and CBP^+/− controls. Expected WT and targeted mutant (MUT) alleles are indicated by arrows. (D) p300 and CBP Western blot analysis of protein extracts from plasmacytomas arising in multiple transplant recipients (T2, T5, T6), compared with equivalent amounts of protein extract from spleens of WT and CBP^+/− controls.

Figure 4

LOH at the CBP locus in tumors. Southern blot analysis of tumors isolated from a mediastinal mass in an animal with myelogenous leukemia (A) (Table 2, mouse 18); HS arising in a WT recipient transplanted with bone marrow from a CBP^+/− mouse (B); and plasmacytomas arising in multiple recipients (T2, T5, T6) of bone marrow from a CBP^+/− mouse (C). Comparisons are made to WT, and CBP^+/− controls. Expected WT and targeted mutant (MUT) alleles are indicated by arrows. (D) p300 and CBP Western blot analysis of protein extracts from plasmacytomas arising in multiple transplant recipients (T2, T5, T6), compared with equivalent amounts of protein extract from spleens of WT and CBP^+/− controls.

Figure 4

LOH at the CBP locus in tumors. Southern blot analysis of tumors isolated from a mediastinal mass in an animal with myelogenous leukemia (A) (Table 2, mouse 18); HS arising in a WT recipient transplanted with bone marrow from a CBP^+/− mouse (B); and plasmacytomas arising in multiple recipients (T2, T5, T6) of bone marrow from a CBP^+/− mouse (C). Comparisons are made to WT, and CBP^+/− controls. Expected WT and targeted mutant (MUT) alleles are indicated by arrows. (D) p300 and CBP Western blot analysis of protein extracts from plasmacytomas arising in multiple transplant recipients (T2, T5, T6), compared with equivalent amounts of protein extract from spleens of WT and CBP^+/− controls.