Point mutation in the gene encoding p300 suppresses thrombocytopenia in Mpl-/- mice

Abstract

In an N-nitroso-N-ethylurea (ENU) mutagenesis screen using Mpl(-/-) mice, we isolated a semidominant suppressor of thrombocytopenia, termed Plt6. The gene mutated in Plt6 mice encodes the transcriptional coregulator p300, and the mutation, a tyrosine to asparagine substitution at amino acid 630 (Y630N), disrupts the interaction between p300 and c-Myb. Mpl(-/-) p300(Plt6/+) mice displayed elevated platelet counts relative to Mpl(-/-) p300(+/+) controls, whereas mice homozygous for the Plt6 mutation produced supraphysiological levels of circulating platelets. On a wild-type genetic background, mice homozygous for the p300(Plt6) mutation, or recipients of Mpl(+/+) p300(Plt6/Plt6) bone marrow, also exhibited thrombocytosis as well as deficiencies in B-lymphoid cells. Increased platelet numbers in Plt6 mutant mice were accompanied by significant increases in megakaryocyte progenitor cells within the bone marrow and spleen with concomitantly elevated numbers of megakaryocytes. The expansion of megakaryocytopoiesis and suppression of Mpl(-/-) thrombocytopenia in Plt6 mutants is highly reminiscent of that observed in mice with mutations affecting the p300 partner protein c-Myb, suggesting an indispensable repressive role for the c-Myb/p300 transcriptional regulatory complex in megakaryocyte development, the inhibition of which allows substantial thrombopoietin (TPO)-independent platelet production.

Figure 1

Mutation in p300 causes suppression of Mpl^−/− thrombocytopenia. (A) To map the chromosomal location of Plt6, a (C57BL/6 × 129/Sv)F₂ cohort was bled and phenotypically categorized as having platelet counts typical of unmutated Mpl^−/−, Plt6/+, or Plt6/Plt6 mice and then genotyped using simple sequence length polymorphisms (SSLPs) spaced evenly throughout the genome. Markers found to be homozygous 129/Sv are shown in white; heterozygous, in gray; and homozygous C57BL/6, in black. The number of animals with each haplotype is shown below. Plt6 was localized between D15AahA10 and D15AahA3. (B) Sequence of PCR-amplified genomic DNA from representative Plt6/Plt6, Plt6/+, and wild-type mice showing a T to A mutation in exon 10 of Plt6 mice resulting in a Tyr to Asn substitution at amino acid 630. (C) Model of the p300 KIX domain indicating the Plt6 mutation site (red) and potentially disrupted contacts (blue), as well as residues previously mutated in mice (Y631, A635, and Y639; green). Modeled using FUGUE by homology with pdb file, 1KDX, the solution structure of mouse CREB-binding protein KIX domain.

Figure 2

The Plt6 mutation reduces p300 affinity for c-Myb. GST, GST-p300 KIX (Y630N), and GST-p300 KIX (wild type; 2 μg each) were used as baits for pull-down experiments with ³⁵S-labeled, in vitro–transcribed/translated c-Myb. Pull-down reactions were resolved by SDS-PAGE followed by detection of bound ³⁵S-c-Myb by autoradiography. (A) Typical autoradiogram. (B) Coomassie blue staining to determine bait levels. (C) The amount of bound ³⁵S-c-Myb was determined by densitometric analysis of the autoradiographs of 3 independent experiments, and the ³⁵S-c-Myb bound by GST and GST-p300 KIX (Y630N) is shown relative to the amount bound by GST-p300 KIX (wild type). Data represent the mean of 3 independent experiments with error bars corresponding to SD.

Figure 3

Expanded megakaryocytopoiesis in Mpl^−/−Plt6 mutants. Elevated platelet counts in Plt6 homozygous (6/6) and heterozygous (6/+) mice were accompanied by (A) increased numbers of megakaryocytes (megs), shown as number per microscopic field (×600, BM; ×200, spleen), (B) megakaryocyte colony-forming cells (meg-CFCs), shown as number per 2.5 × 10⁴ bone marrow (BM) or 10⁵ spleen cells, and (C) colony-forming units–spleen (CFU-s's), shown as number per 1.5 × 10⁵ donor bone marrow cells at days 12 or 8 following transplantation. Means plus or minus SD are shown. * P value less than .05 for comparison of data from Mpl^−/− Plt6/Plt6 or Mpl^−/− Plt6/+ mice with that of Mpl ^−/−+/+ mice. n = 3 to 7 mice per genotype.

Figure 4

Expanded megakaryocytopoiesis in recipients of Mpl^+/+p300^Plt6/Plt6 bone marrow. Recipients of Mpl^+/+ p300^Plt6/Plt6 (6/6) and Mpl^+/+ p300^+/+ control (+/+) bone marrow exhibited (A) increased numbers of megakaryocytes (megs), shown as number per microscopic field (×600, BM; ×200, spleen), and (B) megakaryocyte colony-forming cells (meg-CFCs), shown as number per 2.5 × 10⁴ bone marrow (BM) or 10⁵ spleen cells. Means plus or minus SD are shown. * P value less than .05 for comparison of data from recipients of Mpl^+/+ p300^Plt6/Plt6 marrow with that of Mpl^+/+ p300^+/+ marrow recipients. n = 4 mice per genotype.

Figure 5

B lymphocyte deficit in recipients of Mpl^+/+p300^Plt6/Plt6 bone marrow. (A) Flow cytometry plots of bone marrow, lymph node, and spleen cells from recipients of Mpl^+/+ p300^Plt6/Plt6 (Plt6/Plt6) and Mpl^+/+ p300^+/+ control (+/+) bone marrow stained with antibodies to B220 and IgM showing reduced proportions of B220⁺ cells, including IgM⁺ B cells in each tissue (percentages of cells in specific quadrants is shown). (B) Percentages of B220⁺ cells in bone marrow (BM), spleen (spl), mesenteric lymph node (LN), and blood of recipients of Mpl^+/+ p300^Plt6/Plt6 (6/6) and Mpl^+/+ p300^+/+ control (+/+) bone marrow. Means plus or minus SD are shown. *P < .05 for comparison of data from recipients of Mpl^+/+ p300^Plt6/Plt6 marrow with that of Mpl^+/+ p300^+/+ marrow recipients. n = 4 mice per genotype.