Monoallelic RUNX1 mutations cause familial platelet disorder with predisposition for acute myelogenous leukemia (FPD/AML). Sporadic mono-and biallelic mutations are found at high frequencies in AML M0, in radiation-associated and therapy-related myelodysplastic syndrome and AML, and in isolated cases of AML M2, M5a, M3 relapse, and chronic myelogenous leukemia in blast phase. Mutations in RUNX2 cause the inherited skeletal disorder cleidocranial dysplasia (CCD). Most hematopoietic missense mutations in Runx1 involve DNA-contacting residues in the Runt domain, whereas the majority of CCD mutations in Runx2 are predicted to impair CBFb binding or the Runt domain structure. We introduced different classes of missense mutations into Runx1 and characterized their effects on DNA and CBFb binding by the Runt domain, and on Runx1 function in vivo. Mutations involving DNA-contacting residues severely inactivate Runx1 function, whereas mutations that affect CBFb binding but not DNA binding result in hypomorphic alleles. We conclude that hypomorphic RUNX2 alleles can cause CCD, whereas hematopoietic disease requires more severely inactivating RUNX1 mutations.
The family of core-binding factors includes the DNA-binding subunits Runx1-3 and their common non-DNA-binding partner CBF. We examined the collective role of core-binding factors in hematopoiesis with a hypomorphic Cbfb allelic series. Reducing CBF levels by 3-or 6-fold caused abnormalities in bone development, megakaryocytes, granulocytes, and T cells. T-cell development was very sensitive to an incremental reduction of CBF levels: mature thymocytes were decreased in number upon a 3-fold reduction in CBF levels, and were virtually absent when CBF levels were 6-fold lower. Partially penetrant consecutive differentiation blocks were found among early T-lineage progenitors within the CD4 ؊ CD8 ؊ double-negative 1 and downstream double-negative 2 thymocyte subsets. Our data define a critical CBF threshold for normal T-cell development, and situate an essential role for corebinding factors during the earliest stages of T-cell development. IntroductionHypomorphic alleles have long been known to cause developmental disorders in model organisms and in humans, and can reveal additional functions for genes in pathways that are completely obliterated when the gene's function is eliminated. For example, the many different spontaneous, chemically induced, and targeted mutant alleles of the Kit gene have illuminated c-kit's multiple roles in gametogenesis, melanogenesis, and hematopoiesis, and in the interstitial cells of Cajal. [1][2][3] Hypomorphic alleles can reveal differences in the requirements of certain developmental pathways for a protein's concentration, and help pinpoint lineage decisions that are influenced by that protein. Many mutations in cancercausing genes may cause a functional dosage reduction as part of their overall activity, and the study of hypomorphic alleles may allow an assessment of this contribution.In this study, we used a hypomorphic allele of the core-binding factor  (Cbfb) gene to unveil new developmental requirements for all 3 core-binding factors. Core-binding factors (CBFs) are a small family of transcription factors consisting of a DNA-binding subunit encoded by the Runx1, Runx2, or Runx3 genes, and a common non-DNA-binding CBF subunit. Runx1 is required for hematopoietic stem cell (HSC) emergence in the fetus, 4 and during postnatal hematopoiesis for megakaryocyte, B-, and T-lymphocyte development. [5][6][7] Runx1 participates in CD4 silencing during the CD4 Ϫ CD8 Ϫ double-negative (DN) and CD8 ϩ stages of T-cell development and is necessary at the DN2 to DN3 and DN3 to DN4 transitions. [5][6][7][8] Runx2 is required for bone formation, both for osteoblast differentiation and chondrocyte hypertrophy. [9][10][11][12] Runx3 contributes to the maturation of chondrocytes during bone formation 13 and is necessary for CD4 silencing at the CD8 ϩ stage of T-cell development, for Langerhans-cell development, and its deletion accelerates the maturation of dendritic cells resulting in an allergic airway inflammation. 7,8,14 We used a hypomorphic Cbfb allele in conjunction with a nonfunction...
Runx1 is expressed in skeletal elements, but its role in fracture repair has not been analyzed. We created mice with a hypomorphic Runx1 allele (Runx1L148A) and generated Runx1L148A/− mice in which >50% of Runx1 activity was abrogated. Runx1L148A/− mice were viable but runted. Their growth plates had extended proliferating and hypertrophic zones, and the percentages of Sox9‐, Runx2‐, and Runx3‐positive cells were decreased. Femoral fracture experiments revealed delayed cartilaginous callus formation, and the expression of chondrogenic markers was decreased. Conditional ablation of Runx1 in the mesenchymal progenitor cells of the limb with Prx1‐Cre conferred no obvious limb phenotype; however, cartilaginous callus formation was delayed following fracture. Embryonic limb bud–derived mesenchymal cells showed delayed chondrogenesis when the Runx1 allele was deleted ex vivo with adenoviral‐expressed Cre. Collectively, our data suggest that Runx1 is required for commitment and differentiation of chondroprogenitor cells into the chondrogenic lineage. © 2012 American Society for Bone and Mineral Research.
The family of core binding factors includes the DNA-binding subunits Runx1-3 and the common non-DNA binding partner CBFβ. Runx1 and CBFβ are essential for the emergence of hematopoietic stem cells during fetal development, but not for stem cell maintenance during later ontogeny. Runx1 is also required for megakaryocyte differentiation, B cell development, and for the DN2 to DN3 transition in thymocyte development. Runx2/CBFβ are critical for normal osteogenesis, and Runx3 for CD4 silencing in CD8+ T cells, but their contribution to other steps of hematopoietic development is unknown. To examine the collective role of core binding factors in hematopoiesis, we generated a hypomorphic Cbfb allele (Cbfbrss). CBFβ protein levels were reduced by approximately 2–3 fold in fetuses homozygous for the Cbfbrss allele (Cbfbrss/rss), and 3–4 fold in fetuses carrying one hypomorphic and one knockout allele (Cbfbrss/−). Cbfbrss/rss and Cbfbrss/− fetuses had normal erythroid and B cell development, and relatively mild abnormalities in megakaryocyte and granulocyte differentiation. In contrast, T cell development was very sensitive to an incremental reduction of CBFβ levels: mature thymocytes were decreased in Cbfbrss/rss fetuses, and virtually absent in Cbfbrss/−fetuses. We next assessed the development of Cbfbrss/rss and Cbfbrss/− fetal liver progenitors after transplantation to irradiated adult recipients, in competition with wild-type (wt) bone marrow cells. Wt, Cbfbrss/rss and Cbfbrss/− fetal progenitors replenished the erythroid, myeloid and B cell compartments equally well. The overall development of Cbfbrss/rss T cells was preserved, although CD4 expression was derepressed in double negative thymocytes. In Cbfbrss/− chimeras, mature thymocytes were entirely derived from competitor cells. Furthermore, the developmental block in Cbfbrss/− progenitors was present at the earliest stages of T cell development within the DN1 (ETP) and DN2 subsets. Our data define a critical CBFβ threshold for normal T cell development, and they situate an essential role of core binding factors during the earliest stages of T cell development. In addition, early thymopoiesis appeared more severely affected by reduced CBFβ dosage than by the lack of Runx1 (Ichikawa et al., Nat Med 2004; Growney et al., Blood 2005), suggesting that Runx2/3 may contribute to core binding factor activity in the T cell lineage.
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