T-lymphoid, megakaryocyte, and granulocyte development are sensitive to decreases in CBFβ dosage.

Abstract

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...

AML1/Runx1 Negatively Regulates Quiescent Hematopoietic Stem Cells in Adult Hematopoiesis

“…It is known that mice that lack CBF␤, a functional partner of AML1, phenotypically recapitulate AML1 knockout mice, showing total loss of definitive hematopoiesis. A recent report using CBF␤ hypomorphic mice revealed that 15% of the wildtype level of CBF␤ is sufficient for emergence of definitive hematopoiesis (30). In this report, attenuated doses of CBF␤ between 15 and 30% of wild type cause expansion of hematopoietic stem/ progenitor cells in neonates, which return to normal when CBF␤ is restored up to the hemizygous level.…”

Differentiation-Dependent Interactions between RUNX-1 and FLI-1 during Megakaryocyte Development

“…RUNX-1-deficient megakaryocytes have hypolobulated nuclei, underdeveloped cytoplasm, low DNA ploidy, and enhanced replating activity in semisolid medium culture assays. Haploinsufficiency of CBF-␤ also perturbs megakaryopoiesis in mice (54). These findings indicate that RUNX-1/CBF-␤ is required for terminal megakaryocyte maturation.…”

The Transcription Factor Zbtb7b Promotes CD4 Expression by Antagonizing Runx-Mediated Activation of the CD4 Silencer

“…Because Runx transcription factors act in association with the structurally unrelated Cbf␤ protein (29), which participates in Runx-mediated CD4 repression (24,30), it was possible that Zbtb7b might antagonize Runx function by reducing the expression of endogenous Cbf␤. To address this possibility, we examined whether Zbtb7b would antagonize Runx3-mediated silencer activation in RLM-11 cells cotransfected with Cbf␤.…”