Terryl Stacy

Runx1 Expression Marks Long-Term Repopulating Hematopoietic Stem Cells in the Midgestation Mouse Embryo

Hematopoietic stem cells (HSCs) are first found in the aorta-gonad-mesonephros region and vitelline and umbilical arteries of the midgestation mouse embryo. Runx1 (AML1), the DNA binding subunit of a core binding factor, is required for the emergence and/or subsequent function of HSCs. We show that all HSCs in the embryo express Runx1. Furthermore, HSCs in Runx1(+/-) embryos are heterogeneous and include CD45(+) cells, endothelial cells, and mesenchymal cells. Comparison with wild-type embryos showed that the distribution of HSCs among these various cell populations is sensitive to Runx1 dosage. These data provide the first morphological description of embryonic HSCs and contribute new insight into their cellular origin.

The CBFβ Subunit Is Essential for CBFα2 (AML1) Function In Vivo

Abstract The CBFβ subunit is the non-DNA-binding subunit of the heterodimeric core-binding factor (CBF). CBFβ associates with DNA-binding CBFα subunits and increases their affinity for DNA. Genes encoding the CBFβ subunit ( CBFB ) and one of the CBFα subunits ( CBFA2 , otherwise known as AML1 ) are the most frequent targets of chromosomal translocations in acute leukemias in humans. We and others previously demonstrated that homozygous disruption of the mouse Cbfa2 ( AML1 ) gene results in embryonic lethality at midgestation due to hemorrhaging in the central nervous system and blocks fetal liver hematopoiesis. Here we demonstrate that homozygous mutation of the Cbfb gene results in the same phenotype. Our results demonstrate that the CBFβ subunit is required for CBFα2 function in vivo.

Failure of Embryonic Hematopoiesis andLethal Hemorrhages in Mouse Embryos Heterozygousfor a Knocked-In Leukemia Gene CBFB–MYH11

The fusion oncogene CBFB-MYH11 is generated by a chromosome 16 inversion in human acute myeloid leukemia subtype M4Eo. Mouse embryonic stem (ES) cells heterozygous for this oncogene were generated by inserting part of the human MYH11 cDNA into the mouse Cbfb gene through homologous recombination (knock-in). Chimeric mice were leukemia free, but the ES cells with the knocked-in Cbfb-MYH11 gene did not contribute to their hematopoietic tissues. Mouse embryos heterozygous for Cbfb-MYH11 lacked definitive hematopoiesis and developed multiple fatal hemorrhages around embryonic day 12.5. This phenotype is very similar to that resulting from homozygous deletions of either Cbfb or Cbfa2 (AML1), consistent with a dominant negative function of the Cbfb-MYH11 fusion oncogene. An impairment of primitive hematopoiesis was also observed, however, suggesting a possible additional function of Cbfb-MYH11.

The core-binding factor β subunit is required for bone formation and hematopoietic maturation

Core-binding factor β (Cbfβ) is the common non-DNA-binding subunit of the Cbf family of heterodimeric transcription factors. Mice deficient in Cbfβ have a severe block in fetal liver hematopoiesis at the stage of hematopoietic stem cell (HSC) emergence. Here we show that by providing Cbfβ function in endothelial cells and hematopoietic progenitors we can rescue fetal liver hematopoiesis in Cbfβ-deficient embryos. The rescued mice die at birth, however, with severe defects in skeletal development, though intramembranous ossification occurs to some extent. Fetal liver hematopoiesis is restored at embryonic day (E) 12.5, but by E17.5 significant impairments in lymphopoiesis and myelopoiesis are observed. Thus, we conclude that the Cbfβ subunit is required for HSC emergence, bone formation and normal differentiation of lymphoid and myeloid lineage cells.

The Leukemic Protein Core Binding Factor β (CBFβ)–Smooth-Muscle Myosin Heavy Chain Sequesters CBFα2 into Cytoskeletal Filaments and Aggregates

ABSTRACT The fusion gene CBFB-MYH11 is generated by the chromosome 16 inversion associated with acute myeloid leukemias. This gene encodes a chimeric protein involving the core binding factor β (CBFβ) and the smooth-muscle myosin heavy chain (SMMHC). Mouse model studies suggest that this chimeric protein CBFβ-SMMHC dominantly suppresses the function of CBF, a heterodimeric transcription factor composed of DNA binding subunits (CBFα1 to 3) and a non-DNA binding subunit (CBFβ). This dominant suppression results in the blockage of hematopoiesis in mice and presumably contributes to leukemogenesis. We used transient-transfection assays, in combination with immunofluorescence and green fluorescent protein-tagged proteins, to monitor subcellular localization of CBFβ-SMMHC, CBFβ, and CBFα2 (also known as AML1 or PEBP2αB). When expressed individually, CBFα2 was located in the nuclei of transfected cells, whereas CBFβ was distributed throughout the cell. On the other hand, CBFβ-SMMHC formed filament-like structures that colocalized with actin filaments. Upon cotransfection, CBFα2 was able to drive localization of CBFβ into the nucleus in a dose-dependent manner. In contrast, CBFα2 colocalized with CBFβ-SMMHC along the filaments instead of localizing to the nucleus. Deletion of the CBFα-interacting domain within CBFβ-SMMHC abolished this CBFα2 sequestration, whereas truncation of the C-terminal-end SMMHC domain led to nuclear localization of CBFβ-SMMHC when coexpressed with CBFα2. CBFα2 sequestration by CBFβ-SMMHC was further confirmed in vivo in a knock-in mouse model. These observations suggest that CBFβ-SMMHC plays a dominant negative role by sequestering CBFα2 into cytoskeletal filaments and aggregates, thereby disrupting CBFα2-mediated regulation of gene expression.

Runx1 is expressed in adult mouse hematopoietic stem cells and differentiating myeloid and lymphoid cells, but not in maturing erythroid cells.

The transcription factor Runx1 marks all functional hematopoietic stem cells (HSCs) in the embryo and is required for their generation. Mutations in Runx1 are found in approximately 25% of acute leukemias and in familial platelet disorder, suggesting a role for Runx1 in adult hematopoiesis as well. A comprehensive analysis of Runx1 expression in adult hematopoiesis is lacking. Here we show that Runx1 is expressed in functional HSCs in the adult mouse, as well as in cells with spleen colony‐forming unit (CFU) and culture CFU capacities. Additionally, we document Runx1 expression in all hematopoietic lineages at the single cell level. Runx1 is expressed in the majority of myeloid cells and in a smaller proportion of lymphoid cells. Runx1 expression substantially decreases during erythroid differentiation. We also document effects of reduced Runx1 levels on adult hematopoiesis.