CBFB Is Required for Early T Cell Development in the Thymus.
Authors: Zhao Ling , Cannons Jennifer L. , Anderson Stacie , Kirby Martha R. , Xu Liping , Castilla Lucio , Schwartzberg Pamela L. , Bosselut Rémy , Liu P. Paul 
Published: October 2019

Abstract
Runx1 and Runx3 play important roles in early T cell development. Runx1 is required in the development of double negative cells. Runx1 is also required to repress CD4 expression in DN cells while Runx 3 is essential for epigenetic silencing of CD4 expression in CD8 cells. Both Runx1 and Runx3 are required for CD8 cell development. Because Cbfβ heterodimerizes with both Runx1 and Runx3, we hypothesized that Cbfb is also important in T cell development.
To address this issue we analyzed transgenic mouse models with three Cbfb alleles. The first is a null allele for Cbfb and embryos homozygous for this allele die in midgestation due to failure of definitive hematopoiesis and hemorrhage. The second one is a GFP knockin. This Cbfb-GFP allele is a hypomorphic one in that the fusion protein Cbfβ-GFP produced from the allele behaves similarly as the wildtype Cbfβ protein but RNA and protein production from the allele is lower than that of the wildtype allele. Interestingly, AGM hematopoiesis is relatively normal and there is no hemorrhage in the CbfbGFP/GFP embryos, which die at birth due to bone formation defects. The third model is our knock-in mouse model expressing Cbfb-MYH11, the fusion gene found in human AML MeEo with inv (16)(p13; q22). Heterozygous knock-in mice had a phenotype identical to that of the Cbfb and Runx1 null mice, suggesting that the fusion gene Cbfb-MYH11 functions in a dominant-negative manner. We conditionally expressed the Cbfb-MYH11 fusion gene in T cells by using Cre-lox recombination with a floxed Cbfb-MYH11 allele and a Lck-Cre transgene, which starts to express the Cre enzyme at the DN2 stage.
By analyzing embryos compound-heterozygous for the null and the hypomorphic GFP knockin alleles (Cbfb−/GFP), we found that CD4 expression was derepressed and thymocyte development was blocked at DN1 and DN2 stages in E17.5 Cbfb−/GFP embryos, which also had much smaller thymi with reduced cellularity compared to their litter mate controls. Further studies on cell proliferation and apoptosis indicated that increased cell death might account for the reduced cellularity. The compound heterozygous Cbfb−/GFP mice died at birth with severe bone formation defects.
The Tg(Lck-Cre)/conditional Cbfb-MYH11 mice were viable. In adult thymus, Cbfb-MYH11 expression led to a 10-fold reduction in thymocyte numbers, resulting from both impaired survival of CD4+CD8+ thymocytes (similar as in Cbfb−/GFP embryos) and a differentiation block at DN3 stage. The reduced cellularity could be rescued by over expression of Bcl2 through crossing with Tg(Lck-hBcl2) mice. Cbfb-MYH11 did not derepress CD4 expression in the thymus even though it did so in reporter assays in vitro, which could be due to incomplete Cre-lox reaction, or that Cbfb-MYH11 acts more than just a pure dominant negative. Our data suggest that Cbfβ is critical for several stages of T cell development and may help to explain why CBFB-MYH11+ cells cannot be detected in the T cell lineage in AML patients with this fusion gene.