Wheat CBF gene family: identification of polymorphisms in the CBF coding sequence

Expression of cold-regulated genes needed for protection against freezing stress is mediated, in part, by the CBF transcription factor family. Previous studies with temperate cereals suggested that the CBF gene family in wheat was large, and that CBF genes were at the base of an important low temperature tolerance trait. Therefore, the goal of our study was to identify the CBF repertoire in the freezing-tolerant hexaploid wheat cultivar Norstar, and then to examine if the coding region of CBF genes in two spring cultivars contain polymorphisms that could affect the protein sequence and structure. Our analyses reveal that hexaploid wheat contains a complex CBF family consisting of at least 65 CBF genes of which 60 are known to be expressed in the cultivar Norstar. They represent 27 paralogous genes with 1–3 homeologous copies for the A, B, and D genomes. The cultivar Norstar contains two pseudogenes and at least 24 additional proteins having sequences and (or) structures that deviate from the consensus in the conserved AP2 DNA-binding and (or) C-terminal activation-domains. This suggests that in cultivars such as Norstar, low temperature tolerance may be increased through breeding of additional optimal alleles. The examination of the CBF repertoire present in the two spring cultivars, Chinese Spring and Manitou, reveals that they have additional polymorphisms affecting conserved positions in these domains. Understanding the effects of these polymorphisms will provide additional information for the selection of optimum CBF alleles in Triticeae breeding programs.

Human CD34+ Cells Expressing CBFβ-MYH11 Exhibit a Myelomonocytic Phenotype with Greatly Enhanced Proliferative Ability.

The Core Binding Factor (CBF) transcription factor complex is required for normal hematopoiesis. Knockout of either component of the complex, AML1 or CBFβ, results in embryonic lethality in mice due to a lack of definitive hematopoiesis. Chromosomal rearrangements involving the CBF genes are commonly associated with leukemia. The t(8:21) fuses the AML1 gene to ETO (AE) and is predominantly observed in leukemias of the FAB M2 subtype of acute myeloid leukemia (AML). The t(16:16) and inv(16) are associated with FAB M4Eo leukemias and result in fusion of the CBFβ gene to MYH11 (CM). While knockin of either AE or CM is not sufficient to cause disease, expression of either protein increases the incidence of leukemia in mice when combined with mutagens or specific secondary genetic manipulations. We have previously shown that retroviral transduction of primary human CD34+ cells with AE causes a dramatic accumulation of CD34+ progenitor cells with increased CFU-C and CAFC activity during an increased lifespan in vitro. These cultures retain the ability to differentiate to myeloid, lymphoid, and erythroid lineages and the ability to engraft NOD/SCID mice. Consistent with mouse studies, these cells fail to cause disease in mice or become transformed in vitro. These cultures serve as a preleukemic model for human AML associated with AML1-ETO expression. We show here that retroviral transduction of CM into human CD34+ hematopoietic cells similarly mimics a putative pre-leukemic condition initiated by inv(16). Transduced cells proliferate for up to 7 months in culture with retention of CD34 expression on a subset of cells. Cells from CM cultures display a more mature morphology than AE cells, consistent with the association with M4Eo AMLs, and have a greatly reduced ability to differentiate to the erythroid lineage in methylcellulose assays. Nevertheless, long-term CM cultures retain CFU-C activity, the ability to engraft NOD/SCID-B2M mice, and the potential for B-cell development in vitro, implicating a primitive progenitor as the target cell rather than a committed granulocyte/monocyte progenitor. Microarray analysis of AML leukemias has identified specific genes with altered expression in inv(16) patient samples. We have found that a number of these inv(16)-associated transcripts are likewise altered in CD34+ cells from our CM cultures when compared to control CD34+ cells, while others are not affected. These data suggest that our system will be useful in examining the effects of CBFβ-MYH11 on gene expression in the human preleukemic cell, in characterizing the effect of this oncogene on human stem cell biology and in defining its contribution to the development of leukemia.

Cooperative binding of Ets-1 and core binding factor to DNA.

Two phorbol ester-inducible elements (beta E2 and beta E3) within the human T-cell receptor beta gene enhancer each contain consensus binding sites for the Ets and core binding factor (CBF) transcription factor families. Recombinant Ets-1 and purified CBF bound individually to beta E2 and beta E3, in which the Ets and core sites are directly adjacent. In this report, we show that CBF and Ets-1 bind together to beta E2 and beta E3 and that Ets-1-CBF-DNA complexes are favored over the binding of either protein alone to beta E2. Formation of Ets-1-CBF-DNA complexes increased the affinity of Ets-1-DNA interactions and decreased the rate of dissociation of CBF from DNA. Ets-1-CBF-DNA complexes were not observed when either the Ets or core site was mutated. The spatial requirements for the cooperative interaction of Ets-1 and CBF were analyzed by oligonucleotide mutagenesis and binding site selection experiments. Core and Ets sites were coselected, and there appeared to be little constraint on the relative orientation and spacing of the two sites. These results demonstrate that CBF and Ets-1 form a high-affinity DNA-binding complex when both of their cognate sites are present and that the relative spacing and orientation of the two sites are unimportant. Ets and core sites are found in several T-cell-specific enhancers, suggesting that this interaction is of general importance in T-cell-specific transcription.