Core binding factor

More than twenty years ago, Nancy Speck and David Baltimore identified a DNA binding activity that bound to the core site (TGTGGTAA) in the enhancer of Moloney Murine Leukemia Virus that, when mutated, altered disease specificity to produce thymic leukemia instead of erythroleukemia [16, 17]. This DNA binding activity, which was named Core Binding Factor, was identified in a variety of cell lines [16]. Dr. Speck's laboratory purified several peptides that had core- binding activity from calf thymus nuclei [18]. The Speck laboratory then went on to sequence 5 peptides and used these sequences to isolate 3 cDNA

clones from a murine thymus library that encoded the three mammalian isoforms of CBFp (CBFp p22.0, CBFp p21.5, and CBFp p17.6). [19]. The Speck study demonstrated that CBFp did not bind to DNA itself but, instead, partnered with a DNA binding protein, at that time termed acute myeloid leukemia-1 (AML-1), since one of their peptides appeared to be contained in the bovine homologue of the human AML-1. AML-1 had been identified by virtue of its involvement in the t(8;21) chromosomal translocation in 1991 [20]. A similar DNA binding activity was also isolated via interaction with the polyomavirus enhancer and was called polyomavirus enhancer binding protein 2 (PEBP2) [21]. CBF also binds to the Type B leukemogenic virus enhancer [22]. In 1993, Scott Hiebert's laboratory demonstrated that AML-1 selected a site related to the enhancer core motif (TGT/cGGT) and identified the DNA binding domain [23]. Later, Dr. Hiebert's group identified a larger isoform of AML-1 (termed AML-1B) produced from the AML-1 gene using a homology screen of a human B-cell library [24]. Two other AML-1 family members expressed from independent genes were identified; AML-2 and AML-3 [25]. Following these studies, the AML-1 family of proteins underwent a revision in nomenclature with guidance from the Human Genome Organization [26]. AML-1 is now termed RUNX1, AML-2 is now termed RUNX3, and AML-3 is now termed RUNX2. The murine nomenclature is written in small case. This nomenclature will be used for the remainder of the chapter.

Mammalian CBF is a heterodimeric complex consisting of RUNX1, RUNX2, or RUNX3. As the Speck laboratory suggested, these three proteins bind to promoters and enhancers of target genes (or viral LTRs) as a heterodimer with CBFp [10, 27]. DNA binding is achieved with a central domain (runt domain), consisting of an S-type immunoglobulin fold resembling the DNA binding domains of p53 and NF-kB [23, 28]. Although CBFp does not contact DNA it regulates and enhances RUNX protein DNA binding via interactions with the Runt domain [28]. Complexity in CBF-regulated transcription comes about not only through co-expression in many tissues and a highly conserved DNA binding domain and recognition sequence, but also through the existence of multiple isoforms. For example, the RUNX1 gene produces three main isoforms, all of which contain the DNA binding domain. These isoforms are thought to have both overlapping and unique functions. For example, RUNX1 isoforms are differentially expressed during hematopoietic differentiation of human embryonic stem cells (ESCs) and the RUNX1c isoform is expressed at the time of emergence of definitive HSCs [29]. Such complexity makes it difficult to assign function to each RUNX isoform and clearly, we are just at the beginning of understanding the distinct roles played by each protein. CBFp is encoded on one gene in mammals but, as noted above, multiple isoforms are produced that may have distinct functions [19].

CBF is conserved in all multicellular organisms examined but is not present in yeast or any nonmetazoan studied to date. RUNX and CBFp genes were identified in the nematode C. elegans, the fruit fly Drosophila melanogaster, which contains two CBFp genes and four RUNX genes, the sea urchin (Strongylocentrotus purpuratus), sponges, puffer fish (Takifugu rubripes), and the zebrafish (Danio rerio) [30-32]. In Drosophila, RUNT, the first RUNX gene identified in that organism, is required for segmentation [33]. RUNT gene mutations produce fly embryos with segmentation defects while Lozenge, a second RUNX gene in fruit flies, is required for eye development (Coffman 2009). In sea urchin, the spRunt-1 gene is required throughout development for cellular proliferation, cell survival, and tissue-specific gene expression [30]. Unlike mammals, two CBFp homologs exist in Drosophila. Big brother and Brother (Bgb and Bro) display high homology to human CBFp and are required for RUNX gene function in flies [34]. Studies in these model organisms have clearly demonstrated that CBF coordinates cellular proliferation, stem cell fate and terminal differentiation [30, 35].

Mouse genetics further demonstrate specific requirements for CBF in development and stem cell function. For example, RUNX1 is required for hematopoietic development and Runxl null animals die in utero by day E12.5 due to a complete absence of fetal-liver derived hema-topoiesis [36]. Runx2 is critical for skeletal morphogenesis and Runx2 null mice survive until birth but die shortly thereafter due to a complete lack of bone formation [37]. Interestingly, Runx1 and Runx3 are also expressed in bone cells and support skeletal development [27, 38]. Runx3 null mice were reported to display gut hyperplasia due to an increase in cell proliferation and a reduced rate of apoptosis [39]. However, a second study showed that Runx3-deficient mice develop severe limb ataxia due to a defect in the dorsal root ganglion (DRG) proprioceptive neurons [40]. Runx3 is also important for hematopoiesis [27, 41]. Similar genetic studies demonstrated that CBFp is required for RUNX protein function. For example, CBFp knockdown mice recapitulate the Runxl null phenotype and hematopoietic-specific rescue of CBFp null animals has demonstrated that CBFp, like Runx2, is required for skeletal development [42, 43]. Thus, CBF functions as a master regulator of genes required for development, differentiation and stem cell maintenance [44, 45]. The requirement for CBFp is likely due to it's ability to enhance RUNX DNA binding and, therefore, to augment the transcriptional strength of the RUNX factors [46].

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