Mutagenesis of the Runt Domain Defines Two Energetic Hot Spots for Heterodimerization with the Core Binding Factor β Subunit

Abstract

Core-binding factors (CBFs) 1 are a small family of heterodimeric transcription factors that play critical roles in development and in human disease. CBFs contain a DNA-binding CBF␣ subunit and a CBF␤ subunit that does not contact DNA directly (1-3). Three related genes encode CBF␣ subunits: RUNX1 (CBFA2/AML1/Pebpa2b), RUNX2 (CBFA1/AML3/ Pebpa2a), and RUNX3 (CBFA3/AML2/Pebpa2c). The common CBF␤ subunit is encoded by one gene, CBFB. RUNX1 and CBFB are required for hematopoiesis and are frequently targeted by chromosomal rearrangements and point mutations in human leukemia (4 -9). RUNX2 and CBFB are required for bone development, and mutations in RUNX2 cause the human skeletal disorder cleidocranial dysplasia (10 -15). Disruption of the Runx3 gene in mice causes hyperplasia of gastric mucosa (16) and severe limb ataxia (17,18). Runx3 is thought to be required for the controlled proliferation and apoptosis of gastric epithelial cells, the survival of dorsal root ganglia proprioreceptive neurons, and for epigenetic silencing of the CD4 gene in cytotoxic T lymphocytes (16 -19). Hemizygous deletion and hypermethylation of RUNX3 is found in a significant number of primary gastric cancers (16).The DNA binding domain in CBF␣ is known as the "Runt domain" (20). The Runt domain is responsible for DNA binding as well as for heterodimerizing with the CBF␤ subunit (2, 20, 21). The Runt domain of the CBF␣ subunits is an s-type immunoglobulin fold in the p53 family of DNA-binding transcription factors, whose other members include STAT3␤, p53, NF-B, NFAT, and the Brachyhury T box family of proteins (22)(23)(24). DNA binding by the Runt domain is mediated by loops and ␤-strands at one end of the immunoglobulin ␤-barrel (25, 26). The DNA is bent toward the protein by ϳ20°(25-27). CBF␤ increases the DNA binding affinity of the Runt domain by ϳ7-10-fold (25, 28). CBF␤ binds to a region of the Runt domain distinct from the DNA-binding sequences and contacts neither the DNA nor amino acids in the Runt domain that are directly involved in DNA binding (25,26,29,30).Comparison of the Runt domain-DNA and Runt domain-CBF␤-DNA complexes (25,26,31) with the recently determined crystal structures of the Runt domain in the free state (30, 32) suggests a model for allosteric regulation of DNA binding by CBF␤. The Runt domain contains several regions that are likely to equilibrate between at least two conformations in the free state, including the ␤C-D, ␤EЈ-F, and ␤G-GЈ loops. CBF␤ appears to stabilize a specific conformation of the Runt domain. The most dramatic change occurs in the ␤G-GЈ loop that assumes two conformations in the free and complexed state crystal structures and has been referred to as the "Sswitch" (32). The dramatic change in the ␤G-GЈ loop and the changes in the ␤C-D loop are in agreement with an earlier study by NMR chemical shift perturbation that showed dramatic changes in the C␣ chemical shift for residues in the ␤G-GЈ loop indicative of a significant conformational change (28).Previous studies in our laboratory and oth...

PRMT1 interacts with AML1-ETO to promote its transcriptional activation and progenitor cell proliferative potential

“…However, it has been reported that R142A mutation weakens DNA-binding ability of the RUNT domain without disturbing its overall structure and affinity to CBF␤. 43,44 In fact, we also have data to support the negative effect of R142A mutation on DNA binding of AE9a (supplemental Figure 6B) as well as cell replating capability (supplemental Figure 6C). The negative effect of R142A mutation on AE9a-driven transactivation or transformation may be contributed by methylation deficiency and/or weaker DNA affinity.…”

RUNX1 DNA-Binding Mutants, Associated with Minimally Differentiated Acute Myelogenous Leukemia, Disrupt Myeloid Differentiation

“…It is important to note that neither of the two CBFh-binding mutations tested disrupted RDB function. Due to its direct hydrogen bonding interactions with CBFh, the T161A mutation would be expected to specifically disrupt binding to CBFh but not disrupt the overall Runt domain structure (15,33,36). In contrast, however, the N109D leads to a more radical disruption by perturbing the overall folding architecture of the Runt domain (15,33,36,37).…”

“…To investigate the possibility that the RDB effect on replating and immortalization was due to sequestering of CBFh and thus resulting in the reduced activity of Runx1 (or one of its homologues), we mutated either one or both of two amino acid residues (T161 and N109), which specifically reduce CBFh equilibrium binding constants by 40-and 60-fold, respectively (33). The activity of either K83N or wt Runx1 proteins containing one of these mutations was determined.…”

“…Due to its direct hydrogen bonding interactions with CBFh, the T161A mutation would be expected to specifically disrupt binding to CBFh but not disrupt the overall Runt domain structure (15,33,36). In contrast, however, the N109D leads to a more radical disruption by perturbing the overall folding architecture of the Runt domain (15,33,36,37). This has been shown to lead to loss not only of CBFh binding but also of low-affinity DNA binding (15,25,37) and thus may also disrupt other protein-protein interactions occurring within the Runt domain.…”

ETO, but Not Leukemogenic Fusion Protein AML1/ETO, Augments RBP-Jκ/SHARP-Mediated Repression of Notch Target Genes

“…Different potential scenarios might explain our results at the molecular level. (i) The Runx domain of AML1 is the binding domain of CBF␤, and this heterodimer has been characterized extensively (47). The heterodimer of AML1/ETO and CBF␤ could potentially activate Notch target genes.…”