Structural Analyses of DNA Recognition by the AML1/Runx-1 Runt Domain and Its Allosteric Control by CBFβ

Abstract

The core binding factor (CBF) heterodimeric transcription factors comprised of AML/CBFA/PEBP2alpha/Runx and CBFbeta/PEBP2beta subunits are essential for differentiation of hematopoietic and bone cells, and their mutation is intimately related to the development of acute leukemias and cleidocranial dysplasia. Here, we present the crystal structures of the AML1/Runx-1/CBFalpha(Runt domain)-CBFbeta(core domain)-C/EBPbeta(bZip)-DNA, AML1/Runx-1/CBFalpha(Runt domain)-C/EBPbeta(bZip)-DNA, and AML1/Runx-1/CBFalpha(Runt domain)-DNA complexes. The hydrogen bonding network formed among CBFalpha(Runt domain) and CBFbeta, and CBFalpha(Runt domain) and DNA revealed the allosteric regulation mechanism of CBFalpha(Runt domain)-DNA binding by CBFbeta. The point mutations of CBFalpha related to the aforementioned diseases were also mapped and their effect on DNA binding is discussed.

TheC. elegansCBFβ homologue BRO-1 interacts with the Runx factor, RNT-1, to promote stem cell proliferation and self-renewal

“…The RUBY assay indicates a direct interaction between BRO-1 and RNT-1 and EMSA studies demonstrate that BRO-1 is required for robust binding of RNT-1 to the Runx consensus DNA binding site, consistent with other studies of CBF␤ function (Bushweller, 2000;Li et al, 2003;Nagata and Werner, 2001;Tahirov et al, 2001;Yan et al, 2004). Furthermore, our experiments show that BRO-1 3913 RESEARCH ARTICLE CBF␤ factor in C. elegans development dramatically increases the specificity of RNT-1-DNA binding, thereby extending previous studies of CBF␤/Runx interactions.…”

“…1) (reviewed by Kagoshima et al, 2007). In addition to the low overall homology, it also lacks the conserved region in fly and mammalian CBF␤ corresponding to the sequences between the beta sheets, ␤3 and ␤6, which might be expected to interfere with the positioning of the Runx interaction domain around ␤3 and ␤5 (Tahirov et al, 2001) (Fig. 1B).…”

“…However, the L2-L3 V lineage trace shown in Fig. 3C indicates that the penetrance of division defects is slightly higher in double mutant animals than 3907 RESEARCH ARTICLE CBF␤ factor in C. elegans development (Tahirov et al, 2001), indicating the RUNX1-interacting regions, and including the particular amino acids that would be expected to participate in the direct interaction. is the case for either single mutant.…”

“…Next we tested the rnt-1(e1241) mutant protein RNT-1 Runt domain I 112 K (substitution of Lys for Ile 112 ). The I 112 residue of RNT-1 is equivalent to V 152 of murine Runx1, which is located within the exposed ␤ sheet region of Runx1, known as ␤10, situated at the Runx1-CBF␤ heterodimerisation interface (Tahirov et al, 2001). However, alanine scanning mutagenesis experiments have suggested a role for V 152 in DNA binding, rather than CBF␤ interaction (Li et al, 2003).…”

Genetic evidence of PEBP2β-independent activation of Runx1 in the murine embryo

“…The phenotype of PEBP2b -/-mice is almost identical to that of Runx1 -/-mice [9][10][11], which suggests that PEBP2b is required for Runx1 function in vivo. While PEBP2b does not bind to DNA, it increases the DNAbinding affinity of Runx1 in an allosteric manner, thus elevating the transcriptional activity of the PEBP2/CBF complex [12,13]. However, it has also been shown that the Runx1/PEBP2b heterodimer does not form readily.…”