The c-myb protooncogene encodes a sequence-specific DNA-binding protein (c-Myb) that induces transcriptional activation or repression. We have identified three functional domains of the mouse c-Myb protein that are responsible for DNA binding, transcriptional activation, and negative regulation, respectively. In addition to the DNAbinding domain, which is located near the N terminus, an adjacent region (the transcriptional activation domain) containing about 80 amino acids was found to be essential for transcriptional activation. Deletion of a region spanning about 175 amino acids of the C-proximal portion increased transcriptional activation markedly,-revealing that this domain normally represses activation. Differences between the transcriptional activation and repression functions of c-Myb and v-Myb are discussed in the light of these functional domains. Our results suggest that transcriptional activation may be involved in transformation by myb gene products. When the sequences of the MBS-I and MBS-II sites were compared, 11 of 19 base pairs (bp) were identical. MBS-I is a high-affinity site and was shown to be a c-Myb-dependent enhancer element. MBS-II is a low-affinity site, and tandem repeats of the sequence containing this site induce cMyb-dependent transcriptional repression (unpublished results). Here we report the identification of three functional domains of c-Myb: a DNA-binding domain, a transcriptional activation domain, and a negative regulatory domain. The functional differences between c-Myb and v-Myb are discussed in the light of the structures of these functional domains.MATERIALS AND METHODS Plasmid Construction. The effector plasmids pact-c-myb, in which the 5' regulatory region of the chicken cytoplasmic 13-actin gene is linked to the mouse c-myb gene, and pactl, which was constructed by deletion of the c-myb sequence from pact-c-myb, have been described (14). The reporter plasmids pMFcolCAT6MBS-I and pMFcolCAT6SV-II contain six tandem repeats of the MBS-I and the SV-II sequence, respectively, in the BamHI site of the plasmid pMFcolCAT, in which the bacterial chloramphenicol acetyltransferase (CAT) gene is linked to the mouse a2(I)-collagen promoter. The SV-II sequence contains the MBS-II site and corresponds to positions 184-218 in the SV40 genome. All plasmids designed to express mutant c-Myb proteins in cultured cells were generated from the plasmid pact-c-myb. To make the CT1, CT5, CT6, and CT7 mutants, termination codons were introduced at nucleotides 1537, 616, 463, and 307, respectively, by site-specific mutagenesis as described by Kunkel et al. (15). Nucleotide numbers are as in ref. 5. To obtain the NT1, NT3, and NT4 mutants, the sequence recognized by the restriction enzyme Nco I was introduced at nucleotides 147, 2%, and 455, respectively, by site-specific mutagenesis, and the regions between the introduced Nco I sites and the Nco I site at nucleotide 36 that overlaps the normal c-myb initiation codon were deleted. In-frame deletion mutants ADB, ANR, and ATA were construc...

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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.

The DNA-binding domain of Myb consists of three imperfect repeats, R1, R2 and R3, each containing a helix-turn-helix motif variation. Among these repeats, R2 has distinct characteristics with high thermal instability. The NMR structure analysis found a cavity inside the hydrophobic core of R2 but not in R1 or R3. Here, we show that R2 has slow conformational fluctuations, and that a cavity-filling mutation which stabilizes the R2 structure significantly reduces specific Myb DNA-binding activity and trans-activation. Structural observations of the free and DNA-complexed stages suggest that the implied inherent conformational flexibility of R2, associated with the presence of the cavity, could be important for DNA recognition by Myb.