Highly conserved features of DNA binding between two divergent members of the Myb family of transcription factors.

Bas1p, a divergent yeast member of the Myb family of transcription factors, shares with the proteins of this family a highly conserved cysteine residue proposed to play a role in redox regulation. Substitutions of this residue in Bas1p (C153) allowed us to establish that, despite its very high conservation, it is not strictly required for Bas1p function: its substitution with a small hydrophobic residue led to a fully functional protein in vitro and in vivo. C153 was accessible to an alkylating agent in the free protein but was protected by prior exposure to DNA. The reactivity of cysteines in the first and third repeats was much lower than in the second repeat, suggesting a more accessible conformation of repeat 2. Proteolysis protection, fluorescence quenching and circular dichroism experiments further indicated that DNA binding induces structural changes making Bas1p less accessible to modifying agents. Altogether, our results strongly suggest that the second repeat of the DNA-binding domain of Bas1p behaves similarly to its Myb counterpart, i.e. a DNA-induced conformational change in the second repeat leads to formation of a full helix-turn-helix-related motif with the cysteine packed in the hydrophobic core of the repeat.

Nitric oxide (NO) disrupts specific DNA binding of the transcription factor c-Myb in vitro.

In an attempt to elucidate signal transduction pathways which may modulate DNA binding of the transcription factor c-Myb, we investigated whether c-Myb could be a target for the signaling molecule nitric oxide (NO) in vitro. NO-generating agents severely inhibited specific DNA binding of the c-Myb minimal DNA-binding domain R2R3. This inhibition was readily reversible upon treatment with excess DTT. A redox-sensitive cysteine (C130) was required for this NO sensitivity. Moreover, a DNA-binding domain carrying two of the avian myeloblastosis virus (AMV)-specific mutations (L106H, V117D) appeared to be less sensitive to S-nitrosylation than the wild-type c-Myb. This difference in NO sensitivity may influence the regulation of wild type versus AMV v-Myb protein function.

Oncogenic point mutations induce altered conformation, redox sensitivity, and DNA binding in the minimal DNA binding domain of avian myeloblastosis virus v-Myb.

c-Myb is the founder member of a class of transcription factors with tryptophan-rich repeats responsible for DNA binding. Activated oncogenic forms of Myb are encoded by the avian retroviruses, avian myeloblastosis virus (AMV) and E26. AMV v-Myb encodes a truncated protein with 11 point mutations relative to c-Myb. The mutations in the DNA binding domain (DBD) were reported to impose distinct phenotypes of differentiation on transformed myeloid cells (Introna, M., Golay, J., Frampton, J., Nakano, T., Ness, S. A., and Graf, T. (1990) Cell 63, 1287-1297). The molecular mechanism operating has remained elusive since no change in sequence specificity has been found. We introduced AMV-specific point mutations in the minimal DBD of chicken c-Myb and studied their effect on structure and function of the purified protein. Fluorescence emission spectra and fluorescence quenching experiments showed that the AMV-specific point mutations had a significant effect on the conformation of the DBD, giving rise to a more compact structure, a change that was accompanied by a reduced sensitivity toward cysteine-specific alkylation and oxidation. The DNA binding properties were also altered by the AMV-specific point mutations, leading to protein-DNA complexes with highly reduced stability. This reduction in stability was, however, more severe with certain subtypes of binding sequences than with others. This differential behavior was also observed in an in vivo model system where DBD-VP16 fusions were coexpressed with various reporters. These findings imply that different subsets of Myb-responsive promoters may react differentially toward the AMV-specific mutations, a phenomenon that could contribute to the altered patterns of gene expression induced by the AMV v-Myb relative to wild type c-Myb.

Flexibility in the second half-site sequence recognised by the c-Myb R2 domain--in vitro and in vivo analysis.

The oncoprotein c-Myb is a transcription factor that recognises its specific target sequences through two subdomains. The R3-domain binds the first half-site, YAAC, and plays a dominant role in sequence recognition, while the homologous R2-domain interacts with a more loosely defined sequence in the second half-site. The difficulty in precisely defining a preferred second half-site sequence might reflect the flexible nature of R2 which only attains its fully folded structure upon binding to DNA, a process that might allow the protein to adapt to different half-site sequences. Here we report that shifting the most conserved base in the second half-site, the G6, into position 5 resulted only in a minor reduction of complex stability in vitro. From an analysis of a series of second half-site variants by EMSA and DMS-interference, we conclude that the preferred recognition sequence should be revised to read [YAACNG or YAACGN]. Modeling the structure of c-Myb R2R3 in complex with a GT half-site variant revealed specific interactions with G5. When second half-site variants were tested in vivo using a sensitive yeast effector-reporter system, both the TG and GT half-site variants were functional mediating c-Myb-dependent transactivation. Unexpectedly, we observed large differences between the best second half-site variants at low levels of c-Myb-effector, the GG variant being five- to fifteen-fold more active in vivo than the single-G half-sites, the GH or HG variants.