Dissecting the transactivation domain (tAD) of the transcription factor c-Myb to assess recent models of tAD function.

Transcription factors use a DNA-binding domain to localize their action and a transactivation domain (tAD) to stimulate activation of the associated gene. Recent work has renewed interest in how tADs activate genes, which remains poorly understood. Key features in the new models are exposure of short linear motifs (SLMs) and liquid-liquid phase separation (LLPS). Inspired by the new models for tAD function, we decided to revisit the tAD of the haematopoietic transcription factor c-Myb by performing a mutational analysis to see how these new models fit and potentially explain the tAD behaviour of this master regulator. We know that c-Myb has an acidic tAD, which contains a well-characterized SLM in the form of a LxxLL motif. By testing 12 alanine-scanning mutants and three mutants with major reorganization of its tAD in two mammalian reporter systems, we found a pattern of effects very close to what would be expected from the SLM-exposure model, with strong effects exerted by both acidic replacements and SLM mutation. When the same mutants were tested in a yeast system, the pattern of effects was dramatically different, with the SLM mutation exerting no effect, and tAD behaviour was much less affected by small alterations, as would be expected from a LLPS model. These observations are discussed in the light of the two new tAD models, and a two-step hypothesis for transactivation, combining both models, is proposed.