The role of Med15 sequence features in transcription factor interactions

Abstract

Med15 is a general transcriptional regulator and subunit within the tail module of the RNA Pol II Mediator complex. The S. cerevisiae Med15 protein has a well-structured N-terminal KIX domain, three Activator Binding Domains (ABDs), several naturally variable polyglutamine (poly-Q) tracts (Q1, Q2, Q3) embedded in an intrinsically disordered central region, and a C-terminal Mediator Association Domain (MAD). We investigated how the presence of ABDs and changes in length and composition of poly-Q tracts influences Med15 activity and function using phenotypic, gene expression, transcription factor interaction and phase separation assays of truncation, deletion, and synthetic alleles. We found that individual Med15 activities were influenced by the number of activator binding domains (ABDs) and adjacent polyglutamine tract composition. Robust Med15 activity required at least the Q1 tract and the length of that tract modulated activity in a context-dependent manner. We found that loss of Msn2-dependent transcriptional activation due to Med15 Q1 tract variation correlated well with a reduction in Msn2:Med15 interaction strength, but that interaction strength did not always mirror the propensity for phase separation. We also observed that distant glutamine tracts and Med15 phosphorylation affected the activities of the KIX domain, suggesting that intramolecular interactions may affect some Med15-transcription factor interactions. Further, two-hybrid based interaction studies revealed intramolecular interactions between the N-terminal KIX domain and the Q1R domain of Med15. Author SummaryGlutamine tracts are relatively uncommon, but are a feature of many transcriptional regulators including the Med15 subunit of the Mediator Complex which is a large protein complex that plays an important role in gene expression in eukaryotic organisms including yeast and animals. Strains lacking Med15 are compromised in their ability to grow on many kinds of media, under stress conditions, and in fermentation, reflecting its importance in gene expression. Naturally occurring yeast strains specialized for growth in specific environments (e.g., wine, beer, clinical) vary in their glutamine tract lengths, suggesting that the length of glutamine tracts may influence Med15 function in a manner that is adaptive for a specific environment. In this study, we intentionally manipulated the length of the glutamine tracts in Med15 and found that these changes have subtle effects on Med15 interactions with transcription factors, target gene expression and growth. Taken together, our data suggests that glutamine tracts do not themselves mediate critical interactions with partner proteins, but instead may influence the shape of the Med15 protein, thus indirectly affecting the nature of these interactions.