To activate eukaryotic genes, several pathways which modify chromatin and recruit general factors of the transcriptional machinery are utilized. We investigated the factors required for activation of yeast phospholipid biosynthetic genes, depending on activator protein Ino2 which binds to the inositol/choline-responsive element (ICRE) upstream promoter motif together with its partner protein Ino4. We used a set of 15 strains each defective for one of the non essential subunits of yeast mediator complex and identified med2, med3, med15, med18 and med19 as impaired for inositol biosynthesis. In these mutants, ICRE-dependent gene activation was reduced to 13-22% of the wild-type level. We also demonstrate synthetic growth and activation defects among mediator mutants and mutants lacking defined histone modifications (snf1, gcn5) and transcriptional coactivators (sub1). Analysis of mutants defective for histone methylation (set1, set2 and dot1) and demethylation (jhd1, jhd2, gis1, rph1 and ecm5) revealed the importance of the H3 Lys36-specific Set2 methyltransferase for ICRE-dependent gene expression. Although defined mediator subunits are critical for gene activation, we could not detect their interaction with Ino2. In contrast, Ino2 directly binds to the Set2 histone methyltransferase. Mapping of interaction domains revealed the importance of the SET core domain which was necessary and sufficient for binding Ino2.
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