Unique Role of Med8 in Ace2 Recruitment and Target Gene Expression in <i>Schizosaccharomyces pombe</i>

The Mediator, an essential RNA polymerase II coactivator, is a conserved multi-subunit protein complex present in organisms ranging from yeast to humans. Although its role in transcription is well-characterized, the distinct functions of its subunits remain largely unclear. In this study, we aimed to investigate the roles of Med8, Med14, and Med17 in Ace2-dependent transcriptional regulation in <i>Schizosaccharomyces pombe</i>. Transcriptome analysis revealed that depletion of Med14 and Med17 caused widespread transcriptional repression, consistent with their structural roles in maintaining Mediator integrity. In contrast, depletion of Med8 specifically impaired the transcription of Ace2 target genes. Chromatin-binding analysis revealed that despite stable Ace2 protein levels, Med8 depletion led to a remarkable decrease in Ace2 occupancy at the target promoters, indicating that Med8 plays a crucial role in the recruitment of Ace2. In contrast, the binding of Ace2 was largely unaffected by the depletion of Med14 or Med17, underscoring their involvement in the transcriptional activation steps following Ace2 recruitment. Moreover, depletion of Med8 did not influence the expression or genome binding of Med14 and Med17, suggesting that Med8 plays a distinct role, rather than maintaining Mediator stability. Co-immunoprecipitation further revealed a physical association between Med8 and Ace2, suggesting that Med8 may be involved in Ace2-dependent transcriptional regulation. Overall, our findings highlighted that Med8 uniquely promotes Ace2-dependent transcriptional initiation by enhancing the recruitment of transcription factors. The study could advance our understanding of how individual Mediator subunits fine-tune gene expression through direct interactions with specific transcription factors.