DNA Topoisomerases Maintain Promoters in a State Competent for Transcriptional Activation in Saccharomyces cerevisiae

To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in Saccharomyces cerevisiae cells deficient for topoisomerases I and II and performed single-gene analyses to support our findings. The genome-wide studies show a general transcriptional down-regulation upon lack of the enzymes, which correlates with gene activity but not gene length. Furthermore, our data reveal a distinct subclass of genes with a strong requirement for topoisomerases. These genes are characterized by high transcriptional plasticity, chromatin regulation, TATA box presence, and enrichment of a nucleosome at a critical position in the promoter region, in line with a repressible/inducible mode of regulation. Single-gene studies with a range of genes belonging to this group demonstrate that topoisomerases play an important role during activation of these genes. Subsequent in-depth analysis of the inducible PHO5 gene reveals that topoisomerases are essential for binding of the Pho4p transcription factor to the PHO5 promoter, which is required for promoter nucleosome removal during activation. In contrast, topoisomerases are dispensable for constitutive transcription initiation and elongation of PHO5, as well as the nuclear entrance of Pho4p. Finally, we provide evidence that topoisomerases are required to maintain the PHO5 promoter in a superhelical state, which is competent for proper activation. In conclusion, our results reveal a hitherto unknown function of topoisomerases during transcriptional activation of genes with a repressible/inducible mode of regulation.
Author Summary Gene expression is controlled at many different levels to assure appropriate responses to internal and environmental changes. The effect of topological changes in the DNA double helix on gene transcription in vivo is a poorly understood factor in the regulation of eukaryotic gene expression. Topological changes are constantly generated by DNA tracking processes and may influence gene expression if not constantly removed by DNA topoisomerases. For decades it has been generally accepted that these enzymes regulate transcription by removing excess topological strain generated during tracking of the RNA polymerase, but we still lack a more holistic view of how these enzymes influence gene transcription in their native environment. Here, we examine both global and gene-specific changes in transcription following lack of DNA topoisomerases in budding yeast. Taken together, our findings show that topoisomerases play a profound role during transcriptional activation of genes with a repressible/inducible mode of regulation. For the PHO5 gene, which is investigated in more detail, we demonstrate that topoisomerases are required for binding of a transcription factor, which is crucial for promoter opening during PHO5 activation. Our data thus suggest that inducible gene promoters are highly sensitive to changes in DNA superhelicity.