1. DNA Topoisomerases maintain promoters in a state competent for transcriptional activation in Saccharomyces cerevisiae. 21 June 2013 Ph.D. student Jacob Fredsøe Laboratory of Genome Research Supervisor: Anni Hangaard Andersen 1

2. Introduction Topoisomerases are enzymes which catalyzes the relaxation of supercoiled DNA 2 Topoisomerase I J.J. Champoux et al. “Science. 1998 Mar 6;279(5356):1534-41.” Topoisomerase II J. C. Wang et al. “Nature. 1996 Jan 18;379(6562):225-32.”

3. Introduction 3 Koster, D.A., Croquette, V., Dekker, C., Shuman, S. & Dekker, N.H. 2005, "Friction and torque govern the relaxation of DNA supercoils by eukaryotic topoisomerase IB", Nature, vol. 434, no. 7033, pp. 671-674. Topoisomerase I Topoisomerase II

4. Introduction According to the twin-supercoiled-domain- model, topological challenges will arise during transcription. 4

5. Topoisomerase dependency is reflected by transcriptional activity The transcriptional response to lack of topoisomerases in S. cerevisiae was investigated using microarray technology 5

6. Genes dependent on topoisomerases are regulated on a chromatin level 6

7. Topoisomerases are required for transcriptional induction of a range of inducible genes Selected a number of genes which had high transcriptional plasticity and sensitivity to chromatin regulation Tested their requirement for topoisomerases 7

8. PHO5 is regulated by a change in chromatin structure 8 Induced by lack of phosphate Cytoplasm Nucleus

9. PHO5 requires topoisomerases during transcriptional activation mRNA levels are measured by qPCR on cDNA 9

10. Topoisomerases are required for Pho4p binding prior to promoter nucleosome removal during PHO5 activation Nucleosome ChIP 10 Pho4 ChIP

11. Lack of induction is caused by a perturbance of promoter superhelicity 11 TopA preferential relax negative superhelicity Gyrase preferential relax positive superhelicity The loss of PHO5 induction capabilities is most likely caused by a change in promoter superhelicity Time after phosphate depletion (minutes) PHO5 mRNA levels (induced/uninduced) log2 wild type top1∆top2ts top1∆top2ts + topA top1∆top2ts + gyrase S. cerevisiae Topoisomerase I and II - - + + E. coli gyrase E. coli TopA

12. Acknowledgements 12 Supervisor: Anni Hangaard Andersen Jakob Madsen Pedersen The entire LGR lab You for listening

13. Questions 13