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21 June 2013 Ph.D. student Jacob Fredsøe Laboratory of Genome Research

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. Introduction.

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21 June 2013 Ph.D. student Jacob Fredsøe Laboratory of Genome Research

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  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: AnniHangaard Andersen

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

  3. Introduction Topoisomerase II Topoisomerase I 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.

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

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

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

  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

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

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

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

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

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

  13. Questions

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