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DNA Topoisomerases maintain promoters in a state competent for transcriptional

activation in Saccharomyces cerevisiae.

21 June 2013Ph.D. student Jacob Fredsøe

Laboratory of Genome ResearchSupervisor: Anni Hangaard Andersen

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Introduction• Topoisomerases are enzymes which catalyzes

the relaxation of supercoiled DNA

Topoisomerase IJ.J. Champoux et al. “Science. 1998 Mar

6;279(5356):1534-41.”

Topoisomerase IIJ. C. Wang et al. “Nature. 1996 Jan

18;379(6562):225-32.”

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Introduction

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

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Introduction• According to the twin-supercoiled-domain-

model, topological challenges will arise during transcription.

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Topoisomerase dependency is reflected by transcriptional activity

• The transcriptional response to lack of topoisomerases in S. cerevisiae was investigated using microarray technology

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Genes dependent on topoisomerases are regulated on a chromatin level

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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

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PHO5 is regulated by a change in chromatin structure

• Induced by lack of phosphate

Cytoplasm

Nucleus

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PHO5 requires topoisomerases during transcriptional activation

• mRNA levels are measured by qPCR on cDNA

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Topoisomerases are required for Pho4p binding prior to promoter nucleosome removal during

PHO5 activation

• Nucleosome ChIP • Pho4 ChIP

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Lack of induction is caused by a perturbance of promoter superhelicity

• 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)PHO

5 m

RNA

leve

ls (i

nduc

ed/u

nind

uced

) log

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wild type

top1∆top2ts

top1∆top2ts + topA

top1∆top2ts + gyrase

S. cerevisiaeTopoisomerase I and II

- - - - + + + +

E. coligyrase

E. coliTopA

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Acknowledgements

• Supervisor: Anni Hangaard Andersen

• Jakob Madsen Pedersen

• The entire LGR lab

• You for listening

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Questions