The importance of topoisomerases for chroman regulated genes AARHUS UNIVERSITY Fredsoe J, Pedersen JM, Roedgaard M, Andersen AH Laboratory of Genome Research, Department of Molecular Biology and Genecs, University of Aarhus, Denmark. DNA topoisomerases are enzymes, which funcon to relieve torsional stress in the DNA helix by introducing transient breaks into the DNA molecule. By use of Saccharomyces cerevisiae and microarray technology we have previously shown that topoisomerases are required for the acvaon of chroman regulated genes. A further study of the PHO5 gene belonging to this gene class demonstrated a lack of binding of the transcripon factor Pho4 to the PHO5 promoter in the absence of topoisomerases, most likely due to changes in the overall superhelical state of the promoter. The GAL genes also require topoisomerases for opmal acvaon, but in contrast to the PHO5 gene, topoisomerases are not required for chroman remodeling of the GAL1/10 promoter region, indicang a different role of the enzymes. We are currently performing a detailed invesgaon of the GAL genes to elucidate the precise role of topoisomerases during the acvaon process, and the obtained results will be presented. ABSTRACT INTRODUCTION Transcripon elongaon generates negave and posive supercoiling in front of and behind the RNA polymerase, respecvely. In the absence of funconal topoisomerases in S. cerevisiae, a large percentage of genes are de-regulated 2-fold or more. This de-regulaon correlates with transcriponal acvity (A), but not transcript length (B). Furthermore, genes de-regulated by lack of topoisomerases show high sensivity to chroman regulaon (C). -1 -0.8 -0.6 -0.4 -0.2 0 0.2 -4 -2 0 2 4 Transcriptional activity (log2) top1Δtop2ts Gene expression changes (log2) Transcript length (nt) top1Δtop2ts Gene expression changes (log2) -1 -0.8 -0.6 -0.4 -0.2 0 0.2 100 1000 10000 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 100 1000 10000 -0.2 -0.1 0.0 0.1 0.2 -2 -1 0 1 Sensitivity to chromatin regulation (log2) top1Δtop2ts gene expression changes (log2) RNA polymerase RNAPII DNA topoisomerases Overwinding Underwinding A B C THE GAL GENES In the absence of glucose and galactose, the promoters of the GAL genes are found in a poised state, where the transcripon factor Gal4 is bound to the promoter along with Gal80. When galactose becomes available, Gal80 will dissociate and allow Gal4 to recruit chroman remodelers, which in turn exposes the TATA box, so that the TATA box binding protein (TBP) can bind and facilitate the formaon of the general transcripon machinery (A, B). Topoisomerases are required for opmal transcriponal acvaon of GAL genes (C) but not for nucleosome evicon (D). Topoisomerases are dispensable in the absence of Gal80, suggesng that the enzymes are required only during gene acvaon (E). Gal2p gal gal gal gal gal gal gal Gal80p Gal80p Gal3p Gal3p gal Gal3p Gal3p Gal80p Gal80p Gal80p Gal80p Gal80p Gal80p Gal4p Gal4p Gal4p Gal4p Galactose Poised state (Glycerol, raffinose) Acve state (Galactose) Chroman remodelers General transcripon factors Gal1p Gal1p Gal1p only when induced. Binding of Gal1p results in higher affinity for galactose p, Mig2p, and Mig3p o the nucleus and bind oter (1, 2, and 3) Gal2p is a galatose permadase which transports galatose into the cell Gal3p share ~70% homology with Gal1p and are able to bind galactose. GAL3 null strains takes 3-4 days to induce the GAL genes When Gal3p has bound galatose it is able to interact with Gal80p, in an ATP dependent way. This will keep Gal80p in the cytoplasm In the absence of galactose Gal80p binds as a dimer to Gal4p, thus prevenng Gal4p in recruing other factors Acve Gal4p recruits a variety of acvang factors 0,01 0,1 1 10 100 1000 0 20 40 60 80 100 120 mRNA levels (%) Time aſter inducon (minutes) GAL1 wild type gal80∆ gal80∆top1∆top2ts 0,01 0,1 1 10 100 1000 0 20 40 60 80 100 120 Time aſter inducon (minutes) GAL10 Galactose (acvaon) GAL10 ORF GAL1 ORF Gal4p concensus TATA box TATA box GAL10 ORF GAL1 ORF Gal4p concensus TATA box TATA box 0,0 20,0 40,0 60,0 80,0 100,0 120,0 0 30 90 GAL1 0,0 20,0 40,0 60,0 80,0 100,0 120,0 0 30 90 GAL2 0,0 20,0 40,0 60,0 80,0 100,0 120,0 0 30 90 GAL7 0,0 20,0 40,0 60,0 80,0 100,0 120,0 0 30 90 GAL10 0 0,2 0,4 0,6 0,8 1 1,2 0 30 90 GAL2 0 0,2 0,4 0,6 0,8 1 1,2 0 30 90 GAL7 0 0,2 0,4 0,6 0,8 1 1,2 0 30 90 GAL1 0 0,2 0,4 0,6 0,8 1 1,2 0 30 90 GAL10 Percentage induction Inducon me (minutes) Inducon me (minutes) H3 enrichment mRNA levels H3 ChIP wild type top1∆top2ts A C D E B MODEL Aſter successful binding of transcripon factor (1), chroman remodelers are recruited (2) facilitang nucleosome evicon (3). Exposure of the TATA box allows for binding of the TATA box binding protein (TBP)(4), which will recruit the general transcripon machinery (5), allowing acve transcripon (6). Topoisomerases can be required for inial transcripon factor binding (in the case of PHO5, step 1), or in the binding of TBP (GAL genes, step 5). Future invesgaon of promoter superhelicity combined with studies of TBP binding will be used to assess the validity of the model. Transcripon factor binding Recruitment of chroman remodelers Ac Ac Ac Ac Ac Ac Histone evicon TPB recruitment Transcripon complex recruitment/formaon Acve transcripon 1 2 3 4 5 6 THE PHO5 GENE In the absence of funconal topoisomerases, the phosphate regualted gene PHO5 is unable to undergo transcriponal acvaon (A). However, if PHO5 is constuve expressed, topoisomerases become dispensable for connued transcripon (B). The PHO5 promoter is covered by four posioned nucleosomes (C). These nucleosomes are removed following binding of the transcripon factor Pho4. Without topoisomerase acvity the nucleosomes remain bound to the promoter (D), due to lack of Pho4 binding (E). Time after phosphate depletion (min) 0 2 4 6 8 10 12 0 45 90 135 180 wild type pho80Δ pho80Δtop1Δtop2ts mRNA levels (log2) 252 Time after phosphate depletion (min) wild type top1Δ top2ts top1Δtop2ts 45 0 20 40 60 80 100 120 0 90 135 180 mRNA levels (%) 232 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 45 90 135 180 Histone H3 enrichment wild type top1Δtop2ts Time after phosphate depletion (min) -1 -0.5 0 -4 -3 -2 -1 UAS1 -750 -600 -450 -300 -150 0 Distance from PHO5 bp) Nucleosome occupancy (log2) TSS ( -4 -3 -2 -1 UAS2 TATA PHO5 0 1 2 3 4 5 6 0 45 90 135 180 Pho4-13xcMyc enrichment wild type top1Δtop2ts Time after phosphate depletion (min) A B C D E