DNA Topoisomerases Maintain Promoters in a State Competent for Transcriptional Activation in <em>Saccharomyces cerevisiae</em>

<div><p>To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in <em>Saccharomyces cerevisiae</em> 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 <em>PHO5</em> gene reveals that topoisomerases are essential for binding of the Pho4p transcription factor to the <em>PHO5</em> promoter, which is required for promoter nucleosome removal during activation. In contrast, topoisomerases are dispensable for constitutive transcription initiation and elongation of <em>PHO5</em>, as well as the nuclear entrance of Pho4p. Finally, we provide evidence that topoisomerases are required to maintain the <em>PHO5</em> 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.</p> </div

Transcriptional activity and not transcript length reflects topoisomerase dependency.

<p>(A) <i>top1Δ, top2ts</i>, and <i>top1Δtop2ts</i> gene expression changes plotted against mRNA abundance in wild-type cells as a 200 gene moving average. (B) <i>top1Δtop2ts</i> gene expression changes plotted against transcriptional activity in wild-type cells as a 200 gene moving average. (C) <i>top1Δtop2ts</i> gene expression changes plotted against transcript length <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003128#pgen.1003128-David1" target="_blank">[27]</a> as a 200 gene moving average. Nt, nucleotides.</p

Experimental design.

<p>*Three animals from each control and four animals from each hypoxic subgroup were used for GeneChip analysis. Four animals from each group either pulmonary trunk banded (PTB) or sham at week 5 were used for GeneChip analysis.</p

Topoisomerases are essential for binding of the Pho4p transcription factor.

<p>(A) Nucleosome occupancy profile of the <i>PHO5</i> promoter <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003128#pgen.1003128-Lee1" target="_blank">[35]</a>. Positioned nucleosomes (yellow), which are removed upon <i>PHO5</i> induction, are denoted −1 to −4 relative to the transcription start site (TSS). Red box indicates TATA box and green boxes indicate upstream activating sequences, UAS1 and UAS2, which both contain a Pho4p binding site. Black and grey arrows indicate the primers used in the H3 and Pho4-13xcMyc ChIP experiments, respectively. (B) Time course ChIP analysis of nucleosome removal from the <i>PHO5</i> promoter in wild-type and <i>top1Δtop2ts</i> cells following transcriptional activation. Experimental setup was as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003128#pgen-1003128-g005" target="_blank">Figure 5A</a>. The plot depicts average levels of histone H3 in nucleosome regions −1 to −2 and −3 to −4, respectively, in the <i>PHO5</i> promoter. H3 binding levels were normalized relative to the binding under un-induced conditions at the 0 min time point (set to 1). (C) Localization of Pho4-GFP was investigated by fluorescence microscopy in wild-type and <i>top1Δtop2ts</i> cells grown under high phosphate conditions (+P_i) at 25°C, and 90 and 180 min after shifting cells to phosphate-free medium (−P_i) at 37°C for inhibition of Top2p. The experimental setup was as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003128#pgen-1003128-g005" target="_blank">Figure 5A</a>. Differential interference contrast (DIC) and fluorescence (GFP) images of representative cells are shown, and the nuclei are indicated by DAPI (4′,6-diamidino-2-phenylindole) staining of DNA. Scale bars represent 2 µm. (D) Time course ChIP analyses of Pho4-13xcMyc recruitment kinetics in the <i>PHO5</i> promoter in wild-type and <i>top1Δtop2ts</i> cells following transcriptional activation. The experimental setup was as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003128#pgen-1003128-g005" target="_blank">Figure 5A</a>. The plots depict levels of Pho4-13xcMyc binding to UAS1 (<i>left panel</i>) and UAS2 (<i>right panel</i>) in the <i>PHO5</i> promoter, although the resolution of the ChIP assay may be insufficient to discriminate between the two sites, and Pho4p binding at the low affinity UAS1 site may be below the detection threshold of the assay. Pho4-13xcMyc binding levels were normalized relative to the binding under un-induced conditions at the 0 min time point (set to 1). In B and D averages from three individual experiments are shown and error bars represent ± one standard deviation.</p

Correlation of the gene chip and qPCR gene expression results.

<p>The correlation is tested by comparing three upregulated, four downregulated and two not regulated genes (according to the gene chip, tsum analysis). There was found significant correlation between qPCR and gene chip analysis. Values are means ± SE. *P<0.05 vs. normoxia, gene chip at same time point, †P<0.05 vs. normoxia, qPCR at same time point.</p

Topoisomerases are required for transcriptional induction of a range of inducible genes.

<p>(A) Experimental setup. α indicates α-factor. (B) Time-course experiments of induced gene expression in wild-type and <i>top1Δtop2ts</i> cells. The mRNA levels of the indicated genes were quantified by qPCR at the indicated time points after transfer of cells to inducible conditions and normalized to the mRNA level obtained in the wild-type at the latest time point (set to 100%). Averages from two individual experiments are shown with error bars representing ± one standard deviation. Numbers indicate the mean fold increase in wild-type cells at the latest time point.</p

Immunoblottings of right ventricle samples from normoxic and hypoxic rats.

<p>Proteins participating in metabolism A: ACAA2, B: HADHA and C: aquaporin 7 show tendency to downregulation by hypoxia. D: monoamine oxidase A, E: tissue transglutaminase and F: endothelin receptor B were all upregulated by hypoxia. ACAA2: acetyl-Coenzyme A acyltransferase 2, HADHA: hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (trifunctional protein), alpha subunit. Values are means ± SE and are calculated as percent of normoxia 1 week. n = 6 in both groups at all time points. *P<0.05 vs. normoxia at same time point.</p

Changes in global DNA supercoiling levels affect <i>PHO5</i> transcription.

<p>(A) Time-course experiment of <i>PHO5</i> transcriptional activation in wild-type, <i>top1Δ</i>, <i>top2ts</i>, and <i>top1Δtop2ts</i> cells. The experimental setup was as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003128#pgen-1003128-g005" target="_blank">Figure 5A</a>. The quantified <i>PHO5</i> mRNA levels were normalized to the wild-type level at the 180 min time point (set to 100%). (B) Time course of <i>PHO5</i> transcriptional activation in wild-type cells with and without expression of TopA from a high-copy YEp-TopA plasmid <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003128#pgen.1003128-Gartenberg1" target="_blank">[8]</a>. The experimental setup was as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003128#pgen-1003128-g005" target="_blank">Figure 5A</a> except for TopA expression. The <i>PHO5</i> mRNA levels were quantified at the indicated time points, normalized to the mRNA level at the 0 min time point (set to 1) and presented on a log2-scale. In A and B averages from three individual experiments are shown with error bars representing ± one standard deviation. Numbers indicate the mean fold increase in wild-type cells at the latest time point.</p

Topoisomerases are dispensable for transcription once the <i>PHO5</i> promoter is activated, and they are not required during <i>PHO5</i> inactivation.

<p>(A) Time-course experiments of <i>PHO5</i> transcription in wild-type, <i>pho80Δ</i>, and <i>pho80Δtop1Δtop2ts</i> cells after transfer from high phosphate to phosphate-free conditions. <i>Upper panel</i>, experimental setup. <i>Lower panel</i>, the <i>PHO5</i> mRNA levels were quantified at the indicated time points, normalized to the wild-type level at the 0 min time point (set to 1) and presented on a log2-scale. Number indicates the mean fold increase in wild-type cells at the latest time point. (B) Time course experiment of <i>PHO5</i> transcriptional inactivation in wild-type and <i>top1Δtop2ts</i> cells. <i>Upper panel</i>, experimental setup. <i>Lower panel</i>, the <i>PHO5</i> mRNA levels were quantified at the indicated time points and normalized to the level at the 0 min time point (set to 100%). In A and B the averages from three and two individual experiments, respectively, are shown. Error bars represent ± one standard deviation. α indicates α-factor and +P_i and -P_i indicate high and no phosphate, respectively.</p

Global reduction in mRNA levels occurs due to lack of topoisomerases I and II.

<p>(A) Experimental setup showing the timing of G1 arrest by α-factor (α) and inhibition of Top2p at 37°C. (B) Distribution of gene expression changes (between mutant and wild-type) in topoisomerase single and double mutants. (C) Relative mRNA levels were calculated using the total microarray signal intensities in mutants and wild-type. mRNA levels in wild-type were set to 100%. Error bars represent ± one standard deviation from biological triplicates. (D) Percentage of genes up- and down-regulated 2-fold or more (open and filled columns, respectively). Error bars represent ± one standard error of the means from biological triplicates.</p