19 research outputs found
Endogenous single-strand DNA breaks at RNA polymerase II promoters in <i>Saccharomyces cerevisiae</i>
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<i>GAL</i> gene transcription requires topoisomerase activity.
<p>(A) Organization of the <i>GAL</i> genes. <i>GAL1</i> and <i>GAL10</i> are located on Chromosome II (Chr. II) and share a promoter with two UASs and three nucleosomes. The <i>GAL7</i> promoter, located immediately after the <i>GAL10</i> open reading frame on Chr. II, contains a single UAS and a single nucleosome. The <i>GAL2</i> promoter is similar to the <i>GAL7</i> promoter, but is located on Chr. XII. Promoter nucleosomes are illustrated in yellow. A promoter nucleosome occupancy profile [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132739#pone.0132739.ref006" target="_blank">6</a>] is shown below each gene (black line) indicating chromosomal coordinates of nucleosomes. Primers used in ChIP experiments are indicated by black arrows. UAS, Upstream Activating Sequence. TATA, TATA box. (B) Experimental setup. Cells were grown at 25°C in raffinose media (de-repressive conditions), and α-factor was added to arrest cells in G1. After 1.5 hours, cells were shifted to the restrictive temperature. After Top2 inactivation for 15 minutes, cells were treated with galactose to induce the <i>GAL</i> genes, α-factor was added again to keep cells in G1, and samples were collected at the indicated time points. α, α-factor. (C) Induction of <i>GAL1</i>, <i>GAL2</i>, <i>GAL7</i>, and <i>GAL10</i> in wild type and <i>top1Δtop2</i><sup><i>ts</i></sup> cells. Cells were treated as illustrated in (B), and samples were collected 0 and 150 minutes after galactose treatment. mRNA was isolated, and the levels of the individual <i>GAL</i> genes and two control genes (<i>GAPDH</i> and <i>ACT1</i>) were quantified by qPCR. mRNA levels of the <i>GAL</i> genes were calculated relative to the mean of the mRNA levels for <i>GAPDH</i> and <i>ACT1</i> for each time point and the value obtained in wild type at the latest time point was set to 100. Numbers indicate relative mRNA levels. Averages from two individual experiments are shown with error bars representing ± one standard deviation. (D) Induction of <i>GAL1</i>, <i>GAL2</i>, <i>GAL7</i>, and <i>GAL10</i> in wild type, <i>top1Δ</i>, <i>top2</i><sup><i>ts</i></sup>, and <i>top1Δtop2</i><sup><i>ts</i></sup> cells. Cells were treated as shown in (B), and samples were collected at the indicated time points for qPCR measurements of <i>GAL</i> gene mRNA levels. mRNA levels are presented as fold increase relative to the level at time point 0. Averages from three individual experiments are shown with error bars representing ± one standard deviation.</p
Topoisomerase activity is required for RNA polymerase II recruitment.
<p>ChIP analysis of RNA polymerase II enrichment in <i>GAL</i> gene promoters of wild type and <i>top1Δtop2</i><sup><i>ts</i></sup> cells following transcriptional activation. Experimental setup was as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132739#pone.0132739.g001" target="_blank">Fig 1B</a>, and ChIP was performed using antibodies targeting RNA polymerase II. RNA polymerase II binding levels were normalized relative to the binding under uninduced conditions at the 0 min time point (set to 1). Averages from two individual experiments are shown, and error bars represent ± one standard deviation. Positions of primers used in the ChIP experiments for the individual <i>GAL</i> genes are indicated with arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132739#pone.0132739.g001" target="_blank">Fig 1A</a>.</p
TBP binding to the TATA box requires topoisomerase activity.
<p>(A) ChIP analysis of TBP enrichment in <i>GAL</i> gene promoters of wild type and <i>top1Δtop2</i><sup><i>ts</i></sup> cells following transcriptional activation. Experimental setup was as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132739#pone.0132739.g001" target="_blank">Fig 1B</a>. ChIP was performed on cells having the endogenous TBP protein fused to a HA epitope tag using anti-HA antibodies. TBP binding levels were normalized relative to the binding under uninduced conditions at the 0 min time point (set to 1). Averages from three individual experiments are shown, and error bars represent ± one standard deviation. Positions of primers used in the ChIP experiments for the individual <i>GAL</i> genes are indicated with arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132739#pone.0132739.g001" target="_blank">Fig 1A</a>. (B) TBP protein levels in wild type and <i>top1Δtop2</i><sup><i>ts</i></sup> cells. Cells used in (A) were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132739#pone.0132739.g001" target="_blank">Fig 1B</a>, and samples taken at the indicated time points were processed for Western Blot analysis with antibodies targeting the HA tag on TBP. Mcm2 was used as loading control.</p
Topoisomerase activity has a direct role in <i>GAL</i> gene activation but is not required for transcriptional elongation and reinitiation.
<p>(A) Overview of promoter changes during transcriptional induction of the <i>GAL</i> genes. (<i>left</i>) In raffinose, the <i>GAL</i> gene promoter is covered by nucleosomes except at the UAS, which binds Gal4 having its activation domain blocked by Gal80. (<i>right</i>) Upon galactose addition, Gal3 binds Gal80, leaving the activation domain of Gal4 free to bind chromatin remodelers. Subsequent removal of promoter nucleosomes allows recruitment of TBP and RNA polymerase II. UAS, Upstream Activating Sequence. TATA, TATA box. TSS, Transcription Start Site. gal, galactose. Light coloring and dashed borders of Gal3 and Gal80 indicate that the enzymes do not block the Gal4 activation domain, either due to dissociation or rearrangement of the complex. (B) Time course experiment of <i>GAL1</i>, <i>GAL2</i>, <i>GAL7</i>, and <i>GAL10</i> transcription in wild type, <i>gal80Δ</i>, and <i>gal80Δtop1Δtop2</i><sup><i>ts</i></sup> cells. Cells were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132739#pone.0132739.g001" target="_blank">Fig 1B</a>, and mRNA levels of the individual genes were quantified by qPCR, normalized to the wild type level at the latest time point (set to 100%), and presented on a log10-scale. The average from two individual experiments is shown, and error bars represent ± one standard deviation. (C) Time course experiment with ChIP analysis of RNA polymerase II enrichment in the coding regions of the <i>GAL</i> genes and two control genes, <i>GAPDH</i> and <i>ACTI</i>, following transcriptional activation. Cells were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132739#pone.0132739.g001" target="_blank">Fig 1B</a>, and ChIP was performed with antibodies recognizing the C-terminal domain of the Rpb1 subunit of RNA polymerase II. RNA polymerase II binding levels were normalized relative to the binding at the 0 min time point (set to 1). (D) Time course experiment with ChIP analysis of Top1 and Top2 enrichment in the promoters of the <i>GAL</i> genes following transcriptional activation. Cells expressing the endogenous Top1 or Top2 enzymes fused to a cMyc tag were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132739#pone.0132739.g001" target="_blank">Fig 1B</a>, and ChIP was performed with antibodies recognizing the cMyc tag. Top1 and Top2 binding levels were normalized as in (C). In (C) and (D) averages from three individual experiments are shown, and error bars represent ± one standard deviation. Positions of primers used in the ChIP experiments for the individual <i>GAL</i> genes are indicated with arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132739#pone.0132739.g001" target="_blank">Fig 1A</a> and presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132739#pone.0132739.t002" target="_blank">Table 2</a>.</p
<i>S</i>. <i>cerevisiae</i> strains used in this study.
<p><i>S</i>. <i>cerevisiae</i> strains used in this study.</p
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
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
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