Additional file 1 of epiG: statistical inference and profiling of DNA methylation from whole-genome bisulfite sequencing data

Additional information and figures. Additional information about the statistical method, in particular, the optimization and implementation of epiG, as well as additional figures referenced in the main text. (PDF 1880 kb

Top2 and Sgs1-Top3 Act Redundantly to Ensure rDNA Replication Termination

<div><p>Faithful DNA replication with correct termination is essential for genome stability and transmission of genetic information. Here we have investigated the potential roles of Topoisomerase II (Top2) and the RecQ helicase Sgs1 during late stages of replication. We find that cells lacking Top2 and Sgs1 (or Top3) display two different characteristics during late S/G2 phase, checkpoint activation and accumulation of asymmetric X-structures, which are both independent of homologous recombination. Our data demonstrate that checkpoint activation is caused by a DNA structure formed at the strongest rDNA replication fork barrier (<i>RFB</i>) during replication termination, and consistently, checkpoint activation is dependent on the <i>RFB</i> binding protein, Fob1. In contrast, asymmetric X-structures are formed independent of Fob1 at less strong rDNA replication fork barriers. However, both checkpoint activation and formation of asymmetric X-structures are sensitive to conditions, which facilitate fork merging and progression of replication forks through replication fork barriers. Our data are consistent with a redundant role of Top2 and Sgs1 together with Top3 (Sgs1-Top3) in replication fork merging at rDNA barriers. At <i>RFB</i> either Top2 or Sgs1-Top3 is essential to prevent formation of a checkpoint activating DNA structure during termination, but at less strong rDNA barriers absence of the enzymes merely delays replication fork merging, causing an accumulation of asymmetric termination structures, which are solved over time.</p></div

Replication termination structures accumulate at <i>RFB</i> and at other barriers in the rDNA in <i>sgs1Δtop2</i>^<i>ts</i> cells.

<p>(A) Schematic illustration representing the expected Neutral-Alkaline 2D gel migration behavior of replicating rDNA corresponding to the 4.577bp <i>Bgl</i>IIB-fragment encompassing the <i>RFB</i> (grey box) with reference to the migration of the corresponding DNA in Neutral-Neutral 2D gels. Migration of the DNA strands shown with black lines is indicated. <i>RFB</i> as well as the positions of the 1N and 2N dots are indicated. (B) Genomic DNA was isolated from wt, <i>sgs1Δtop2</i>^<i>ts</i>, <i>sgs1Δtop2</i>^<i>ts</i><i>pif1Δ</i> and <i>sgs1Δtop2</i>^<i>ts</i><i>fob1Δ</i> cells at the indicated time points after release from α-factor, digested with <i>Bgl</i>II, and subjected to Neutral-Alkaline 2D gel analysis and Southern blotting using the P3 probe (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005697#pgen.1005697.g002" target="_blank">Fig 2C</a>). FACS profiles of samples taken throughout the experiments are shown to the right. (C) Quantification of forks converging at <i>RFB</i> at the indicated time points, where the signal at time point 0 is set to 1. Error bars represent STDEV from two to four independent experiments. (D) Quantification of forks converging outside <i>RFB</i> (“<”-smear) at the indicated time points, where the signal at time point 0 is set to 1. Error bars represent STDEV from two to four independent experiments.</p

Structures causing checkpoint activation and X-spike formation in <i>sgs1Δtop2</i>^<i>ts</i> cells are formed independently of homologous recombination.

<p>ISA analysis of Rad53 was performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005697#pgen.1005697.g001" target="_blank">Fig 1</a> with <i>sgs1Δrad52Δ</i> and <i>sgs1Δtop2</i>^<i>ts</i><i>rad52Δ</i> cells (A) or with <i>sgs1Δrad51Δ</i> and <i>sgs1Δtop2</i>^<i>ts</i><i>rad51Δ</i> cells (B). FACS profiles of samples taken throughout the experiments are shown below. Genomic DNA was isolated from <i>sgs1Δtop2</i>^<i>ts</i><i>rad52Δ</i> (C) or <i>sgs1Δtop2</i>^<i>ts</i><i>rad51Δ</i> cells (D) and processed for 2D gel analysis as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005697#pgen.1005697.g003" target="_blank">Fig 3</a> using the P1 probe recognizing the <i>Bgl</i>IIB fragment in the Southern blots. In the quantifications of Xs to Ys, the Xs/Ys obtained at time point 0 is set to 1. Error bars represent STDEV from three (in C) and two (in D) independent experiments.</p

Asymmetric X-structures are formed independent of Fob1.

<p>(A) Genomic DNA was isolated from <i>sgs1Δtop2</i>^<i>ts</i><i>fob1Δ</i> cells at the indicated time points after release from α-factor and processed for 2D gel analysis and Southern blotting using either probe P1, recognizing <i>Bgl</i>IIB (upper panel), or probe P2, recognizing <i>Bgl</i>IIA (lower panel). Quantification of the Xs to Ys obtained with the <i>Bgl</i>IIB fragment is shown to the right, where the Xs/Ys at time point 0 is set to 1. Error bars represent STDEV from three independent experiments. FACS profiles are shown to the right. (B) Chromosomes were prepared from <i>fob1Δ</i>, <i>sgs1Δtop2</i>^<i>ts</i> and <i>sgs1Δtop2</i>^<i>ts</i><i>fob1Δ</i> cells at the indicated time points after release from α-factor and visualized after pulsed-field gel electrophoresis by EtBr staining (upper panel) or by Southern blotting with a probe (P1) specific for chr. XII (middle panel) or chr. II (lower panel). <i>M</i>, Chromosomal marker with indication of individual chromosomes to the left.</p

X-spike generating structures are formed in the rDNA in <i>sgs1Δtop2</i>^<i>ts</i> cells.

<p>(A) The experimental setup was as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005697#pgen.1005697.g001" target="_blank">Fig 1A</a> except that cells were released from α-factor at 34°C into nocodazole. Genomic DNA was isolated from wt, <i>sgs1Δ</i>, <i>top2</i>^<i>ts</i> and <i>sgs1Δtop2</i>^<i>ts</i> cells at the indicated time points after release, digested with <i>Bgl</i>II and subjected to 2D gel analysis and Southern blotting using the P1 probe recognizing the <i>Bgl</i>IIB fragment. FACS profiles of samples taken throughout the experiments are shown to the right. (B) Schematic illustration representing the expected migration behavior of replicating rDNA corresponding to the 4.577bp <i>Bgl</i>IIB-fragment encompassing the <i>RFB</i>. Forks converging at <i>RFB</i> are indicated by a horizontal symmetric “X” and makes up the “top of the X-spike”. The stippled area below represents asymmetric X-structures, which make up the “main X-spike” in <i>sgs1Δtop2</i>^<i>ts</i> cells. (C) Quantification of Xs to Ys at the indicated time points, where the Xs/Ys obtained at time point 0 was set to 1. Error bars represent STDEV from two to four independent experiments. (D) Quantification of the <i>RFB</i> signal relative to all Ys at the indicated time points, where the ratio at time point 0 was set to 1. Error bars represent STDEV from three to four independent experiments. (E) Genomic DNA was isolated from <i>sgs1Δtop2</i>^<i>ts</i> cells and treated as in (A) except that probe P2 recognizing the <i>Bgl</i>IIA fragment (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005697#pgen.1005697.g002" target="_blank">Fig 2C</a>) was used in the Southern blot. FACS profiles and quantifications of Xs to Ys are shown to the right. The arrowheads shown in (A) and (E) represent replication forks stalling at sites other than <i>RFB</i>.</p

Checkpoint activation in <i>sgs1Δtop2</i>^<i>ts</i> cells is dependent on Fob1.

<p>(A) The experimental setup was as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005697#pgen.1005697.g001" target="_blank">Fig 1A</a>, except that cells were released at 34°C into nocodazole to block further cell cycling. Chromosomes were prepared from wt, <i>sgs1Δ</i>, <i>top2</i>^<i>ts</i> and <i>sgs1Δtop2</i>^<i>ts</i> cells and visualized after pulsed-field gel electrophoresis by EtBr staining (upper panels) and by Southern blotting with a probe (P1) specific for chr. XII (middle panels) or chr. II (lower panels). <i>M</i>. Chromosomal marker with indication of individual chromosomes to the left. Migration of individual chromosomes in the used strains is indicated to the right. FACS profiles of samples taken throughout the experiment are shown below each strain. (B) Quantification of the ratio of chr. XII to chr. II re-entering the gel. (C) Schematic illustration of the rDNA repeat unit in <i>S</i>. <i>cerevisiae</i>, indicating the 5S and 35S transcription units. <i>RFB</i>, Replication Fork Barrier. <i>ARS</i>, Autonomously Replicating Sequence. <i>Bgl</i>II restriction sites generating the <i>Bgl</i>IIA and B fragments as well as probes used for Southern blotting are indicated. (D) The experimental setup was as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005697#pgen.1005697.g001" target="_blank">Fig 1A</a>. Proteins were isolated from <i>sgs1Δtop2</i>^<i>ts</i> and <i>sgs1Δtop2</i>^<i>ts</i><i>fob1Δ</i> cells at the indicated time points and processed for ISA analysis of Rad53 as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005697#pgen.1005697.g001" target="_blank">Fig 1</a>. FACS profiles are shown below each strain.</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

Loss of Top2 and Sgs1-Top3 leads to chromosome segregation-independent checkpoint activation.

<p>(A) Schematic illustration of the experimental setup. ISA analysis of Rad53 was performed with wt, <i>sgs1Δ</i>, <i>top2</i>^<i>ts</i>, and <i>sgs1Δtop2</i>^<i>ts</i> cells (B), with <i>top2</i>^<i>ts</i> and <i>sgs1Δtop2</i>^<i>ts</i> cells treated with nocodazole (noc) as indicated (C), or with <i>top3</i>^<i>ts</i> and <i>top2</i>^<i>ts</i><i>top3</i>^<i>ts</i> cells (D). At the indicated time points after cells were released into YPD-media at 34°C, the restrictive temperature for <i>top2</i>^<i>ts</i> (B and C) or at 37°C, a restrictive temperature for both <i>top2</i>^<i>ts</i> and <i>top3</i>^<i>ts</i> (D), samples were taken and processed for ISA analysis. The positive control (+control) represents ISA analysis on extract from MMS-treated wt cells. Mcm2 was used as a loading control. FACS profiles of samples taken throughout the experiment are shown below each strain. <i>1C</i> and <i>2C</i> indicate DNA content in G1 and G2, respectively, and <i>asyn</i>. refers to asynchronously growing cells.</p

Stalling at <i>RFB</i> in the absence of Top2 and Sgs1 leads to checkpoint activation whereas stalling at other rDNA barriers causes X-spike formation.

<p>(A) Genomic DNA was isolated from <i>sgs1Δtop2</i>^<i>ts</i><i>pif1Δ</i> at the indicated time points after release from α-factor and processed for 2D gel analysis and Southern blotting using either probe P1, recognizing <i>Bgl</i>IIB (upper panel), or probe P2, recognizing <i>Bgl</i>IIA (lower panel). Quantification of the Xs to Ys obtained with the <i>Bgl</i>IIB fragment is shown to the right, where the Xs/Ys at time point 0 is set to 1. Error bars represent STDEV from three independent experiments. FACS profiles are shown to the right. (B) ISA analysis of Rad53 was performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005697#pgen.1005697.g001" target="_blank">Fig 1</a> with protein isolated from <i>sgs1Δtop2</i>^<i>ts</i> and <i>sgs1Δtop2</i>^<i>ts</i><i>pif1Δ</i> cells.</p