Cooperation of protosilencers ORC-BS, Abf1-BS and Rap1-BS with the <i>HML-E</i> silencer in forming heterochromatin at <i>HML</i>.

<p>(A) The schematics of the original <i>HML</i> locus and the modified <i>HML</i> locus (<i>HML′</i>) in strains 17 through 21 are shown on the left. In strains 17 through 21, the BstBI-BstBI fragment containing the divergent promoters and a portion of coding regions of the <i>α1</i> and <i>α2</i> genes at <i>HML</i> was replaced by a sequence designated β2 from the coding region of the <i>E. coli lacZ</i> gene, as has been described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037092#pone.0037092-Bi1" target="_blank">[8]</a>, and two FRTs in the same orientation were inserted at <i>HML</i>. The <i>HML-I</i> silencer was replaced by <i>HMR-E</i> (strain 17), ORC-BS (strain 18), Abf1-BS (strain 19), or Rap1-BS (strain 20). <i>HML-I</i> was replaced by <i>HMR-E</i> lacking all three binding sites in strain 21. Right, growth phenotypes of strains 17 through 21 on SC, -Ura and FOA media. (B) Left, schematics of <i>HML′</i> in strains 17n through 21n. Right, growth phenotypes of strains 17n through 21n. (C) Method for examining the structure and stability of <i>HML</i> heterochromatin. Top, <i>HML</i> locus excluding silencers is flanked by two FRTs. Recombination between the FRTs by Flp1 excises the <i>HML</i> circle without silencers. Heterochromatin on the circle is subject to disruption (changing to derepressed chromatin) during cell growth (cell cycle progression) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037092#pone.0037092-Bi1" target="_blank">[8]</a>. Nucleosomes in heterochromatin and derepressed chromatins are marked by filled and shaded circles, respectively. (D) Analysis of <i>HML</i> DNA supercoiling. Cells of each strain grown in YPR to late log phase were treated with 2% galactose for 2.5 hr. Nucleic acids were isolated and fractionated in the presence of 26 µg/ml chloroquine. Topoisomers of <i>HML</i> circles from strains 17 and 17s were labeled <i>SIR</i>⁺ and <i>sir</i>⁻, respectively. The nicked form of <i>HML′</i> circle is marked N. The profile of topoisomers in each strain was examined using NIH image software, and presented on the right. Open dots denote the centers of distribution of topoisomers in the samples.</p

Protosilencers do not modulate chromatin structure in the absence of Sir proteins.

<p>Top, the modified <i>HML</i> locus in strain 17s. The black bar indicates the sequence corresponding to the probe used in indirect end labeling. Bottom, chromatin mapping in strains 17s through 21s by MNase digestion and indirect end labeling. MNase treated chromatin in each strain was digested with SnaBI and EcoNI and fractionated on an agarose gel. After Southern-blotting, DNA fragments ending at the SnaBI site were detected by hybridization with the probe shown at the top. The positions of the <i>HMR-E</i> silencer and FRT site are shown on the left of the blot. M, DNA markers. N, naked genomic DNA from strain 17s treated with MNase.</p

<i>ARS1</i> can promote the establishment of a <i>SIR</i>-dependent chromatin on extra-chromosomal circles in a S-phase dependent fashion.

<p>(A) Method for investigating the <i>de novo</i> establishment of heterochromatin on <i>HML</i> circle. The <i>HML</i> locus including the <i>E</i> and <i>I</i> silencers is flanked by two FRTs in a <i>sir3-8</i> strain. <i>HML</i> circle is excised in cells grown at 30°C. Cells are then shifted to fresh YPD medium and grown further at 23°C, which activates <i>sir3-8</i> and allows the formation of heterochromatin on the <i>HML</i> circle. Shaded and filled circles represent nucleosomes in derepressed chromatin and heterochromatin, respectively. Cells of strain YXB141 (B), strain 25 (C) or strain 26 (D) were grown to log phase and then further incubated for 2.5 hr in the presence of 2% galactose. Cells were pelleted and resuspended in fresh YPD medium, and were incubated for 20 hr at 23°C. Samples were taken for DNA isolation at the indicated time points. DNA isolated from cells was fractionated by agarose gel electrophoresis in the presence of 17 µg/ml chloroquine. (E) Top, modified <i>HML</i> allele in strain 27. The <i>ARS1</i> sequence inserted at <i>HML′</i> is indicated. Cells of strain 27 were initially grown at 30°C. An aliquot of the culture was incubated for 2.5 hr in the presence of 2% galactose to induce the excision of the <i>HML′</i> circle. Cells were then pelleted and resuspended in fresh YPD medium and further grown for 20 hr at 23°C. Samples were taken for DNA isolation after the indicated times (samples d through h). Another aliquot was treated with α-factor for 2.5 hr at 30°C to arrest cells in G1 phase of the cell cycle. Galactose was then added to this culture that was incubated at 30°C for another 2.5 hr. A third of this culture was used to isolate DNA (sample i). The rest was shifted to fresh YPD medium, and half of it was grown in the presence of 0.2 M HU (sample j) and the other in the presence of 20 µg/ml nocodazole (sample k), for 8 hr at 23°C. DNA isolated from each sample of cells was fractionated by agarose gel electrophoresis in the presence of 17 µg/ml of chloroquine. Dots indicate the Gaussian center of the topoisomer distribution of <i>HML</i>′ circles. (F) The distribution of topoisomers in each sample examined in (E) was determined using the NIH image software. The centers of distribution are marked by dots. Note that all the strains examined here have a BstBI restriction fragment containing the promoters and part of the coding regions of the <i>HMLα</i> genes replaced by a sequence from the bacterial <i>lacZ</i> gene (designated β1) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037092#pone.0037092-Bi1" target="_blank">[8]</a>.</p

Effects of deleting the ORC-, Abf1- or Rap1-binding site from <i>HMR-E</i> on transcriptional silencing at <i>HMR</i>.

<p>(A) Effects of <i>HMR-E</i> mutations on <i>HMR</i>-silencing in the presence of <i>HMR-I</i> silencer. The silencing reporters <i>Tel V-R-URA3</i> and <i>HMR::TRP1</i> are illustrated at the top. The intact and mutant <i>HMR-E</i> silencers are shown on the left. Serial 10 fold dilutions of two independent clones of each of strains 1 through 8 were incubated at 30°C for two overnights on synthetic complete (SC), SC + 5-fluoroorotic acid (FOA), and SC lacking tryptophan (-Trp) media. The growth phenotypes are shown on the right. (B) Effects of <i>HMR-E</i> mutations on <i>HMR</i>-silencing in the absence of <i>HMR-I</i>. Growth phenotypes of strains 9 through 16 on SC, FOA and -Trp media are shown on the right. Note growth phenotypes of strains 13 and 14 on –Trp medium after one overnight (1 o.n.) incubation are also shown.</p

Additive effects of Abf1-BSs on the stability of heterochromatin.

<p>(A) Left, schematics of the modified <i>HML</i> locus in strains 17, 19, 22 and 23. Right, growth phenotypes. (B) Left, schematics of the modified <i>HML</i> locus in strains 17n, 19n, 22n and 23n. Right, growth phenotypes. (C) The topoisomers of <i>HML′</i> circles excised in strains 17, 19, 22, 23 and 17s were fractionated in the presence of 26 µg/ml chloroquine. The profiles of topoisomers were presented on the right.</p

Fun30 contributes to cellular tolerance to genotoxins MMS, HU and CPT.

<p>Shown are growth phenotypes of strains 1–12 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121341#pone.0121341.s006" target="_blank">S1 Table</a>) on indicated media. Strains bearing the <i>rad5-535</i> mutation are marked with asterisks. Cells were grown to late log phase and serial 10-fold dilutions were spotted on SC (synthetic complete medium) with or without MMS, HU or CPT. The plates were incubated for 3 days unless otherwise indicated.</p

Fun30 does not affect MMS-induced PCNA modifications.

<p>(A). Exponentially growing BY4741 and BY4741 <i>fun30Δ</i> strains were treated with 0.02% MMS for 2 hours. Proteins in the cell extracts were analyzed by Western blot using an anti-PCNA antibody. Image of the ponceau-stained blot is also shown. The asterisk denotes a cross-reacting species. (B). The relative level of PCNA-Ub₁ in each sample was measured as the ratio of the intensity of PCNA-Ub₁ band over that of the PCNA band. PCNA-Ub₁ level in <i>FUN30</i> cells without MMS treatment was taken as 1. (C). Quantification of PCNA-Ub₂/SUMO relative to that of PCNA. PCNA-Ub₂/SUMO level in <i>FUN30</i> cells without MMS treatment was taken as 1.</p

MMS- and HU-hypersensitivity of cells lacking Rad5/Ubc13/Mms2 is suppressed by <i>fun30Δ</i> but not <i>exo1Δ</i>.

<p>Shown are growth phenotypes of strains 3, 4, 19, 20, 27, 28, and 34 through 43 on indicated media.</p

Suppression of MMS- and HU-hypersensitivity of <i>rad5Δ</i> mutant by <i>fun30Δ</i> is dependent on Rad51 but not Rad9 or Pol32.

<p>Shown are growth phenotypes of strains 3, 4, 33, 34, and 69 through 80 on indicated media.</p

Fun30 delays the recovery of <i>rad5Δ</i> mutant from MMS induced G2/M arrest.

<p>Shown are FACS profiles of indicated strains with or without a 4 hr exposure to 0.003% MMS. FACS profiles of strains 33 and 34 at indicated time points after release from a 4 hr 0.003% MMS treatment are also shown.</p