6 research outputs found
Hydroxyurea (HU) treatment enhances centromeric PEV.
<p><b>(A)</b> HU promotes switching in expression states of <i>cnt2</i>::<i>ade6</i><sup>+</sup> in a dosage-dependent manner. Cell suspensions of a red (upper panel) or a white (lower panel) colony are grown in liquid media with HU as labeled for 24 hours, washed and spread onto YE+4S (low adenine) plates, with >500 cells/plate. The red or white colonies are counted at 5th day when incubated at 25°C. Each experiment has been performed by at least triple biological repeats. Each biological repeat has its unique composition of red/white cell ratio in the initial culture. One biological experiment result is shown here. The error bars are 1 SD of percentages for four plates. <i>p</i> values are calculated by Fisher exact test. <b>(B)</b> The red colony exhibits enhanced sectoring pattern upon HU treatment. Cells derived from the above red colony (before HU treatment) are planted evenly at one cell/cm<sup>2</sup> by microscopic manipulation on YE+4S plates containing HU as labeled, incubated at 25°C for eight days. Two representative colonies for each are shown. The number shown here means the number of red colonies in the total colonies. <b>(C)</b> Elevated frequencies of <i>cnt2</i>::<i>ade6</i><sup><i>+</i></sup> red-to-white switching measured by pedigree analysis in wild type cells with HU treatment and <i>cdc22-3</i> mutants. Pedigree analyses are performed on “hybrid” plates for measuring switching rate of HU treated cells, each with a drug-free zone and a drug-containing zone. For each pedigree, three cell generations are tracked at the zone containing HU as labeled. The eight progeny cells are then moved to the drug-free zone, incubated at 25<sup>°</sup>C for five days. For each experimental condition (wild type with or without HU treatment and <i>cdc22-3</i> mutant), colony color switching events are scored in 400–1000 cell division events in total. The error bars are 1 SD of percentages for four independent experiments. <b>(D)</b> Colonies maintain the wild type level of sectoring after short-term HU treatment. Cell suspensions of a red colony in (A) grown under 2mM HU is washed and planted evenly at 1 cell/cm<sup>2</sup> in YE4S media, and incubated at 25°C for eight days.</p
HU affects PEV at heterochromatin region.
<p><b>(A)</b> HU affects PEV within the mating type locus. Diagram illustrates the location of <i>ade6</i><sup>+</sup> insertion at the mating type locus, between the centromere-proximal boundary and the <i>mat2-P</i> gene (strain AP144—<i>mat_L(BglII)</i>::<i>ade6</i><sup>+</sup>). Dark green box indicates a normal silencing domain. <b>(D)</b> HU treatment affects PEV induced by <i>cenH</i> at an ectopic site. Diagram illustrates the genetic construct of the strain (LW75- <i>cenH- ade6</i><sup><i>+</i></sup>). <i>cenH</i> and <i>ade6</i><sup><i>+</i></sup>are inserted in the <i>ura4</i><sup><i>+</i></sup> locus, replacing the majority of the <i>ura4</i><sup><i>+</i></sup> ORF. <b>(B-C, E-F)</b> For each strain, cell suspensions of a predominantly white colony (B, E) and red colony (C, F) are treated with HU as labeled for 24 hours, washed and plated onto YE+4S solid media, and incubated at 25<sup>°</sup>C for 5 days. The red and white colonies are counted respectively, with 300–400 total/per plate. The error bars are 1 SD of percentages for four plates. <i>p</i> values are calculated by Fisher exact test.</p
Replication stress affects white gene PEV in <i>D</i>.<i>melanogaster</i> and vulva development in <i>C</i>.<i>elegans</i>.
<p><b>(A)</b> HU treatment increases the likelihood of <i>In(1)wm4</i> silencing in flies. Male or female flies grown without drug (gray bars) or 6 mM HU (black bars) were separated into five bins based on the degree of eye pigmentation. Bar height is the percentage of the population in that bin for all pooled trials. The error bars are 1 SD of percentages for four or five independent trials. Total flies assayed: > 500. (B-F) HU treatment induces a <i>SynMuv</i> phenotype in an <i>hpl-2</i> mutant worm. <b>(B)</b> Montage of DIC images of a wild-type with one normal valva (arrowhead) and <b>(C)</b> an hpl-2 mutant adult raised on 10mM HU plate, with three vulva-like structures (arrows). Scale bars are 50 mms. <b>(D-F)</b> Synchronized L1 larvae were placed on NGM plates containing HU as labeled, the multivulva phenotype was scored after worms reached adulthood. Each data point is represented as mean ± SD. In (D), the animals for scoring and their mothers were raised at 20°C; in (E), mothers were raised at 20°C, and the animals for scoring were raised at 25°C, start at the embryonic stage; in (F), the animals for scoring and their mothers were all raised at 25°C.</p
Mutations in the replication machinery enhance centromeric PEV correlates with excessive formation of ssDNA.
<p><b>(A)</b> Increased levels of Ssb2-GFP signal in the mutant S phase cells. Cells are grown in the liquid rich media except for Psf1-HBD cells in YE+5S media plus 100nM β-estradiol (proficient condition) or 0.1nM β-estradiol (deficient condition), respectively. <i>mcm4-M68</i> cells are grown in 29<sup>°</sup>C for 6 hours. Ssb2-GFP signal is measured as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006900#pgen.1006900.g002" target="_blank">Fig 2A</a>. Left panels, representative images of S phase cells. <b>(B)</b> Quantification of <i>cnt2</i>::<i>ade6</i><sup>+</sup> red-to-white switching frequency in the replication mutants. For <i>psf1</i>-HBD (deficient) cells, pedigree analyses are performed on “hybrid” plates, on which three cell generations are tracked at the “deficient”-zone containing 0.1nM β-estradiol. The eight progeny cells are then moved to the “proficient”-zone, containing 100nM β-estradiol, incubated at 25<sup>°</sup>C for five days. For <i>mcm4-M68</i> cells, pedigree analysis is performed at 29<sup>°</sup>C, and incubated at 25<sup>°</sup>C for 5 days before scoring. For each test, 500–800 cell division events are scored. The error bars are 1 SD of percentages for four independent experiments. <i>p</i> values are calculated by t test.</p
Replication stress promotes spreading of heterochromatin throughout the yeast genome.
<p><b>(A)</b> H3K9me2 enrichment DNA reads in wild type and <i>cdc22-3</i> mutant cells are plotted over sub-telomeric region of all three chromosomes. Chromosome coordinates are indicated above the peaks. Scale bars on the right denote H3K9me2 enrichment DNA reads numbers normalized per one million reads. <b>(B)</b> H3K9me2 enrichment DNA reads in wild type and <i>cdc22-3</i> mutant cells were plotted over mating type locus. The location of the <i>ade6</i><sup>+</sup> gene (orange), the <i>IRL</i> repeats (yellow), <i>mat2-P</i> (light green), <i>cenH</i> (blue), the <i>IRR</i> (purple) repeats, and the <i>ura4</i><sup>+</sup> gene (pink) are indicated below the DNA peaks. Shaded areas highlight the <i>ade6</i><sup>+</sup>, <i>IRL</i>, <i>IRR</i> and <i>ura4</i><sup>+</sup> genes. <b>(C)</b> H3K9me2 enrichment DNA reads in three biological <i>cdc22-3</i> mutant cells were plotted over sub-telomeric region of all three chromosomes. Chromosome coordinates are indicated above the peaks. Scale bars on the right denote H3K9me2 enrichment DNA reads numbers normalized per one million reads. <b>(D)</b> Multiplex PCR is performed on immune-precipitated DNA from 7 independent yeast colonies of <i>cdc22-3</i> mutant to amplify a fragment from the gene (<i>SPBPB2B2</i>.<i>08</i>) nearby sub-telomere right region in chromosome II, as well as a fragment from <i>tel2R</i> locus. For the normalization control, PCR is performed in parallel from non-immunoprecipitated DNA (WCE panel). The numbers shown are calculated by the gray value of PCR product from ChIP samples divided by the gray value of that from WCE samples.</p
Replication stalling affects RC nucleosome assembly.
<p>Left: normal RC nucleosome assembly. Individual nucleosomes are distinguished by colors. Accurate duplication of chromatin is exemplified by the “red” nucleosomes maintaining association with the “green” DNA segment after replication. Right: during replication stalling (HU), helicase continues unwinding DNA, accumulating evicted parental histones and forming ssDNA, which cause errors in parental histone recycling after replication resumes. Mutant such as <i>mcm4-84c</i>, that causes excessive formation of ssDNA, aggravates the defect in nucleosome inheritance.</p