Interplay between the 500-bp interspersed homology 4H-7N_1 and different patterns of interspersed homology in the adjacent 100-bp region.

<p>(a) Reference/test combinations are created for a region derived from the 100-bp insert (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006015#pgen.1006015.g002" target="_blank">Fig 2D</a>: construct v). All interspersed homologies (as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006015#pgen.1006015.g001" target="_blank">Fig 1</a>) have a homologous unit length of 6 base-pairs and are varied with respect to periodicity (from 8 to 15) and sequence position (from 1 to 12, for the selected periodicity of 10). We note that the last homologous unit in 6H-4N_2, 6H-4N_3 and 6H-4N_4 was inadvertently extended by 3, 2 and 1 base-pairs, respectively. (b) The mean number of RIP mutations (measured as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006015#pgen.1006015.g002" target="_blank">Fig 2C</a>) for repeat constructs described in (Panel a). (c) RIP mutation profiles for selected repeat constructs with periodicities of 8, 9, 10 and 11, corresponding to homology patterns 6H-2N_1, 6H-3N_1, 6H-4N_1 and 6H-5N_1, respectively, as described in (Panel a). (d) RIP mutation profiles for selected repeat constructs with sequence positions of 3, 5, 9, and 11, and corresponding to homology patterns 6H-4N_3, 6H-4N_5, 6H-4N_9 and 6H-4N_11, respectively, as described in (Panel a).</p

Short regions of perfect homology promote stronger mutation in combination with the 500-bp interspersed homology 5H-6N_1.

<p>(a) The mean number of RIP mutations (measured as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006015#pgen.1006015.g002" target="_blank">Fig 2C</a>) for repeat constructs with 0–100 base-pairs of perfect homology adjacent to the interspersed homology 5H-6N_1 or 4H-7N_1. Data for the interspersed homology 4H-7N_1 are replotted from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006015#pgen.1006015.g002" target="_blank">Fig 2C</a>. (b) The mean number of RIP mutations detected across the 500-bp reference region for repeat constructs with 0–100 base-pairs of perfect homology adjacent to the interspersed homology 5H-6N_1 or 4H-7N_1. (c) RIP mutation profiles for selected repeat constructs containing 0, 15, 50 and 100 base-pairs of perfect homology adjacent to the interspersed homology 5H-6N_1.</p

Identification of a specific triplet sequence that modulates RIP.

<p>(a) The number of triplets included in the homologous units of 4H-7N_1 and 4H-7N_7. Forward and reverse-complemented instances of each triplet are counted together. (b) Variants of the interspersed homology 4H-7N_7 that lack two GAC triplets (Δ2GAC), or all six GAC triplets (Δ6GAC), or seven TGA triplets (Δ7TGA). (c) The mean number of RIP mutations (measured as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006015#pgen.1006015.g002" target="_blank">Fig 2C</a>) for repeat constructs described in (Panel b). (d) RIP mutation profiles for repeat constructs described in (Panel b). (e) The role of GAC triplets in the interspersed homology 6H-4N_11 was examined using the repeat construct described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006015#pgen.1006015.g004" target="_blank">Fig 4A</a>. “ΔGAC”: both GAC triplets in the original sequence are deleted. "Mock": analogous mutations are made to delete two non-GAC triplets, as described in the text. (f) The mean number of RIP mutations (measured as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006015#pgen.1006015.g002" target="_blank">Fig 2C</a>) for repeat constructs in (Panel e). (g) RIP mutation profiles for repeat constructs described in (Panel e).</p

A sensitive genetic system for assaying RIP in regions of interspersed homology.

<p>(a) Tester constructs comprise pairs of closely-positioned DNA repeats: the base-pair sequence of the “left” repeat (“Reference”) is held constant and corresponds to an arbitrarily chosen segment of the Neurospora genome, the base-pair sequence of the “right” repeat (“Test”) can be varied as desired. (b) Patterns of interspersed homology (relative to the reference sequence) are defined by the three distances: <b>X</b>—the length of the homologous unit, <b>Y</b>—the separation between homologous units, and <b>Z</b>—the sequence position of the first homologous unit relative to the reference sequence. The sum of <b>X</b> and <b>Y</b> defines the periodicity of interspersed homology. (c) Alignment of a reference sequence and three different test sequences. A minimally effective pattern of interspersed homology is "3H-8N" [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006015#pgen.1006015.ref014" target="_blank">14</a>]. One such pattern, with the sequence position parameter set to 1 (<b>Z</b> = 1), is shown as 3H-8N_1. Two homology patterns examined in this study are 4H-7N_1 and 4H-7N_7.</p

Homology recognition and ensuing mutation are not coupled with respect to the involved base-pairs.

<p>(a) RIP mutation profiles of the two diagrammed repeat constructs are compared along their entire lengths. A 200-bp portion of the reference sequence that appears to have similar levels of RIP in both cases is outlined. We note that a distal portion of the same reference sequence (marked with “*”) escapes RIP in the case of 4H-7N_1 but not in the case of 4H-7N_7. (b) RIP mutation profiles along the 200-bp portion of the reference sequence at the base-pair resolution. “●●●●” indicates the positions, in the reference sequence, of the 4-bp homologous units defined by the corresponding test sequences 4H-7N_1 (upper profile) and 4H-7N_7 (lower profile). (c) Correlation of mutations observed for individual sites within the 200-bp segment (Panel b). <i>r</i> is the Pearson correlation coefficient.</p

Short regions of perfect homology promote RIP mutation in combination with the 500-bp interspersed homology 4H-7N_1.

<p>(a) Partially-homologous repeats containing 500 base-pairs of the interspersed homology 4H-7N_1 (as defined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006015#pgen.1006015.g001" target="_blank">Fig 1A</a>) are created by replacing the <i>cyclosporin-resistant-1</i> gene (<i>csr-1</i>) with synthetic DNA. (b) Derivatives of the basic repeat construct include 0–100 base-pairs of perfect homology created by integrating additional DNA between restriction sites <i>Not</i>I and <i>Nde</i>I. The repeat construct with 0 base-pairs of perfect homology was reported previously [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006015#pgen.1006015.ref014" target="_blank">14</a>]. (c) The mean number of RIP mutations detected across the entire sequenced region for repeat constructs described in (Panels b and d). X-axis: Arabic numerals refer to the length of perfect homology inserted between <i>Not</i>I and <i>Nde</i>I sites, as shown in (Panel b); Roman numerals refer to additional repeat constructs tested in (Panel d). Error bars represent the standard error of the mean. The total number of sequenced spores and replica crosses are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006015#pgen.1006015.s002" target="_blank">S1 Table</a>. (d) RIP mutation profiles for selected repeat constructs. Roman numerals correspond to the following RepeatIDs (provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006015#pgen.1006015.s002" target="_blank">S1 Table</a>): i—“XIQ”, ii—“XIO”, iii—“XIN”, iv—“XJG”, v—“XIM”, vi—“XKE”, vii—“XJH”, viii—“XJY”. The number of mutations per site is expressed as percent of all spores sequenced for a given repeat construct.</p

RIP is modulated by the underlying DNA sequence.

<p>(a) The 500-bp interspersed homologies 4H-7N_1 and 4H-7N_7 are tested in the absence of any adjacent homology (“none”, same as “0 bps”) or in combination with the 100-bp interspersed homologies 6H-4N_1 and 6H-4N_7. The last homologous unit in 4H-7N_1 was inadvertently extended by one base-pair. Roman numerals correspond to the following RepeatIDs (provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006015#pgen.1006015.s002" target="_blank">S1 Table</a>): i—“XIR” ii—“XKO”, iii—“XJJ”, iv—“XKM”, v—“XKC”, vi—“XKN”. (b) The mean number of RIP mutations (measured as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006015#pgen.1006015.g002" target="_blank">Fig 2C</a>) for repeat constructs described in (Panel a). (c) RIP mutation profiles for repeat constructs described in (Panel a).</p

Unique requirements for the inducible <i>trans</i> association.

<p><u>A. Map of new constructs in comparison to the original constructs.</u> Position 1 contains either NatMX cassette (Nat ORF flanked by TEF1 promoter and terminator), or Nat ORF in direct or inverted orientation, or one of its five subsequences, or nothing; position 2 contains a selectable marker for transformation: KanMX or LEU2. <u>B. 3C assay in the new constructs.</u> Different combinations of the two constructs were integrated in each strain, at the same loci as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075895#pone-0075895-g001" target="_blank">Figure 1B</a>. Light grey bars – uninduced cultures; dark grey bars – cultures induced with β-estradiol. Content of positions 1 and 2 is shown below each pair of bars, with color-coding being consistent with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075895#pone-0075895-g002" target="_blank">Figure 2A</a>. See text for details. <u>C. 3C assay in the new constructs, continued.</u> Different combinations of the two constructs with different sub-sequences of Nat ORF. Light grey bars – uninduced cultures; dark grey bars – induced cultures. Content of positions 1 and 2 is shown below each pair of bars, with color-coding being consistent with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075895#pone-0075895-g002" target="_blank">Figure 2A</a>. See text for details.</p

Identification of a system that exhibits inducible <i>trans</i> association between chromosomes.

<p><u>A. Map of constructs 1 and 2.</u> Both constructs contain promoter pGal1, transcription terminator tADH1, selectable marker NatMX and bacterial <i>bla</i> gene and replication origin, flanked by segments from bacteriophage lambda: construct 1 is flanked by lambda segments 1and 2, construct 2 – by segments 3 and 4. X – XhoI restriction sites used in 3C assay. Arrows – primers used in 3C assay (one primer anneals to lambda 1 segment in construct 1, the second one – to lambda 3 segment in construct 2). <u>B. Schematic representation of genomic integration of the constructs.</u> Construct 1 (flanked with sequences 1 and 2) was integrated near telomere of chromosome XIV. Construct 2 (flanked with sequences 3 and 4) was integrated near telomere of chromosome XVI. Stars – positions of integration; circles – centromeres; numbers indicate kilo base pairs of DNA. <u>C. Schematic representation of the 3C assay.</u> 3C assay relies on chromatin crosslinking and subsequent PCR analysis of ligation junctions between crosslinked segments. <i>Trans</i> PCR assays spatial proximity of constructs 1 and 2, while <i>cis</i> PCR, which assays spatial proximity between two randomly chosen chromosomal segments, is used for normalization. <u>D. Example of a representative gel showing result of the 3C assay.</u> “−” – culture not treated with β-estradiol, “+” – culture treated with β-estradiol. 3C-PCR using primers that assay constructs 1 and 2 (<i>trans</i>) is significantly stronger in the induced compared to uninduced culture, while the normalization PCR (<i>cis</i>) is not visibly changed. Each PCR reaction is performed in triplicate. <u>E. Kinetics of induction.</u> Logarithmic culture of yeast was divided into two halves, one half was induced with 1 mkM β-estradiol, and samples from both cultures were taken and crosslinked every 30 minutes. 3C assay was done simultaneously for all crosslinked samples from one experiment. Light grey – uninduced culture, dark grey – induced culture. X-axis – minutes after induction, Y-axis – quantified 3C-signal (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075895#s2" target="_blank">Materials and Methods</a>).</p

Visualization of inducible <i>trans</i> association in 3D in whole cells.

<p><u>A, B</u> – spot counting in fixed cells. <u>A. Representative examples of cells with 1 spot and 2 spots.</u> Cells with one round spot were classified as “1 spot – cells”. Cells with two discernable spots, no matter how close to one another, were classified as “2 spot – cells”. <u>B. Quantitation of the microscopic analysis.</u> Percentages of cells with 1 spot, 2 spots and >2 spots (3 or 4 spots) are plotted for a culture without β-estradiol (light grey bars) and a culture with β-estradiol (dark grey bars). Three independent cultures were analyzed; 500 cells per each sample were scored. <u>C–H. Spot localization analysis in live cells. C. A cartoon showing z-planes.</u> The z-plane with the brightest spot is highlighted. <u>D. Examples of z-planes with the brightest spots.</u> The spots were computationally enhanced for publication purposes. <u>E. Measurements.</u> The definition of the nuclear periphery was based on the perinuclear localization of Nup49-GFP, which manifested itself as a close curve enveloping the nucleus with an average 250 nm thickness (green oval); the true nuclear perimeter was assumed to be centered within this band. In the z-plane with the brightest spot, three parameters were measured: the shortest distance from the center of the spot to the estimated true nuclear periphery (L), long axis of the nucleus (d1) and short axis of the nucleus (d2). The average diameter was calculated by taking the arithmetic mean of the long and short axis. Each spot distance to the nuclear periphery was then normalized to the corresponding average diameter, and divided by two to bring values to the 0–1 scale, where 0 is the periphery and 1 is the center. <u>F. Analysis of nuclear diameters.</u> Left – histogram of d1/d2, where d1 is the longest nuclear axis and d2 is the shortest nuclear axis. Relationship between d1 and d2 is a measure of roundness of nuclei. Right – distribution of average diameters, which were calculated as arithmetic mean of the long and short axis for all categories of cells combined. <u>G. Equal area zones.</u> Normalized distances (L′) were binned into three zones with equal areas. The borders of the zones were defined as follows: 0.184, 0.422, 1 (where 0 is nuclear periphery and 1 is nuclear center). <u>H. Percent of cells with spots in each of the three zones.</u> Four categories of cells were analyzed: 1 spot cells from the culture without β-estradiol (light grey bars); 1 spot cells from the culture with β-estradiol (dark grey bars); 2 spot cells from the culture without β-estradiol (light brown bars); 2 spot cells from the culture with β-estradiol (dark brown bars). Dotted line illustrates the hypothetical scenario on which spots are randomly distributed between the three equal area zones.</p