Telomere-adjacent breaks trigger BES loss or replacement.

<p>(A) Immunofluorescence analysis of VSG^down survivors. The schematic maps indicate the regions of the BES deleted/replaced in each case (grey) as determined using PCR assays (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003260#ppat-1003260-g003" target="_blank">FIG. 3B–C</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003260#ppat.1003260.s002" target="_blank">FIG. S2</a>). DNA was counter-stained with DAPI. Scale bar, 5 µm. (B) <i>RFP</i> status of switched survivors (n = 8) from the VSG^down strain, as determined by PCR assay (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003260#ppat.1003260.s002" target="_blank">FIG. S2C</a>). Also see the schematic maps in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003260#ppat-1003260-g002" target="_blank">Figure 2A</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003260#ppat.1003260.s002" target="_blank">Figure S2A</a>. (C) Examples of switched DSB-survivors. The Coomassie-stained gel indicates the abundant, clone-specific VSGs (yellow dots). The western blots were generated using a VSG221-specific or a VSG cross-reacting (VSG XR) antibody.</p

Distinct subtelomeric DNA double-strand break responses.

<p>(A) Cell-cycle phase was determined by DAPI-staining and defined by the number of nuclei (N) and kinetoplasts (K); G₂, a single nucleus and two separate kinetoplasts (n = 200 at each time point). Error bars, SD. (B) γH2A accumulates at sub-nuclear foci in response to a DSB at a BES. Proportions of nuclei with foci were counted in uninduced (0 h) cells and 12 h after I-<i>Sce</i>I-induction (n = 200 at each time point). Error bars, SD. (C) γH2A foci are enriched in S-phase and G₂. Cell-cycle phase was defined as above; G₁, a single nucleus and a single rounded kinetoplast; S-phase, a single nucleus and an elongated kinetoplast; post-mitotic (post-M), two nuclei and two kinetoplasts (n = 100 for each bar). Error bars for BES-break strains, SD. Control; uninduced cells. (D) Immunofluorescence microscopy analysis of γH2A. Gallery of representative images showing cells with focal accumulation of γH2A during the cell-cycle 12 h after I-<i>Sce</i>I induction. Scale bar, 5 µm.</p

RAD51-dependent and independent BES repair and antigenic variation.

<p>(A) Nuclei with RAD51 foci were scored following a DSB at a chromosome-internal locus (Control) or at the active or silent BES. I-<i>Sce</i>I expression was induced for 12 h. n = 200 for each bar. Error bars for BES-break strains, SD. The inset shows a representative example of a nuclear RAD51 focus, red; DNA, blue; scale-bar, 5 µm. (B) Western blot analysis of <i>rad51</i> null strains, the Coomassie panels serve as loading controls. (C) Clonogenic assays. <i>rad51</i> null strains were distributed in 96-well plates under I-<i>Sce</i>I inducing conditions. Survivors were assessed after 7 days. Two independent clones were assayed in triplicate plates for each strain. A wild-type control displayed close to 100% survival (data not shown). Error bars, SD. *, <i>P</i><0.05 based on Student's t-test. (D). PCR assays were used to check for the presence of the <i>ESAG1</i> gene in VSG^up<i>rad51</i>-null survivors following I-<i>Sce</i>I induction (n = 20).</p

<i>T. brucei</i> subtelomeres are fragile sites.

<p>(A) The schematic shows the subtelomeric <i>VSG221</i> (also known as <i>VSG2</i>) locus and indicates the locations (i–iii) of the specific LM-PCR primers (arrowheads) and probes (black bars). 70, 70-bp repeats. (B) Southern blotting of LM-PCR products derived from both the active and silent BES. A chromosome internal primer-probe combination was used as a control (Tb11.02.2110 locus). Grey triangles represent increasing number of cells surveyed in each PCR reaction (4,000, 20,000 and 100,000). The LM-PCR approach is prone to a loss of signal as the number of cells in the sample is increased.</p

Probability and mechanism of antigenic variation are highly dependent upon subtelomeric break site.

<p>(A) DSB-induced survivors were assessed by <i>VSG221</i> immunofluorescence microscopy and scored as either VSG221 active or switched. VSG^pro, n = 24; VSG^up, n = 22; VSG^down, n = 32. (B) PCR assays demonstrate <i>VSG221</i> and <i>ESAG1</i> gene status following I-<i>Sce</i>I-mediated cleavage in switched survivors from VSG^up cells. See the schematic maps in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003260#ppat-1003260-g002" target="_blank">Figure 2A</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003260#ppat.1003260.s002" target="_blank">Figure S2A</a> for details. (C) As in B above but for VSG^down cells. (D) Comparison of <i>ESAG1</i> status of switched survivors from VSG^up (n = 22) and VSG^down strains (n = 9) as determined by PCR assay. The schematic shows the BES and DSB-sites, red arrowheads. <i>E</i>, <i>ESAG1</i>; 70, 70-bp repeats; <i>VSG</i>, <i>VSG221</i>.</p

Summary of outputs and model for <i>VSG</i> replacement.

<p>(A) The schematic shows the active BES with the relevant markers. Left, DNA repair leaves the active <i>VSG</i> intact; middle, 70-bp repeat recombination replaces the <i>VSG</i> through duplicative transposition; right, loss or replacement of the entire BES. Arrow, BES promoter; <i>R</i>, <i>RFP:PAC</i>; <i>E</i>, <i>ESAG1</i>; 70, 70-bp repeats; <i>221</i>, <i>VSG221</i>; black arrowheads, T₂AG₃-repeats. Red arrowheads, sites of induced DSBs in VSG^pro, VSG^up and VSG^down strains. Percentages of survivors that displayed each outcome are indicated. (B) Model to explain antigenic variation via subtelomere fragility and 70-bp repeat recombination at the active BES. 1. Breaks may be more frequent closer to the telomeric-repeats (blue wedge). 2. Breaks adjacent to the telomeric-repeats initiate a distinct DNA damage response and typically fail to use the 70-bp repeats for recombination, while breaks within the 70-bp repeats would be expected to be repaired by single-strand annealing (grey wedges). The remaining breaks (blue region) are productive, in that they initiate resection that progresses towards the 70-bp repeats from the telomeric side of these repeats and also allow for recombination within these repeats. 3. A DSB triggers resection (blue bar) and reveals ssDNA, which initiates a search for recombination templates. Recombination in the 70-bp repeats would terminate further resection in this region (dashed blue bar). 4. The 70-bp repeats provide a template for the initiation of (micro)homologous recombination and duplicative transposition (blue bar). We propose that recombination is favored within these repeats because they are highly repetitive and widely dispersed.</p

DNA double-strand breaks initiate DNA resection at active and silent <i>VSG</i> expression sites.

<p>(A) The schematic indicates the location of the I-<i>Sce</i>I cleavage sites (*) and probes (bars). Other details as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003260#ppat-1003260-g002" target="_blank">Figure 2A</a>. (B) Assays in strains with an active <i>VSG221</i> BES. Genomic DNA samples were extracted at the times indicated following I-<i>Sce</i>I induction, and ssDNA was monitored on slot-blots. Ninety percent of each sample was ‘native’ (n) and the remaining 10% denatured (d) as a ‘loading’ control. (C) Assays in strains with a silent <i>VSG221</i> BES. Other details as in B above.</p

DNA double-strand breaks at an active <i>VSG</i> expression site are often lethal.

<p>(A) The schematic shows the <i>VSG221</i> BES on chromosome 6a with the I-<i>Sce</i>I sites (*) and reporters (<i>NPT</i>, Neomycin PhosphoTransferase; <i>RFP-PAC</i>, Red Fluorescent Protein-Puromycin <i>N-</i>ACetyltransferase; and <i>PAC</i>) incorporated using pESP-<i>R^SP</i>, pES-70 or pTMF-Sce to give VSG^pro, VSG^up or VSG^down, strains respectively. Relevant restriction sites are shown. <i>P_ES</i>, BES promoter; <i>P_RRNA</i>, ribosomal RNA promoter (allows for low-level <i>NPT</i> transcription when the BES is ‘silent’). (B) DSB-induction is rapid and efficient. I-<i>Sce</i>I expression was induced with tetracycline (1 µg/ml), genomic DNA was extracted after different periods of time and digested with <i>Hpa</i>I (VSG^down strains) or <i>Xho</i>I (VSG^up strain); a plasmid control (P) was digested with <i>Hpa</i>I/I-<i>Sce</i>I. The probes are indicated and blots were rehybridized with either a ‘<i>2190</i>’ probe or a <i>28S</i> probe to show loading. Terminal restriction fragments (closed arrowheads) and <i>in vivo</i> cleaved fragments (open arrowheads) are indicated. (C) Clonogenic assays. Cells in which I-<i>Sce</i>I expression was induced were distributed in 96-well plates. Survivors were assessed after 7 days. Error bars, SD from three or more 96-well plates.</p

An RNAi library screen to identify <i>T</i>. <i>b</i>. <i>brucei</i> apoL1 sensitivity determinants.

<p>(A) <i>T</i>. <i>b</i>. <i>brucei</i> (MITat1.2; strain 2T1) is similarly sensitive to human (<i>H</i>. <i>sapiens</i>) and baboon (<i>P</i>. <i>hamadryas</i> and <i>P</i>. <i>papio</i>) apoL1. EC₅₀ assays were carried out in quadruplicate; error bars, standard deviation. (B) Schematic showing selection of apoL1-resistant parasites from the RNAi library. (C) Selection of populations with reduced sensitivity to apoL1. RNAi induced in 1 μg.ml^-1 tetracycline (Tet) for 24 hours prior to selection (initiated at day-0); arrows indicate culture dilution, and addition of fresh apoL1 and tetracycline at 2X EC₅₀ and 1 μg.ml^-1, respectively. (D) RNAi target fragment-specific PCR amplification from <i>T</i>. <i>b</i>. <i>brucei</i> genomic DNA extracted after eight days’ selection in apoL1 (Hs, <i>H</i>. <i>sapiens</i>; Ph, <i>P</i>. <i>hamadryas</i>; Pp, <i>P</i>. <i>papio</i>).</p

RIT-seq profiles of candidate novel apoL1-sensitivity determinants.

<p>ApoL1 sensitivity determinants include (A) putative ubiquitin modifiers and (B) putative membrane trafficking proteins. RIT-seq profiles show predicted transcripts (open reading frames and untranslated regions of interest in black) and the RNAi targeting fragment reads mapped; total reads (red) and tagged reads (blue; containing the 14-base RNAi construct barcode sequence) mapped to each predicted transcript are shown in the top right corner of each panel. Known or putative annotations based on domain organisation are included beneath each accession number and derived from GeneDB (<a href="http://www.genedb.org/Homepage/Tbruceibrucei927" target="_blank">http://www.genedb.org/Homepage/Tbruceibrucei927</a>). Ub, ubiquitin; DUB, deubiquitinase; VAMP, vesicle-associated membrane protein; VAP, VAMP-associated protein.</p