TbRPA1 is SUMOylated in <i>T. brucei</i>.

<p>(A) Immunoprecipitation (IP) of SUMOylated proteins revealed that TbRPA1 is SUMOylated. Nuclear fraction from BF cell extracts was lysed in urea containing buffer. SUMOylated proteins were immunoprecipitated with anti-TbSUMO mAb or unspecific antiserum (mock) and probed with anti-TbRPA1 (arrow). As control the same blot was probed with anti-TbSUMO (bottom) (B) Reciprocal IP experiment was performed using anti-TbRPA1 antiserum and probed with anti-TbSUMO mAb. As control the same blot was probed with anti-TbRPA1 (bottom). Loading information: input (Inp): 0.3%, IPs: 50%. (C) TbRPA1 is SUMOylated in a TbSIZ1 dependent manner. Immunoprecipitation experiments were carried out using protein extracts from the parental cell line and compared with a TbSIZ1 depleted extract (48 h RNAi induced). SUMOylated proteins were immunoprecipitated with anti-TbSUMO mAb or anti-TOR4, as control. Western blot with anti-TbRPA1 shows a reduction of the SUMOylated TbRPA1 detected upon TbSIZ1 depletion (arrowhead). Loading information: input 2× (Inp 2×): 0.1%, input 1× (Inp 1×): 0.05%, IPs: 50%.</p

Expression pattern and subcellular localization of <i>T. brucei</i> SUMOylated proteins.

<p>(A) Multiple bands corresponding to SUMO conjugated proteins are reduced upon TbSUMO knockdown. Western blot analysis of SUMOylated proteins with a monoclonal antibody generated against TbSUMO (mAb 1C9H8). Whole cell extracts from bloodstream trypanosomes were prepared in the presence of 20 mM NEM and separated in 4–20% precast polyacrylamide gels (Bio-Rad). (5×10⁶ cells/lane); Parental, uninduced (−) and induced 48 h (+). Anti-tubulin was used as a loading control. (B) Expression pattern of SUMOylated proteins in two developmental forms of <i>T. brucei</i>. Bloodstream form (BF) and insect procyclic form (PF) total cell extracts were prepared in the presence of 20 mM NEM and analyzed by Western blot using the mAb 1C9H8. (C) SUMO conjugated proteins are diffusely distributed in the nucleoplasm including a Highly SUMOylated Focus (HSF) (arrowhead) in the bloodstream form of the parasite. Double indirect three-dimensional immunofluorescence (3D-IF) analysis was carried out using the anti-TbSUMO mAb 1C9H8 (green). (C′) Higher magnification of the nucleus showing anti-SUMO and DAPI fluorescence signals. DNA in the nucleus (N) and the kinetoplast (K) was stained with DAPI (blue). Statistical analysis showed the presence of a highly SUMOylated focus in 74.9% of the cells (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004545#ppat.1004545.s002" target="_blank">Figure S2B</a>). (D) SUMOylated proteins in the nucleus of procyclic form are diffusely distributed in many foci. 3D-IF analysis was carried out using the anti-TbSUMO mAb (green). (D′) Higher magnification of the nucleus showing anti-SUMO and DAPI fluorescence signals. DNA was stained with DAPI (blue). Scale bars 1 µm. Maximum intensity projections of deconvolved two-channel 3D stack are shown.</p

SUMOylation of chromatin-associated proteins occurs at the active <i>VSG-</i>ES in the bloodstream form.

<p>(A) Schema of tagged cell lines used in ChIP experiments. SALR cell line: <i>firefly luciferase</i> gene (FLuc) inserted downstream of the active <i>VSG221</i>-ES promoter and SILR: FLuc gene inserted downstream of an inactive <i>VSG</i>-ES. <i>Renilla</i>-luciferase reporter gene (RLuc) was inserted in the tubulin locus of both cell lines, as RNA pol II control. Fragments amplified by qPCR using ChIPed DNA are indicated (primers are listed in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004545#ppat.1004545.s010" target="_blank">Table S1</a>). (B), (C) TbRPA1 ChIP analysis in SALR and SILR shows the occupancy of RNA pol I in these cell lines. High TbRPA1 enrichment is found in SALR along the active <i>VSG</i>-ES at FLuc gene, pseudo-VSG (pseVSG) and the active <i>VSG221</i> in contrast to inactive <i>VSGs</i>. Similar occupancy is detected in SILR except for the FLuc gene inserted in an inactive <i>VSG-ES</i> promoter of the BES5 in this cell line. The RNA pol I occupancies between FLuc SALR and FLuc SILR were found significantly different (p-value<0.01). Sequences present in rDNA promoter and 18S gene transcribed by RNA pol I were analyzed as positive controls and RNA pol II or pol III transcribed genes as negative controls. (D), (E) ChIP analysis using anti-TbSUMO mAb in SALR and SILR cell lines. Enrichment of SUMOylated proteins is found at the active <i>VSG</i>-ES chromatin from the promoter to the telomeric <i>VSG221</i>. SUMO ChIP levels between active (FLuc SALR) and inactive (FLuc SILR) reporters are significantly different (p-value<0.01). Similarly, SUMO enrichment on the active <i>VSG221</i> versus the inactive <i>VSGs</i> (<i>121</i>, <i>JS1</i> and <i>VO2</i>) is significantly different (p-values<0.05). ChIP analyses show the average from at least three independent experiments with standard error (SE). Data are represented as percentage of input immunoprecipitated (% input) after background subtraction of the negative control ChIP using the pre-bleed antiserum. Tubulin (Tub), Splicer Leader (SL), EP Procyclin promoter (EP pro), Procyclin gene (EP cds), rDNA promoter (rDNA pro), rDNA spacer (rDNA sp).</p

Chromatin upstream of active <i>VSG</i>-ES promoter is highly enriched for SUMOylated proteins.

<p>(A) Schema of <i>VSG</i>-ES promoter region mapping indicating fragments amplified by qPCR (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004545#ppat.1004545.s010" target="_blank">Table S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004545#ppat-1004545-g003" target="_blank">Figure 3A</a>). (B) ChIP analysis of sequences upstream of the <i>VSG</i>-ES promoters using anti-TbRPA1 and anti-TbSUMO antibodies. Fragments 5 and 6 are highly enriched for SUMOylated proteins and the fragments 1–4 show moderate enrichment. In contrast, TbRPA1 is no detected in the region upstream and enriched at the promoter and downstream sequences. PCR fragment 4 from genomic and ChIPed DNA was cloned and sequenced showing that SUMO-ChIPed chromatin corresponds to the active <i>VSG221</i>-ES (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004545#ppat.1004545.s005" target="_blank">Figure S5B</a>). (C) Schema of ribosomal DNA (rDNA) promoter showing mapped fragments amplified by qPCR (primers listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004545#ppat.1004545.s010" target="_blank">Table S1</a>). (D) Chromatin upstream of the rDNA promoter is not significantly SUMOylated. ChIP analysis of rDNA promoter region shows no SUMO enrichment detected above background. The results show the average from at least three independent experiments with standard error (SE). Data are represented as percentage of input immunoprecipitaded (% input) after background subtraction of the pre-bleed antiserum ChIP.</p

SUMOylation of chromatin-associated proteins by TbSIZ1 is important for efficient recruitment of RNA pol I and <i>VSG</i>-ES expression.

<p>(A) Reduced <i>VSG</i>-ES chromatin SUMOylation upon TbSIZ1 depletion. ChIP analysis carried out in TbSIZ1 depleted cells (48 h) and parental cell line (SALR) shows that SUMOylated chromatin is significantly reduced along all active <i>VSG</i>-ES, from the upstream promoter to the active <i>VSG221</i> gene. (B) Reduced RNA pol I occupancy upon TbSIZ1 depletion. Occupancy of RNA pol I was determined by TbRPA1 ChIP. Statistical analysis shows a significant difference of TbRPA1 levels between parental and TbSIZ1 depleted cells at the active <i>VSG</i>-ES. Data from three independent TbSIZ1 RNAi clones and parental controls are represented as percentage of input immunoprecipitated after background subtraction of the pre-bleed antiserum ChIP. The results show the average from at least three independent experiments with standard error (SE). Statistical analysis (Student's t-test), *p<0.05, **p<0.01. (C) Reduced RNA pol I occupancy results in reduced <i>VSG</i>-ES derived transcripts. Quantitative RT-PCR analysis shows reduced amounts of <i>FLuc</i> reporter gene and <i>VSG221</i> mRNA without significant effect in rDNA transcripts or RNA pol II derived transcripts <i>RLuc</i> and myosin. Results are the average from three independent clones. Data were normalized with U2 mRNA, transcribed by RNA pol III. Statistical analysis (Student's t-test) *p<0.05, **p<0.01.</p

Alignment of Tf binding sites (ESAG6/7) and polymorphic ESAG3 regions.

<p>Amino acid alignment of polymorphic regions of ESAG6/7 (A) and ESAG3 (B). Tf binding sites are highlighted with red squares. Other polymorphic regions of ESAG6/7 are marked with black boxes. Polymorphic regions of ESAG3 are shown. I: S103-E119; II: L171-K189; III: A/G215-D/E244; IV: A309-R/E321. <i>T</i>. <i>b. brucei</i> ESAGs 6, 7 and 3 are shown in red.</p

Antigenic variation in ACLs.

<p>(A) Immunofluorescence analysis of different <i>T</i>. <i>b. gambiense</i> ACLs. Monoclonal antibodies anti-LiTat 2.1 VSG and rabbit antiserum anti-LiTat 3.1 VSG were used. White bar represents the scale, set at 5 µm. (B) Frequency of expressed VSG (LiTat 2.1, LiTat 3.1 or other) in ACLs from goat, pig, human and calf sera. Cells were manually counted by IF (n≈500 per sample). FCS: foetal calf serum, GS1/2/3: goat serum (adaptation experiments 1, 2 or 3), HS1/2/3/4: human serum (adaptation experiments 1, 2, 3 or 4), PS1/2/3: pig serum (adaptation experiments 1, 2 or 3).</p

Tf uptake and mRNA <i>ESAG6/7</i> expression levels.

<p>(A) Tf uptake of different ACLs. Mean ± SD of three (goat and pig) or four (human) sera independent adaptation experiments and three independent measures of calf serum ACL are shown. Uptake is expressed in FAU (fluorescence arbitrary units). One-way ANOVA test was performed (* p< 0.05; ** p<0.01). (B) Normalized Tf uptake of different ACLs. Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085072#pone-0085072-g006" target="_blank">Figure 6A</a> were normalized with dextran uptake at 10 minutes of incubation (data not shown). One-way ANOVA test was performed (* p< 0.05; ** p<0.01). Human holo-Tf (iron saturated) was used at final concentration of 20 μg/ml. (C) Histogram showing relative expression of mRNA measured by qRT-PCR of <i>ESAG6</i>, <i>ESAG7</i> and <i>TbHpHbR</i> in all ACLs. Mean ± SD of two different measures is shown. Expression values are plotted in relative expression units, calculated relative to <i>ATM</i> (<i>PI3Kinase-</i>like -Tbb927.2.2260-) expression. <i>Myosin</i> housekeeping gene is also shown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085072#B25" target="_blank">25</a>]. ACL: adapted cell line, <i>TbHpHbR</i>: Haptoglobin-Hemoglobin receptor, FCS: foetal calf serum, GS1/2/3: goat serum (adaptation experiments 1, 2 or 3), HS1/2/3/4: human serum (adaptation experiments 1, 2, 3 or 4), PS1/2/3: pig serum (adaptation experiments 1, 2 or 3).</p

<i>ESAG</i> diversity in ACLs.

<p>Graphic representation of <i>ESAG</i> genotype diversity. Each ring is divided in 2-4 inner circles representing each independent experiment. Unless otherwise is indicated, ACLs distribution in each ring is as indicated in <i>ESAG7</i> row. Each genotype is represented in a different colour according to indicated in the graph. Partial ORF was used for DNA genotyping (673-676 bp in <i>ESAG6/7</i> and 853 bp in <i>ESAG3</i>). FCS: foetal calf serum, GS1/2/3: goat serum (adaptation experiments 1, 2 or 3), HS1/2/3/4: human serum (adaptation experiments 1, 2, 3 or 4), PS1/2/3: pig serum (adaptation experiments 1, 2 or 3). </p

Duplication time of ACLs after adaptation.

<p>Histogram showing the duplication time of <i>T</i>. <i>b. gambiense</i> (ELIANE strain) before and after 25 passages (75-105 days) in HMI9 supplemented with 20% of different mammalian sera. Mean ± SD of at least three independent experiments is shown. Student´s t-test showed significant differences in human and pig serum ACLs between before and after adaptation (* p<0.05; ** p<0.01). FCS: Foetal Calf Serum, ACL: adapted cell line.</p