To investigate the role of PIP5Pase activity in VSG gene expression and switching, we performed gene expression analysis in T. brucei bloodstream forms that exclusively express a wildtype (WT) or a catalytic mutant D360A/N362A (Mut) PIP5Pase; the latter is unable to dephosphorylate PI(3,4,5)P3 (Cestari et al., 2019). The exclusive expression cells have the endogenous PIP5Pase alleles replaced by drug-selectable markers, a tetracycline (tet)-regulatable PIP5Pase allele introduced in the silent rDNA spacer, and a V5-tagged WT or Mut PIP5Pase allele in the constitutively expressed tubulin loci (Cestari et al., 2019). In the absence of tet, the cells exclusively express the WT or Mut PIP5Pase allele (Cestari et al., 2019). We performed RNA-seq after 24 hr exclusive expression of the WT or Mut PIP5Pase with Oxford nanopore sequencing (~500 bp reads) to distinguish the VSG genes expressed. The expression of the Mut PIP5Pase resulted in 1807 genes upregulated and 33 downregulated (Figure 1B,≥2 fold change, p-value ≤0.01, Supplementary file 1). Notably, all silent ES VSG genes were upregulated (Figure 1B, C), consistent with their transcription. In contrast, there was a~10-fold decrease in the active VSG (VSG2) and ESAG mRNAs (Figure 1B, C), implying decreased expression of the active ES (BES1) genes, perhaps resulting from competition among ESs for polymerase or factors required for their transcription. The remaining upregulated genes were primarily from silent subtelomeric arrays, largely VSG genes and pseudogenes (Figure 1B and D), indicating that PIP5Pase activity is also required for subtelomeric ES repression. Other upregulated genes included primarily retrotransposons, endonucleases, and RNA polymerases, but most chromosome core genes (Pol II and Pol III transcribed genes) were not significantly affected (Figure 1B). Re-establishing expression of WT PIP5Pase for 60 hr after its 24 hr withdrawal restored VSG monogenic expression. However, analysis of this population by VSG-seq revealed the expression of several VSGs other than the initially expressed VSG2 indicating a switch in VSG expression (Figure 1E). In contrast, only a few VSGs were detected in cells expressing WT PIP5Pase by VSG-seq (Figure 1E). To verify PIP5Pase role in VSG switching, we knocked down PIP5Pase for 24 hr (Tet -), then restored its expression (Tet +) and isolated clones by limit dilution and growth for 5–7 days (Figure S1). Analysis of isolated clones after temporary PIP5Pase knockdown (Tet -/+) confirmed VSG switching in 93 out of 94 (99%) analyzed clones (Figure 1F, Figure 1—figure supplement 1). The cells switched to express VSGs from silent ESs or subtelomeric regions, indicating switching by transcription or recombination mechanisms. Moreover, no switching was detected in 118 isolated clones from cells continuously expressing WT PIP5Pase (Tet +, Figure 1F). The data imply that PIP5Pase activity can control the transcription and switching of VSGs.

The active VSG gene is expressed in bloodstream forms but silenced during parasite development to the insect stage procyclic forms. To determine whether PIP5Pase is required for VSG developmental silencing, we generated procyclic conditional null cells expressing a tet-regulatable V5-tagged PIP5Pase (Figure 1—figure supplement 2). Immunofluorescence analysis showed that PIP5Pase-V5 localizes in the nucleus of procyclic forms (Figure 1—figure supplement 2), as in bloodstream forms (Cestari and Stuart, 2015). Western analysis showed that PIP5Pase-V5 proteins were eliminated 72 hr after knockdown (Figure 1G), and growth arrest was detected after 5 days of knockdown (Figure 1—figure supplement 2). Gene expression analysis showed a 5–15 log2-fold upregulation of all ES VSG genes at 72 hr (Figure 1H) at a time when cells are viable and dividing. The data indicate that VSG switching and developmental silencing depend on PIP5Pase activity.

Because PIP5Pase and its substrate PI(3,4,5)P3 interact with RAP1 (Cestari et al., 2019), we postulated that VSG silencing might involve the regulation of RAP1’s repressive function. We showed that RAP1 binds telomeric or 70 bp repeats (Cestari et al., 2019), but it is unknown if it binds to other ES sequences or genomic loci. We performed ChIP-seq with a cell line expressing an in-situ HA-tagged RAP1 and nanopore sequencing (~500 bp reads). We found that RAP1-HA binds primarily to silent ESs at the 70 bp and telomeric repeats, which are sequences upstream and downstream of the VSG genes in bloodstream-form ESs, respectively (Figure 2A–B and E and Figure 2—figure supplement 1; p-values<10^–4). There was also a slight enrichment of RAP1-HA in the 50 bp repeat sequences preceding the ES promoters (Figure 2A and Figure 2—figure supplement 1; p-values <10^–4). However, RAP1-HA did not bind significantly to genes, including VSG genes or pseudogenes in the ESs or subtelomeric regions (Figure 2A–B and F). Notably, analysis of uniquely mapped reads showed that RAP1-HA was depleted from the active ES (Figure 2C–D and Figure 2—figure supplement 2), consistent with RAP1 having a repressive function. The RAP1-HA bound sequences are rich in TAAs and GTs, including the 70 bp (TTA)₇(GT/A)₉ and telomeric (TTAGGG)_n repeats (Figure 2A), but also T and G/T-rich sequences downstream of VSG genes. RAP1-HA was also bound to metacyclic ESs at AT/GC-rich or (TAACCC)n repeat sequences near VSG genes (Figure 2E p-values < 10^–4, Figure 2—figure supplement 3). RAP1-HA also bound sparsely to subtelomeric regions, including centromeres (Figure 2F p-values < 10^–4), which are AT-rich in trypanosomes (Obado et al., 2007), and some binding sites overlapped with the centromeric protein kinetoplastid kinetochore 2 (KKT2) (Akiyoshi and Gull, 2014). Other poorly enriched RAP1-HA peaks in the subtelomeric regions reflect residual ES sequences from recombination (Figure 2F). Hence, RAP1-HA binds primarily to telomeric and 70 bp repeats within silent ESs. The 70 bp repeats are ES-specific sites for RAP1 binding near silent VSG genes.

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To confirm RAP1 binding to ES sequences, we expressed and purified from E. coli recombinant 6xHis-tagged T. brucei RAP1 (rRAP1-His) (Figure 3A–B). We performed electrophoretic mobility shift assays (EMSA) using rRAP1-His and biotinylated telomeric repeats, 70 bp repeat, or a scrambled telomeric repeat sequence as a control. rRAP1-His bound to telomeric and 70 bp repeats but not to the scrambled DNA sequence (Figure 3C). We obtained similar results by microscale thermophoresis (MST) kinetics using rRAP1-His and Cy5-labelled DNA sequences (Figure 3D, Figure 3—figure supplement 1). The MST analysis revealed a Kd of 24.1 and 10 nM of rRAP1-His for telomeric and 70 bp repeats, respectively (Table 1). RAP1 has two DNA binding domains, a central Myb (position E426-Q500) and a C-terminal Myb-like (MybL, position E639-R761) domain (Figure 3A), and a divergent BRCT domain (S198-P385) (Yang et al., 2009), which is involved in RAP1 self-interaction (Afrin et al., 2020b) and perhaps other protein interactions (Cestari and Stuart, 2015; Cestari et al., 2019). To identify which RAP1 domain mediates DNA interactions, we performed EMSA with rRAP1^1-300-His (aa positions in superscript), rRAP1^301-560-His, and rRAP1^561-855-His, the last two fragments encompass the Myb and MybL domains, respectively (Figure 3A–B). Both rRAP1^301-560-His and rRAP1^561-855-His proteins bound to telomeric or 70 bp repeats (Figure 3E–F, Figure 3—figure supplement 1), but no DNA binding was detected with the N-terminal rRAP1^1-300-His (Figure 3G). Because all proteins were His-tagged and no binding was detected to scrambled DNA nor with rRAP1^1-300-His, unspecific DNA binding by the His-tag is ruled out. Our data show that both Myb and MybL can independently and directly bind to 70 bp and telomeric repeats.

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We found in the N-terminus of RAP1 a villin headpiece (VHP) domain (Figure 3A, position Y59 to F94, e-value <10^–4), which is typically present in Villin proteins and binds phosphoinositides (Kumar et al., 2004). T. brucei RAP1 VHP domain is conserved (~27% aa identity) with other VHPs in Villin proteins forming a three helices fold over a hydrophobic core (Chiu et al., 2005; Meng and McKnight, 2009; Figure 4A), which is essential for phosphoinositide binding (Kumar et al., 2004). We performed binding assays with rRAP1-His and biotinylated phosphoinositides (Cestari, 2019) and confirmed that rRAP1-His binds specifically to PI(3,4,5)P3 (Figure 3—figure supplement 1; Cestari et al., 2019). Moreover, we found that rRAP1^1-300-His binds to PI(3,4,5)P3 (Figure 4B), but not other phosphoinositides or inositol phosphates tested (Figure 4C) and the binding was competed by a molar excess of unlabelled PI(3,4,5)P3 (Figure 4D). However, no PI(3,4,5)P3 binding was detected with rRAP1^301-560-His or rRAP1^561-855-His proteins, indicating that RAP1 binds to PI(3,4,5)P3 via its N-terminus containing the VHP domain. Moreover, binding kinetics by differential scanning fluorescence with unlabelled phosphoinositides showed that rRAP1-His binds to PI(3,4,5)P3 with a Kd of 19.7 µM (Figure 4E, Table 1). To determine if RAP1 binds to PI(3,4,5)P3 in vivo, we in-situ HA-tagged RAP1 in cells that express the WT or Mut PIP5Pase and analyzed endogenous PI(3,4,5)P3 levels associated with immunoprecipitated RAP1-HA. There were ~100 fold more PI(3,4,5)P3 associated with RAP1-HA in cells expressing the Mut than the WT PIP5Pase (Figure 4F, Figure 4—figure supplement 1). In contrast, there was a mild but not significant difference in the total cellular PI(3,4,5)P3 levels comparing cells expressing WT versus Mut PIP5Pase (Figure 4F), implying that PIP5Pase activity controls a localized pool of PI(3,4,5)P3 in the nucleus available for RAP1 binding. Hence, RAP1 binds specifically to PI(3,4,5)P3 via its N-terminus in a PIP5Pase activity-dependent fashion.

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Because RAP1 binds to PI(3,4,5)P3 via its N-terminus and to ESs via its central and C-terminus Myb and Myb-L domains, we posited that PI(3,4,5)P3 binds to RAP1 and controls its association with ESs. We performed EMSA binding assays and found that PI(3,4,5)P3 inhibited rRAP1-His binding to telomeric repeats in a dose-dependent fashion, but no inhibition was detected with PI(4,5)P2 (Figure 5A–B). Similar PI(3,4,5)P3 binding inhibition of rRAP1-His was observed for 70 bp repeats (Figure 5B). Moreover, MST binding kinetics with 30 µM of PI(3,4,5)P3 increased rRAP1-His Kd to telomeric and 70 bp repeats in ~6.5-fold and ~18.5-fold, respectively (Figure 5C, Table 1). Notably, PI(3,4,5)P3 did not affect rRAP1^301-560-His or rRAP1^561-855-His binding to telomeric or 70 bp repeats (Figure 3—figure supplement 1), implying that PI(3,4,5)P3 does not compete with Myb or MybL domains direct binding to DNA. PI(3,4,5)P3 inhibition of RAP1-DNA binding might be due to its association with RAP1 N-terminus causing conformational changes that affect Myb and MybL domains association with DNA.

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Our data suggest a model in which PI(3,4,5)P3 levels control RAP1 binding to ESs and thus silencing and activation of VSG genes. To evaluate this model, we performed ChIP-seq with RAP1-HA in cells that exclusively express WT or Mut PIP5Pase. The Mut PIP5Pase expression results in local PI(3,4,5)P3 available for RAP1 binding (Figure 4F), whereas WT PIP5Pase dephosphorylates PI(3,4,5)P3 into PI(3,4)P2, which cannot bind RAP1 (Cestari et al., 2019). The expression of Mut PIP5Pase abolished RAP1-HA binding to 70 bp, 50 bp, and telomeric repeats in all bloodstream and metacyclic ESs (Figure 5D–G). Notably, the decreased RAP1-HA binding to ESs correlates with increased expression of VSGs and ESAG genes (Figure 5D–E, RNAseq), indicating that PIP5Pase controls RAP1 binding to ESs and thus VSG gene expression. The increased level of VSG and ESAG mRNAs detected in cells expressing Mut PIP5Pase (Figure 5D) may reflect increased Pol I transcription. It is possible that the low levels of RAP1-HA at the 50 bp repeats affect Pol I accessibility to the BES promoter; alternatively, RAP1 association to telomeric or 70 bp repeats may affect chromatin compaction or folding impairing VSG and ESAG genes transcription. The expression of Mut PIP5Pase also removed RAP1-HA peaks in subtelomeric regions (Figure 2F), which might impact subtelomeric chromatin organization and thus VSG expression and recombination (Figure 1D–F). We showed that RAP1 interacts with PIP5Pase within a 0.9 MDa complex, and this association is stable in cells expressing the Mut PIP5Pase (Cestari et al., 2019). Hence, RAP1 dissociation from ESs is unlikely the result of PIP5Pase mutations affecting the complex integrity. Our data indicate that PIP5Pase activity regulates RAP1 binding to DNA via PI(3,4,5)P3, thus controlling the reversible silencing of telomeric VSG genes.

T. brucei bloodstream forms (BF) single marker 427 conditional null (CN) and V5- or HA-tagged cell lines were generated and maintained in HMI-9 at 37 °C with 5% CO₂ as described (Cestari and Stuart, 2015). T. brucei were cultured at McGill University under permit B-07840 (for I. Cestari laboratory). Cell lines that exclusively express wildtype (WT) or mutant D360A/N360A (Mut) PIP5Pase gene (Tb927.11.6270) were generated as previously described (Cestari et al., 2019). The WT or Mut PIP5Pase mRNAs exclusively expressed from tubulin loci are 1.6 and 1.3-fold the WT PIP5Pase mRNA levels expressed from endogenous alleles in the single marker 427 strain. The fold-changes were calculated from RNA-seq counts per million from this work (WT and Mut PIP5Pase, Supplementary file 1) and our previous RNA-seq from single marker 427 strain (Cestari et al., 2019). T. brucei 29.13 were maintained in SDM-79 medium supplemented with 10% FBS, G418 (2.5 µg/mL), and hygromycin (50 µg/mL) at 27 °C. To generate T. brucei procyclic forms (PF) CN PIP5Pase, PF 29.13 was transfected with a V5-tagged PIP5Pase gene cloned into pLEW100-3V5 (Cestari and Stuart, 2015). The plasmid was integrated into the rRNA silent spacer, and transgenic cells were selected by resistance to phleomycin (5 µg/mL). The PIP5Pase endogenous alleles were then sequentially replaced by homologous recombination with PCR constructs containing about 500 bp of the PIP5Pase 5'UTR and 3'UTR flanking a puromycin or blasticidin drug resistance gene (Cestari and Stuart, 2015). The CRISPR/Cas9 ribonucleoprotein complex was used to cut the second endogenous allele and increase homologous recombination rates. Guides targeting 5' and 3' regions (see primer sequences in Supplementary file 6) of the PIP5Pase coding sequence were produced by in vitro transcription with T7 polymerase (Promega), and co-transfected with recombinant saCas9 Nuclease NLS Protein (Applied Biological Materials Inc), and a PCR-based puromycin resistance construct containing 5'- and 3’-UTRs of the targeted gene. Note that this procedure increased the efficiency of recombination by about 20 times. The T. brucei 427 strain, or mutants derived from this strain, were verified by PCR analysis and DNA sequencing. Cumulative growth curves were performed by diluting T. brucei PFs to 2×10⁶ parasites/mL in the absence or presence of tetracycline (tet, 0.5 µg/mL). The culture growth was counted every two days using a Coulter Counter (Beckman Coulter) for twelve days. Cell lines generated in this work are available upon request.

Immunofluorescence: Immunofluorescence was done using T. brucei PF PIP5Pase CN, which expresses V5-tagged WT PIP5Pase in the presence of 0.1 µg/mL of tet. Mid-log growth phase parasites were washed three times in PBS with 6 mM glucose (PBS-G) for 10 minutes (min). Then, 2.0x10⁶ cells were fixed in 2% paraformaldehyde in PBS for 10 min at room temperature (RT) onto poly L-lysine treated 12 mm glass coverslips (Fisher Scientific). The coverslips were washed three times in PBS, and cells permeabilized in 0.2% Nonidet P-40 in PBS for 10 min. Cells were washed five times in PBS, and then blocked for 1 hour (hr) in 10% nonfat dry milk in PBS. After, cells were incubated in ɑ-V5 mouse monoclonal antibodies (mAb) (Thermo Life Technologies) diluted 1:500 in 1% milk in PBS for 2 hr at RT. The cells were washed five times in PBS and incubated in goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488 (Thermo Fisher Scientific, catalog number A11001) 1:1000 in 1% milk in PBS for 2 hr at RT. Cells were washed five times in PBS and stained in 10 µg/mL of 4′,6-diamidino-2-phenylindole (DAPI) diluted in PBS for 15 min at RT. Cells were washed four times in PBS, twice in water, and then mounted onto microscope glass slides (Fisher Scientific) using a mounting medium (Southern Biotech). Cells' images were acquired using a Nikon E800 Upright fluorescence microscope (Nikon). Western blotting: Western analyses were performed as previously described (Cestari, 2019). Briefly, cleared lysates of T. brucei PFs were prepared in 1% Triton X-100 in PBS with 1 X protease inhibitor cocktail (Bio-Vision) and mixed in 4 X Laemmli buffer (Bio-Rad) with 710 mM β-mercaptoethanol and heated for 5 min at 95 °C. Proteins were resolved in 10% SDS/PAGE and transferred to nitrocellulose membranes (Sigma Aldrich). Membranes were probed for 2 h at RT (or overnight at 4 °C) with mAb α-V5 (BioShop Canada Inc, catalog number TAG006.100) 1:2500 in 6% milk in PBS 0.05% Tween (PBS-T). Membranes were incubated in 1:5,000 goat anti-mouse IgG (H+L)-HRP (Bio-Rad, catalog number 1706516) in 6% milk in PBS-T, washed in PBS-T, and developed by chemiluminescence using Supersignal West Pico Chemiluminescent Substrate (Thermo scientific). Images were acquired on a ChemiDoc MP imaging system (Bio-Rad). Membranes were stripped in 125 mM glycine, pH 2.0, and 1% SDS for 30 min, washed in PBS-T, and re-probed with mAb anti-mitochondrial heat shock protein (HSP) 70 of T. brucei (gift from Ken Stuart, Center for Global Infectious Diseases Research, Seattle Children’s) 1:25 in 3% milk in PBS-T, followed by 1:5,000 goat anti-mouse IgG (H+L)-HRP (Bio-Rad, catalog number 1706516) in 3% milk in PBS-T and developed as described above.

The T. brucei RAP1 (Tb927.11.370) recombinant protein was expressed and purified as described before (Cestari et al., 2019). The RAP1 fragments rRAP1^1-300-His (aa 1–300), rRAP1^301-560-His (aa 301–560), or rRAP1^561-855-His (aa 561–855) were amplified by PCR (see Supplementary file 6 for primers) from the T. brucei 427 genome and cloned into the pET-29a(+) vector (Novagen) using NdeI and XhoI restriction sites for the expression of proteins with a C-terminal 6xHis tag. Proteins were expressed and purified from 2 to 4 liters of Escherichia coli NiCo21(DE3) (New England Biolabs) as previously described (Cestari et al., 2019). Briefly, lysates were sonicated in PBS supplemented with 5 mM dithiothreitol (DTT), 0.2 mg/mL lysozyme, 0.05% NP-40, 10% glycerol, and 1 mM PMSF. For rRAP1^1-300-His, this buffer was supplemented with 7 M of urea (Fisher Scientific). After sonication, His-tagged proteins in the cleared lysate were purified using Profinity IMAC Nickel Charged Resin (Bio-Rad) and eluted in 50 mM sodium phosphate buffer with 300 mM NaCl pH 8 and 300 mM imidazole (Fischer Scientific). Eluted proteins were dialyzed overnight at 4 °C in binding buffer (25 mM HEPES, 150 mM NaCl, 10% glycerol, pH 7.5).

Phosphoinositide binding assays were performed as previously described (Briggs et al., 2018). Briefly, 1 μg of His-tagged recombinant protein (rRAP1-His, rRAP1^1-300-His, rRAP1^301-560-His, or rRAP1^561-855-His) was incubated with 1 μM of biotin-conjugated dioctanoylglycerol (diC8) PI(3,4,5)P3, diC8 PI(4,5)P2, InsP(1,4,5)P3 or InsP(1,3,4,5)P4 (Echelon Biosciences) rotating at RT for 1 hr. Afterward, magnetic streptavidin beads (Sigma Aldrich) (previously blocked overnight at 4 °C with 3% BSA in PBS) were added and incubated at 4 °C for 1 hr rotating. Using a magnetic stand, the mix was washed 5–6 times in 25 mM HEPES pH 7.5, 300 mM NaCl, 0.2% NP-40 and 0.1% Tween 20, and then eluted in 50 μL of 2 x Laemmli buffer supplemented with 710 mM β-mercaptoethanol. Samples were boiled at 95 °C for 5 min. Samples were resolved in 10% SDS-PAGE, transferred onto a nitrocellulose membrane (Sigma Aldrich), and probed with ɑ-His mAb (Thermo Scientific) diluted 1:1000 in 6% milk in PBS-T followed by α-mouse IgG HRP (Bio-Rad) diluted 1:5000 in 6% milk in PBS-T. Membranes developed as indicated above.

Synthetic single-stranded 5’-biotin-conjugated DNA sequences (Supplementary file 6) of telomeric repeats (TTAGGG)₁₀, 70 bp repeats (one repeat), and scrambled DNA (derived from telomeric repeats) were annealed (1:1 v/v, 10 µM) to reverse complementary unlabeled sequences (Supplementary file 6) (Integrated DNA Technologies) in a thermocycler by incubation at 95 °C for 10 min, 94 °C for 50 s, and a gradual decrease of 1 °C every 50 s for 72 cycles in 200 mM Tris–HCl pH 7.4, 20 mM MgCl₂, 500 mM NaCl. A total of 100 nM of annealed DNA were mixed with 1 μg of recombinant protein, and 2 mg/mL yeast tRNA (Life Technologies) in 20 mM HEPES, 40 mM KCl, 10 mM MgCl₂, 10 mM CaCl₂, and 0.2% N P-40, and incubated on a thermomixer (Eppendorf) at 37 °C for 1 hr. Then, 10 μL of the reaction was resolved on a 6% native gel (6% acrylamide in 200 mM Tris, 12,5 mM ethylenediaminetetraacetic acid (EDTA), pH 7.8 adjusted with acetic acid) at 100 V in 0.5 X TAE buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA). DNAs were transferred onto a nylon membrane (Life Technologies) at 100 V for 30 min in 0.5 x TAE. The membrane was blocked for 1 hr at RT with nucleic acid detection blocking buffer (Life Technologies), then probed for 1 hr at RT with streptavidin-HRP 1:5000 (Genescript) diluted in PBS 3% BSA. The membrane was washed in PBS-T and developed using Supersignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). Images were acquired with a ChemiDoc MP imaging system (Bio-Rad).

Binding assays were performed using synthetic single-stranded 5’-Cy5 conjugated DNA sequences of telomeric repeats (TTAGGG)10, 70 bp repeats (one repeat), or scrambled DNA (derived from telomeric repeats) (see Supplementary file 6 for sequences). Sequences were annealed (1:1 v/v, 10 µM) to reverse complementary unlabeled sequences (Supplementary file 6, all sequences synthesized by Integrated DNA Technologies) in a thermocycler by incubation at 95 °C for 10 min, 94 °C for 50 s, and a gradual decrease of 1 °C every 50 s for 72 cycles in 200 mM Tris–HCl pH 7.4, 20 mM MgCl₂, 500 mM NaCl. Then, 1 µM rRAP1-His was diluted in 16 twofold serial dilutions in 250 mM HEPES pH 7.4, 25 mM MgCl₂, 500 mM NaCl, and 0.25% (v/v) N P-40 and incubated with 20 nM telomeric or 70 bp repeats for 2 hr at 37 °C, gently shaking. Afterward, samples were centrifuged at 2000 xg for 5 min, loaded into Monolith capillary tubes (NanoTemper Technologies), and analyzed using the Monolith NT.115 MiscroScale Thermophoresis instrument with MO.Control software (NanoTemper Technologies). For binding assays in the presence of phosphatidylinositol phosphates (PIPs), the binding reaction was prepared as above but in the presence of 30 µM of diC8 PI(3,4,5)P3 or diC8 PI(4,5)P2. Four to six replicates were performed and presented as mean ± standard deviation. Data were analyzed using MO.Affinity Analysis (NanoTemper Technologies).

T. brucei rRAP1-His at 10 µM was incubated with 10 mM SYPRO Orange dye (ThermoFisher Scientific), filtered before use with a 0.22 μm sterile filter (Fisher scientific), in 15 mM HEPES, 150 mM NaCl, pH 7.0, and 10, 20, 30, or 40 µM of diC8 PI(3,4,5)P3, diC8 PI(4,5)P2, or no PIPs in 96-well microplates (Life Technologies). The plate was sealed with MicroAmp Optical Adhesive Film (Life Technologies), and the reaction mixture was placed on ice for 1 hr. Afterward, 20 µL of the reaction was added to a differential scanning fluorimetry plate (Life Technologies) and run using the Applied Biosystems StepOnePlus instrument (ThermoFisher Scientific) according to the manufacturer’s instructions. Briefly, samples were run using continuous ramp mode with two thermal profiles: Step 1 at 25 °C for 2 min and step 2 at 99 °C for 2 min, with a ramp rate of 100% at step 1 and 1% at step 2. Experiments were performed in triplicate and presented as mean ± standard deviation. Data analysis was performed using the DSFworld (https://gestwickilab.shinyapps.io/dsfworld/) platform, and melting temperatures were calculated as the midpoint of the resulting fluorescence versus temperature curve.

Poly-A enriched RNAs were extracted from 1.0x10⁸ T. brucei BFs CN PIP5Pase exclusively expressing V5-tagged PIP5Pase WT or mutant D360A/N362A for 24 hr using the magnetic mRNA isolation kit (New England Biolabs) according to manufacturer’s instructions. For procyclic forms, total RNAs were extracted from 5.0x10⁸ T. brucei CN PIP5Pase growing in Tet + (0.5 µg/mL, no knockdown) or Tet – (knockdown) at 5 hr, 11 hr, 24 hr, 48 hr, and 72 hr using TRIzol (Thermo Fisher Scientific) according to manufacturer’s instructions. The isolated mRNA samples were used to synthesize cDNA using ProtoScript II Reverse Transcriptase (New England Biolabs) according to the manufacturer’s instructions. Real-time PCRs were performed using VSG primers as previously described (Cestari and Stuart, 2015). For RNA-seq, cDNA samples were purified using Totalpure NGS mag-bind (Omega-BioTek) at a 1.0 x ratio (beads to cDNA volume). cDNA samples were eluted in 55 µL of water, then fragmented to a size range of 300 bp – 1 kb (average 700 bp) using an M220 Focused-ultrasonicator (Covaris) with 75 peak incidence power, 10% duty factor, and 200 cycles per burst for 35 s at 20 °C. The fragmented cDNAs were then used for RNA-seq library preparation for Oxford nanopore sequencing (indicated below). Fifty fmol of barcoded libraries were sequenced in a MinION (Oxford Nanopore Technologies) using an FLO-MIN106 flow cell (Oxford Nanopore Technologies). Three biological replicates were performed.

ChIP-seq was performed with T. brucei BFs CN PIP5Pase expressing endogenously HA-tagged RAP1 and exclusively expressing V5-tagged PIP5Pase WT or mutant D360A/N362A. Cells were grown for 24 hr in the absence of tet, which results in the exclusive expression of WT or mutant PIP5Pase, in HMI-9 media supplemented with 10% FBS, 2 µg/mL of G418, 2.5 µg/mL of phleomycin, 0.1 µg/mL of puromycin, and 25 µg/mL of nourseothricin. A total of 3.0x10⁸ cells at mid-log growth were fixed in 1% paraformaldehyde at 4 °C for 10 min, then quenched for 10 min in 140 mM glycine. Fixed cells were spun down at 2000 xg for 10 min, washed twice in PBS, and processed for ChIP using truChIP Chromatin Shearing Kit with Formaldehyde (Covaris) according to the manufacturer’s instructions. DNA was sheared to a size range of 0.5–1.5 kb (average 700 bp) using an M220 Focused-ultrasonicator (Covaris) with 75 peak incidence power, 5% duty factor, and 200 cycles per burst for 7 min at 7 °C. ChIP was performed with a total of 15 µg of sonicated chromatin and 10 µg of Mab anti-V5 (BioShop Canada Inc). The antibodies were cross-linked to Protein G Mag Sepharose Xtra (GE Healthcare) according to the manufacturer’s instructions before immunoprecipitations. Antibody eluted chromatin was incubated with 20 units of Proteinase K (Thermo Fisher Scientific) and reverse cross-linked at 65 °C overnight, followed by DNA extraction by phenol:chloroform:isoamyl alcohol (25:24:1, ThermoFisher Scientific) and isopropanol (v/v) precipitation. Samples were resuspended in water, and the DNA was selected for fragments above 300 bp using Totalpure NGS mag-bind (Omega-BioTek) at 0.85 x ratio (beads to DNA volume). The DNA was eluted in water and used to prepare Oxford nanopore DNA sequencing libraries (indicated below). Fifty fmol of barcoded libraries was sequenced in a MinION (Oxford Nanopore Technologies) using an FLO-MIN106 flow cell (Oxford Nanopore Technologies). Three biological replicates were performed.

T. brucei BFs PIP5Pase CN cell lines exclusively expressing V5-tagged PIP5Pase WT or mutant D360A/N362A were seeded at 1.0x10³ parasites/mL in 50 mL of HMI-9 media supplemented with 10% FBS, 2 µg/mL of G418, 2.5 µg/mL of phleomycin, and grown for 24 hr in the absence of tet, which results in the exclusive expression of WT or PIP5Pase mutant. Then, tet (0.5 µg/mL) was added to Mut PIP5Pase cells to rescue WT PIP5Pase expression, and cells were grown for an additional 60 hr at 37 °C and 5% CO₂ until they reached a concentration of 1.0x10⁶ parasites/mL. RNA was then isolated using Trizol (Sigma-Aldrich) according to the manufacturer’s instructions. cDNA was synthesized using random primers with 5 x Protoscript II Reverse Transcriptase (New England BioLabs Ltd). cDNA was amplified using the VSG Splice Leader and SP6-VSG14mer primers (Mugnier et al., 2015; Supplementary file 6) with denaturing at 94 °C for 3 min, followed by 22 cycles of 94 °C for 1 min, 42 °C for 1 min, and 72 °C for 2 min (Mugnier et al., 2015). Amplified fragments were purified using 0.55 x beads/sample ratio with Mag-Bind TotalPure NGS (Omega Bio-Tek). Amplicons were prepared for Oxford Nanopore sequencing using the ligation sequencing kit SQK-LSK110 (Oxford Nanopore Technologies) and PCR Barcoding Expansion kit (EXP-PBC001) according to the manufacturer’s instructions (described below). Five fmol pooled barcoded libraries were sequenced in a MinION using a Flongle FLO-FLG001 flow cell (Oxford Nanopore Technologies). Experiments were performed in three biological replicates.

T. brucei BFs PIP5Pase CN cell lines seeded at 1.0x10⁴ parasites/mL in 10 mL of HMI-9 media supplemented with 10% FBS and 2 µg/mL of G418 and 2.5 µg/mL of phleomycin in the absence of tet (tet -) for PIP5Pase knockdown, or in the presence of tet (tet +, 0.5 µg/mL) for PIP5Pase expression. After 24 hr, tet (0.5 µg/mL) was added to the tet – cells to restore PIP5Pase expression and cells were cloned by limited dilution. A protocol for clonal VSG switching is available (Touray et al., 2023). Briefly, the cells were diluted to a concentration of 1 parasite/300 µL and grown in 200 µL of media in 96-well culture plates (Thermo Scientific Inc) and grown for 5–7 days at 37 °C and 5% CO₂. Clones were collected from the plates and transferred to 24-well culture plates (Bio Basic Inc) containing 2 mL of HMI-9 media with 10% FBS, 2 µg/mL of G418, 2.5 µg/mL of phleomycin, and 0.5 µg/mL of tet for 24 hr to reach 1.0x10⁶ parasites/mL. Afterward, cells were harvested by centrifugation at 2,000 xg, and pellets were collected for RNA extraction using the 96 Well Plate Bacterial Total RNA Miniprep Super Kit (Bio Basic Inc) according to the manufacturer’s instructions. cDNA was synthesized from extracted RNA samples using M-MuLV reverse transcriptase kit (New England Biolabs Ltd) based on the manufacturer’s instructions and using customized primers splice leader (SL_F) and VSG barcoded primers (BCA_Rd_3'AllVSGs 1–96) (see Supplementary file 6 for primer sequences). The forward SL F primer is complementary to the splice leader sequence and has an Oxford nanopore barcode adapter sequence. The reverse primer contains a sequence complementary to the 3’-region conserved among VSG genes, a unique 6 random nucleotide barcode sequence, and an Oxford nanopore barcode adapter sequence for library preparation. The synthesized cDNAs were pooled in a 1.5 mL Eppendorf tube and amplified using the Oxford nanopore library barcoding primers (PCR Barcoding Expansion kit EXP-PBC001, Oxford Nanopore Technologies) with denaturing at 95° for 3 min, followed by 22 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 2.30 min. Amplified fragments were purified using 0.55 x beads/sample ratio NucleoMag NGS magnetic beads (Takara Bio). Amplicons were prepared for Oxford Nanopore sequencing using the ligation sequencing kit SQK-LSK110 (Oxford Nanopore Technologies) according to the manufacturer’s instructions (details described below). Five fmol of the barcoded library were sequenced in a MinION (Oxford Nanopore Technologies) using a Flongle FLO-FLG001 flow cell (Oxford Nanopore Technologies). A total of 118 and 94 clones of CN PIP5Pase cells tet + (PIP5Pase expression) and tet – (PIP5Pase knockdown), respectively, were analyzed.

DNA libraries were prepared using the ligation sequencing kit SQK-LSK110 (Oxford Nanopore Technologies), according to the manufacturer’s instructions. Briefly, the fragmented nucleic acids were end-repaired and A-tailed using the NEBNext Ultra II End Repair and A-Tailing Module (New England Biolabs). The samples were cleaned with Totalpure NGS mag-bind (Omega-BioTek) at 0.85 x ratio (beads to DNA volume), then ligated with nanopore barcode adapters. The product was subsequently used for PCR amplification with barcoding primers (Oxford Nanopore Technologies). The PCR product was cleaned with Totalpure NGS mag-bind (Omega-BioTek) at a 0.85 x ratio (beads to DNA volume) and ligated to motor proteins for DNA sequencing. The libraries were sequenced in a MinION Mk1C sequencer (Oxford Nanopore Technologies) using FLO-MIN106 flow cells (unless otherwise stated), and sequences were basecalled using the Guppy software (Oxford Nanopore Technologies). Sequencing information is available in Supplementary file 5 and fastq data is available in the Sequence Read Archive (SRA) with the BioProject identification PRJNA934938.

RNA-seq or ChIP-seq fastq data from nanopore sequencing were mapped to T. brucei 427–2018 reference genome using minimap2 (https://github.com/lh3/minimap2; Li, 2023; Li, 2018) and the alignment data were processed with SAMtools (https://github.com/samtools/samtools; Danecek et al., 2021; samtools, 2023). Reads with a nanopore sequencing Q-score >7 were used for analysis, and the mean Q-score was 12. RNA-seq and VSG-seq (including clonal VSG-seq) mapped reads were quantified using featureCounts from package Subread and used for differential expression analysis using EgdeR. Alignments were filtered to remove supplementary alignments using samtools flags. Because RNA-seq reads were detected mapping to subtelomeric regions in cells expressing Mut PIP5Pase, differential expression analysis was also performed with alignments filtered for primary reads with a stringent mapping probability of 99,9% (mapQ 30) to eliminate potential multiple mapping reads. Read counts were obtained with featureCounts counting primary alignments only and processed using EgdeR. For ChIP-seq, aligned reads mapped with minimap2 were processed with deepTools (https://deeptools.readthedocs.io/en/develop/) for coverage analysis using bamCoverage and enrichment analysis using bamCompare comparing RAP1-HA ChIP vs Input. Peak calling and statistical analysis were performed for broad peaks with Model-based Analysis of ChIP-Seq MACS3 (https://github.com/macs3-project/MACS, Zhang et al., 2008; MACS3 project team, 2023) and data were visualized using the integrated genomics viewer tool (Broad Institute). To identify if RAP1-HA was mapping specifically to silent vs active ESs, mapped reads were filtered to remove supplementary and secondary alignments, that is only primary alignments were considered. Analyses were also performed with a minimum mapping probability of 90% and 99% (mapQ 10 and 20, see Supplementary file 4). This stringent analysis removes reads aligning to multiple regions of the genome including reads mapping to multiple ESs, that is it retains reads aligning to specific regions of the ESs. Filtered reads were processed as described above using deepTools and MACS3 analysis. Scripts used for ChIP-seq, RNA-seq, and VSG-seq analysis are available at https://github.com/cestari-lab/lab_scripts (copy archived at Cestari, 2023a). A specific pipeline was developed for clonal VSG-seq analysis, available at https://github.com/cestari-lab/VSG-Bar-seq (copy archived at Cestari, 2023b; Touray et al., 2023).

T. brucei BFs PIP5Pase CN cell lines exclusively expressing V5-tagged PIP5Pase WT or mutant D360A/N362A were grown in 100 mL HMI-9 media supplemented with 10% FBS, 2 µg/mL of G418, 2.5 µg/mL of phleomycin, and grown for 24 hr in the absence of tet, which results in the exclusive expression of WT or Mut PIP5Pase. A total of 1.0x10⁸ cells were used for PI(3,4,5)P3 quantification using Echelon’s PIP3 Mass ELISA Kit (Echelon Biosciences), according to the manufacturer’s instructions. Briefly, the cells were centrifuged at 2000 xg at RT for 5 min, washed in PBS 6 mM Glucose, and the supernatant was discarded. The cell pellets were resuspended in 10 mL of ice-cold 0.5 M Trichloroacetic Acid (TCA), incubated on ice for 5 min, then centrifuged at 1000 xg for 7 min at 4 °C. The supernatant was discarded, and the pellets were washed twice using 3 mL of 5% TCA/1 mM EDTA per wash at RT. Neutral lipids were extracted by adding 3 mL of MeOH: CHCl₃ (2:1) to the pellets and vortexed for 10 min at RT, followed by centrifugation at 1000 xg for 5 min, and the supernatant discarded. This step was repeated once more to remove neutral lipids. 2.25 mL of MeOH:CHCl₃:HCl (80:40:1) was added to the pellets and vortexed for 25 min at RT, then centrifuged at 1000 xg for 5 min to extract the acidic lipids. The supernatants were transferred to new 15 mL centrifuge tubes. 0.75 mL CHCl₃ and 1.35 mL 0.1 N HCl were added to the supernatants, vortexed for 30 s, then centrifuged at 1000 xg for 5 min to separate the organic and aqueous phases. 1.5 mL of the organic (lower) phase was transferred to new 2 mL vials and vacuum dried using a SpeedVac (Thermo Scientific) at RT. The dried lipids were stored at –20 °C until used. To quantify PI(3,4,5)P3 interacting with RAP1-HA, 300 mL of T. brucei BFs CN PIP5Pase expressing endogenously HA-tagged RAP1 and exclusively expressing V5-tagged PIP5Pase WT or mutant D360A/N362A were grown for 24 hr in absence of tet, which results in the exclusive expression of WT or mutant PIP5Pase, in HMI-9 media supplemented with 10% FBS, 2 µg/mL of G418, 2.5 µg/mL of phleomycin, 0.1 µg/mL of puromycin, and 25 µg/mL of nourseothricin. A total of 3x10⁸ cells (PIP5Pase WT and Mut) were centrifuged at 2,000 xg at RT for 5 min, washed in 20 mL of PBS 6 mM glucose and then lysed in 5 mL of T. brucei cell lysis buffer (50 mM Tris, 150 mM NaCl, 2 X protein inhibitor cocktail, 0.1% NP-40, and 1% Triton-X100), rotating at 4 °C for 30 min. Lysates were centrifugated at 15,000 xg, 4 °C for 30 min, and the cleared lysate was transferred to a new 15 mL falcon tube. Two percent (100 µL) of the cleared lysate was collected for Western blot of HA-tagged RAP1 (input). The remaining cleared lysate was used for RAP1-HA immunoprecipitation with anti-HA monoclonal antibodies (ABClonal). Twenty µg of anti-HA monoclonal antibodies crosslinked to protein G MicroBeads (GE Healthcare) were added to the cleared lysate and incubated overnight at 4 °C. Immunoprecipitated samples on beads were washed five times in wash buffer (50 mM Tris, 150 mM NaCl, 2 X protein inhibitor cocktail, 0.1% NP-40). An aliquote corresponding to 20% of the beads-bound samples was collected and eluted in 6 M Urea/100 mM Glycine pH 2.9 for western blot analysis of immunoprecipitated RAP1-HA protein. The remaining samples on the beads (80%) were used for acidic lipids extraction, as indicated above. The dried lipids were resuspended in PBS-T 0.25% Protein Stabilizer (PBS-T 0.25% PS), and PI (3,4,5)P3 content was measured using Echelon’s PIP3 Mass ELISA Kit (Echelon Biosciences), according to the manufacturer’s instructions.

Data are shown as means ± SEM from at least three biological replicates. Comparisons among groups were made by a two-tailed t-test using GraphPad Prism. P-values <0.05 with a confidence interval of 95% were considered statistically significant. Graphs were prepared using Prism (GraphPad Software, Inc), MATLAB (Mathworks), or Integrated Genome Viewer (Broad Institute).

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

This research was enabled in part by computational resources provided by Calcul Quebec (https://www.calculquebec.ca/en/) and the Digital Research Alliance of Canada (alliancecan.ca). Funding: Canadian Institutes of Health Research grant CIHR PJT-175222 (IC); The Natural Sciences and Engineering Research Council of Canada grant RGPIN-2019–05271 (IC); Fonds de Recherche du Québec - Nature et Technologie grant 2021-NC-288072 (IC); Canada Foundation for Innovation grant JELF 258389 (IC); McGill University fund 130251 (IC); Islamic Development Bank Scholarship 600042744 (AOT); FRQNT-Ukraine postdoctoral fellowship BUKX:2022–2023 337989 (OK); The Natural Sciences and Engineering Research Council of Canada CGS M fellowship (ML).

1. Preprint posted: May 11, 2023 (view preprint)
2. Sent for peer review: May 17, 2023
3. Preprint posted: July 12, 2023 (view preprint)
4. Preprint posted: August 31, 2023 (view preprint)
5. Preprint posted: November 7, 2023 (view preprint)
6. Version of Record published: November 29, 2023 (version 1)

You can cite all versions using the DOI https://doi.org/10.7554/eLife.89331. This DOI represents all versions, and will always resolve to the latest one.

© 2023, Touray et al.

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