Inositol Phosphate Pathway Controls Transcription of Telomeric

Inositol phosphate pathway controls transcription of PNAS PLUS telomeric expression sites in trypanosomes

Igor Cestaria and Ken Stuarta,b,1

aSeattle Biomedical Research Institute, Seattle, WA 98109; and bDepartment of Global Health, University of Washington, Seattle, WA 98195

Edited by Paul T. Englund, Johns Hopkins University, Baltimore, MD, and approved April 10, 2015 (received for review January 19, 2015) African trypanosomes evade clearance by host antibodies by period- nositols (PIs) occur in various cellular compartments, where they ically changing their variant surface glycoprotein (VSG) coat. They act as cofactors in the regulation of the functions or interactions transcribe only one VSG gene at a time from 1 of about 20 telomeric of proteins (22, 23). Knowing this, we explored the role of the IP expression sites (ESs). They undergo antigenic variation by switching pathway in the control of telomeric ESs transcription and VSG transcription between telomeric ESs or by recombination of the VSG switching (see Tables S1 and S2 for gene IDs). We found that gene expressed. We show that the inositol phosphate (IP) pathway conditional knockdown of TbPIP5K or TbPIP5Pase or over- controls transcription of telomeric ESs and VSG antigenic switching in expression of TbPLC resulted in transcription of all known Trypanosoma brucei. Conditional knockdown of phosphatidylinositol telomeric VSG and ESAG genes but no change in transcription of 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5- procyclin or genes that are transcribed by Pol II or Pol III. Pase) or overexpression of phospholipase C (TbPLC) derepresses nu- Reexpression of TbPIP5K after a temporary knockdown restored merous silent ESs in T. brucei bloodstream forms. The derepression is monoallelic VSG gene transcription and was accompanied by a specific to telomeric ESs, and it coincides with an increase in the high frequency of VSG switching. The IP enzymes and metabolites number of colocalizing telomeric and RNA polymerase I foci in the are primarily on the plasma membrane, but TbPIP5Pase is in the nucleus. Monoallelic VSG transcription resumes after reexpression of nucleus, proximal to telomeres, and is associated with TbRAP1, TbPIP5K; however, most of the resultant cells switched the VSG gene and their silencing function is altered by TbPIP5K knockdown. expressed. TbPIP5K, TbPLC, their substrates, and products localize to Thus, the IP pathway controls ES transcription and antigenic the plasma membrane, whereas TbPIP5Pase localizes to the nucleus switching by regulation of telomere silencing. proximal to telomeres. TbPIP5Pase associates with repressor/activator MICROBIOLOGY protein 1 (TbRAP1), and their telomeric silencing function is altered by Results TbPIP5K knockdown. These results show that specific steps in the IP Specific Steps of the IP Pathway Affect Transcription of Subtelomeric T. brucei pathway control ES transcription and antigenic switching in ESs. The T. brucei genome encodes ∼26 genes that predict en- by epigenetic regulation of telomere silencing. zymes for the IP pathway including those for IP and PI kinases and phosphatases, a PLC, and enzymes for the synthesis or transcription | trypanosomes | antigenic variation | telomere silencing | recycling of inositol and PIs (24), although the specificities of all inositol phosphates these enzymes have not yet been directly determined. A portion of the IP pathway that is relevant to this paper is shown in Fig. nly one of the ∼20 telomeric expression sites (ESs) is tran- 1B. We made conditional null (CN) BF cell lines of IP pathway Trypanosoma brucei Oscribed at a time in in the mammalian genes by replacing the endogenous alleles of the selected genes infectious stage bloodstream (BF) or metacyclic forms (MFs), with drug resistance markers in cells in which we inserted a copy whereas no ES is transcribed in the insect stage procyclic forms of the gene that has its transcription dependent on the presence (PFs) (1). Each ES contains 1 telomeric variant surface glycopro- of tetracycline (tet). Tet-induced expression of the target gene in tein (VSG) gene, whose expression confers a distinct cellular an- CNs resulted in mRNA levels similar to the endogenous levels tigenic type and up to 12 expression site-associated genes (ESAGs) whose functions are incompletely understood (Fig. 1A)(2–4). The Significance parasites evade immune clearance by periodically changing antigenic type by switching transcription between ESs or by ES recombination with the ∼2,500 non-ES VSG genes and pseudo- African trypanosomes cause human and animal diseases and genes (5, 6). ESs are transcribed by RNA polymerase I (Pol I), evade the host immune systems by periodically switching which initiates at the single promoter at all ESs but terminates which variant surface glycoprotein (VSG) they express. The within a few kilobases except at one fully transcribed ES (7, 8). mechanisms that result in expression of one of the numerous RNAi knockdown of expression of the nuclear protein TbRAP1 VSG genes at a time and the switching of expression between that interacts with the TTAGGG-binding factor (TbTRF) or of the different VSG genes are poorly understood. We show that nuclear lamina protein 1 (TbNUP1) or deletion of the histone- specific steps in the inositol phosphate (IP) pathway control lysine N-methyltransferase DOT1B (TbDOT1B) alleles each results both monoallelic VSG gene transcription and the switching of in transcription of silent ESs (9–11). Similarly, RNAi knockdown of VSG gene expression. The data indicate that the cellular amounts, the transcription facilitating histone chaperone suppressor of ty locations, and molecular interactions of specific IP pathway en- 16 (TbSPT16) or the SWI2/SNF2-related chromatin-remodel- zymes and their metabolites control both processes. The results offer new drug targets and insights into the epigenetic control ing protein TbISWI or deletion of histone deacetylase sirtuin of gene expression by this biologically ubiquitous pathway. 2-related protein 1 (TbSIR2RP1) each increases transcription – near the promoter but not the VSG gene of silent ESs (12 14). Author contributions: I.C. and K.S. designed research; I.C. performed research; I.C. and K.S. These results indicate that the control of ES transcription involves contributed new reagents/analytic tools; I.C. and K.S. analyzed data; and I.C. and K.S. telomeric silencing (15), which entails the functions and interactions wrote the paper. of multiple proteins and chromatin remodeling (16, 17), but im- The authors declare no conflict of interest. portantly, what regulates these processes is unknown. This article is a PNAS Direct Submission. The inositol phosphate (IP) pathway regulates multiple cellular 1To whom correspondence should be addressed. Email: [email protected]. processes in eukaryotes including chromatin remodeling and gene This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. expression (18–21). Soluble IPs or lipid-conjugated phosphatidyli- 1073/pnas.1501206112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1501206112 PNAS Early Edition | 1of10 Downloaded by guest on September 29, 2021 A

TbPIP5K TbPI3K TbPIP5Pase

TbPIP5Pase TbPI3Pase TbPIP5K Lipid associated PI TbCDS TbDK TbPLC CTD- PA DAG DAG

TbIMPase TbIPMK TbIPMK IMP I(1,4)P2 Soluble C Fig. 1. IP pathway genes that affect ES transcription 14 TbPIP5K in BF T. brucei.(A)DiagramofaT. brucei BF ES. The TbPIP5K CN tet+ ′ TbPIP5K CN tet - SM427 promoter (flag) that is 3 to 50-bp repeats drives Pol I 12 TbPIP5Pase CN tet + TbPIP5Pase transcription of ESAGs and the downstream VSG CN Tet + TbPIP5Pase CN tet - gene, which is flanked by 70 bp and TTAGGG telo- 10 TbIPMK CN tet + TbIPMK CN Tet- meric repeats (closed circles). Ψ indicates pseudo- TbIPMK CN tet - 8 genes. Fifteen BF and 5 MF ESs have been sequenced TbCDS CN tet + TbCDS in the strain Lister 427 (47, 48). MFs ESs lack ESAG (Parasites/ml) TbCDS CN tet - 6 genes. (B) Part of the predicted IP metabolic pathway

Cumulative growth Cumulative TbIMPase CN tet + TbIMPase in T. brucei. Enzyme names are colored to correspond 4 TbIMPase CN tet - 0 1 2 3 4 5 6 7 8 to C, and those whose genes were not manipulated 1.2 0.0 0.4 1.6 0.8 D Days Relative RNA expression (24h) are in gray. See Tables S1 and S2 for enzyme and 12 metabolite abbreviations and gene IDs. (C)Invitro growth of CN BF cells (Left) and qPCR analysis (Right) TbPLC WT OX tet + 10 of gene expression 24 h after conditional knockdown TbPLC WT OX tet - of IP pathway genes; tet+=0.5 μg/mL. (D)Invitro (H306L/ 8 growth (Left) of BF cells that overexpress (OX) WT or TbPLC ΔR510L) OX tet + (H306L/ mutant (H306L/R510L) PLC and Western analysis

(Parasites/ml) 6 TbPLC ΔR510L) OX tet - (Right) of the cells at 24 h. The membrane was probed with anti-V5 Mabs, stripped, and reprobed with anti- Cumulative growth Cumulative 4 HSP70 Mab; tet+=1 μg/mL. Growth data in C and D 0 1 2 3 4 5 6 are the means (and SEM) of five and three experi- Days ments, respectively. See Fig. S2 for cell viability.

in the parental cell line SM427 (Fig. 1C). Removal of tet resulted recombination between the ESs and VSG genes, which occurs in 95–99% reduction in the target gene mRNA levels and in some within the cell populations. The knockdowns did not affect ex- cases resulted in inhibition of cell growth after 48 h. We also made a pression of procyclins or rRNAs, both of which, like the ES, are BF cell line that conditionally overexpresses V5-tagged TbPLC in transcribed by Pol I, or the expression of genes that are tran- the presence of tet by inserting into the rRNA intergenic region scribed by Pol II or Pol III (Fig. 2A and Table S5). VSG de- an additional copy of the gene that is transcribed by a tet- repression was detected as early as 3 h after the knockdowns, and dependent promoter. Addition of tet resulted in a 3.7- and 3.3-fold immunofluorescence (IF) analysis with monoclonal antibodies increase, respectively, of WT or catalytically inactive mutated (Mabs) for two different VSGs (6) showed that individual cells (H306L/R510L) (25) TbPLC mRNA as measured by quantita- express both VSGs on their surface following TbPIP5K knock- tive PCR (qPCR) and additional V5-tagged TbPLC protein as seen down (Fig. 2 C and D). Overexpression of V5-tagged TbPLC also by Western analysis, but had no effect on cell growth (Fig. 1D). resulted in derepression of VSG genes in BF and MF ESs and Conditional knockdown of TbPIP5K or TbPIP5Pase resulted mutation of the TbPLC catalytic site (H306L/R510L) ablated in the expression, i.e., derepression, of all known silent BF and this effect (Fig. 2E). Thus, multiple ESs are derepressed upon per- MF ESs (Fig. 2A). The abundances of the VSG mRNAs were turbation of expression of IP enzymes, the cells express multiple increased by up to 10,000-fold relative to cells that express these VSGs, and the derepression is specific to ESs. genes (i.e., cells growing in tet). ESAG mRNAs are highly con- Conditional knockdown of other IP pathway genes including served among BF ESs, but the abundances of those that could be inositol polyphosphate multikinase (TbIPMK), inositol (1, 4) distinguished between ESs were increased by up to two orders of monophosphatase (TbIMPase), and cytidine diphosphate-diac- magnitude (Fig. 2 A and B and Fig. S1). The differences in the ylglycerol synthase (TbCDS) (Fig. 1B) for 24 h had no effect on relative increases among the VSG and ESAG mRNAs are likely the relative abundances of ES mRNAs (Fig. 2C). The conditional due to their differential posttranscriptional regulation (7). Cal- knockdowns of TbPIP5K, TbPIP5Pase, TbIPMK, and TbCDS culation of the cellular amounts of the VSG and ESAG mRNAs resulted in growth inhibition of T. brucei BFs, with most parasites indicated that the absolute levels of these mRNAs per cell were dying by 72 h, but null mutants of TbIMPase are viable (Fig. 1C). increased after 18 h of TbPIP5Pase knockdown (Fig. 2A, Inset Lethality that ultimately occurs by 72 h is likely due to multiple and Tables S3 and S4). The knockdowns did not affect expres- factors including the loss of essential IP metabolites, e.g., which sion of non-ES VSG genes and pseudogenes. The marginal in- function in protein anchoring, and the disruption of their “second creases in non-ES VSG gene expression may be due to periodic messenger” functions. Nevertheless, the effects on ES transcription

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1501206112 Cestari and Stuart Downloaded by guest on September 29, 2021 A PNAS PLUS TbPIP5K TbPIP5Pase RNA molecules/cell after TbPIP5Pase knockdown 10 5 10000 VSG Tet+ 1000 VSG Tet - BF ES VSGs ESAG Tet+ 100 ESAG Tet- 10 10 4 18S Tet+ 1 18S Tet- RNA 0.1 TERT Tet+ MF ES TERT Tet- VSGs 0.01 3 β-Tubulin Tet+ 10 molecules/cell) (Avg 0.001 β-Tubulin Tet-

ESAGs 18S TERT VSG8 VSG9 VSG3 VSG6 VSG2 VSG11 VSG14 VSG19 VSG13 VSG15 VSG16 VSG18 VSG21 VSG17 -Tubulin VSG531 VSG639 VSG653 2 VSG397 β 10 BF ESs VSG1954 MF ESs ESAG7 BES15 ESAG2 BES12 ESAG4 BES11 ESAG5 BES11 ESAG1 BES2 ESAG3 BES11 ESAG6 BES7 10 1 Non ES VSGs Procyclins Pol II Pol III

10 0 * ns (p<0.05) (ns) Relative RNA expression (Tet+/Tet-) expression RNA Relative Ile Tyr Thr Ser Arg 5S -1 Met 18S EP2 EP3 10 EP1 VSG2 VSG3 VSG6 VSG9 VSG8 tRNA VSG14 VSG11 VSG13 VSG17 VSG18 VSG19 VSG15 VSG16 VSG21 GPEET tRNA tRNA tRNA tRNA tRNA VSG397 VSG639 VSG531 VSG653 MC VSG MC VSG1954 Tb927.2.4840 Tb927.5.2820 Tb927.8.1100 Tb927.6.1610 Tb927.4.3160 ESAG2 BES12 ESAG3 BES11 ESAG4 BES11 ESAG5 BES11 ESAG7 BES15 ESAG1 BES2 ESAG6 BES7 Tb427.01.5060 Tb927.9.12510 Tb427.05.4990 Tb427.06.5320 Tb427.10.16370 Tb927.11.15760 Tb427tmp.v2.0230 Tb427tmp.21.0011 Tb427tmp.01.4560 Tb427tmp.160.5350 B Tb427tmp.354.0120 3.3 58 55 39 226 1.6 6.2 7.3 7.6 862 7 6 548 3 4 9 70 bp repeats VSG BF ES11 ESAGs ψ ψ MICROBIOLOGY TbPLC-V5

ns WT VSG2 CDE ΔH306L/R510L VSG3 VSG6 TbPIP5K CN VSG8 DIC+MERGE 4F6-FITC VSG9 BF ES VSGs VSG11 ns (p<0.5) VSG13 VSG14 9F7-CF595 MERGE VSG15 Tet + ns VSG16 VSG17 VSG18 DIC+MERGE 4F6-FITC VSG19 VSG21 VSG397 VSG531 ns MERGE ns MF ES Tet - 9F7-CF595 VSG639 ns VSGs VSG653 VSG1954 ns

10-1 10 0 101 10 2 103 10 4 Relative RNA expression (Tet+/Tet-)

Fig. 2. IP pathway gene functions affect subtelomeric ESs silencing. (A) Relative mRNA levels by qRT-PCR analysis after knockdown of TbPIP5K or TbPIP5Pase genes for 24 or 18 h, respectively, by withdrawal of tet from CN cell lines; tet+,0.5μg/mL. CN cell lines express only the VSG2 gene in tet+.(Inset) Average RNA molecules per cell 18 h after knockdown of TbPIP5Pase (Tables S3 and S4). The gray line indicates one RNA molecule per cell, and the blue line indicates the average RNA molecules per cell of several Pol II transcribed genes. See Table S5 for additional expression analysis of Pol II transcribed genes and Dataset S1 for primers. (B) Fold increase of mRNAs from ES11 24 and 18 h after conditional knockdown, respectively, of TbPIP5K (red) and TbPIP5Pase (black). See Fig. S1 for other ESs. The ES gene organization shown is from strain 427 (47) and may differ in CNs cell lines. (C) Time course of VSG gene expression after knockdown of TbPIP5K or TbPIP5Pase and after 24-h knockdown of TbIPMK, TbCDS, or TbIMPase in CN cell lines. (D) IF analysis following knockdown of TbPIP5K for VSG2

using FITC-conjugated anti-VSG 4F610–1 Mab (green) and for a VSG that is recognized by 9F715–1 Mab using CF-594–conjugated antibody (red). DNA is stained with DAPI (blue). (Scale bar, 2.5 μm.) (E) Relative levels of ES VSG mRNAs 24 h after conditional overexpression (OX) of V5-tagged WT or mutant PLC. The qPCR data are the means (SEM) of two to six experiments, normalized relative to telomerase reverse transcriptase (TERT) and β-tubulin and significant at P < 0.05, except as indicated as not significant (ns).

of TbPIP5K and TbPIP5Pase knockdown or TbPLC overexpression morphology and cell cycle were unaffected, and flow cytometry are not due to cell lethality. Overexpression of V5-tagged TbPLC analysis of live/dead cells showed that 89–91% of the cells were alive did not affect cell growth (Fig. 1D). In addition, over the 24-h cell (Fig. S2). As detailed below, ∼90% of the cells were also viable growth period after knockdown, TbPIP5K and TbPIP5Pase CN cell when tet was added back 24 h after knockdowns in CN cells. Thus,

Cestari and Stuart PNAS Early Edition | 3of10 Downloaded by guest on September 29, 2021 steps in the IP pathway that affect phosphorylation at inositol po- from the nucleolus. Thus, expression of multiple ESs correlated sition 5 specifically impact the control of ES transcription. with an increase in the number of telomeric and Pol I foci, and their distribution implies their repositioning within the nucleus at Temporary Knockdown of TbPIP5K Increases the Frequency of VSG sites where transcription occurs. Switching. The release from 24-h conditional knockdown by adding tet back to the parasite culture restored ES monoalellic expression; Subcellular Distribution of IP Pathway Enzymes and Metabolites. We however, most cells switched to expression of a different VSG gene. generated tet-inducible V5-tagged cell lines of IP pathway enzymes Expression of TbPIP5K or TbIPMK was temporarily knocked down in BF T. brucei. IF analysis of V5-tagged TbPIP5K revealed it to be by withdrawal of tet for 24 h, and then tet was added back, and predominantly located at the inner face of the plasma membrane numerous clones were made by limiting dilution. Five serial 10-fold and to be distributed over much of the cell and flagellar membranes dilutions resulted in clones in about one of three wells, as is typical, and to colocalize with its product PI(4,5)P2 (Fig. 4A). Similarly, V5- and only about 4% of these clones failed to grow, indicating a high tagged TbPLC was predominantly located at the inner face of the level of viability after 24 h in the absence of tet. The qPCR analysis of plasma and flagellar membranes and colocalized with its substrate 104 randomly isolated clones following the temporary knockdown of PI(4,5)P2. PI(4,5)P2 was detected in permeabilized cells with anti- TbPIP5K revealed that 45 clones (43%) expressed the same PI(4,5)P2 monoclonal antibodies, showing its location at the inner VSG as before the knockdown, i.e.,VSG2. Analysis of 44 clones faceoftheplasmamembrane(Fig. S5), and expression of a GFP- that did not express VSG2 for the expression of the 19 VSGs that tagged PH domain probe that recognizes PI(4,5)P2 confirmed its are in the BF or MF ESs in the strain 427 (Fig. 2) showed that 17 location. In addition, IF analysis with specific monoclonal anti- clones (16% of the total) transcribed VSG13, 2 (2%) VSG8, 2 bodies showed that PI(4)P and PI(3,4,5)P3 were also predominately (2%) VSG21, and 1 (1%) transcribed VSG19 (Fig. S3). None of at the plasma membrane (Fig. 4B). TbPIP5K knockdown resulted the clones analyzed expressed VSGs that were in the MF ESs. in reduced PI(4,5)P2 and PI(3,4,5)P3 signal from the plasma The other 22 clones (21%) did not express any of the original membrane, indicating that these metabolites result from TbPIP5K VSGs in the ESs, and they likely expressed a VSG gene that activity (Fig. S5). In contrast, V5-tagged TbPIP5Pase was primarily arose by recombination involving the numerous non-ES VSG located in the nucleus and was adjacent to and partially colocalized genes in the genome. Analysis of 50 clones derived from with telomeres (Fig. 4C). A similar pattern of colocalization with TbPIP5K CN cells growing in tet (no knockdown) showed no telomeres was found for hemagglutinin (HA)-tagged TbRAP1 and switch from the original VSG2 expression. In addition, none of TbTRF, which are proteins known to localize at the telomeres (Fig. the 82 clones obtained from TbIPMK CN switched from VSG2 4D). The location of most TbPIP5Pase-V5 was distinct from that of expression whether they were derived from temporary knock- the nuclear envelope protein TbNUP1-HA and from the nucleolar down cells (44 clones) or not (38 clones), indicating that the switch Pol I (Fig. 4E). Overall, TbPIP5K and TbPLC and the metabolites was not due to nonspecific effects of knockdown. The switching rate PI(4)P, PI(4,5)P2, and PI(3,4,5)P3 are primarily located at the inner − following TbPIP5K reexpression was 1.6 × 10 2 cells per cell cycle, face plasma membrane, whereas TbPIP5Pase is primarily a nuclear which is ∼103-fold higher than reported for in vitro switching (26). protein that is located near telomeres. Overall, following the temporary knockdown of TbPIP5K, about two-thirds of the clones expressed the original VSG or VSGs that TbPIP5Pase Is a PI 5 Phosphatase with Preference for Substrates with were in known BF ESs. The other clones did not express either of a 4 Phosphate. The TbPIP5ase is conserved in kinetoplastids these types of VSGs and presumably expressed VSGs that resulted (∼30–50% amino acid identity) but shares less than 12% overall from recombination involving the numerous non ES VSG genes. sequence identity with the human and yeast 5-phosphatase en- Thus, release from the temporary knockdown of TbPIP5K restored zymes. Nevertheless, it conserves the K292 and G293 residues that VSG monoalellic expression and revealed a high switching fre- are required for recognition of the inositol ring and 1-phosphate, quency of the VSG gene expressed. (green box, Fig. 5A), which is characteristic of all phosphoinositides and the H312, D360, and N362 residues that form the 5-phosphate Derepression of VSG Genes Is Associated with an Increase in the binding pocket in human 5-phosphatases (red boxes) (27). Native Number of Telomeric and Pol I Foci. FISH analysis using a telo- TAP tagged TbPIP5Pase was purified from T. brucei BF by affinity meric repeat probe and 3D deconvolution microscopy showed chromatography with IgG-Sepharose via the protein-A domain, and that the nuclei of ∼80% of BF parental or CN cells that express the activity of the TEV-eluate was assayed with several IP and PI IP pathway genes (i.e., tet+) had a few prominent telomeric foci substrates (Fig. 5 B and C). The purified enzyme had negligible and generally less than five foci (Fig. 3 A–C and Fig. S4). These activity with several soluble IPs but preferential activity with PI(4,5)P2 foci likely represent clusters of telomeres because T. brucei has and PI(3,4,5)P3. There was some activity with PI(5)P and PI(3,5)P2 ∼200 telomeres [from ∼100 minichromosomes and 11 large but essentially no activity with potential PI substrates that lack a chromosomes (24)]. Knockdown of TbPIP5K or TbPIP5Pase for 5-phosphate. The presence of a 4-phosphate in PI substrates that 24 h resulted in ∼80% of the cells having 6 to >10 telomeric foci contain a 5-phosphate enhanced the enzyme activity. These results per nucleus, with an apparently broadened intranuclear distri- indicate that TbPIP5Pase dephosphorylates 5-phosphate substrates bution. Knockdown of TbIPMK, which does not result in ES with preference for those that also contain a 4-phosphate (Fig. 5D). derepression, did not affect the number of telomeric foci, which Kinetic analysis showed higher activity with PI(4,5)P2 compared was similar to that in WT BF or PF cells (Fig. S4). The TbPIP5K with PI(3,4,5)P3 and only low activity with I(1,4,5)P3 and primarily and TbPIP5Pase knockdowns did not affect parasite growth, cell at exceptionally high substrate concentrations (Fig. 5E). Enzymo- cycle and morphology or cell viability over this 24-h period (see logic analysis revealed that the Km and the Vmax was slightly dif- above), suggesting that these effects are not due to nonspecific ferent for PI(4,5)P2 and PI(3,4,5)P3, but the enzyme efficiency was factors. Similarly, knockdown of TbPIP5Pase also resulted in similartobothsubstrates(Fig.5D and F). This enzyme has been multiple extranucleolar Pol I foci. These foci became evident annotated as an I(1,4,5)P3 5-phosphatase in GeneDB based on 12 h after knockdown, which correlates with the early ES dere- sequence homology, but these analyses indicate that it is a PI pression and some of the Pol I foci partially colocalized with 5-phosphatase with specificity toward PI(4,5)P2 and PI(3,4,5)P3. telomeric foci (Fig. 3 D and E). Because these cells transcribe multiple ESs, the multiple Pol I foci likely correspond to dif- TbPIP5Pase Interacts with the Telomeric Protein TbRAP1. Due to the ferent sites of ES transcription by Pol I. Longer periods of nuclear location of TbPIP5Pase near telomeres, we analyzed TbPIP5Pase knockdown resulted in less coherent nucleolar Pol I whether it associates with known telomeric proteins. TbRAP1 is a fluorescence (Fig. 3D at 24 h), perhaps due to Pol I depletion telomeric protein that associates with the TbTRF, and TbRAP1

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1501206112 Cestari and Stuart Downloaded by guest on September 29, 2021 PNAS PLUS ABTbPIP5K CN TbPIP5Pase C TbIPMK CN CN Tet + Tet + Tet + Tet - Tet - Tet -

30 30 n = 1,140 30 n = 1,099 n = 644 Tet + * * Tet + Tet + * Tet - Tet - Tet - * * * 20 * * 20 * 20 * * * * * * * Cell (%) Cell Cell (%) 10 Cell (%) 10 10

0 0 0 1 2 3 4 5 6 7 8 9 >10 1 2 3 4 5 6 7 8 9 >10 1 2 3 4 5 6 7 8 9 >10 Number of foci/nucleus Number of foci/nucleus Number of foci/nucleus D Tet + Tet - 6h 12h 18h 24h

Fig. 3. Effect of TbPIP5K and TbPIP5Pase knockdown on the number of telomeric and Pol I foci.

(A–C) FISH analysis with the PNA-(CCCTAA)3-FITC probe Pol I for telomeres after 24 h knockdown of TbPIP5K, n TbPIP5Pase or TbIPMK expression; tet +,0.5μg/mL. Quantification of telomeric foci per nucleus are

shown below the images. See also Fig. S4.(D)IF MICROBIOLOGY analysis of Pol I foci after conditional knockdown of TbPIP5Pase. (E) Pol I and telomeric foci 24 h after E Pol I + telomeres knockdown of TbPIP5Pase. Pol I was detected with DAPI Pol I DAPI Pol I Mab 16B1a (8) followed by anti–IgG-AlexaFluor 594

(red), telomeres with a FITC-conjugated PNA-(CCCTAA)3 probe (green), and DNA stained with DAPI (blue). Ar- rowheads indicate regions of Pol I foci (D)orPolIand

TetTelomeres + MERGE Telomeres MERGE telomeric foci colocalization (E). Data in A–C are the

Tet – (24h) means (SEM) of three experiments. *Significance at P < 0.05. (Scale bar, 1 μm.)

knockdown has been shown to result in ES derepression (9). We cell line that has an HA-tagged TbRAP1 allele also resulted in replaced an endogenous allele of TbRAP1 with an HA-tagged increased numbers of TbRAP1-HA foci (Fig. 7D). These results allele in a cell line that expresses V5-tagged TbPIP5Pase in a tet- indicate that positioning of TbRAP1 and TbTRF is affected in a dependent fashion. IF analysis with Mabs that are specific for the TbPIP5K- or TbPIP5Pase-dependent fashion, similarly to the tags showed that TbPIP5Pase-V5 and TbRAP1-HA colocalize effect on telomeres as analyzed by FISH (Fig. 3). Because no (Fig. 6A). TbRAP1-HA was coimmunoprecipitated with TbPIP5- obvious effect was detected on TbNUP1-HA after TbPIP5K Pase-V5 that had been pulled down from total BF lysate with anti- knockdown, it indicates that the IP pathway metabolites affect V5 Mabs, as revealed by Western analysis using anti-HA Mabs specifically telomeres and telomeric proteins. (Fig. 6B). In addition, TbPIP5Pase-TAP and TbRAP1-HA pre- To analyze whether the IP pathway controls the associations of dominantly sedimented at ∼10S as shown by Western analysis of the telomeric proteins, we analyzed TbRAP1 and TbTRF sedi- fractions collected from 10% to 30% (vol/vol) glycerol gradients mentation after knockdown of TbPIP5K. TbPIP5K knockdown for (Fig. 6C). However, a discrete peak was also consistently detected 18 h shifted TbRAP1-HA to a higher sedimentation coefficient in at ∼20S for both proteins. Analysis of TbTRF-HA also showed 10–30% (vol/vol) glycerol gradients (Fig. 7E). In contrast, TbPIP5K that it sediments predominantly at ∼10S, but we did not detect an knockdown shifted TbTRF-HA to a slightly lower sedimentation ∼20S peak. These results indicate that TbPIP5Pase, TbRAP1, and coefficient. These results imply that TbRAP1 and TbTRF in- TbTRF interact, directly or indirectly. TbRAP1 and TbPIP5Pase teractions and their effect on telomeric silencing function in a may be in two complexes that may include TbTRF and differ in their TbPIP5K-dependent fashion, which may be due to the effect of abundance and sedimentation coefficient, possibly due to their as- specific 5-phosphate metabolites, e.g., PI(4,5)P2, on their interactions sociations with additional proteins. and function. In addition, the increase in the coefficient of sedimentation of TbRAP1-HA (Fig. 7E)correlateswithsimultaneous IP Pathway Controls TbRAP1 Interactions and Telomeric Silencing. To transcription of multiple ESs, which suggests that the ∼20S TbRAP1- assess the effect of manipulating the IP pathway genes on these HA peak (Fig. 6C) may be related to transcriptionally active ESs. telomeric proteins, we replaced an endogenous allele of TbRAP1, Overall, specific IP pathway enzymes and presumably their metabo- TbTRF, or TbNUP1 with HA-taggedversionsinaTbPIP5KCN lites control ES transcription by affecting the interactions and func- cell line and examined the effects of knockdown of TbPIP5K by IF tions of proteins involved in telomeric silencing. (Fig. 7 A–C). TbPIP5K knockdown for 24 h resulted in increased numbers of TbRAP1-HA and TbTRF-HA foci but had no obvious Discussion effect on numbers or apparent locations of the nuclear envelope We found that specific steps in the IP pathway function in the protein TbNUP1-HA. Similarly, knockdown of TbPIP5Pase in a control of ES transcription and antigenic switching in T. brucei.

Cestari and Stuart PNAS Early Edition | 5of10 Downloaded by guest on September 29, 2021 A B DIC+MERGE K5PIPbT PI(4,5)P2 EGREM DIC+MERGE PI(3,4,5)P3 + PI(4,5)P2 TbPIP5K PCC = 0.88 PI(3,4,5)P3 DIC+MERGE TbPLC PI(4,5)P2 MERGE DIC+MERGE

+ PI(4)P TbPLC PI(4,5)P2 PI(4)P

PCC = 0.87 C TbPIP5Pase + telomeres D TbRAP1 + telomeres TbTRF + telomeres DIC+MERGE TbPIP5Pase + DAPI DIC+MERGE Telomere DIC+MERGE Telomere

TbPIP5Pase Telomeres TbRAP1 MERGE TbTRF MERGE

PCC = 0.65 PCC = 0.75 MERGE E TbPIP5Pase + Pol I TbPIP5Pase + TbNUP1 DIC+MERGE Pol I DIC+MERGE TbNUP1

TbPIP5Pase MERGE TbPIP5Pase MERGE

PCC = 0.68 PCC = 0.37 PCC = 0.47

Fig. 4. Subcellular locations of IP pathway proteins and metabolites. (A) IF analysis of (Upper) V5-tagged TbPIP5K (red) and its product PI(4,5)P2 (green) and (Lower) V5-tagged TbPLC (red) and its substrate PI(4,5)P2 (green). (B) PI(4)P and PI(3,4,5)P3 metabolites. (C) V5-tagged TbPIP5Pase (red) and telomeres (green). (D)(Left) HA-tagged TbRAP1 (red) and telomeres (green) and (Right) HA-tagged TbTRF (red) and telomeres (green). (E)(Left) V5-tagged TbPIP5Pase (green) and Pol I (red) and (Right) V5-tagged TbPIP5Pase (green) and HA-tagged TbNUP1 (red). Arrowheads indicates extranucleolar Pol I. Metabolites were detected with FITC-

conjugated Mabs that are specific to each metabolite. A FITC-conjugated PNA-(CCCTAA)3 probe was used to detect telomeres by FISH, and DNA was stained with DAPI (blue). V5-tagged and HA-tagged proteins were detected with anti-V5 and anti-HA antibodies (see SI Materials and Methods for details). The mean of the Pearson coefficient of correlation (PCC) from multiple cells are indicated for colocalization. See Fig. S5 for the effects of TbPIP5K knockdown on metabolites and staining of nonpermeabilized cells. (Scale bars: A, B, D,andE,2.5μm; C,1μm.)

Conditional knockdown of TbPIP5K or TbPIP5Pase or over- transcribed in procyclic forms and ESs that lack ESAGs are se- expression of TbPLC resulted in transcription of multiple ESs lectively transcribed in MFs (29, 30). The silencing of all ESs in and cells that expressed more than one VSG protein on their PFs and the selective silencing of all but one ES in MF and BF life surface. This transcription was specific to telomeric ESs and did cycle stages likely entails responses of the regulatory system to not affect transcription of procyclin genes or genes that are stage-specific differences in parasite metabolism, physiology, and transcribed by Pol II or Pol III. The derepression of multiple ESs environment, i.e., 28 °C vs. 37 °C and different nutrients. It would was accompanied by an increase in the number of telomeric foci not be surprising if the IP pathway contributed to the stage-specific and the appearance of multiple extranucleolar Pol I foci. Reex- regulation of ES transcription. pression of TbPIP5K after temporary knockdown restored mono- The process by which all but one of the ESs are silenced in- alellic VSG transcription, and the majority of resultant cells volves multiple proteins that include TbRAP1 and TbTRF (9, changed which VSG was expressed. TbPIP5K, TbPLC, and their 31). TbPIP5Pase associates, directly or indirectly, with TbRAP1 metabolites are primarily located at the plasma membrane. How- and TbTRF as shown here by the results of our IF colocalization, ever, TbPIP5Pase is in the nucleus near telomeres. TbPIP5Pase is in pull down, and sedimentation experiments (Figs. 6 and 7). In a complex with TbRAP1 and TbTRF, and knockdown of TbPIP5K addition, we found that knockdown of TbPIP5K shifts the sedi- altered their association and their distribution within the nucleus. mentation of TbRAP1 and TbTRF, which implies that specific The results indicate that specific steps in the IP pathway control IP metabolite levels affect the associations among these proteins. monoallelic ES transcription via telomeric silencing and VSG The proteins TbDOT1B and TbNUP1, which function in histone switching and hence antigenic variation in T. brucei. H3 methylation and heterochromatin organization, respectively, The IP pathway transcriptional control reported here is spe- also participate in ES regulation (10, 11). Hence, remodeling cific to telomeric ESs. The specificity is likely due to a combi- of the chromatin at telomeric regions and associations among nation of recognition of the ES promoters by Pol I and their telomeric proteins contribute to ES silencing, and this process subtelomeric locations, which provides for the process of telo- appears to be controlled, at least in part, by the IP pathway. meric silencing (15, 28). The sequences of the ES promoters are The overall control of transcription of a single ES may also highly conserved (∼90% nucleotide identity), and they are func- involve the relocation of ESs within the nucleus. The appearance tionally related to promoters of the rRNA and procyclin genes, of increased numbers of telomeric, TbRAP1, TbTRF, and Pol I which are also transcribed by Pol I but are not at telomeric sites (2, foci in the nucleus when multiple ESs are transcribed, as shown 28). The specificity of the ES transcriptional control is maintained here, suggests that the control of ES transcription may entail through the life cycle of the parasite, during which no ESs are repositioning of telomeres in the nucleus. The fewer telomeric foci

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TEV- TEV- 1500 )

B kDa Mr C 4 Substrates at 50 μM eluate eluate 1250 *** 250 37°C for 1h *** 200 1000 100 75 750 55 * 500 37 25 250 **

20 Activity (pmol PO 17 0 10 WB: anti-CBP 4-20% SDS/PAGE PI(3)P PI(4)P PI(5)P I(1,4)P2 PI(3,4)P2 PI(4,5)P2 PI(3,5)P2 I(1,4,5)P3 No enzyme PI(3,4,5)P3 I(1,3,4,5)P4 No substrate D I(1,2,4,5,6)P5

Kcat /Km Kcat /Km 1.8x104 2.1x104 MICROBIOLOGY

PI(4,5)P2 EFI(1,4,5)P3 PI(3,4,5)P3 PI(4,5)P2 PI(3,4,5)P3 2000 2000 K = ) m 82 μM 4 V max = 2032 pmol 1500 1500

1000 1000 pmol/min) (pmol PO 4 Velocity 500 500 K = (PO m 49 μM

Activity V max = 1401 pmol 0 0 0 50 100 150 200 0 50 100 150 200 Substrate [ μM ] Substrate [ ] μM

Fig. 5. Enzymatic analysis of TbPIP5Pase. (A) Alignment of TbPIP5Pase sequence with the human and yeast 5 phosphatases showing conservation of key residues. The green box indicates residues that are conserved in the 5 phosphate pocket and the red boxes those that recognize the 1 phosphate and inositol ring. (B) SDS/PAGE (4–20%) imperial stained TEV-eluate of TAP-tagged enzyme (asterisk) purified from T. brucei BF (Left) and Western analysis with Mab anti-CBP (calmodulin-binding peptide) (Right). (C) Activity of TEV-elute of the native enzyme with various IP and PI substrates (Materials and Methods). -1 −1 (D) Diagram showing conversion of the preferred substrates and enzyme efficiency (Kcat/Km) in [(mol/liter) /S ] obtained from data in F.(E) Kinetic analyses of TEV-eluted TbPIP5Pase. (F) Enzymologic analyses of TEV-eluted TbPIP5Pase comparing velocities with PI(4,5)P2 and PI(3,4,5)P3. Data in C, E, and F are the mean (SEM) of three experiments in triplicate; significance is *P < 0.05 or ***P < 0.0001.

when a single ES is expressed also suggests that the silent telo- generate the 5 position phosphorylated lipid-associated and soluble meres may be clustered, a process that has been associated with metabolites that correlate with the control of ES transcription. telomeric position effect silencing in other organisms (16, 32). Our IF experiments show that these enzymes and their lipid- Telomere clustering in yeast is accompanied by the association of associated metabolites are located at the plasma membrane. the telomeres with the nuclear envelope and the formation of However, the PI and the soluble IP metabolites (for which we heterochromatin (33). The single active ES forms a discrete have no antibodies) may be present in the nucleus but at lower extranucleolar Pol I focus that is detectable by IF, named the levels, perhaps as a result of their flux (34). In yeast and mam- expression site body (ESB) (8). The appearance of several Pol I malian cells, the IP pathway enzymes and metabolites are found foci when multiple ESs are fully transcribed suggests that these are at the plasma membrane and also in intracellular organelles, multiple sites of complete ES transcription by Pol I, which have vesicles, and the nucleus, where they exert different regulatory more Pol I associated with them than do ESs where transcription is roles (23, 35). The nuclear and telomeric location of TbPIP5Pase abortive. Thus, there can be multiple subnuclear sites where ESs and its association with TbRAP1 and TbTRF suggests that can be transcribed, and the single ESB that is seen in normal cells TbPIP5Pase functions via physical association in a fashion that is is likely due to its substantial content of associated Pol I. responsive to the 5 phosphate metabolites PI(4,5)P2 or PI(3,4,5) Enzymologic analysis of the TbPIP5Pase shows that it is a P3, and it may exert a local control of these metabolite levels. 5-phosphatase that is specific for PI(4,5)P2 and PI(3,4,5)P3, which TbPIP5Pase, TbRAP1, and TbTRF all primarily sediment at are the metabolic products of TbPIP5K, and PI(4,5)P2 is also the ∼10S in glycerol gradients, but another less abundant peak is substrate of TbPLC (Fig. 1B). TbPIP5K and TbPLC, respectively, consistently seen at ∼20S. TbPIP5K knockdown resulted in the

Cestari and Stuart PNAS Early Edition | 7of10 Downloaded by guest on September 29, 2021 A recovered cell lines did not express any of the VSG genes that were originally in the telomeric ESs, and thus they likely expressed a DIC+MERGE TbPIP5Pase+DAPI MERGE VSG gene that arose by recombination. This range of frequencies following temporary knockdown is reminiscent of the preferential order of expression that occurs during antigenic variation following infection (36). The differences in ES sequences and/or epigenetic TbPIP5Pase TbRAP1 chromatin marks may contribute to their probability of not being silenced and hence the order of expression during antigenic variation. None of the five VSG genes that are in the MF ESs were expressed following the temporary knockdown, which may be due PCC = 0.81 to a low probability of them being selected for expression or it may reflect a mechanism that selects them for preferential expression in the metacyclic stage as indicated above. The frequent occurrence of B switching to expression of VSGs that appear to have arisen by re- Imput

α-V5:IP α-IgG:IP Tet combination implies that the conditions that enabled transcription + - + - + of the ESs may have increased the probability of recombination because the temporary perturbation only spanned four to five cell TbRAP1-HA divisions. Active transcription is known to favor homologous re- anti-HA combination (37). RNAi knockdown of the telomeric proteins TbTRF or TbTRF-interacting factor 2 (TbTIF2) increased VSG TbPIP5Pase-V5 switching rates in T. brucei BF (38, 39). The loss of these proteins anti-V5 appears to have enhanced repair and recombination. Overall, these C results indicate that perturbation of the IP pathway may precipitate 10S 20S antigenic switching by both switching transcription among telomeric

1 3 5 7 9 11 13 15 17 19 21 23 Imput kDa ESs or by recombination, perhaps by affecting associations and functions of proteins that associate with telomeric ESs. 62 TbPIP5Pase-TAP A general model that embraces knowledge from other systems (19, 20, 22) is that certain IP metabolites may specifically bind to proteins that have roles in telomeric silencing and affect their interactions with other proteins and/or their functions, e.g., histone 91 TbRAP1-HA modification and chromatin remodeling. The ultimate consequence of the derepression is that the chromatin at multiple ESs is per- missive for the elongation of transcription by Pol I through the VSG 37 TbTRF-HA genes. Our results suggest that the control of VSG gene allelic exclusion is the result of the properties of the cells molecular com- ponents, which are functionally integrated within the cell. The Fig. 6. TbPIP5Pase associates with telomeric proteins. (A) IF showing colocali- locations and levels of IP enzymes and metabolites are determined zation of V5-tagged TbPIP5Pase (green) and HA-tagged TbRAP1 (red). (Scale bar, by these properties and may maintain a dynamic equilibrium that 1 μm.) (B) Western analysis with anti-V5 and anti-HA Mabs of V5-tagged results in silencing of all but one ES. Disruption of the equilibrium TbPIP5Pase immunoprecipitated with Mab anti-V5 from total lysate of cells alters the associations and/or functions of proteins, perhaps in- that express tet-inducible TbPIP5Pase-V5 and constitutively express HA-tagged volving specific interactions with the IP metabolites, resulting in the TbRAP1. (C) Western analysis of 10–30% (vol/vol) glycerol gradient fractions of loss of silencing of all ESs. Reestablishment of the equilibrium re- lysates from T. brucei BFs that express TAP tagged TbPIP5Pase (Top), HA-tagged sults in silencing of all but one ES but a likely switch to a different TbRAP1 (Middle), or HA-tagged TbTRF (Bottom) using the rPAP reagent and anti-HA Mab, respectively. ES. This hypothesis implies that the equilibrium maintains the BF cells in a state that is poised for antigenic switching. By way of perspective, the IP pathway functions to regulate shift of TbRAP1 to a higher sedimentation coefficient but had silencing of telomeric ESs and antigenic switching in T. brucei minor effects on TbTRF sedimentation. The higher TbRAP1 and thus controls allelic exclusion of VSG genes. The stage- sedimentation correlates with the derepression of multiple ESs specific control of VSG gene expression implies that the IP and may result from TbRAP1 association with additional pro- pathway may also participate in the control and coordination of teins or modifications that affect the conformation of TbRAP1 stage-specific processes during the life cycle in African try- or other chromatin-associated proteins or DNA. The data imply panosomes. In addition, the IP pathway is conserved from yeast that the IP metabolites may directly affect TbRAP1 interaction to humans (18); hence, the IP pathway may function in telomeric with TbTRF and perhaps other telomere-associated proteins. silencing and allelic exclusion in other organisms such as the var Plasmodium Specific IP metabolites may repress ES transcription by con- expression of genes in spp., or perhaps in the al- T. cruzi trolling the association of proteins that function in telomeric lelic exclusion of odorant receptors in mammals (40, 41). Leishmania T. brucei silencing, e.g., TbRAP1. Perturbation of these metabolite levels and , which are related to , conserve the IP may alter the associations of these telomeric proteins and thus pathway but lack telomeric ESs and may have adapted the pathway result in the transcription of numerous ESs. for alternate functions, e.g., control the expression of numerous Reexpression of TbPIP5K after temporary knockdown restored surface proteins. monoallelic VSG expression. Analysis of the cloned cell lines re- Materials and Methods covered after the knockdown revealed that most cells switched the VSG gene expressed at a frequency that is three orders of magni- Cell Lines. Cell lines that conditionally express IP pathway genes, i.e., CN cells, were generated as previously described (42); see Dataset S1 for primers. Cells tude higher than the normal in vitro rate (26). There was a range of that conditionally express C-terminal V5-tagged TbPLC, TbPIP5K, or TbPIP5Pase frequencies for the VSG expressed with the VSG2, which was were generated using the pLEW100-3V5 vector, and cells that constitutively expressed before knockdown, being the most frequent followed by express HA-tagged TbRAP1, TbTRF, or TbNUP1 were generated by replacing an VSG13 and others at lower frequency (Fig. S3). About 21% of the endogenous allele with one that has a 3′ terminal tag using PMOTag2H vector as

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1501206112 Cestari and Stuart Downloaded by guest on September 29, 2021 ABD PNAS PLUS TbPIP5K CN_TbRAP1-HA TbPIP5K CN_TbTRF-HA TbPIP5Pase CN_TbRAP1-HA + tet + tet tet + tet – (24h) tet – (24h) tet – (24h)

n = 710 Tet+ ** 30 n = 259 Tet+ 30 n = 215 Tet+ * 30 Tet- ** Tet- Tet- *** * ** 20 20 * * ** ** * ** 20 *** ** * * * ** Cell (%) Cell (%) 10 10 Cell (%) 10

0 0 0 1 2 3 4 5 6 7 8 9 >10 1 2 3 4 5 6 7 8 9 >10 1 2 3 4 5 6 7 8 9 >10 Number of foci/nucleus Number of foci/nucleus Number of foci/nucleus

10S 20S

C E 1 3 5 7 9 11 13 15 17 19 21 23 Imput TbPIP5K_TbNUP1-HA TbRAP1-HA

tet - tet + TbRAP1-HA tet + TbPIP5K CN MICROBIOLOGY 10S 20S

1 3 5 7 9 11 13 15 17 19 21 23 Imput

TbTRF-HA tet – (24h)

tet - tet + TbTRF-HA TbPIP5K CN

Fig. 7. Effects of TbPIP5K knockdown on telomeric proteins. (A–D) IF analysis with anti-HA Mab of HA-tagged TbRAP1, TbTRF, or TbNUP1 before and after TbPIP5K or TbPIP5Pase knockdown for 24 h. Quantification of the foci is shown below the images except in C and are the means (SEM) of three experiments each (*P < 0.05 and **P < 0.001). (E) Western analysis with anti-HA Mab of 10–30% (vol/vol) glycerol gradient fractions of cellular lysates from CN TbPIP5K cells that express a TbRAP1-HA (Upper) or TbTRF-HA (Lower) tagged gene. Knockdown was for 18 h by withdrawal of the 0.5 μg/mL tet. (Scale bars, 2.5 μm.)

previously published (43, 44). Growth curves in the presence and absence of tet by limited dilution into 96-well plates after five times 10-fold serial dilution. were determined by seeding 25-cm2 cell culture flasks with 5.0 × 104 parasites/mL The clones were expanded for an additional 3–5 d, and the RNAs were in 10 mL HMI-9 medium supplemented with 10% (vol/vol) FBS, 2.0 μg/mL isolated as above. The VSG expressed was assayed by qRT-PCR (SI Materials G418, and 2.5 μg/mL of phleomycin to select for the tet regulatable allele. and Methods). VSG switching was calculated as previously published (26). Parasites were counted using a Neubauer chamber. Western and Glycerol Gradient. Western and glycerol gradient analyses were RNA Quantification. CN cell lines were grown with or without 0.5 μg/mL tet performed as described previously (43). Briefly, lysates from 5.0 × 108 cells for 18 or 24 h and harvested at room temperature (10 min, 1,300 × g), RNA were obtained with 1% Triton-X-100 in Tris 50 mM/NaCl 150 mM with EDTA- was extracted using TRIzol (Life Technologies), and cDNA preparation free protease inhibitor mixture (Roche), loaded on 10–30% (vol/vol) con- and qRT-PCR were done as previously described (42). The absolute RNA tinuous glycerol gradients, and centrifuged for 10 h at 38,000 × g in a SW40 levels were determined by qRT-PCR relative to a standard curve using the Beckman ultracentrifuge. Fractions of 500 μL each were collected from the β-tubulin gene fragment in the pHD1344-tub vector, and the amounts of top and analyzed by Western analysis. VSG, ESAG, and Pol II mRNAs and 18S rRNA were calculated as previously described (45). Immunoprecipitation. T. brucei BF cells (5.0 × 107) that express tet-regulatable TbPIP5Pase-V5, and endogenous TbRAP1-HA were lysed in 50 mM Tris, IF and FISH. T. brucei midlog phase cells were fixed with 2% (wt/vol) 150 mM NaCl, 1% Triton-X-100, 0.5% sodium deoxycolate, and 0.2% Non- paraformaldehyde in PBS and adhered to poly-L-lysine–treated 2-mm cover idet P-40 with EDTA-free protease inhibitor mixture (Roche). The cleared glass (Fisher) and permeabilized with 0.2% Nonidet P-40 in PBS for 5 min. Cells lysates were obtained after a 5-min centrifugation at 16,000 × g and in- were blocked for 1 h with 3% (wt/vol) BSA in PBS and incubated for 2 h at cubatedwith2μg/mL Mabs anti-V5 (Life Technologies) cross-linked to Protein A room temperature or overnight at 4 °C with specific antibodies (SI Materials Mag Sepharose according to the manufacturer’s instructions (GE Healthcare Life and Methods). An FITC-conjugated PNA (CCCTTA)3 telomere C FISH probe Sciences). An anti-mouse IgG (Bio-Rad) cross-linked to Protein A Mag Sepharose (Biosynthesis) was used for FISH as previously described (11). DNA was stained was used as a negative control. After a 2-h incubation at 4 °C, the mix was with 1 μg/mL DAPI (Sigma), and slides were mounted with ProLong Gold washed in washing buffer (50 mM Tris, 150 mM NaCl, and 0.2% Nonidet P-40) Antifade Reagent (Life Technologies). Images were obtained using a Deltavi- andelutedin2× Tricine Sample Buffer (Bio-Rad) without reducing agent. The sion 3D deconvolution microscope (Olympus IX70) and analyzed with Soft- eluted fractions were supplemented with 1.4 M β-mercaptoethanol and ana- works software (Applied Precision). lyzed by Western analysis.

VSG Switching. Clonal TbPIP5K and TbIPMK CN cells that express VSG2 were Enzymology. BF cells that conditionally express TAP-tagged TbPIP5Pase were grown for 24 h without tet for gene knockdown, followed by reexpression by produced, and TbPIP5Pase-CBP (250 ng) was expressed, purified, and assayed adding 0.5 μg/mL tet to medium. Cells (1.0 × 105) were immediately cloned with soluble 50 μM inositol phosphates or diC8 phosphatidylinositol phosphates

Cestari and Stuart PNAS Early Edition | 9of10 Downloaded by guest on September 29, 2021 (Echelon) as previously described (27, 43). Enzyme kinetic reactions were pre- tervals of 95% were considered statistically significant. Pearson coefficient pared using 1–300 μM of I(1,4,5)P3, diC8-PI(4,5)P2 or diC8-PI(3,4,5)P3 and in- of correlation for colocalization analysis was performed with Softworks cubating for 30 min at 37 °C, and kinetics were calculated by nonlinear software (Applied Precision) according to the manufacturer’s instructions. regressions using GraphPad Prism 5.03 (GraphPad Software). ACKNOWLEDGMENTS. We thank members of the K.S. laboratory, John Statistical Analysis. Data are shown as means ± SEM, and a two-tailed Stu- Aitchison, and Peter Myler for helpful comments on this work; Anna Sokolov for administrative support; and Keith Gull for anti-Pol I antibody. The work dent t test was calculated using GraphPad Prism 5.03 (GraphPad Software). received support from National Institutes of Health Grant R01AI078962 and Statistical analyses of gene expression were performed by a pairwise fixed Supplement R01AI014102-37S1 (to K.S. and I.C.), American Heart Association reallocation randomization test (46). P values of 0.05 with confidence in- Fellowship 14POST18970046 (to I.C.), and the Seattle Biomedical Research Institute.

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