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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 an- tigenic 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 B 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.
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