Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes

Abstract

African trypanosomes evade clearance by host antibodies by periodically changing their variant surface glycoprotein (VSG) coat. They transcribe only one VSG gene at a time from 1 of about 20 telomeric expression sites (ESs). They undergo antigenic variation by switching transcription between telomeric ESs or by recombination of the VSG gene expressed. We show that the inositol phosphate (IP) pathway controls transcription of telomeric ESs and VSG antigenic switching in Trypanosoma brucei. Conditional knockdown of phosphatidylinositol 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5-Pase) or overexpression of phospholipase C (TbPLC) derepresses numerous silent ESs in T. brucei bloodstream forms. The derepression is specific to telomeric ESs, and it coincides with an increase in the number of colocalizing telomeric and RNA polymerase I foci in the nucleus. Monoallelic VSG transcription resumes after reexpression of TbPIP5K; however, most of the resultant cells switched the VSG gene expressed. TbPIP5K, TbPLC, their substrates, and products localize to the plasma membrane, whereas TbPIP5Pase localizes to the nucleus proximal to telomeres. TbPIP5Pase associates with repressor/activator protein 1 (TbRAP1), and their telomeric silencing function is altered by TbPIP5K knockdown. These results show that specific steps in the IP pathway control ES transcription and antigenic switching in T. brucei by epigenetic regulation of telomere silencing.O nly one of the ∼20 telomeric expression sites (ESs) is transcribed at a time in Trypanosoma brucei in the mammalian infectious stage bloodstream (BF) or metacyclic forms (MFs), whereas no ES is transcribed in the insect stage procyclic forms (PFs) (1). Each ES contains 1 telomeric variant surface glycoprotein (VSG) gene, whose expression confers a distinct cellular antigenic type and up to 12 expression site-associated genes (ESAGs) whose functions are incompletely understood ( Fig. 1A) (2-4). The 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 pseudogenes (5, 6). ESs are transcribed by RNA polymerase I (Pol I), which initiates at the single promoter at all ESs but terminates within a few kilobases except at one fully transcribed ES (7,8).RNAi knockdown of expression of the nuclear protein TbRAP1 that interacts with the TTAGGG-binding factor (TbTRF) or of the nuclear lamina protein 1 (TbNUP1) or deletion of the histonelysine N-methyltransferase DOT1B (TbDOT1B) alleles each results in transcription of silent ESs (9-11). Similarly, RNAi knockdown of the transcription facilitating histone chaperone suppressor of ty 16 (TbSPT16) or the SWI2/SNF2-related chromatin-remodeling protein TbISWI or deletion of histone deacetylase sirtuin 2-related protein 1 (TbSIR2RP1) each increases transcription near the promoter but not the VSG gene of silent . These results indicate that the control of ES transcription involves telomeric silencing (15), which entails...

Chemogenetic Characterization of Inositol Phosphate Metabolic Pathway Reveals Druggable Enzymes for Targeting Kinetoplastid Parasites

“…In cases where null cells could not be obtained, we generated conditional null (CN) cells by replacing both endogenous alleles in cells that transcribe an ectopic copy of the target gene under tetracycline (tet) control. Using this approach we showed previously that the genes encoding TbPIP5K1, TbPIP5Pase1, TbIPMK and TbCDS enzymes were essential for T. brucei BF growth in vitro , whereas TbIMPase1 was not essential (Cestari and Stuart, 2015) (Table 1). We show here that genes encoding a predicted TbPIP5Pase2 and TbIP5Pase are not essential for growth of T. brucei BF, although knockdown of TbPIP5Pase2 slightly reduced parasite growth (Table 1 and Fig S1).…”

“…Thus, although the TbIMPase2 null cells did not exhibit severe growth defects in vitro , they exhibited reduced infectivity in vivo . The reasons underlying this recrudescent parasitemia after a subpatent period are unknown, but it may be due to the IP pathway role in regulating VSG gene expression (Cestari and Stuart, 2015) or IP requirements for GPI synthesis (Martin and Smith, 2006). Overall, at least four of the eight IP pathway genes essential for T. brucei BF in vitro are also essential for infection of mice, whereas one nonessential gene (TbIMPase2) is important for infectivity.…”

“…Moreover, genes involved in the synthesis of IPs or phosphatidylinositol (PIs) are essential for parasite growth (Martin and Smith, 2005; Martin and Smith, 2006). We and others have also shown that some PI kinases and phosphatases are essential for bloodstream forms (BF) of T. brucei (Cestari and Stuart, 2015; Hall, et al, 2006; Rodgers, et al, 2007) (Fig 1). The essentiality of these enzymes is likely due to the variety of processes that their metabolic products control (Millard, et al, 2013; Watson, et al, 2012).…”

“…We recently showed that this pathway controls transcription and allelic exclusion of variant surface glycoprotein (VSG) genes in T. brucei (Cestari and Stuart, 2015), allowing T. brucei to evade the host immune response. The IP pathway also regulates Ca 2+ homeostasis (Huang, et al, 2013) and Golgi biogenesis (Hall, et al, 2006; Rodgers, et al, 2007) in T. brucei , differentiation and infectivity of T. cruzi (Hashimoto, et al, 2013), and its enzymes are also targets for malaria drugs (Mbengue, et al, 2015).…”

VEX1 controls the allelic exclusion required for antigenic variation in trypanosomes

“…For example, a histone H3 variant, a bloodstream stagespecific modified DNA base known as J or hydroxymethyluracil (22,23), the chromatin remodeling ISWI complex (24), the histone H3K76 trimethyltransferase DOT1B (25), and the telomere-associated protein RAP1 (26) all facilitate VSG-ES silencing. In addition, cohesin function facilitates maintenance of the active VSG-ES (27), and inositol phosphate signaling impacts VSG-ES regulation (28). Allelic exclusion, however, requires the establishment of differential expression states and coordination among members of a gene family, which are not understood (1)(2)(3).…”