PI(3,4,5)P3 allosteric regulation of repressor activator protein 1 controls antigenic variation in trypanosomes

Abdoulie O. Touray; Rishi Rajesh; Tony Isebe; Tamara Sternlieb; Mira Loock; Oksana Kutova; Igor Cestari

doi:10.1101/2023.05.11.540368

Abstract

African trypanosomes evade host immune clearance by antigenic variation, causing persistent infections in humans and animals. These parasites express a homogeneous surface coat of variant surface glycoproteins (VSGs). They transcribe one out of hundreds of VSG genes at a time from telomeric expression sites (ESs) and periodically change the VSG expressed by transcriptional switching or recombination. The mechanisms underlying the control of VSG switching and its developmental silencing remain elusive. We report that telomeric ES activation and silencing entail an on/off genetic switch controlled by a nuclear phosphoinositide signaling system. This system includes a nuclear phosphatidylinositol 5-phosphatase (PIP5Pase), its substrate PI(3,4,5)P3, and the repressor-activator protein 1 (RAP1). RAP1 binds to ES sequences flanking VSG genes via its DNA binding domains and represses VSG transcription. In contrast, PI(3,4,5)P3 binds to the N-terminus of RAP1 and controls its DNA binding activity. Transient inactivation of PIP5Pase results in the accumulation of nuclear PI(3,4,5)P3, which binds RAP1 and displaces it from ESs, activating transcription of silent ESs and VSG switching. The system is also required for the developmental silencing of VSG genes. The data provides a mechanism controlling reversible telomere silencing essential for the periodic switching in VSG expression and its developmental regulation.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

This revised version of the manuscript addresses the critiques and recommendations from the reviewers. Briefly, we showed that HA-tagged RAP1 protein has correct telomeric localization, shown by fluorescence in situ hybridization (FISH) and immunofluorescence (Cestari and Stuart, 2015, PNAS), and interacts with telomeric proteins and DNA (Cestari et al. 2019, Mol Cell Biol). We also address that the His-tagged RAP1 does not bind unspecifically to DNA as sown by electrophoretic mobility shift assay (EMSA) and microscale thermophoresis. There was no binding of rRAP1-His to scrambled telomeric DNA sequences (Fig 3C-G, Fig 5A-B, and Fig S6), and His-tagged rRAP1 N-terminal domain protein does not bind to DNA (Fig 3G). The data rule out His-tag unspecific binding to DNA. We also added information on expression levels of WT and Mut PIP5Pase (from tubulin loci) compared to endogenous PIP5Pase (they have similar levels). We modified Fig 1B, E, and F to increase the clarity of data presentation and added Table S1 (related to Fig 1E) to include VSG gene IDs. Other minor changes were made to improve the clarity of Fig 2 and 5 (e.g., figure labelling). We indicated scripts used in the analysis (https://github.com/cestari-lab) and highlighted sequencing metrics (previously available in Fig S3). We included Fig S4 to show ChIP-seq analysis of silent vs active ES with multiple mapping stringency conditions. We changed the title to reflect a direct role of PI(3,4,5)P3 regulation of RAP1, which better describes the manuscript and may reach a readership interested in telomeric gene regulation.
https://github.com/cestari-lab

Abbreviations

PI(3,4,5)P3: phosphatidylinositol-(3,4,5)-triphosphate
PIP5Pase: phosphatidylinositol phosphate 5-phosphatase
RAP1: repressor activator protein 1
ES: expression site
VSG: variant surface glycoproteins.

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