TbSAP is a novel chromatin protein repressing metacyclic variant surface glycoprotein expression sites in bloodstream form Trypanosoma brucei

Carys Davies,Gloria Rudenko,Sam Alsford,Falk Butter,Bill Wickstead,Belinda S Hall,Haneesh Sidhu,Cher-Pheng Ooi,Georgios Sioutas

doi:10.1093/nar/gkab109

Abstract

The African trypanosome Trypanosoma brucei is a unicellular eukaryote, which relies on a protective variant surface glycoprotein (VSG) coat for survival in the mammalian host. A single trypanosome has >2000 VSG genes and pseudogenes of which only one is expressed from one of ∼15 telomeric bloodstream form expression sites (BESs). Infectious metacyclic trypanosomes present within the tsetse fly vector also express VSG from a separate set of telomeric metacyclic ESs (MESs). All MESs are silenced in bloodstream form T. brucei. As very little is known about how this is mediated, we performed a whole genome RNAi library screen to identify MES repressors. This allowed us to identify a novel SAP domain containing DNA binding protein which we called TbSAP. TbSAP is enriched at the nuclear periphery and binds both MESs and BESs. Knockdown of TbSAP in bloodstream form trypanosomes did not result in cells becoming more ‘metacyclic-like'. Instead, there was extensive global upregulation of transcripts including MES VSGs, VSGs within the silent VSG arrays as well as genes immediately downstream of BES promoters. TbSAP therefore appears to be a novel chromatin protein playing an important role in silencing the extensive VSG repertoire of bloodstream form T. brucei.

Highlights

The African trypanosome Trypanosoma brucei is an early-branching unicellular eukaryote with highly unusual features to its molecular biology [1]
We found that TbSAP is enriched at the T. brucei nuclear periphery, and using Chromosome immunoprecipitation (ChIP), established that it binds metacyclic ESs (MESs) as well as immediately upstream of bloodstream form expression sites (BESs) promoters
Progress has been made in identifying factors involved in BES repression in bloodstream form T. brucei including a range of chromatin proteins and remodelers [13]

Summary

Introduction

The African trypanosome Trypanosoma brucei (causative agent of African Sleeping sickness) is an early-branching unicellular eukaryote with highly unusual features to its molecular biology [1]. The T. brucei genome consists primarily of extensive polycistronic transcription units containing functionally unrelated assortments of genes which are constitutively transcribed by RNA polymerase II (Pol II) [2]. T. brucei is an extracellular parasite of the mammalian bloodstream and tissue spaces [7,8]. Within the host, it is covered with a dense protective VSG coat comprised of ∼107 molecules corresponding to ∼10% total protein [9]. A single trypanosome has a vast repertoire of thousands of VSG genes and pseudogenes, but only one is transcribed at a time from one of 15 bloodstream form VSG expression sites (BES) [10,11]. Switching the active VSG entails replacement with another VSG through DNA rearrangements including gene conversion, or a transcriptional switch to a different BES [13,14,15,16]

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Results

Conclusion