Promoter occupancy of the basal class I transcription factor A differs strongly between active and silent VSG expression sites in Trypanosoma brucei

Abstract

Monoallelic expression within a gene family is found in pathogens exhibiting antigenic variation and in mammalian olfactory neurons. Trypanosoma brucei, a lethal parasite living in the human bloodstream, expresses variant surface glycoprotein (VSG) from 1 of 15 bloodstream expression sites (BESs) by virtue of a multifunctional RNA polymerase I. The active BES is transcribed in an extranucleolar compartment termed the expression site body (ESB), whereas silent BESs, located elsewhere within the nucleus, are repressed epigenetically. The regulatory mechanisms, however, are poorly understood. Here we show that two essential subunits of the basal class I transcription factor A (CITFA) predominantly occupied the promoter of the active BES relative to that of a silent BES, a phenotype that was maintained after switching BESs in situ. In these experiments, high promoter occupancy of CITFA was coupled to high levels of both promoter-proximal RNA abundance and RNA polymerase I occupancy. Accordingly, fluorescently tagged CITFA-7 was concentrated in the nucleolus and the ESB. Because a ChIP-seq analysis found that along the entire BES, CITFA-7 is specifically enriched only at the promoter, our data strongly indicate that monoallelic BES transcription is activated by a mechanism that functions at the level of transcription initiation.

Transcription is initiated on silent variant surface glycoprotein expression sites despite monoallelic expression in Trypanosoma brucei

“…The occupancy by this factor and RNA pol I was several times higher in the first few hundred base pairs of the active ES than in the equivalent region of a silent ES. However, significant CITFA occupancy also was found on silent ESs (43,44). Thus, these analyses, together with the results presented herein, allow us to claim that transcription initiation is not the determinant level of ES control.…”

“…For instance, the active expression site is uniquely devoid of nucleosomes (9, 10), probably favoring transcription elongation and correlating with a higher occupancy by RNA pol I and RNA pol I transcription factor at the beginning of the active ES (44). Also, rates of transcript processing and cytoplasmic export and stability are different in transcripts of the active and inactive ESs (29).…”

DNA Recombination Strategies During Antigenic Variation in the African Trypanosome

“…The observation that some transcripts can in fact be detected from silent BES, even in the absence of the above perturbations, suggests that selective RNA Pol I elongation may be the key (100). However, other studies have shown that RNA Pol I occupation levels are higher at the promoter of the active BES, suggesting that selective transcription initiation may also contribute (101).…”

DNA Double-Strand Breaks and Telomeres Play Important Roles in Trypanosoma brucei Antigenic Variation

“…This monoallelic VSG expression ensures that after a VSG switching event, the originally active VSG no longer is expressed on the cell surface. Several mechanisms of VSG expression regulation have been identified, including specialized localization of the active ES at an extranucleolar ES body (ESB) that is enriched with RNA polymerase I (24), regulated transcription elongation along ESs (25,26), modulation of ES chromatin structure (27)(28)(29)(30)(31)(32), modulation of ES promoter activities (33)(34)(35)(36)(37), and telomere protein-mediated telomeric silencing (38,39). VSG expression regulation has been reviewed elsewhere recently (40, 41) and will not be discussed here in detail.…”