Nucleosomes Are Depleted at the VSG Expression Site Transcribed by RNA Polymerase I in African Trypanosomes

Abstract

In most eukaryotes, RNA polymerase I (Pol I) exclusively transcribes long arrays of identical rRNA genes (ribosomal DNA [rDNA]). African trypanosomes have the unique property of using Pol I to also transcribe the variant surface glycoprotein VSG genes. VSGs are important virulence factors because their switching allows trypanosomes to escape the host immune system, a mechanism known as antigenic variation. Only one VSG is transcribed at a time from one of 15 bloodstream-form expression sites (BESs). Although it is clear that switching among BESs does not involve DNA rearrangements and that regulation is probably epigenetic, it remains unknown why BESs are transcribed by Pol I and what roles are played by chromatin structure and histone modifications. Using chromatin immunoprecipitation, micrococcal nuclease digestion, and chromatin fractionation, we observed that there are fewer nucleosomes at the active BES and that these are irregularly spaced compared to silent BESs. rDNA coding regions are also depleted of nucleosomes, relative to the rDNA spacer. In contrast, genes transcribed by Pol II are organized in a more compact, regularly spaced, nucleosomal structure. These observations provide new insight on antigenic variation by showing that chromatin remodeling is an intrinsic feature of BES regulation.Chromatin structure is dynamic, adopting a more condensed conformation at transcriptionally silent regions (closed chromatin) than at transcriptionally active regions (open chromatin) (6). Studies of genes transcribed by RNA polymerase II (Pol II) have shown that open and closed chromatin structures are dynamically regulated through multiple mechanisms, including histone modifications, histone variant incorporation, and DNA methylation (reviewed in reference 17).

Trypanosoma brucei TIF2 suppresses VSG switching by maintaining subtelomere integrity

“…The active BES differs from silent BESs in that it is actively transcribed by RNA polymerase I [59]. In addition, the active BES forms an open chromatin structure depleted of most nucleosomes, while silent BESs are packed with nucleosomes [60,61]. Our data suggest that the transcriptional status and/or the chromatin structure of the DSB site may influence the choice of DSB repair mechanism.…”

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

“…The mechanism controlling the selective activity of a single ES at a time is still unknown, although several factors have been found to be involved, such as differential chromatin/histone acetylation and methylation between active and inactive ESs (6-8); nucleosome depletion in the active ES (9,10); and the influence of various negative transcription modulators such as a histone methylase, a high-mobility group protein, histone H1, NLP (a nucleoplasm-like protein), a FACT subunit (a chromatin remodeler), a SWI/SNF (a chromatin remodeling factor), ORC1 and MCM-BP (proteins involved in the initiation of DNA replication), RAP1 (a telomere-binding protein), histone chaperones, or a lamin-like protein (8,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). However, no master ES regulator has been identified so far.…”

TbISWI Regulates Multiple Polymerase I (Pol I)-Transcribed Loci and Is Present at Pol II Transcription Boundaries in Trypanosoma brucei

“…In the case of the ESs, it has recently been shown that the active ES in bloodstream-form T. brucei is depleted of nucleosomes (13,51), indicating that chromatin remodeling plays a key role in ES activation. This is supported by the discovery that a number of proteins involved in epigenetic control play a role in ES regulation, including the T. brucei imitation switch homologue TbISWI (21), the telomere-binding protein RAP1 (64), and the histone methyltransferase DOT1B (14).…”