RAP1 Is Essential for Silencing Telomeric Variant Surface Glycoprotein Genes in Trypanosoma brucei

Department of Biological, Geological, and Environmental Sciences, Center for Gene Regulation in Health and Diseases, Cleveland State University, Cleveland, OH 44115, USA.
Cell (Impact Factor: 32.24). 05/2009; 137(1):99-109. DOI: 10.1016/j.cell.2009.01.037
Source: PubMed


Trypanosoma brucei expresses variant surface glycoprotein (VSG) genes in a strictly monoallelic fashion in its mammalian hosts, but it is unclear how this important virulence mechanism is enforced. Telomere position effect, an epigenetic phenomenon, has been proposed to play a critical role in VSG regulation, yet no telomeric protein has been identified whose disruption led to VSG derepression. We now identify tbRAP1 as an intrinsic component of the T. brucei telomere complex and a major regulator for silencing VSG expression sites (ESs). Knockdown of tbRAP1 led to derepression of all VSGs in silent ESs, but not VSGs located elsewhere, and resulted in stronger derepression of genes located within 10 kb from telomeres than genes located further upstream. This graduated silencing pattern suggests that telomere integrity plays a key role in tbRAP1-dependent silencing and VSG regulation.

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Available from: Bibo Li, May 22, 2014
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    • "We found the telomeric protein TbRAP1 to be SUMOylated. This protein has been shown to generate a silencing gradient from the telomere ends, both in PCF (Pandya et al., 2013) and in BSF (Yang et al., 2009). It is intriguing, nevertheless, how SUMO correlates with this effect. "
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    ABSTRACT: SUMOylation is an important post-translational modification conserved in eukaryotic organisms. In Trypanosoma brucei, SUMO is essential in procyclic and bloodstream forms. Furthermore, SUMO has been linked to the antigenic variation process, as a highly SUMOylated focus was recently identified within chromatin-associated proteins of the active VSG expression site. We aimed to establish a reliable strategy to identify SUMO conjugates in T. brucei. We expressed various tagged variants of SUMO from the endogenous locus. His-HA-TbSUMO was useful to validate the tag functionality but SUMO conjugates were not enriched enough over contaminants after affinity purification. A Lys-deficient SUMO version, created to reduce contaminants by Lys-C digestion, was able to overcome this issue but did not allow mapping many SUMOylation sites. This cell line was in turn useful to demonstrate that polySUMO chains are not essential for parasite viability. Finally, a His-HA-TbSUMO(T106K) version allowed the purification of SUMO conjugates and, after digestion with Lys-C, the enrichment for diGly-Lys peptides using specific antibodies. This site specific proteomic strategy led us to identify 45 SUMOylated proteins and 53 acceptor sites unambiguously. SUMOylated proteins belong mainly to nuclear processes such as DNA replication and repair, transcription, rRNA biogenesis, and chromatin remodelling, among others. This article is protected by copyright. All rights reserved.
    Cellular Microbiology 06/2015; DOI:10.1111/cmi.12467 · 4.92 Impact Factor
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    • "Telomeric proteins have been found in many organisms, such as vertebrates, yeasts and ciliates [4] [5] [6] [7]. In trypanosomatids, in spite of the presence of LaRbp38, only the orthologs of TRF and Rap-1 reported in other eukaryotes were found to interact with parasite doublestranded telomeres [8] [9] [10] [11], and very few typical 3 0 G-overhang binding proteins have been reported in these protozoa [12] [13] [14]. Some of them, including LaRPA-1 [13] [14], were isolated in Leishmania amazonensis telomerase-positive nuclear extracts using affinity chromatography on the telomeric G-rich strand DNA. "
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    ABSTRACT: Replication protein A-1 (RPA-1) is a single-stranded DNA-binding protein involved in DNA metabolism. We previously demonstrated the interaction between LaRPA-1 and telomeric DNA. Here, we expressed and purified truncated mutants of LaRPA-1 and used circular dichroism measurements and molecular dynamics simulations to demonstrate that the tertiary structure of LaRPA-1 differs from human and yeast RPA-1. LaRPA-1 interacts with telomeric ssDNA via its N-terminal OB-fold domain, whereas RPA from higher eukaryotes show different binding modes to ssDNA. Our results show that LaRPA-1 is evolutionary distinct from other RPA-1 proteins and can potentially be used for targeting trypanosomatid telomeres. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
    FEBS Letters 11/2014; 588(24). DOI:10.1016/j.febslet.2014.11.005 · 3.17 Impact Factor
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    • "Telomeres consisting of tandemly repeated G-rich sequences are specialized protein–DNA complexes located at the end of linear chromosomes [1]. Proteins that bind to duplex or single-stranded telomere DNA are indispensable components of the telomere complexes and play critical roles in telomere length regulation, end protection and maintenance of specialized telomere chromatin structure [2]. To date, several species of telomeric DNA binding proteins have been identified in parasites. "
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    ABSTRACT: Coccidiosis is considered to be a major problem for the poultry industry, and coccidiosis control is yet urgent. Due to the roles in telomere length regulation and end protection, telomere-binding proteins have been considered as a good target for drug design. In this work, a putative Gbp1p that is similar to telomeric DNA-binding protein Gbp (G-strand binding protein) of Cryptosporidium parvum, was searched in the database of Eimeria tenella. Sequence analysis indicated Eimeria tenella Gbp1p (EtGbp1p) has significant sequence similarity to other eukaryotic Gbps in their RNA recognition motif (RRM) domains. Electrophoretic mobility shift assays (EMSAs) demonstrated recombinant EtGbp1p bound G-rich telomeric DNA, but not C-rich or double-stranded telomeric DNA sequences. Competition and antibody supershift assays confirmed the interaction of DNA-protein complex. Chromatin immunoprecipitation assays confirmed that EtGbp1p interacted with telomeric DNA in vivo. Collectively, these evidences suggest that EtGbp1p represents a G-rich single-stranded telomeric DNA-binding protein in Eimeria tenella.
    Biochemical and Biophysical Research Communications 09/2014; 451(4). DOI:10.1016/j.bbrc.2014.08.030 · 2.30 Impact Factor
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