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Silencing subtelomeric VSGs by Trypanosoma brucei RAP1 at the insect stage involves chromatin structure changes.

ABSTRACT: Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen variant surface glycoprotein (VSG) to evade mammalian host immune responses at the bloodstream form (BF) stage. Monoallelic expression of BF Expression Site (BES)-linked VSGs and silencing of metacyclic VSGs (mVSGs) in BF cells are essential for antigenic variation, whereas silencing of both BES-linked and mVSGs in the procyclic form (PF) cells is important for cell survival in the midgut of its insect vector. We have previously shown that silencing BES-linked VSGs in BF cells depends on TbRAP1. We now show that TbRAP1 silences both BES-linked and mVSGs at both BF and PF stages. The strength of TbRAP1-mediated BES-linked VSG silencing is stronger in the PF cells than that in BF cells. In addition, Formaldehyde-Assisted Isolation of Regulatory Elements analysis and MNase digestion demonstrated that depletion of TbRAP1 in PF cells led to a chromatin structure change, which is significantly stronger at the subtelomeric VSG loci than at chromosome internal loci. On the contrary, no significant chromatin structure changes were detected on depletion of TbRAP1 in BF cells. Our observations indicate that TbRAP1 helps to determine the chromatin structure at the insect stage, which likely contributes to its strong silencing effect on VSGs.

SUBMITTER: Pandya UM

PROVIDER: S-EPMC3763547 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Silencing subtelomeric VSGs by Trypanosoma brucei RAP1 at the insect stage involves chromatin structure changes.

Pandya Unnati M UM Sandhu Ranjodh R Li Bibo B

Nucleic acids research 20130626 16

Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen variant surface glycoprotein (VSG) to evade mammalian host immune responses at the bloodstream form (BF) stage. Monoallelic expression of BF Expression Site (BES)-linked VSGs and silencing of metacyclic VSGs (mVSGs) in BF cells are essential for antigenic variation, whereas silencing of both BES-linked and mVSGs in the procyclic form (PF) cells is important for cell survival in the midgut of ...[more]

PMID: 23804762

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Project description:Trypanosoma brucei ssp., unicellular parasites causing human and animal trypanosomiasis, are transmitted between mammals by tsetse flies. Periodic changes in variant surface glycoproteins (VSG), which form the parasite coat in the mammal, allow them to evade the host immune response. Different isolates of T. brucei show heterogeneity in their repertoires of VSG genes and have single nucleotide polymorphisms and indels that can impact on genome editing. T. brucei brucei EATRO1125 (AnTaR1 serodeme) is an isolate that is used increasingly often because it is pleomorphic in mammals and fly transmissible, two characteristics that have been lost by the most commonly used laboratory stocks. We present a genome assembly of EATRO1125, including contigs for the intermediate chromosomes and minichromosomes that serve as repositories of VSG genes. In addition, de novo transcriptome assemblies were performed using Illumina sequences from tsetse-derived trypanosomes. Reads of 150 bases enabled closely related members of multigene families to be discriminated. This revealed that the transcriptome of midgut-derived parasites is dynamic, starting with the expression of high affinity hexose transporters and glycolytic enzymes and then switching to proline uptake and catabolism. These changes resemble the transition from early to late procyclic forms in culture. Further metabolic reprogramming, including upregulation of tricarboxylic acid cycle enzymes, occurs in the proventriculus. Many transcripts upregulated in the salivary glands encode surface proteins, among them 7 metacyclic VSGs, multiple BARPs and GCS1/HAP2, a marker for gametes. A novel family of transmembrane proteins, containing polythreonine stretches that are predicted to be O-glycosylation sites, was also identified. Finally, RNA-Seq data were used to create an optimised annotation file with 5' and 3' untranslated regions accurately mapped for 9302 genes. We anticipate that this will be of use in identifying transcripts obtained by single cell sequencing technologies.

Project description:Repressor-activator protein 1 (scRap1) is the major binding activity at Saccharomyces cerevisiae telomeres, with roles in telomere length regulation and establishment of subtelomeric silencing by recruiting the Sir proteins. scRap1 also acts as a transcription factor controlling the expression of ribosomal proteins and glycolytic enzymes. A homolog of scRap1 exist in mammals, Rap1 (also known as Terf2ip), however, its roles in telomere biology and transcriptional regulation are largely unknown. We have employed microarrays to obtain gene expression signatures related to Rap1-deleted mice when compared to the wild-type. Construction of the Rap1 targeting vector: The strategy for disrupting the Rap1 locus was designed to conditionallty delete exon 3 (E3) through a Cre-mediated excision. Deletion of E3 leads to the removal of coding sequences encoding for the RCT domain and the NLS sequence, as well as the whole 3’UTR region and poly-adenylation signal (Fig.1A,B). Deletion of E3 was expected to destabilize the Rap1 transcript and thereby to prevent translation of any truncated RAP1 protein. The targeting vector contains homology regions isogenic with the ES cell line used (129Sv/Pas). The short homology region (SA) harbors a 2.4 kb DNA fragment encompassing exon 2 (E2) and intron 2 (Fig. 1A). The long homology region (LA) is a 5.6 kb DNA fragment downstream the end of E3 (Fig. 1A). The central part contains E3 flanked by two loxP sites and a positive selection neomycin gene (PGK-Neo) flanked by two Frt sites (Fig. 1A). At the 3’-end of the LA a Diphteria Toxin (DTA) selection marker was cloned (Fig. 1A). The vector contains a unique NruI linearization site. The targeting vector was quality controlled by sequencing of the coding exons, the junctions between the homology arms and the selection cassettes, the selection cassettes and the junctions between the homology arms and the plasmid backbone. The analysis of the sequencing results showed no polymorphis between the C57BL/6 and 129Sv/Pas genetic background within the isolated Rap1 sequences. The sequence of the primers used for the PCR amplification of the different targeting vector parts and the details of the cloning steps are available upon request. The construction of the targeting vector was performed by genOway (www.genoway.com; Lyon, France). Generation of conditional Rap1 knockout mice 129Sv/Pas ES cells were transfected with 40 µg of linearized plasmid. Positive selection was started 48 hours after electroporation by addition of 200 µg/ML G418. Approximately 230 positive resistant clones were isolated and amplified. They were PCR screened for homologous recombination first at the 5’ end of the Rap1 locus. The 6 positive identified 5’ targeted ES cells were further investigated by PCR amplification over the 3’ long homology arm to amplify the region of the targeted locus containing the distal loxP site. The direct sequencing of the PCR products amplified from the 6 ES clones revealed that 3 of them contained the distal loxP site. The recombinant clones identified by PCR were further verified by Southern blot analysis of AflII and PciI restricted genomic DNA using a 5’-internal and a 3’-external probes, respectively (data not shown). The Southern blots confirmed the correct homologous integration over the 5’ and the 3’ homology arm and the absence of any additional randomly integrated copies of the targeting vector. Chimeric mice were generated by microinjection of three independently targeted ES clones into C57BL/6J host blastocyst. The resulting offspring showed a high level of chimerism as shown by coat color, and were mated to C57BL/6J mice to assess germ line transmission. The resulting heterozygous Rap1+/flox-Neo mice were then bred to transgenic mice expressing the Flpe recombinase (Rodriguez et al., 2000) to induce excision of the Neo marker. The Rap1+/flox heterozygous mice were then intercrossed to generate Rap1flox/flox, and Rap1+/flox mice. Homozygous Rap1flox/flox mice were crossed with transgenic mice expressing the Cre recombinase under the control of the keratine 5 promoter (Tarutani et al., 1997) (Fig. 5A). Heterozygous Rap1+/D K5-Cre were crossed either to Rap1flox/flox or Rap1+/flox to generate Rap1Δ/Δ K5-Cre. The removal of exon 3 by Cre-mediated recombination was confirmed by PCR analysis using primers F and R (Fig. 1A,C and Fig. 5A). Amplification of the wild type, flox and knockout alleles renders a 3.2 kb, 3.3 kb and 0.5 kb fragments, respectively. The breeding to the F1 generation and characterization of heterozygous Rap1+/flox-Neo F1 animals was performed by geneOway (www.genoway.com; Lyon, France). All mice were generated and maintained at the Spanish National Cancer Centre (CNIO) under specific pathogen-free conditions in accordance with the recommendation of the Federation of European Laboratory Animal Science Associations.

Project description:RAP1 is a telomere protein that is well conserved from protozoa to mammals. It plays important roles in chromosome end protection, telomere length control, and gene expression/silencing at both telomeric and nontelomeric loci. Interaction with different partners is an important mechanism by which RAP1 executes its different functions in yeast. The RAP1 ortholog in Trypanosoma brucei is essential for variant surface glycoprotein (VSG) monoallelic expression, an important aspect of antigenic variation, where T. brucei regularly switches its major surface antigen, VSG, to evade the host immune response. Like other RAP1 orthologs, T. brucei RAP1 (TbRAP1) has conserved functional domains, including BRCA1 C terminus (BRCT), Myb, MybLike, and RAP1 C terminus (RCT). To study functions of various TbRAP1 domains, we established a strain in which one endogenous allele of TbRAP1 is flanked by loxP repeats, enabling its conditional deletion by Cre-mediated recombination. We replaced the other TbRAP1 allele with various mutant alleles lacking individual functional domains and examined their nuclear localization and protein interaction abilities. The N terminus, BRCT, and RCT of TbRAP1 are required for normal protein levels, while the Myb and MybLike domains are essential for normal cell growth. Additionally, the Myb domain of TbRAP1 is required for its interaction with T. brucei TTAGGG repeat-binding factor (TbTRF), while the BRCT domain is required for its self-interaction. Furthermore, the TbRAP1 MybLike domain contains a bipartite nuclear localization signal that is required for its interaction with importin α and its nuclear localization. Interestingly, RAP1's self-interaction and the interaction between RAP1 and TRF are conserved from kinetoplastids to mammals. However, details of the interaction interfaces have changed throughout evolution.IMPORTANCETrypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, to evade the host immune response. VSGs are expressed from subtelomeres in a monoallelic fashion. TbRAP1, a telomere protein, is essential for cell viability and VSG monoallelic expression and suppresses VSG switching. Although TbRAP1 has conserved functional domains in common with its orthologs from yeasts to mammals, the domain functions are unknown. RAP1 orthologs have pleiotropic functions, and interaction with different partners is an important means by which RAP1 executes its different roles. We have established a Cre-loxP-mediated conditional knockout system for TbRAP1 and examined the roles of various functional domains in protein expression, nuclear localization, and protein-protein interactions. This system enables further studies of TbRAP1 point mutation phenotypes. We have also determined functional domains of TbRAP1 that are required for several different protein interactions, shedding light on the underlying mechanisms of TbRAP1-mediated VSG silencing.

Dataset Information

Silencing subtelomeric VSGs by Trypanosoma brucei RAP1 at the insect stage involves chromatin structure changes.

Publications

Silencing subtelomeric VSGs by Trypanosoma brucei RAP1 at the insect stage involves chromatin structure changes.

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