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Antigenic variation by switching inter-chromosomal interactions with an RNA splicing locus in trypanosomes

Joana Faria, Vanessa Luzak, Laura S.M. Müller, Benedikt G. Brink, Sebastian Hutchinson, Lucy Glover, David Horn, T. Nicolai Siegel

Preprint posted on January 28, 2020 https://www.biorxiv.org/content/10.1101/2020.01.27.921452v1

A look into the host-pathogen arms race: distinct DNA-DNA interactions ensuring monogenic VSG expression in T. brucei.

Selected by Mariana De Niz

Background

      African trypanosomes are parasites transmitted by tsetse flies, that cause lethal diseases in humans and livestock. In order to escape the host’s immune system, African trypanosomes use an antigenic variation mechanism that involves monogenic antigen expression from a pool of over 2500 antigen-coding genes. In T. brucei, 10 million copies of a single variant surface glycoprotein (VSG) isoform are exposed on the cell surface of the parasite. The exclusive expression of only one VSG gene per cell and the periodic switching of the expressed VSG gene allow the parasite to evade the host immune system and to maintain persistent infections. Until now, the mechanism regulating monogenic expression in these parasites has remained poorly understood. In their work, Faria et al show a novel mechanism of post-transcriptional regulation involving the spatial integration of antigen transcription and mRNA splicing within a dedicated compartment. In this compartment, only the single expressed antigen-coding gene forms a specific inter-chromosomal interaction with a major mRNA splicing locus (1), (summarised in Figure 1).

Figure 1. Schematic model of monogenic VSG expression (Used with permission, from (1)).

Key findings and developments

Context

  • Regulated access to RNA maturation compartments has been suggested to represent an evolutionary conserved strategy for gene expression control (2).
  • In T. brucei, several observations suggest that a specific genome organisation is required for monogenic VSG expression. The single active VSG gene is transcribed in an expression site body (an extranucleolar Pol I compartment) (3). In addition, the transcribed chromosome core regions and the sub-telomeric regions coding for the large reservoir of silent VSG genes, appear to fold into structurally distinct compartments (4).
  • A bipartite VEX protein complex specifically associated with the active VSG gene was identified recently, which maintains mutually exclusive VSG expression (5). In this work, Faria et al (1) aimed to identify the mechanism that connects RNA maturation, genome architecture, and the VEX complex to ensure monogenic antigen expression.

Key overall findings

  • The authors used a T. brucei culture homogeneously expressing a single VSG gene for genome-wide chromosome conformation capture (Hi-C) analyses. These analyses revealed a strong interaction between the Pol-I transcribed active VSG gene and the Pol-II transcribed SL-RNA locus located on a different chromosome. Conversely, VSG genes residing in inactive expression sites interacted less frequently or at background levels with the SL-RNA locus. Moreover, the interaction with the SL-RNA locus is reconfigured upon activation of a different expression-site, demonstrating that the interaction is specific for the active-VSG gene.
  • Super-resolution immunofluorescence microscopy was then used to visualize the spatial proximity between the active VSG gene and the SL-RNA locus at the level of individual cells. During the G1 phase, one of the SL-RNA transcription compartments was adjacent to the VSG transcription compartment in the majority of cells. These results suggest that the Pol I VSG transcription compartment interacts with one of the Pol-II SL-RNA transcription compartments. The interaction is resolved during S phase and re-established after cell division.
  • The authors went on to investigate the relationship between the VEX complex and the transcription compartments in further detail from their previous work (5). Super-resolution microscopy revealed association of VEX1 with the SL-RNA transcription compartment (the splicing locus), and VEX2 with the VSG transcription compartment (the antigen coding locus).
  • Aiming to determine whether the splicing process impacts the connection between the compartments, the authors found that inhibition of trans-splicing disrupted both VEX1 and VEX2 localization, as well as the connection between the VSG and SL-RNA transcription compartments.
  • This led to investigating mechanisms by which the VEX complex ensures monogenic VSG expression, specifically, whether VEX2 functions as a connector or as an exclusion factor. The authors found that VEX2 depletion, which leads to loss of monogenic antigen expression (5), increases interactions between previously silent genes and the splicing locus.
  • Altogether the authors propose a dual function for VEX2: on one hand enhancing mRNA splicing of the VSG gene that is connected to the SL-RNA transcription compartment, and on the other hand, excluding all other VSG expression sites and procyclin genes from the SL-RNA compartment to ensure monogenic VSG expression.

Model proposed

  • VSG choice is intimately associated with an inter-chromosomal interaction, bringing together two nuclear compartments (the VSG and the SL-RNA transcription compartments) to ensure efficient VSG mRNA processing at only one expression site. The close spatial proximity of the two compartments in a single locus provides a sufficiently high concentration of trans-splicing substrate to ensure the efficient maturation of highly abundant VSG transcripts. By shaping a highly selective and specific genome architecture, VEX2 is the key molecule allowing only one VSG gene to productively interact with the mRNA splicing compartment. This ultimately ensures expression of a single VSG gene per cell.

What I like about this paper

I like that this paper addresses a fundamental and highly relevant biological question, with an out-of-the-box approach. The findings of the paper are relevant to infection biology, but in general contribute to our understanding of mechanisms of post-transcriptional regulation. Specific to infection biology, I like the new questions that this finding opens.

 

Open questions

*Questions, and answers from authors at bottom of this page.

  1. You are the first to propose that a selective inter-chromosomal interaction connects transcription and mRNA splicing of a specific gene. Beyond infection biology, were there any fundamental biological questions you thought your finding might impact?

 

  1. You mentioned you found tubulin associated with the SL-RNA locus and with VEX1. What do you think is the relevance of this finding?

 

  1. T. brucei parasites migrate within tissues, causing extensive deformation of their bodies. Previous work in other disciplines has explored the effect of cell deformation on nuclear stress and nuclear functions. Do you expect this to influence key functions such as VSG switching?

 

  1. Based on your model, and taking a natural infection of a mammal as basis, how do the molecular actors and compartments you study, behave upon antigenic switching?

 

  1. Do you think there might be molecules other than VEX2 regulating monogenic VSG expression? From an evolutionary perspective, and particularly in such a sophisticated pathogen as T. brucei, this seems unlikely.

 

 

References

  1. Faria J, Luzak V, Müller LSM, Brink BG, Hutchinson S, Glover L, Horn D, Siegel TN, Antigenic variation by switching inter-chromosomal interactions with an RNA splicing locus in trypanosomes, bioRxiv, (2020).
  2. Chen Y, Belmont AS, Genome organization around nuclear speckles, Current Opinion in Genetics and Development, 55, 91-99, (2019).
  3. Navarro M, Gull K, A pol I transcriptional body associated with VSG mono-allelic expression in Trypanosoma brucei, Nature, 414 (6865), (2001).
  4. Müller LSM, Consentino RO, Förstner KU, Guizetti J, Wedel C, Kaplan N, Janzen CJ, Arampatzi P, Vogel J, Steinbiss S, Otto TD, Saliba AE, Sebra RP, Siegel TN, Genome organization and DNA accessibility control antigenic variation in trypanosomes, Nature, 563 (7729), (2018).
  5. Faria J, et al, Monoallelic expression and epigenetic inheritance sustained by a Trypanosoma brucei variant surface glycoprotein exclusion complex, Nat Comm., 10, 3023, (2019).

Acknowledgements

I thank very much the authors, especially Joana Faria, Vanessa Luzak, David Horn and Nicolai Siegel, for their engagement and time. I thank also Mate Palfy for his input.

 

Posted on: 20th February 2020

doi: https://doi.org/10.1242/prelights.17030

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  • Author's response

    Joana Faria, Vanessa Luzak, David Horn, Nicolai Siegel shared

    Open questions

    1. You are the first to propose that a selective inter-chromosomal interaction connects transcription and mRNA splicing of a specific gene. Beyond infection biology, were there any fundamental biological questions you thought your finding might impact?

    Interactions between highly expressed genes and pools of RNA processing factors might be a common principle in gene expression enhancement. In fact, the interaction we describe is reminiscent of interactions reported in higher eukaryotes between highly transcribed loci and sub-nuclear bodies enriched in splicing components, such as Cajal bodies or nuclear speckles (Quinodoz et al, 2018; PMID 2988737; DOI 10.1016/j.cell.2018.05.024). A recent study supports the view that nuclear speckle association results in gene expression enhancement in human cells but the interactions investigated appeared to be stochastic rather than regulated (Kim et al., J Cell Biol. 2020 doi: 10.1083/jcb.201904046. PMID:31757787). Arguably, the absence of a vast network of super-enhancers, as well as the overall lack of control over transcription initiation, render trypanosomes a good model to study how sub-nuclear compartments can be arranged to post-transcriptionally enhance gene expression.

    As a second aspect, the study provides important information about interactions between different chromosomes. Inter-chromosomal interactions were thought to be very difficult to re-establish after DNA replication, possibly relying on error-prone mechanisms. Indeed, the community has long debated whether such stable and heritable inter-chromosomal interactions were possible and therefore their role in gene expression control has remained elusive. To our knowledge, the only other known stable inter-chromosomal interaction occurs in olfactory neurons between the single active odorant receptor, which is expressed in a monogenic fashion similarly to VSGs, and a super-enhancer regulatory sequence. However, olfactory neurons are terminally differentiated cells whereas trypanosomes replicate rather rapidly, showing that inter-chromosomal interactions can indeed be propagated stably through the cell cycle and play an important role in gene expression regulation.

    2. You mentioned you found tubulin associated with the SL-RNA locus and with VEX1. What do you think is the relevance of this finding?

    An abundant supply of tubulin is required to support cell structure, suggesting that interactions with the SL-RNA compartment play a more general role in enhancing gene expression extending beyond VSG genes. Consistent with this idea, we also found other tandem arrays (which encode for other highly abundant proteins) to interact with the SL-RNA locus. It would be interesting to investigate whether similar interactions take place in other Kinetoplastid parasites.

    3. T. brucei parasites migrate within tissues, causing extensive deformation of their bodies. Previous work in other disciplines has explored the effect of cell deformation on nuclear stress and nuclear functions. Do you expect this to influence key functions such as VSG switching?

    It’s an interesting idea. Mechanical stress could possibly lead to the disruption of the interaction between the one active VSG gene and the SL-RNA locus. Thereby, chances are increased that another VSG gene starts interacting with the splicing locus. This could conceivably increase the rate of switching in tissues relative to the less turbulent culture flask.

    4. Based on your model, and taking a natural infection of a mammal as basis, how do the molecular actors and compartments you study, behave upon antigenic switching?

    Following the activation of a different expression-site, our global Hi-C analysis has demonstrated the establishment of an interaction between the newly activated VSG and the SL-RNA locus, whereas the VSG that was originally active interacts with the SL-locus at background level. At the moment, we can only speculate on how such a rearrangement occurs during switching in a natural infection. Our findings present an intriguing scenario though, whereby DNA breaks or mechanical stress (see above) could liberate VEX2, allowing other VSGs to compete for VEX2 and for the SL-RNA interaction. This is a question worth pursuing, however, switching events are rare and therefore their visualisation at the single cell level has remained technically challenging.

    5. Do you think there might be molecules other than VEX2 regulating monogenic VSG expression? From an evolutionary perspective, and particularly in such a sophisticated pathogen as T. brucei, this seems unlikely.

    Many factors impact nuclear structures and monogenic VSG expression, as described by numerous labs, but VEX2 appears to play a central role. Notably, VEX2-related factors are implicated in X-chromosome inactivation in female mammals.

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