Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

You are viewing this page in draft mode.

Spatial integration of transcription and splicing in a dedicated compartment sustains monogenic antigen expression in African trypanosomes

Abstract

Highly selective gene expression is a key requirement for antigenic variation in several pathogens, allowing evasion of host immune responses and maintenance of persistent infections1. African trypanosomes—parasites that cause lethal diseases in humans and livestock—employ an antigenic variation mechanism that involves monogenic antigen expression from a pool of >2,600 antigen-coding genes2. In other eukaryotes, the expression of individual genes can be enhanced by mechanisms involving the juxtaposition of otherwise distal chromosomal loci in the three-dimensional nuclear space3,4,5. However, trypanosomes lack classical enhancer sequences or regulated transcription initiation6,7. In this context, it has remained unclear how genome architecture contributes to monogenic transcription elongation and transcript processing. Here, we show that the single expressed antigen-coding gene displays a specific inter-chromosomal interaction with a major messenger RNA splicing locus. Chromosome conformation capture (Hi-C) revealed a dynamic reconfiguration of this inter-chromosomal interaction upon activation of another antigen. Super-resolution microscopy showed the interaction to be heritable and splicing dependent. We found a specific association of the two genomic loci with the antigen exclusion complex, whereby VSG exclusion 1 (VEX1) occupied the splicing locus and VEX2 occupied the antigen-coding locus. Following VEX2 depletion, loss of monogenic antigen expression was accompanied by increased interactions between previously silent antigen genes and the splicing locus. Our results reveal a mechanism to ensure monogenic expression, where antigen transcription and messenger RNA splicing occur in a specific nuclear compartment. These findings suggest a new means of post-transcriptional gene regulation.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: The active VSG expression site stably interacts with the SL-RNA array.
Fig. 2: The interaction between the active VSG gene and the SL-RNA locus is dynamic and changes during a switch in VSG expression.
Fig. 3: The VEX complex associates with both the active VSG gene and the spliced leader locus in a splicing-dependent manner.
Fig. 4: The exclusive association between the active VSG gene and the SL-locus is VEX2 dependent.

Data availability

High-throughput sequencing data (Hi-C and RNA-seq) generated for this study have been deposited at GitHub (https://github.com/bgbrink/PRJEB35632) and in the European Nucleotide Archive under primary accession number PRJEB35632, respectively. Previously published ChIP-seq and RNA-seq data that were used for this study are publicly available at the European Nucleotide Archive under accession numbers PRJEB25352 and PRJEB21615, respectively. Processed data and results are available from https://doi.org/10.5281/zenodo.3628212. Source data are provided with this paper.

Code availability

Code generated for this study has been deposited at GitHub (https://github.com/bgbrink/PRJEB35632) and Zenodo (https://doi.org/10.5281/zenodo.3628212).

References

  1. 1.

    Deitsch, K. W., Lukehart, S. A. & Stringer, J. R. Common strategies for antigenic variation by bacterial, fungal and protozoan pathogens. Nat. Rev. Microbiol. 7, 493–503 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Horn, D. Antigenic variation in African trypanosomes. Mol. Biochem. Parasitol. 195, 123–129 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Bashkirova, E. & Lomvardas, S. Olfactory receptor genes make the case for inter-chromosomal interactions. Curr. Opin. Genet. Dev. 55, 106–113 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Dekker, J. & Mirny, L. The 3D genome as moderator of chromosomal communication. Cell 164, 1110–1121 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Schoenfelder, S. & Fraser, P. Long-range enhancer–promoter contacts in gene expression control. Nat. Rev. Genet. 20, 437–455 (2019).

    CAS  PubMed  Google Scholar 

  6. 6.

    Clayton, C. Regulation of gene expression in trypanosomatids: living with polycistronic transcription. Open Biol. 9, 190072 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Muller, L. S. M. et al. Genome organization and DNA accessibility control antigenic variation in trypanosomes. Nature 563, 121–125 (2018).

    PubMed  PubMed Central  Google Scholar 

  8. 8.

    Aresta-Branco, F., Erben, E., Papavasiliou, F. N. & Stebbins, C. E. Mechanistic similarities between antigenic variation and antibody diversification during Trypanosoma brucei infection. Trends Parasitol. 35, 302–315 (2019).

    CAS  PubMed  Google Scholar 

  9. 9.

    Kassem, A., Pays, E. & Vanhamme, L. Transcription is initiated on silent variant surface glycoprotein expression sites despite monoallelic expression in Trypanosoma brucei. Proc. Natl Acad. Sci. USA 111, 8943–8948 (2014).

    CAS  PubMed  Google Scholar 

  10. 10.

    Vanhamme, L. et al. Differential RNA elongation controls the variant surface glycoprotein gene expression sites of Trypanosoma brucei. Mol. Microbiol. 36, 328–340 (2000).

    CAS  PubMed  Google Scholar 

  11. 11.

    Michaeli, S. Trans-splicing in trypanosomes: machinery and its impact on the parasite transcriptome. Future Microbiol. 6, 459–474 (2011).

    CAS  PubMed  Google Scholar 

  12. 12.

    Bertero, A. et al. Dynamics of genome reorganization during human cardiogenesis reveal an RBM20-dependent splicing factory. Nat. Commun. 10, 1538 (2019).

    PubMed  PubMed Central  Google Scholar 

  13. 13.

    Khanna, N., Hu, Y. & Belmont, A. S. HSP70 transgene directed motion to nuclear speckles facilitates heat shock activation. Curr. Biol. 24, 1138–1144 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Navarro, M. & Gull, K. A pol I transcriptional body associated with VSG mono-allelic expression in Trypanosoma brucei. Nature 414, 759–763 (2001).

    CAS  PubMed  Google Scholar 

  15. 15.

    Budzak, J. et al. Dynamic colocalization of 2 simultaneously active VSG expression sites within a single expression-site body in Trypanosoma brucei. Proc. Natl Acad. Sci. USA 116, 16561–16570 (2019).

    CAS  PubMed  Google Scholar 

  16. 16.

    Chaves, I. et al. Subnuclear localization of the active variant surface glycoprotein gene expression site in Trypanosoma brucei. Proc. Natl Acad. Sci. USA 95, 12328–12333 (1998).

    CAS  PubMed  Google Scholar 

  17. 17.

    Glover, L., Hutchinson, S., Alsford, S. & Horn, D. VEX1 controls the allelic exclusion required for antigenic variation in trypanosomes. Proc. Natl Acad. Sci. USA 113, 7225–7230 (2016).

    CAS  PubMed  Google Scholar 

  18. 18.

    Faria, J. et al. Monoallelic expression and epigenetic inheritance sustained by a Trypanosoma brucei variant surface glycoprotein exclusion complex. Nat. Commun. 10, 3023 (2019).

    PubMed  PubMed Central  Google Scholar 

  19. 19.

    Zheng, Y., Ay, F. & Keles, S. Generative modeling of multi-mapping reads with mHi-C advances analysis of Hi-C studies. eLife 8, e38070 (2019).

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Hury, A., Goldshmidt, H., Tkacz, I. D. & Michaeli, S. Trypanosome spliced-leader-associated RNA (SLA1) localization and implications for spliced-leader RNA biogenesis. Eukaryot. Cell 8, 56–68 (2009).

    CAS  PubMed  Google Scholar 

  21. 21.

    Figueiredo, L. M., Janzen, C. J. & Cross, G. A. A histone methyltransferase modulates antigenic variation in African trypanosomes. PLoS Biol. 6, e161 (2008).

    PubMed  PubMed Central  Google Scholar 

  22. 22.

    Takagi, Y., Sindkar, S., Ekonomidis, D., Hall, M. P. & Ho, C. K. Trypanosoma brucei encodes a bifunctional capping enzyme essential for cap 4 formation on the spliced leader RNA. J. Biol. Chem. 282, 15995–16005 (2007).

    CAS  PubMed  Google Scholar 

  23. 23.

    McNally, K. P. & Agabian, N. Trypanosoma brucei spliced-leader RNA methylations are required for trans splicing in vivo. Mol. Cell Biol. 12, 4844–4851 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Ullu, E. & Tschudi, C. Trans splicing in trypanosomes requires methylation of the 5′ end of the spliced leader RNA. Proc. Natl Acad. Sci. USA 88, 10074–10078 (1991).

    CAS  PubMed  Google Scholar 

  25. 25.

    Glover, L., Alsford, S. & Horn, D. DNA break site at fragile subtelomeres determines probability and mechanism of antigenic variation in African trypanosomes. PLoS Pathog. 9, e1003260 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Landeira, D., Bart, J. M., Van Tyne, D. & Navarro, M. Cohesin regulates VSG monoallelic expression in trypanosomes. J. Cell Biol. 186, 243–254 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Yang, X., Figueiredo, L. M., Espinal, A., Okubo, E. & Li, B. RAP1 is essential for silencing telomeric Variant Surface Glycoprotein genes in Trypanosoma brucei. Cell 137, 99–109 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Kornblihtt, A. R., de la Mata, M., Fededa, J. P., Munoz, M. J. & Nogues, G. Multiple links between transcription and splicing. RNA 10, 1489–1498 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Fiszbein, A., Krick, K. S., Begg, B. E. & Burge, C. B. Exon-mediated activation of transcription starts. Cell 179, 1551–1565 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    De Lange, T. et al. Tandem repetition of the 5′ mini-exon of variant surface glycoprotein genes: a multiple promoter for VSG gene transcription? Cell 34, 891–900 (1983).

    CAS  PubMed  Google Scholar 

  31. 31.

    Hnisz, D., Shrinivas, K., Young, R. A., Chakraborty, A. K. & Sharp, P. A. A phase separation model for transcriptional control. Cell 169, 13–23 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Guo, Y. E. et al. Pol II phosphorylation regulates a switch between transcriptional and splicing condensates. Nature 572, 543–548 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Li, X. & Fu, X. D. Chromatin-associated RNAs as facilitators of functional genomic interactions. Nat. Rev. Genet. 20, 503–519 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Hondele, M. et al. DEAD-box ATPases are global regulators of phase-separated organelles. Nature 573, 144–148 (2019).

    CAS  PubMed  Google Scholar 

  35. 35.

    Alsford, S., Kawahara, T., Glover, L. & Horn, D. Tagging a T. brucei RRNA locus improves stable transfection efficiency and circumvents inducible expression position effects. Mol. Biochem. Parasitol. 144, 142–148 (2005).

    CAS  PubMed  Google Scholar 

  36. 36.

    Glover, L. et al. Genome-scale RNAi screens for high-throughput phenotyping in bloodstream-form African trypanosomes. Nat. Protoc. 10, 106–133 (2015).

    CAS  PubMed  Google Scholar 

  37. 37.

    Alsford, S. & Horn, D. Elongator protein 3b negatively regulates ribosomal DNA transcription in African trypanosomes. Mol. Cell Biol. 31, 1822–1832 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Berriman, M. et al. The genome of the African trypanosome Trypanosoma brucei. Science 309, 416–422 (2005).

    CAS  PubMed  Google Scholar 

  39. 39.

    Becker, M. et al. Isolation of the repertoire of VSG expression site containing telomeres of Trypanosoma brucei 427 using transformation-associated recombination in yeast. Genome Res. 14, 2319–2329 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Hertz-Fowler, C. et al. Telomeric expression sites are highly conserved in Trypanosoma brucei. PLoS ONE 3, e3527 (2008).

    PubMed  PubMed Central  Google Scholar 

  41. 41.

    Kolev, N. G., Ramey-Butler, K., Cross, G. A., Ullu, E. & Tschudi, C. Developmental progression to infectivity in Trypanosoma brucei triggered by an RNA-binding protein. Science 338, 1352–1353 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    PubMed  PubMed Central  Google Scholar 

  44. 44.

    Rutherford, K. et al. Artemis: sequence visualization and annotation. Bioinformatics 16, 944–945 (2000).

    CAS  PubMed  Google Scholar 

  45. 45.

    Gu, Z., Gu, L., Eils, R., Schlesner, M. & Brors, B. Circlize implements and enhances circular visualization in R. Bioinformatics 30, 2811–2812 (2014).

    CAS  PubMed  Google Scholar 

  46. 46.

    Ramirez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160–W165 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Siegel, T. N., Hekstra, D. R. & Cross, G. A. Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging. Mol. Biochem. Parasitol. 160, 171–174 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Woodward, R. & Gull, K. Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei. J. Cell Sci. 95, 49–57 (1990).

    PubMed  Google Scholar 

  49. 49.

    Park, J. H., Jensen, B. C., Kifer, C. T. & Parsons, M. A novel nucleolar G-protein conserved in eukaryotes. J. Cell Sci. 114, 173–185 (2001).

    CAS  PubMed  Google Scholar 

  50. 50.

    Barlow, A. L., Macleod, A., Noppen, S., Sanderson, J. & Guerin, C. J. Colocalization analysis in fluorescence micrographs: verification of a more accurate calculation of Pearson’s correlation coefficient. Microsc. Microanal. 16, 710–724 (2010).

    CAS  PubMed  Google Scholar 

  51. 51.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    CAS  Google Scholar 

  52. 52.

    Kraus, A. J., Brink, B. G. & Siegel, T. N. Efficient and specific oligo-based depletion of rRNA. Sci. Rep. 9, 12281 (2019).

    PubMed  PubMed Central  Google Scholar 

  53. 53.

    Aslett, M. et al. TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res. 38, D457–D462 (2010).

    CAS  PubMed  Google Scholar 

  54. 54.

    Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Wedel, C., Forstner, K. U., Derr, R. & Siegel, T. N. GT-rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes. EMBO J. 36, 2581–2594 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Imakaev, M. et al. Iterative correction of Hi-C data reveals hallmarks of chromosome organization. Nat. Methods 9, 999–1003 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Servant, N. et al. HiC-Pro: an optimized and flexible pipeline for Hi-C data processing. Genome Biol. 16, 259 (2015).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the Dundee Imaging Facility and J. Rouse for access to the Zeiss 880 Airyscan and Leica Confocal SP8 HyVolution microscope, respectively, and S. Alsford (London School of Hygiene and Tropical Medicine) for the SNAP42 tagging construct. We thank N. Jones (Oxford Instruments) for advice on image analysis using Imaris 9.5. We thank R. Cosentino and all members of the Siegel, Ladurner, Meissner and Boshart laboratories for valuable discussion, T. Straub (Bioinformatics Core Facility, BMC) for providing server space and help with data analysis, and the Core Unit Systems Medicine, University of Würzburg for next-generation sequencing. This work was funded by a Wellcome Trust Investigator Award to D.H. (100320/Z/12/Z), the German Research Foundation (SI 1610/3-1 and 213249687—SFB 1064), the Center for Integrative Protein Science (CIPSM) and an ERC Starting Grant (3D_Tryps 715466) to T.N.S. The University of Dundee Imaging Facility is supported by the MRC Next Generation Optical Microscopy award (MR/K015869/1). L.S.M.M. was supported by a grant of the German Excellence Initiative to the Graduate School of Life Science, University of Würzburg.

Author information

Affiliations

Authors

Contributions

The experiments were designed by J.F., V.L., L.S.M.M., D.H. and T.N.S. and carried out by J.F., V.L. and L.S.M.M., unless otherwise indicated. The initial observation that the active VSG gene interacts with the SL-RNA locus was made by L.S.M.M. based on Hi-C assays. The initial observation that VEX1 interacts with the SL-RNA locus was made by L.G. based on IFAs and ChIP-seq. RNAi, chemical inhibition, FISH and super-resolution and other microscopy experiments were performed and analysed by J.F. Hi-C experiments were performed by V.L. and L.S.M.M. and computational analysis was carried out by B.G.B., V.L. and L.S.M.M. RNA-seq experiments were performed by V.L. Data analysis was carried out by V.L. and B.G.B. ChIP-seq data analysis was carried out by S.H. Funding was acquired by D.H. and T.N.S. The work was supervised by D.H. and T.N.S. The manuscript was written by J.F., V.L., D.H. and T.N.S. and edited by all other co-authors.

Corresponding authors

Correspondence to David Horn or T. Nicolai Siegel.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Microbiology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Genome-wide interaction frequencies of VSG genes in expression sites and the SL-RNA locus.

a, Hi-C (virtual 4C) analysis with locations of viewpoints marked by pink boxes. Viewpoints VSG ES 4, 7, 11 and 17 are located on intermediate chromosomes that are not depicted in this figure. Interaction frequencies between each viewpoint and the 11 mega-base chromosomes are shown. Chromosome cores, dark grey; subtelomeric regions, light grey. The hemizygous subtelomeric regions of each chromosome are displayed in the following order: 5´(haplotype A)–5´(haplotype B)–diploid chromosome core–3´(haplotype A)–3´(haplotype B). Bin size 50 kb. Virtual 4C analyses in a-b are based on Hi-C experiments of VSG-2 expressing cells (n = 2, the average is shown). As viewpoint control regions, two actively transcribed regions from the diploid cores of chr. 2 and chr 8 and two non-transcribed regions from the subtelomeres of chr. 1 and chr. 10 were chosen. All four control regions were arbitrarily chosen. The coordinates of all viewpoints used for virtual 4C analyses are listed in Supplementary Data 1 (sheet 2). b, Virtual 4C analyses between the SL-RNA locus (chr. 9) as viewpoint and different expression sites. Relative interaction frequencies between the viewpoint and the active VSG ES 1 (cyan) and inactive VSG ESs (magenta) is shown. Each dot represents the average value for one expression site. Bin size 20 kb.

Extended Data Fig. 2 Dynamic association between the active VSG gene and the spliced leader RNA (SL) array transcription compartments.

a, Immunofluorescence-based colocalization studies of tSNAPmyc (SL-RNA transcription compartment) and a nucleolar and active VSG transcription compartment marker (Pol I, largest subunit) using super resolution microscopy - a plot showing signal intensity across a defined area (cyan line) is presented. b-c, The violin plot shows ‘outside edge’ (b) or ‘inside edge’ distance (c) measurements between the SL-RNA and VSG transcription compartments in G1, S phase or G2 nuclei – n values correspond to number of nuclei except in G2 where the n value corresponds to the number of expression-site body (ESB) / tSNAP pairs detected (in a total of 68 and 61 nuclei). d, Immunofluorescence-based colocalization studies of tSNAPmyc and a nucleolar compartment marker (NOG1) using super resolution microscopy. e, The plot shows signal intensity measurements of the SL-RNA compartments, adjacent or non-adjacent to the expression-site-body (ESB). f, The plot shows ‘inside edge’ distance measurements between the SL-RNA compartments, adjacent or non-adjacent to the ESB and the nucleolus. ESB: Pol I extranucleolar reservoir and VSG transcription compartment. a, d, DNA was counter-stained with DAPI; the images correspond to maximal 3D projections of stacks of 0.1 μm slices and are representative of two biological replicates and three independent experiments; scale bars 2 μm. Violin plots (b, c, e, f): white circles show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; polygons represent density estimates of data and extend to extreme values. P values were determined using a two-tailed unpaired (b-c) or paired (e-f) Student’s t-test. Detailed n and p values are provided in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 Changes in DNA-DNA interactions following a change in VSG isoform expression.

a, Virtual 4C analyses between VSG-2 in expression site 1 as viewpoint and the centromere on the subtelomere of chromosome 11. Relative interaction frequencies between the viewpoint and chr. 11 are plotted. Bin size 20 kb. * marks the centromere on chr. 11 (located on subtelomere 3 A). The coordinates of all viewpoints used for virtual 4C analyses are listed in Supplementary Data 1 (sheet 2). b, Hi-C (virtual 4C) analyses between VSG-2, VSG-13 or a subtelomeric control region as viewpoint and chromosomal core or subtelomeric regions (‘VSG-X’ refers to VSG-2, VSG13 or a subtelomeric control region). Each dot represents the ratio of: the average interaction frequency of the viewpoint with the chromosome core / the average interaction frequency with the subtelomeres. One dot per chromosome is plotted. The black bar marks the median ratio per viewpoint. Bin size 50 kb. c, Virtual 4C analyses between the EP1 gene array (chr. 10) as viewpoint and the SL-RNA locus (chr. 9). Relative interaction frequencies between the EP1 array and the SL-RNA locus are plotted. Bin size 20 kb.

Extended Data Fig. 4 The VEX complex associates with both the active VSG gene and the Spliced Leader (SL) locus in a cell cycle and developmental stage-dependent manner.

a-b, Immunofluorescence-based colocalization studies of VEX1myc / Pol I and GFPVEX2 / tSNAPmyc in bloodstream form cells. tSNAP and Pol I were used as markers for the SL-RNA and VSG transcription compartments, respectively. The stacked bar graphs depict proportions of nuclei with overlapping, adjacent or separate signals (these categories were defined by thresholded Pearson’s correlation coefficient – see methods); values are averages of two independent experiments (≥100 nuclei for G1 and S phase cells); error bars, SD. The violin plot (a) shows signal intensity measurements of the VEX1 foci, adjacent or non-adjacent to the expression-site-body (ESB / VSG transcription compartment / Pol I extranucleolar reservoir) – in cells with 2 VEX1 foci and 1 ESB. White circles show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; polygons represent density estimates of data and extend to extreme values. P values were determined using a paired Student’s t-test; detailed n and p values are provided in Source Data Extended Data Fig. 4. c, VEX1myc chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) analysis. The circle plot represents log2 fold change of ChIP versus Input of non-overlapping 1 kb bins of the 11 megabase chromosomes; outside track shows tandem arrays (red) and the SL-RNA locus (black). An inset zooming on the SL-RNA locus is depicted: heat-map of SL-gene loci. Bin size 300 bp. d, Localization of tSNAPGFP and colocalization studies of VEX1myc or mycVEX2 and Pol I and VEX1myc and tSNAPGFP in procyclic forms (insect-stage), using immunofluorescence. Procyclic forms do not express VSGs whereas procyclins are the major surface glycoprotein. Images in a-b and d were obtained using super resolution microscopy and correspond to maximal 3D projections of stacks of 0.1 μm slices; DNA was counter-stained with DAPI; scale bars 2 μm; images are representative of independent experiments using two different biological replicates.

Source data

Extended Data Fig. 5 VEX1 and VEX2, but not tSNAP, delocalize following transcription or splicing inhibition.

a, Western-blot analysis of VEX1myc, mycVEX2 and tSNAPmyc before and after sinefungin treatment (5 μg ml-1 for 30 min at 37 °C), which blocks trans-splicing in trypanosomes. The values in red, green and magenta correspond to the fold change in VEX1, VEX2 and tSNAP abundance, respectively, between non-treated and treated samples (normalized against EF1α, loading control) in four independent experiments (not-statistically significant). b, Immunofluorescence analysis of Pol I, VEX1myc, mycVEX2 and tSNAPmyc localization following actinomycin D (ActD, Pol I + Pol II inhibitor, 10 μg ml-1 for 30 min at 37 °C), BMH-21 (Pol I inhibitor, 1 μM for 30 min at 37 °C) or sinefungin treatment. c, tSNAPmyc before and after ActD and BMH-21 treatment. Cells displaying no detectable signal (<10%) were excluded. Values are averages of two independent experiments (≥200 nuclei each); error bars, SD. Images in b-c were obtained using super resolution microscopy, correspond to maximal 3D projections of stacks of 0.1 μm slices and are representative of multiple biological replicates and independent experiments; DNA was counter-stained with DAPI; scale bars 2 μm. Uncropped blots (a) and detailed n and p values (c) are provided as Source Data Extended Data Fig. 5.

Source data

Extended Data Fig. 6 Pol I and tSNAP expression and localization following knockdown of the VEX complex.

a, Immunofluorescence-based analysis of VSG expression following tetracycline (Tet) inducible VEX1 knockdown, VEX2 knockdown or VEX1/VEX2 knockdown. In unperturbed cells (parental strain), VSG-2 (magenta) is the active VSG and VSG-6 (green) is a silent VSG used to monitor derepression. The stacked bar graph depicts percentages of VSG-2 single positive cells and VSG-2/VSG-6 double positive cells; values are averages of two independent experiments and two biological replicates. DNA was counter-stained with DAPI; scale bar 2 μm. b, Western-blot analysis of VEX2, Pol I, tSNAPmyc, VSG-6 and VSG-2 expression following VEX1, VEX2 or VEX1/VEX2 knockdown. EF1α was used as a loading control. The data is representative of two independent experiments and two biological replicates. c-d, Immunofluorescence-based colocalization studies of tSNAPmyc (SL-RNA transcription compartment) and Pol I (nucleolus and extranucleolar reservoir). The stacked bar graph in c depicts proportions of G1 nuclei with tSNAPmyc / Pol I overlapping, adjacent or separate signals (these categories were defined by thresholded Pearson’s correlation coefficient – see methods) following tetracycline (Tet) inducible VEX1 (48 h), VEX2 (12 h) or VEX1/VEX2 knockdown (12 h). tSNAPmyc / extranucleolar Pol I localization were not monitored beyond 12 h following VEX2 and VEX1/2 knockdown as Pol I signal drops below detection at later time-points. The values are averages of two independent experiments and two biological replicates (≥100 G1 nuclei). In the violin plot in d, the ‘outside edge’ distance between the Pol I extranucleolar focus and tSNAP foci was measured in > 81 G1 nuclei. White circles show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; polygons represent density estimates of data and extend to extreme values. In a/c, error bars, SD. In c-d, knockdown conditions were compared to parental cells using two-tailed paired (c) or unpaired (d) Student’s t-tests; in c, statistical significance is highlighted when applicable: **, p < 0.01; ***, p < 0.001. Uncropped blots (b) and detailed n and p values (c/d) are provided in Source Data Extended Data Fig. 6.

Source data

Extended Data Fig. 7 The exclusive association between the active VSG and the SL-locus is VEX2-dependent.

a-b, DNA fluorescence in situ hybridization (FISH) and super resolution microscopy based colocalization studies of the SL-RNA transcription compartments (probe: digoxigenin labeled SL repeats) and VSG expression sites (probe: biotin-labeled 50 bp repeats) following VEX2 knockdown. a, the box and violin plots depict the average number and the size of the 50 bp repeats foci, respectively, before and after VEX2 knockdown. b, the violin plot represents the distance between both SL-arrays (before and after VEX2 knockdown. c-d, DNA FISH combined with immunofluorescence colocalization studies of the SL-RNA transcription compartments and VSG expression sites before and after VEX2 knockdown using super resolution microscopy. tSNAPmyc (protein marker) and a DNA probe for the SL repeats (biotin-labeled) or DNA probes for the 50 bp repeats (VSG ESs, biotin labeled) were used (c and d, respectively). The violin plot (d) represents the distance between both tSNAP foci before and after VEX2 knockdown. For all violin plots: white circles show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; polygons represent density estimates of data and extend to extreme values. For the box plot in a, centerlines show the medians; box limits indicate the 25th and 75th percentiles whiskers extend between the minimum and maximum values. Two-tailed paired (a, left hand side) or unpaired Student’s t-tests (a, right hand side; b,d) were applied for statistical analysis. Detailed n and p values are provided in Source Data Extended Data Fig. 7. Representative images (c-d): all nuclei are G1; images correspond to maximal 3D projections of stacks of 0.1 μm slices; DNA was counter-stained with DAPI; scale bars 2 μm. The data correspond to (a, box plot) or are representative of (a, violin plot & b-d) two biological replicates and two independent experiments.

Source data

Extended Data Fig. 8 Genome-wide changes in VEX2 depleted cells.

a, Correlation between the average interaction frequency of VSG expression-sites as viewpoint with the SL-RNA locus and VSG expression in reads per kilobase per million (RNA-seq data from18) in control and VEX2-depleted cells. b, Hi-C (virtual 4C) analyses betweeen the SL-RNA locus (chr. 9) as viewpoint and the different expression sites. Relative interaction frequencies between the viewpoint and VSG expression sites are shown. Ticks on the x axes mark the bins used for the vitual 4C analyses. The coordinates of all viewpoints used for virtual 4C analyses are listed in Supplementary Data 1 (sheet 3). c, Virtual 4C analyses between the EP1 gene array as viewpoint and different expression sites (‘VSG ES X’ refers to the different expression sites). Relative interaction frequencies between the viewpoint and the VSG expression sites are shown before and after VEX2 knockdown. Each dot represents the average value for one expression site. Bin size 20 kb.

Extended Data Fig. 9 Tandem arrays interact with the Spliced Leader (SL) locus.

a, Hi-C (virtual 4C) analysis, viewpoint: different tandem gene arrays and control sites. Relative interaction frequencies between the different viewpoints and the SL-RNA locus are plotted. Bin size 20 kb. As viewpoint control regions, two actively transcribed regions from the diploid cores of chr. 4 and chr 7 were arbitrarily chosen. The coordinates of all viewpoints used for virtual 4C analyses are listed in Supplementary Data 1 (sheet 3). b, VEX1myc chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) analysis. Top panel, metagene plot for tandem genes compared to randomly selected genes with no paralogues. Lower left, heat map of tandem genes. Lower right, randomly selected genes with no paralogues.

Supplementary information

Reporting Summary

Peer Review File

Supplementary Data

Sheet 1: ChIP-seq data for VEX1myc-expressing bloodstream-form T. brucei. Sheet 2: coordinates of the viewpoints used in the virtual 4C analyses. Sheet 3: oligos for DNA FISH probes.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 5

Unprocessed western blots.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 6

Unprocessed western blots.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 7

Statistical source data.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Faria, J., Luzak, V., Müller, L.S.M. et al. Spatial integration of transcription and splicing in a dedicated compartment sustains monogenic antigen expression in African trypanosomes. Nat Microbiol 6, 289–300 (2021). https://doi.org/10.1038/s41564-020-00833-4

Download citation

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing