UPF1-like helicases play roles in telomeric heterochromatin formation and X-chromosome inactivation, and also in monogenic variant surface glycoprotein (VSG) expression via VSG exclusion-factor-2 (VEX2), a UPF1-related protein in the African trypanosome. We show that VEX2 associates with chromatin specifically at the single active VSG expression site on chromosome 6, forming an allele-selective connection, via VEX1, to the trans-splicing locus on chromosome 9, physically bridging two chromosomes and the VSG transcription and splicing compartments. We further show that the VEX-complex is multimeric and self-regulates turnover to tightly control its abundance. Using single cell transcriptomics following VEX2-depletion, we observed simultaneous derepression of many other telomeric VSGs and multi-allelic VSG expression in individual cells. Thus, an allele-selective, inter-chromosomal, and self-limiting VEX1-2 bridge supports monogenic VSG expression and multi-allelic VSG exclusion.

Biology Centre Czech Academy of Sciences Institute of Parasitology České Budějovice Czech Republic

Biology Department University of York York UK

Division of Cell Signaling Fujii Memorial Institute of Medical Sciences Institute of Advanced Medical Sciences Tokushima University Tokushima Japan

Faculty of Science Charles University Prague Biocev Vestec Czech Republic

Gene Regulation and Expression School of Life Sciences University of Dundee Dundee UK

Wellcome Centre for Anti Infectives Research Biological Chemistry and Drug Discovery School of Life Sciences University of Dundee Dundee UK

Wellcome Centre for Integrative Parasitology University of Glasgow Glasgow UK

York Biomedical Research Institute University of York York UK

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Williams DL, et al. May the odds be ever in your favor: non-deterministic mechanisms diversifying cell surface molecule expression. Front Cell Dev. Biol. 2021;9:720798. doi: 10.3389/fcell.2021.720798. PubMed DOI PMC

Khamlichi AA, Feil R. Parallels between mammalian mechanisms of monoallelic gene expression. Trends Genet. 2018;34:954–971. doi: 10.1016/j.tig.2018.08.005. PubMed DOI

Faria J, et al. Emergence and adaptation of the cellular machinery directing antigenic variation in the African trypanosome. Curr. Opin. Microbiol. 2022;70:102209. doi: 10.1016/j.mib.2022.102209. PubMed DOI

Florini F, Visone JE, Deitsch KW. Shared mechanisms for mutually exclusive expression and antigenic variation by protozoan parasites. Front. Cell Dev. Biol. 2022;10:852239. doi: 10.3389/fcell.2022.852239. PubMed DOI PMC

Chaconas G, Castellanos M, Verhey TB. Changing of the guard: how the Lyme disease spirochete subverts the host immune response. J. Biol. Chem. 2020;295:301–313. doi: 10.1074/jbc.REV119.008583. PubMed DOI PMC

Müller LSM, et al. Genome organization and DNA accessibility control antigenic variation in trypanosomes. Nature. 2018;563:121–125. doi: 10.1038/s41586-018-0619-8. PubMed DOI PMC

Aresta-Branco F, et al. Mechanistic similarities between antigenic variation and antibody diversification during Trypanosoma brucei Infection. Trends Parasitol. 2019;35:302–315. doi: 10.1016/j.pt.2019.01.011. PubMed DOI

Hertz-Fowler C, et al. Telomeric expression sites are highly conserved in Trypanosoma brucei. PLoS One. 2008;3:e3527. doi: 10.1371/journal.pone.0003527. PubMed DOI PMC

Navarro M, Gull K. A pol I transcriptional body associated with VSG mono-allelic expression in Trypanosoma brucei. Nature. 2001;414:759–763. doi: 10.1038/414759a. PubMed DOI

Monahan K, Horta A, Lomvardas S. LHX2- and LDB1-mediated trans interactions regulate olfactory receptor choice. Nature. 2019;565:448–453. doi: 10.1038/s41586-018-0845-0. PubMed DOI PMC

Pourmorady A, Lomvardas S. Olfactory receptor choice: a case study for gene regulation in a multi-enhancer system. Curr. Opin. Genet Dev. 2022;72:101–109. doi: 10.1016/j.gde.2021.11.003. PubMed DOI

Le Noir S, et al. Functional anatomy of the immunoglobulin heavy chain 3΄ super-enhancer needs not only core enhancer elements but also their unique DNA context. Nucleic Acids Res. 2017;45:5829–5837. doi: 10.1093/nar/gkx203. PubMed DOI PMC

Faria J, et al. Spatial integration of transcription and splicing in a dedicated compartment sustains monogenic antigen expression in African trypanosomes. Nat. Microbiol. 2021;6:289–300. doi: 10.1038/s41564-020-00833-4. PubMed DOI PMC

Melo do Nascimento, L. et al. Functional insights from a surface antigen mRNA-bound proteome. Elife10, e68136 (2021) PubMed PMC

Viegas IJ, et al. N(6)-methyladenosine in poly(A) tails stabilize VSG transcripts. Nature. 2022;604:362–370. doi: 10.1038/s41586-022-04544-0. PubMed DOI PMC

Quinodoz SA, et al. Higher-order inter-chromosomal hubs shape 3D genome organization in the nucleus. Cell. 2018;174:744–757.e24. doi: 10.1016/j.cell.2018.05.024. PubMed DOI PMC

Fare CM, et al. Higher-order organization of biomolecular condensates. Open Biol. 2021;11:210137. doi: 10.1098/rsob.210137. PubMed DOI PMC

Budzak J, et al. An assembly of nuclear bodies associates with the active VSG expression site in African trypanosomes. Nat. Commun. 2022;13:101. doi: 10.1038/s41467-021-27625-6. PubMed DOI PMC

Faria J, et al. Monoallelic expression and epigenetic inheritance sustained by a Trypanosoma brucei variant surface glycoprotein exclusion complex. Nat. Commun. 2019;10:3023. doi: 10.1038/s41467-019-10823-8. PubMed DOI PMC

Glover L, et al. VEX1 controls the allelic exclusion required for antigenic variation in trypanosomes. Proc. Natl Acad. Sci. USA. 2016;113:7225–7230. doi: 10.1073/pnas.1600344113. PubMed DOI PMC

Tihon E, et al. VEX1 influences mVSG expression during the transition to mammalian infectivity in Trypanosoma brucei. Front. Cell Dev. Biol. 2022;10:851475. doi: 10.3389/fcell.2022.851475. PubMed DOI PMC

Kolev NG, et al. Developmental progression to infectivity in Trypanosoma brucei triggered by an RNA-binding protein. Science. 2012;338:1352–1353. doi: 10.1126/science.1229641. PubMed DOI PMC

Cross GA, Kim HS, Wickstead B. Capturing the variant surface glycoprotein repertoire (the VSGnome) of Trypanosoma brucei Lister 427. Mol. Biochem. Parasitol. 2014;195:59–73. doi: 10.1016/j.molbiopara.2014.06.004. PubMed DOI

Kovářová J, et al. CRISPR/Cas9-based precision tagging of essential genes in bloodstream form African trypanosomes. Mol. Biochem. Parasitol. 2022;249:111476. doi: 10.1016/j.molbiopara.2022.111476. PubMed DOI

Ouna BA, et al. Depletion of trypanosome CTR9 leads to gene expression defects. PLoS One. 2012;7:e34256. doi: 10.1371/journal.pone.0034256. PubMed DOI PMC

Yang X, et al. RAP1 is essential for silencing telomeric variant surface glycoprotein genes in Trypanosoma brucei. Cell. 2009;137:99–109. doi: 10.1016/j.cell.2009.01.037. PubMed DOI PMC

Reis H, et al. TelAP1 links telomere complexes with developmental expression site silencing in African trypanosomes. Nucleic Acids Res. 2018;46:2820–2833. doi: 10.1093/nar/gky028. PubMed DOI PMC

Gaurav AK, et al. The RRM-mediated RNA binding activity in T. brucei RAP1 is essential for VSG monoallelic expression. Nat. Commun. 2023;14:1576. doi: 10.1038/s41467-023-37307-0. PubMed DOI PMC

McInnes L, Healy J, Melville J. UMAP: Uniform Manifold Approximation and Projection. J. Open Source Softw. 2018;3:861. doi: 10.21105/joss.00861. DOI

Hutchinson S, et al. The establishment of variant surface glycoprotein monoallelic expression revealed by single-cell RNA-seq of Trypanosoma brucei in the tsetse fly salivary glands. PLoS Pathog. 2021;17:e1009904. doi: 10.1371/journal.ppat.1009904. PubMed DOI PMC

Monahan K, Lomvardas S. Monoallelic expression of olfactory receptors. Annu Rev. Cell Dev. Biol. 2015;31:721–740. doi: 10.1146/annurev-cellbio-100814-125308. PubMed DOI PMC

Vettermann C, Schlissel MS. Allelic exclusion of immunoglobulin genes: models and mechanisms. Immunol. Rev. 2010;237:22–42. doi: 10.1111/j.1600-065X.2010.00935.x. PubMed DOI PMC

Schulz D, Papavasiliou FN. The VEXing problem of monoallelic expression in the African trypanosome. Proc. Natl Acad. Sci. USA. 2016;113:7017–7019. doi: 10.1073/pnas.1608546113. PubMed DOI PMC

López-Escobar L, et al. Stage-specific transcription activator ESB1 regulates monoallelic antigen expression in Trypanosoma brucei. Nat. Microbiol. 2022;7:1280–1290. doi: 10.1038/s41564-022-01175-z. PubMed DOI PMC

Fairman-Williams ME, Guenther UP, Jankowsky E. SF1 and SF2 helicases: family matters. Curr. Opin. Struct. Biol. 2010;20:313–324. doi: 10.1016/j.sbi.2010.03.011. PubMed DOI PMC

Ciaudo C, et al. Nuclear mRNA degradation pathway(s) are implicated in Xist regulation and X chromosome inactivation. PLoS Genet. 2006;2:e94. doi: 10.1371/journal.pgen.0020094. PubMed DOI PMC

Azzalin CM, et al. Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromosome ends. Science. 2007;318:798–801. doi: 10.1126/science.1147182. PubMed DOI

Grunseich C, et al. Senataxin mutation reveals how R-loops promote transcription by blocking DNA methylation at gene promoters. Mol. Cell. 2018;69:426–437.e7. doi: 10.1016/j.molcel.2017.12.030. PubMed DOI PMC

Hatchi E, et al. BRCA1 recruitment to transcriptional pause sites is required for R-loop-driven DNA damage repair. Mol. Cell. 2015;57:636–647. doi: 10.1016/j.molcel.2015.01.011. PubMed DOI PMC

Groh M, et al. Senataxin: genome guardian at the interface of transcription and neurodegeneration. J. Mol. Biol. 2017;429:3181–3195. doi: 10.1016/j.jmb.2016.10.021. PubMed DOI

Ong CT, Corces VG. CTCF: an architectural protein bridging genome topology and function. Nat. Rev. Genet. 2014;15:234–246. doi: 10.1038/nrg3663. PubMed DOI PMC

Dehingia B, et al. CTCF shapes chromatin structure and gene expression in health and disease. EMBO Rep. 2022;23:e55146. doi: 10.15252/embr.202255146. PubMed DOI PMC

Alsford S, et al. Tagging a T. brucei RRNA locus improves stable transfection efficiency and circumvents inducible expression position effects. Mol. Biochem. Parasitol. 2005;144:142–148. doi: 10.1016/j.molbiopara.2005.08.009. PubMed DOI PMC

Rico E, et al. Inducible high-efficiency CRISPR-Cas9-targeted gene editing and precision base editing in African trypanosomes. Sci. Rep. 2018;8:7960. doi: 10.1038/s41598-018-26303-w. PubMed DOI PMC

Glover L, et al. Genome-scale RNAi screens for high-throughput phenotyping in bloodstream-form African trypanosomes. Nat. Protoc. 2015;10:106–133. doi: 10.1038/nprot.2015.005. PubMed DOI

Quintana JF, et al. Instability of aquaglyceroporin (AQP) 2 contributes to drug resistance in Trypanosoma brucei. PLoS Negl. Trop. Dis. 2020;14:e0008458. doi: 10.1371/journal.pntd.0008458. PubMed DOI PMC

Redmond S, Vadivelu J, Field MC. RNAit: an automated web-based tool for the selection of RNAi targets in Trypanosoma brucei. Mol. Biochem. Parasitol. 2003;128:115–118. doi: 10.1016/S0166-6851(03)00045-8. PubMed DOI

Alsford S, Horn D. Single-locus targeting constructs for reliable regulated RNAi and transgene expression in Trypanosoma brucei. Mol. Biochem. Parasitol. 2008;161:76–79. doi: 10.1016/j.molbiopara.2008.05.006. PubMed DOI PMC

Hatos A, et al. FuzDrop on AlphaFold: visualizing the sequence-dependent propensity of liquid-liquid phase separation and aggregation of proteins. Nucleic Acids Res. 2022;50:W337–W344. doi: 10.1093/nar/gkac386. PubMed DOI PMC

Briggs, E. M. et al. Single-cell transcriptomic analysis of bloodstream Trypanosoma brucei reconstructs cell cycle progression and developmental quorum sensing. Nat. Commun.12, 5268 (2021). PubMed PMC

Berriman M, et al. The genome of the African trypanosome Trypanosoma brucei. Science. 2005;309:416–422. doi: 10.1126/science.1112642. PubMed DOI

Eperon IC, et al. The major transcripts of the kinetoplast DNA of Trypanosoma brucei are very small ribosomal RNAs. Nucleic Acids Res. 1983;11:105–125. doi: 10.1093/nar/11.1.105. PubMed DOI PMC

Dobin A, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21. doi: 10.1093/bioinformatics/bts635. PubMed DOI PMC

Clayton C. Regulation of gene expression in trypanosomatids: living with polycistronic transcription. Open Biol. 2019;9:190072. doi: 10.1098/rsob.190072. PubMed DOI PMC

Pita S, et al. The Tritryps comparative repeatome: insights on repetitive element evolution in trypanosomatid pathogens. Genome Biol. Evol. 2019;11:546–551. doi: 10.1093/gbe/evz017. PubMed DOI PMC

Lun ATL, et al. EmptyDrops: distinguishing cells from empty droplets in droplet-based single-cell RNA sequencing data. Genome Biol. 2019;20:63. doi: 10.1186/s13059-019-1662-y. PubMed DOI PMC

Vigneron A, et al. Single-cell RNA sequencing of Trypanosoma brucei from tsetse salivary glands unveils metacyclogenesis and identifies potential transmission blocking antigens. Proc. Natl Acad. Sci. USA. 2020;117:2613–2621. doi: 10.1073/pnas.1914423117. PubMed DOI PMC

Young, M. D. & Behjati, S. SoupX removes ambient RNA contamination from droplet-based single-cell RNA sequencing data. Gigascience9, giaa151 (2020). PubMed PMC

Mendez KM, et al. Toward collaborative open data science in metabolomics using Jupyter Notebooks and cloud computing. Metabolomics. 2019;15:125. doi: 10.1007/s11306-019-1588-0. PubMed DOI PMC

Siegel TN, et al. Four histone variants mark the boundaries of polycistronic transcription units in Trypanosoma brucei. Genes Dev. 2009;23:1063–1076. doi: 10.1101/gad.1790409. PubMed DOI PMC

Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat. Methods. 2012;9:357–359. doi: 10.1038/nmeth.1923. PubMed DOI PMC

Li H, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25:2078–2079. doi: 10.1093/bioinformatics/btp352. PubMed DOI PMC

Rutherford K, et al. Artemis: sequence visualization and annotation. Bioinformatics. 2000;16:944–945. doi: 10.1093/bioinformatics/16.10.944. PubMed DOI

Afgan E, et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 2018;46:W537–W544. doi: 10.1093/nar/gky379. PubMed DOI PMC

Ramírez F, et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 2016;44:W160–W165. doi: 10.1093/nar/gkw257. PubMed DOI PMC

Feng J, et al. Identifying ChIP-seq enrichment using MACS. Nat. Protoc. 2012;7:1728–1740. doi: 10.1038/nprot.2012.101. PubMed DOI PMC

Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30:923–930. doi: 10.1093/bioinformatics/btt656. PubMed DOI

Krzywinski M, et al. Circos: an information aesthetic for comparative genomics. Genome Res. 2009;19:1639–1645. doi: 10.1101/gr.092759.109. PubMed DOI PMC

Obado SO, et al. High-efficiency isolation of nuclear envelope protein complexes from trypanosomes. Methods Mol. Biol. 2016;1411:67–80. doi: 10.1007/978-1-4939-3530-7_3. PubMed DOI

Cox J, Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 2008;26:1367–1372. doi: 10.1038/nbt.1511. PubMed DOI

Tyanova S, et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods. 2016;13:731–740. doi: 10.1038/nmeth.3901. PubMed DOI

Crozier TWM, et al. Prediction of protein complexes in Trypanosoma brucei by protein correlation profiling mass spectrometry and machine learning. Mol. Cell Proteom. 2017;16:2254–2267. doi: 10.1074/mcp.O117.068122. PubMed DOI PMC

Yoshikawa, H. et al. Efficient analysis of mammalian polysomes in cells and tissues using Ribo Mega-SEC. Elife. 7, e36530 (2018). PubMed PMC

Schindelin J, et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods. 2012;9:676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Siegel TN, Hekstra DR, Cross GA. Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging. Mol. Biochem. Parasitol. 2008;160:171–174. doi: 10.1016/j.molbiopara.2008.04.004. PubMed DOI PMC

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. 1990;95:49–57. doi: 10.1242/jcs.95.1.49. PubMed DOI

Barlow AL, et al. Colocalization analysis in fluorescence micrographs: verification of a more accurate calculation of Pearson’s correlation coefficient. Microsc. Microanal. 2010;16:710–724. doi: 10.1017/S143192761009389X. PubMed DOI

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