Authors

Discussion

   Large gene-families often exhibit monogenic expression, with contingent processes including antigenic variation for immune evasion in parasites, and olfaction and B-cell development in mammals. Despite intense study, the mechanisms that underpin these paradigmatic examples of stochastic gene choice and exclusion remain somewhat mysterious. 

      Substantial recent progress yielded Variant-Surface-Glycoprotein (VSG) exclusion factors in African trypanosomes and a new appreciation of the context in which they operate. Specifically, VSG-exclusion-2 (VEX2) accumulates at the active-VSG expression-site and binds VEX1 at a trans-splicing locus on another chromosome; other VSGs are excluded from this sub-nuclear expression-factory (PMIDs: 31289266; 33432154).

    We performed single cell RNA-Seq following VEX1 or VEX2 depletion, revealing a surprisingly complex mixture of simultaneously active VSGs in single cells, and a striking difference between both factors. Further, this analysis showed: 1) the number of simultaneously active VSGs that can be tolerated; 2) a hierarchy of VSG transcriptional derepression. 

     ChIP-Seq indicated strong enrichment of VEX2, which forms a native complex of ∼1 MDa, at the active-VSG expression-site, particularly accumulating at the expression-site associated genes (ESAGs) coding regions. Using super-resolution microscopy, VEX2 N- and C-termini were distinctively visualised extending towards the active-VSG and the splicing locus, respectively, revealing an allele-selective inter-chromosomal bridge, via VEX1, to a trans-splicing locus on another chromosome.

      Through a combination of super resolution microscopy and native gels, we found that most of VEX1 and VEX2 sub-nuclear pools are not in complex with one another, and their interaction is dynamic and cell-cycle regulated. 

      To further dissect VEX interactions, we generated several VEX1 truncated forms, and found that the VEX1 N-terminal fragment (1-289 aa) interacts with VEX2, stabilising it and sustaining exclusion. Additionally, we found that the VEX1 C-terminal fragment, which includes nucleic acid binding domains, also contains regions involved in protein stability and turnover. 

     Finally, VEX2 is a putative DNA:RNA helicase, thus to assess whether this activity was required for VSG-exclusion, we established a CRISPR/Cas9-mediated saturation-mutagenesis assay using FACS followed by amplicon-Seq profiling. Replacement of a critical amino acid with any other amino acid disrupted allelic exclusion. 

     This work begins to reveal the mechanisms by which the VEX complex promotes stochastic VSG gene choice and allelic exclusion in African trypanosomes.