RNA-seq following knockdown of either VEX2 or CAF-1

Expression of a single allele from a large gene family is inherently stochastic rather than deterministic. This allelic exclusion effectively generates diversity and underpins immune evasion by parasitic protozoa 1,2 and olfaction in mammals and insects 3. The molecular mechanisms that allow for activation of one allele, coordinated with the heritable exclusion of others, have remained poorly understood, however. In African trypanosomes, one telomeric and polycistronic variant surface glycoprotein (VSG) gene expression site (ES) is transcriptionally active at a time 2. This active VSG-ES is associated with an RNA-polymerase-I (pol-I) transcription factory, known as the expression-site body (ESB) 4, and with VSG exclusion 1 (VEX1) 5. Here, we identify new proteins responsible for maintaining sequestration of VEX1 at the ESB and for driving this mechanism of allelic exclusion. VEX2, a protein related to UPF1, a conserved helicase involved in nonsense-mediated mRNA decay, and the conserved tripartite DNA replication-associated histone chaperone, chromatin assembly factor 1 (CAF-1) were identified as interacting with VEX1. Both VEX1 and VEX2 are localised, in a transcription-dependent manner, to a single subnuclear focus associated with the ESB. Following VEX2 knockdown, VEX1 is distributed in the nucleus and all pol-I transcribed protein-coding loci in the genome are derepressed. CAF-1 is enriched at VEX1 foci specifically during DNA replication and is also required for sequestration, heritably depriving silent loci of VEX1. This transcription, VEX2 and CAF-1 dependent sequestration of VEX1 provides a 'winner-takes-all' paradigm for the establishment, maintenance and inheritance of nuclear bodies and allele-specific epigenetic states.