Sumoylation in the VSG Expression Site Chromatin

In the mammalian host, bloodstream trypanosomes elude the immune response by periodically changing their main surface antigen, the Variant Surface Glycoprotein (VSG). In Trypanosoma brucei the VSG gene is transcribed by RNA polymerase I (RNAPI) instead of RNAPII. We previously reported that transcription mediated by RNAPI in trypanosomes requires TbRPB7, a dissociable subunit of the RNAPII complex (Peñate, et al. 2009). To identify TbRPB7-interacting proteins, a two hybrid assay was performed. This approach detected several putative interacting proteins, including a protein with a conserved E3 SUMO ligase domain, we named TbSIZ1. In T. brucei a SUMO orthologue was recently identified, however, the sumoylation pathway and functions are mostly unknown in this protozoan parasite Depletion of TbSIZ1 reduced the nuclear signal of SUMO-conjugated proteins analysed by IF using an anti-SUMO antibody. Next, we determined the occupancy of sumoylated proteins within the VSG-ES chromatin by ChIP using an anti-SUMO antibody. Sumoylated proteins were detected along the active VSG transcription unit but not in silent VSGs. Sumoylation of chromatin-associated proteins was not significant at ribosomal DNA, SL promoter, tubulin or U2 loci, suggesting this is a distinct feature of the VSG-ES. Interestingly, conserved sequences upstream of VSG-ES promoters are highly enriched in sumoylated proteins, suggesting that sumoylation is involved in the regulation of the VSG-ES.