Identification of a novel Transcriptional Activator Factor that positively regulates VSG transcription in bloodstream trypanosomes

ANDREU SAURA; DIANA LÓPEZ FARFÁN; PAULA IRIBARREN; VANINA ALVAREZ; MIGUEL NAVARRO

Woods Hole, Massachusetts

Congreso; Kinetoplastid Molecular Cell Biology Meeting; 2015

Marine Biological Laboratory

Antigenic variation in Trypanosoma brucei involves complex genetic mechanisms to exchange the expression of different Variant Surface Glycoprotein (VSG) genes. The VSG gene is mono-allelically transcribed by RNA polymerase I (RNA pol I) from one out of 15 different telomeric loci known as VSG-expression sites (VSG-ES). The active VSG-ES locus is recruited to a single nuclear body named ESB (Expression Site Body), which contains proteins post-transcriptionally modified by the Small Ubiquitin MOdifier (SUMO) peptide (López-Farfán, et al. 2014). Thus, SUMO-conjugated proteins were identified by LC-MS/MS analysis using extracts obtained from bloodstream form trypanosomes that constitutively expressed HA-tagged SUMO. Two independent proteomic screenings led to the identification of a transcriptional activator factor (TAF1) containing a SNF domain that has been previously associated with altering chromatin accessibility in other eukaryotes. To investigate the function of this factor we first developed a monoclonal antibody against TAF1 which detected a single band by Western blot analysis using trypanosome extracts and localized the protein to the nucleus by IF analysis. We next analyzed the effect of TAF1 depletion on gene expression after confirming reduction of TAF1 protein levels. RT-qPCR analysis of TAF1-depleted cells showed a significant reduction of VSG221 mRNA levels, but an increase in Procyclin mRNA, which is repressed in the bloodstream form. However, no significant changes in other analyzed pol I-, pol II- or pol III-transcribed RNAs were detected. Furthermore, ChIP analysis indicated that TAF1 is present in both the active VSG-ES and in the repressed Procyclin chromatin. These data suggest that TAF1 may function as either repressor or activator factor dependent on the respective developmentally-regulated transcribed loci.