Genome-wide overexpression screen reveals a novel transcription termination factor in Trypanosoma brucei

Abstract

Trypanosoma brucei is a protozoan parasite that causes African trypanosomiasis. Understanding essential cellular processes in T. brucei can provide insights into parasite-specific vulnerabilities. We therefore screened for genes whose overexpression impaired parasite growth. Three top-ranking candidates were confirmed to inhibit T. brucei growth upon overexpression. Among them, TbFOP, a previously uncharacterized protein, exhibited distinct phenotypes beyond growth inhibition. TbFOP overexpression caused severe cell-cycle defects, substantially increasing the proportion of sub-G1 cells lacking nuclear DNA. Transcriptome analysis revealed widespread transcriptional dysregulation following TbFOP overexpression. Specifically, antisense transcript levels substantially increased near transcription termination sites (TTSs) across the genome. TbFOP overexpression also increased the expression of normally silent variant surface glycoprotein (VSG) genes across multiple genomic contexts. These phenotypes resemble those observed in trypanosome mutants lacking TTS-associated chromatin marks, including two histone variants and base J, a kinetoplastid-specific DNA modification. The preferential effects at TTSs and VSG loci raise the possibility that TbFOP overexpression interferes with chromatin-associated transcriptional regulation at specific genomic regions. These findings demonstrate that TbFOP overexpression profoundly affects transcription, cell-cycle progression, and parasite growth.