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

Trypanosoma brucei is a protozoan parasite that causes African trypanosomiasis. We performed an overexpression library screen to gain insights into essential cellular processes in T. brucei and identified genes whose overexpression impaired parasite growth. One of the three top-ranking candidates we validated exhibited a notable phenotype. Tb FOP, an uncharacterized protein, has an N-terminal arginine-rich domain and a C-terminal FOP (Friend Of PRMT1) domain. Tb FOP overexpression substantially increases antisense transcription across the genome, particularly at Transcription Termination Sites (TTSs). This phenotype resembles that observed in trypanosome cells lacking a component of the transcription termination complex PJW/PP1 and cells lacking TTS chromatin marks, two histone variants, and base J DNA modification. Base J is synthesized in two steps: hydroxylation of dT to 5-hydroxymethyl-dU (5hmU) by JBP1 & JBP2, and the glucosylation of 5hmU to glucosyl-5hmU (base J) by JGT. Interestingly, human FOP interacts with 5-hydroxymethyl-dC (5hmC) generated by TET enzymes that are orthologs of T. brucei JBP1/2. Our study reveals Tb FOP as a novel transcription termination factor that may interact with TTS-associated 5hmU, thereby interfering with the function of termination factors. We propose that Tb FOP overexpression disrupts termination, leading to widespread transcription defects and trypanosome cell death.