{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,31]],"date-time":"2026-08-31T20:16:29Z","timestamp":1788207389594,"version":"build-2803163510"},"posted":{"date-parts":[[2025,9,5]]},"group-title":"Molecular Biology","reference-count":59,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,9,5]],"date-time":"2025-09-05T00:00:00Z","timestamp":1757030400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"award":["R21 AI151261"],"award-info":[{"award-number":["R21 AI151261"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100001774","name":"New Jersey Health Foundation","doi-asserted-by":"crossref","award":["PC125-23"],"award-info":[{"award-number":["PC125-23"]}],"id":[{"id":"10.13039\/100001774","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,28]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  <jats:italic>Trypanosoma brucei<\/jats:italic>\n                  is a protozoan parasite that causes African trypanosomiasis. Understanding essential cellular processes in\n                  <jats:italic>T. brucei<\/jats:italic>\n                  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\n                  <jats:italic>T. brucei<\/jats:italic>\n                  growth upon overexpression. Among them,\n                  <jats:italic>Tb<\/jats:italic>\n                  FOP, a previously uncharacterized protein, exhibited distinct phenotypes beyond growth inhibition.\n                  <jats:italic>Tb<\/jats:italic>\n                  FOP overexpression caused severe cell-cycle defects, substantially increasing the proportion of sub-G1 cells lacking nuclear DNA. Transcriptome analysis revealed widespread transcriptional dysregulation following\n                  <jats:italic>Tb<\/jats:italic>\n                  FOP overexpression. Specifically, antisense transcript levels substantially increased near transcription termination sites (TTSs) across the genome.\n                  <jats:italic>Tb<\/jats:italic>\n                  FOP 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\n                  <jats:italic>Tb<\/jats:italic>\n                  FOP overexpression interferes with chromatin-associated transcriptional regulation at specific genomic regions. These findings demonstrate that\n                  <jats:italic>Tb<\/jats:italic>\n                  FOP overexpression profoundly affects transcription, cell-cycle progression, and parasite growth.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>IMPORTANCE<\/jats:title>\n                  <jats:p>\n                    <jats:italic>Trypanosoma brucei<\/jats:italic>\n                    , the parasite that causes African trypanosomiasis, survives in mammalian hosts by periodically changing its major surface protein to evade immune responses. While this process is essential for parasite survival and requires strict control of gene expression, many of the underlying regulatory mechanisms remain poorly understood. Here, a genome-wide toxicity screen identified\n                    <jats:italic>Tb<\/jats:italic>\n                    FOP along with other proteins whose overproduction impairs parasite growth.\n                    <jats:italic>Tb<\/jats:italic>\n                    FOP is a previously uncharacterized protein whose overproduction severely disrupts transcriptional regulation as well as parasite growth. Increased\n                    <jats:italic>Tb<\/jats:italic>\n                    FOP levels caused widespread transcriptional dysregulation, including increased antisense transcript levels near transcription termination sites and inappropriate expression of normally silent surface protein genes. These findings reveal a previously unrecognized link among altered\n                    <jats:italic>Tb<\/jats:italic>\n                    FOP expression, transcriptional regulation, and VSG gene silencing. Understanding these unusual regulatory processes is important for defining how trypanosomes maintain persistent infections and may reveal biological processes that distinguish these parasites from their mammalian hosts.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.09.02.673782","type":"posted-content","created":{"date-parts":[[2025,9,6]],"date-time":"2025-09-06T04:00:13Z","timestamp":1757131213000},"source":"Crossref","is-referenced-by-count":0,"title":["A genome-wide overexpression screen identifies\n                  <i>Tb<\/i>\n                  FOP, whose overexpression disrupts transcription termination in\n                  <i>Trypanosoma brucei<\/i>"],"prefix":"10.64898","author":[{"given":"Navina Panneer","family":"Selvan","sequence":"first","affiliation":[{"name":"Public Health Research Institute, Rutgers Biomedical and Health Sciences, Rutgers University, Newark, NJ 07103, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aditi","family":"Mukherjee","sequence":"additional","affiliation":[{"name":"Public Health Research Institute, Rutgers Biomedical and Health Sciences, Rutgers University, Newark, NJ 07103, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vivek","family":"Gupta","sequence":"additional","affiliation":[{"name":"Public Health Research Institute, Rutgers Biomedical and Health Sciences, Rutgers University, Newark, NJ 07103, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Esteban","family":"Erben","sequence":"additional","affiliation":[{"name":"Instituto de Investigaciones Biotecnol\u00f3gicas, Universidad Nacional de San Mart\u00edn (UNSAM) \u2013 Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas (CONICET), Argentina"},{"name":"Escuela de Bio y Nanotecnolog\u00edas (EByN), Universidad Nacional de San Mart\u00edn, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hee-Sook","family":"Kim","sequence":"additional","affiliation":[{"name":"Public Health Research Institute, Rutgers Biomedical and Health Sciences, Rutgers University, Newark, NJ 07103, USA"},{"name":"Department of Microbiology, Biochemistry, and Molecular Genetics, Rutgers Biomedical and Health Sciences, Rutgers University, Newark, NJ 07103, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026083112251037000_2025.09.02.673782v2.1","doi-asserted-by":"publisher","DOI":"10.1038\/273613a0"},{"key":"2026083112251037000_2025.09.02.673782v2.2","first-page":"55","article-title":"Antigenic variation in trypanosomes","volume":"202","year":"1978","journal-title":"Proceedings of The Royal Society London Section B"},{"key":"2026083112251037000_2025.09.02.673782v2.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.mib.2010.08.009"},{"key":"2026083112251037000_2025.09.02.673782v2.4","doi-asserted-by":"publisher","DOI":"10.1042\/ETLS20170104"},{"key":"2026083112251037000_2025.09.02.673782v2.5","doi-asserted-by":"publisher","DOI":"10.1128\/MMBR.00024-10"},{"key":"2026083112251037000_2025.09.02.673782v2.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-018-0619-8"},{"key":"2026083112251037000_2025.09.02.673782v2.7","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(87)90154-1"},{"key":"2026083112251037000_2025.09.02.673782v2.8","doi-asserted-by":"publisher","DOI":"10.1016\/S1097-2765(03)00143-6"},{"key":"2026083112251037000_2025.09.02.673782v2.9","doi-asserted-by":"publisher","DOI":"10.1016\/j.mib.2016.04.018"},{"key":"2026083112251037000_2025.09.02.673782v2.10","doi-asserted-by":"crossref","unstructured":"Clayton C . Regulation of gene expression in trypanosomatids: living with polycistronic transcription. 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