DESIPHERING THE TRYPANOSOMA CRUZI HIGH MOBILITY GROUP B PROTEIN (TcHMGB) ROLE IN GENE TRANSCRIPTION

VARGAS TRINIDAD, ANDREA SUSANA; SMIRCICH, PABLO; DE HERNÁNDEZ, AZUL; CRIBB, PAMELA

Caxambu, MG, Brasil

Congreso; XXXVI Annual Meeting of the Brazilian Society of Protozoology; 2023

Brazilian Society of Protozoology

High Mobility Group Bs (HMGBs) are abundant non-histone chromatin proteins that contribute to chromatin organization and function, impacting various cellular processes including gene expression, DNA replication, repair, and recombination. Through interactions with DNA and other proteins they can influence the accessibility of genes and the assembly of transcriptional complexes. Trypanosoma cruzi HMGB (TcHMGB) is expressed in the nucleus in all the parasite life stages and can alter chromatin structure like its mammalian orthologs. Given the unique characteristics of transcription in trypanosomatids and evidence of epigenetic mechanisms controlling gene expression, our hypothesis is that TcHMGB may have a key role in transcription control.With the aim of investigating TcHMGB functions, we first constructed transgenic parasites capable of overexpressing the protein under tetracycline induction, which showed that TcHMGB can alter chromatin DNA structure making it more sensible to micrococcal nuclease treatment. TcHMGB overexpression affected the parasite fitness, caused a dramatic decrease in epimastigote- and amastigote-proliferation, presumably impairing cell division and lowered trypomastigote infectivity in vitro. Then, we used CRISPR/Cas9 to generate knockout (KO) mutants. We verified gene edition by PCR and lower TcHMGB expression by qRT-PCR and western blot in transient transfectants. However, despite multiple attempts, we were unable to generate stableTcHMGBKOs, which suggests TcHMGB gene is essential or crucial for the parasite ́s normal functioning.Finally, to study TcHMGB role on transcription, we induced its overexpresion on epimastigotes and analized the RNA content using different approaches: labeling and detection of nascent RNAs; qRT-PCR and RNA-seq. Our results suggest that although some genes showed differential expression in overexpressing parasites, TcHMGB seems to have more global effects. Supported by:AGENCIA I+D+I, PICT 2019-4212;.CONICET, PIP 2021- 0848 Keywords:TRYPANOSOMA;HIGH MOBILITY GROUP B;TRANSCRIPTION.