Antunes, L. B. | bioRxiv Science

bioRxiv2025

Chromosome compartment assembly is essential for subtelomeric gene silencing in trypanosomes

Genome three-dimensional organization is essential for the coordination of eukaryote gene expression. The chromosomes of the pathogen Trypanosoma brucei contain hundreds of silent variant surface glycoprotein (VSGs) genes in subtelomeric regions. However, T. brucei transcribes a single VSG gene and periodically changes the VSG expressed by transcriptional or recombination mechanisms, altering its surface coat to escape host antibodies by antigenic variation. We show that VSG-rich silent subtelomeric regions form distinct chromosome compartments from transcribed regions, with subtelomeric compartments of different chromosomes co-interacting. We uncovered chromatin-associating factors at the boundaries of transcribed and silent compartments. Among these, repressor activator-protein 1 (RAP1) marks the compartment boundaries and spreads over silent regions. Inactivation of phosphatidylinositol phosphate 5-phosphatase removed RAP1 from compartment boundaries and subtelomeric regions, disrupting compartment assembly and derepressing all VSG genes. The data show spatial segregation of repressed from transcribed chromatin and phosphoinositides regulation of silent compartment assembly and genome organization.

microbiology↗

bioRxiv2023

High-efficiency transformation and expression of genomic libraries in yeast

Saccharomyces cerevisiae is a powerful system for the expression of genome-wide or combinatorial libraries for diverse types of screening. However, expressing large libraries in yeast requires high-efficiency transformation and controlled expression. Transformation of yeast using electroporation methods is more efficient than chemical methods; however, protocols described for electroporation require large amounts of linearized plasmid DNA and often yield about 106 cfu/{micro}g of plasmid DNA. We optimized the electroporation of yeast cells for the expression of whole-genome libraries to yield up to 108 cfu/{micro}g plasmid DNA. The protocol generates sufficient transformants for 10-100x coverage of diverse genome libraries with small amounts of genomic libraries (0.1{micro}g of DNA per reaction) and provides guidance on calculations to estimate library size coverage and transformation efficiency. It describes the preparation of electrocompetent yeast cells with lithium acetate and dithiothreitol conditioning step and the transformation of cells by electroporation with carrier DNA. We validated the protocol using three yeast surface display libraries and demonstrated using nanopore sequencing that libraries size and diversity are preserved. Moreover, expression analysis confirmed library functionality and the methods efficacy. Hence, this protocol yields a sufficient representation of the genome of interest for downstream screening purposes while limiting the amount of the genomic library required.

molecular biology↗