Biology subjects

Carloni, R.

Publications and source records attributed to Carloni, R..

2 recordsLinked to original sources

bioRxiv2025

Defining the chromatin-associated protein landscapes on Trypanosoma brucei repetitive elements using synthetic TALE proteins

Kinetoplastids, such as Trypansoma brucei, are eukaryotes that likely separated from the main lineage at an exceptionally early point in evolution. Consequently, many aspects of kinetoplastid biology differ significantly from other eukaryotic model systems, including yeasts, plants, worms, flies and mammals. As in many eukaryotes the T. brucei genome contains repetitive elements at various chromosomal locations including centromere- and telomere-associated repeats and interspersed retrotransposon elements. T. brucei also contains intermediate-sized and mini-chromosomes that harbor abundant 177 bp repeat arrays, and 70 bp repeat elements implicated in Variable Surface Glycoprotein (VSG) gene switching. In many eukaryotes repetitive elements are assembled in specialised chromatin such as heterochromatin, however, apart from centromere- and telomere-associated repeats, little is known about chromatin-associated proteins that decorate these and other repetitive elements in kinetoplastids. Here we utilize affinity selection of synthetic TALE DNA binding proteins designed to target specific repeat elements to identify enriched proteins by proteomics. Validating the approach, a telomere repeat binding TelR-TALE identifies many proteins previously implicated in telomere function. Furthermore, the 70R-TALE designed to bind 70 bp repeats indicates that proteins involved in DNA repair are enriched on these elements that reside adjacent to VSG genes. Interestingly, the 177 bp repeat binding 177R-TALE enriches for many kinetochore proteins suggesting that intermediate-sized and mini-chromosomes assemble kinetochores related in composition to those located on the main megabase chromosomes. This provides a first insight into the chromatin landscape of repetitive regions of the trypanosome genome with relevance for their mechanisms of chromosome integrity, immune evasion and cell replication.

cell biology↗

bioRxiv2021

A systematic analysis of Trypanosoma brucei chromatin factors identifies novel protein interaction networks associated with sites of transcription initiation and termination

Nucleosomes composed of histones are the fundamental units around which DNA is wrapped to form chromatin. Transcriptionally active euchromatin or repressive heterochromatin is regulated in part by the addition or removal of histone post-translational modifications (PTMs) by writer and eraser enzymes, respectively. Nucleosomal PTMs are recognised by a variety of reader proteins which alter gene expression accordingly. The histone tails of the evolutionarily divergent eukaryotic parasite Trypanosoma brucei have atypical sequences and PTMs distinct from those often considered universally conserved. Here we identify 68 predicted readers, writers and erasers of histone acetylation and methylation encoded in the T. brucei genome and, by epitope tagging, systemically localize 63 of them in the parasites bloodstream form. ChIP-seq demonstrated that fifteen candidate proteins associate with regions of RNAPII transcription initiation. Eight other proteins exhibit a distinct distribution with specific peaks at a subset of RNAPII transcription termination regions marked by RNAPIII-transcribed tRNA and snRNA genes. Proteomic analyses identified distinct protein interaction networks comprising known chromatin regulators and novel trypanosome-specific components. Notably, several SET-domain and Bromo-domain protein networks suggest parallels to RNAPII promoter-associated complexes in conventional eukaryotes. Further, we identify likely components of TbSWR1 and TbNuA4 complexes whose enrichment coincides with the SWR1-C exchange substrate H2A.Z at RNAPII transcriptional start regions. The systematic approach employed provides detail of the composition and organization of the chromatin regulatory machinery in Trypanosoma brucei and establishes a route to explore divergence from eukaryotic norms in an evolutionarily ancient but experimentally accessible eukaryote.

molecular biology↗