Beati, P. | Search bioRxiv

bioRxiv2025

Beyond the transcript: chromatin implications in trans-splicing in Trypanosomatids

Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, usually known as TriTryps, are the causal agents of animal and human sickness, and are characterized by having complex life cycles, alternating between a mammalian host and an insect vector. Their genes are organized in long transcriptional units that give rise to polycistronic transcripts which maturate into mRNA by a process known as trans-splicing. Among those genes, an important subset is composed of multicopy genes, which play crucial roles in host invasion and immune evasion. Here, we predicted the most likely trans-splicing acceptor sites (TASs) for TriTryps and found that the average chromatin organization is very similar among them with a mild nucleosome depletion at the TASs, and the same layout is observed in most of the genome. A detailed examination of the nucleosome landscapes resulting from different levels of chromatin digestion in T. brucei shows that an MNase-sensitive complex is protecting the TASs, and it is at least partly composed of histones. Additionally, comparative analysis for single and multi-copy genes in T. cruzi revealed a differential chromatin structure at the TASs suggesting a novel mechanism to guarantee the fidelity of trans-splicing in trypanosomatids.

genomics↗

bioRxiv2021

Experimental and Bioinformatic upgrade for genome-wide mapping of nucleosomes in Trypanosoma cruzi

1In Trypanosoma cruzi, as in every eukaryotic cell, DNA is packaged into chromatin by octamers of histone proteins that constitute nucleosomes. Besides compacting DNA, nucleosomes control DNA dependent processes by modulating the access of DNA binding proteins to regulatory elements on the DNA; or by providing the platform for additional layers of regulation given by histone variants and histone post-translational modifications. In trypanosomes, protein coding genes are constitutively transcribed as polycistronic units. Therefore, gene expression is controlled mainly post transcriptionally. However, chromatin organization and the histone code influence transcription, cell cycle progression, replication and DNA repair. Hence, determining nucleosome position is of uppermost importance to understand the peculiarities of these processes in trypanosomes. Digestion of chromatin with micrococcal nuclease followed by deep sequencing has been widely applied for genome-wide mapping of nucleosomes in several organisms. Nonetheless, this parasite presents numerous singularities. On one hand, special growth conditions and cell manipulation are required. On the other hand, chromatin organization shows some uniqueness that demands a specially designed analytical approach. An additional entanglement is given by the nature of its genome harboring a large content of repetitive sequences and the poor quality of the genome assembly and annotation of many strains. Here, we adapted this broadly used method to the hybrid reference strain, CL Brener. Particularly, we developed an exhaustive and thorough computational workflow for data analysis, highlighting the relevance of using its whole genome as a reference instead of the commonly used Esmeraldo-like haplotype. Moreover, the performance of two aligners, Bowtie2 and HISAT2 was tested to find the most appropriate tool to map any genomic read to reference genomes bearing this complexity.

genomics↗