Contrasting study among Trypanosomatids reveals singularities in their chromatin landscape but a conserved nucleosome depletion at the trans-splicing acceptor site

Abstract

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 mammal 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. Here, we have analyzed the genome-wide chromatin organization for the parasite stage present in the insect vector by chromatin digestion with micrococcal nuclease followed by deep sequencing (MNase-seq) using our own data for T. cruzi and public data for T. brucei and L. major. We have predicted the most likely trans-splicing acceptor sites (TAS) and used them as reference points for genomic analysis. We have made a comparative analysis among TriTryps for comparable extensions of MNase digestion. By representing average nucleosome occupancy, we found that the average chromatin organization is very similar between T. cruzi and T. brucei with a mild nucleosome depletion at the TAS. This depletion at the TAS is also conserved in L. major even for less digested samples. A detailed examination of nucleosome landscapes resulting from different levels of digestion shows that an MNase-sensitive complex is protecting the trans-splicing acceptor site in Trypanosoma brucei. Additionally, by analyzing MNase-ChIP-seq data for histone H3 we uphold that the average MNase protection observed at the TAS for earlier digestion points in T. brucei is mainly due to a non-histone complex, underlining the conserved nucleosome depletion at that genomic point in TriTryps. Moreover, we show that this nucleosome organization is not just an average since the same layout is observed in most of the genome. Furthermore, in T. brucei the nucleosome trough observed at TAS colocalizes with a footprint of DNA-RNA duplex. Here, we show that this reciprocity is not only an average but is conserved in the whole genome and there is a correlation between the intensity of the R-loop signal and the TAS protection from MNase. We are currently making a detailed analysis of the genomic regions that present a faster release of the protective complex observed at the TAS in response to the enzymatic digestion and its relationship with their genomic locations epigenetic marks and DNA accessibility, in T. brucei.A deeper analysis of the chromatin organization and the interacting complexes at TAS might open the road for a better understanding on the processing of the polycistronic transcripts into the mature ARNs.