Contrasting study among Trypanosomatids reveals conserved chromatin organization around 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. TriTryps are characterized by having complex life cycles, alternating between a mammal host and an insect vector. One peculiarity of these organisms is that 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. Even though gene expression regulation occurs mainly post-transcriptionally, it has been recently shown that chromatin plays a role in modulation. In this work, we made a comparative analysis of genome-wide chromatin organization and its potential impact on gene expression for the parasite stage present in the insect vector for the TriTryps using MNase-seq and RNAse-seq data, publicly available or generated by our laboratory. To compare average nucleosome positioning and mRNA patterns, we predicted the most likely trans-splicing acceptor sites and used them as reference points to plot average nucleosome or RNA-seq signals. By representing MNase-seq data, we corroborated the presence of a mild nucleosome depleted region (NDR) around trans-splicing acceptor sites (TASs) in T. cruzi and L. major; but not in T. brucei, as previously reported. However, when analyzing H3 ChIP-seq data, we uphold that TAS protection in T. brucei is due to a non-histone complex instead of a well-position nucleosome, as previously claimed. Moreover, we showed that this nucleosome organization around TASs is not just an average, since the same layout is conserved in most of the genome. Furthermore, the strand-specific analysis revealed that the NDR is not exactly at the TAS but a few base pairs upstream of that point. We corroborated that this trough, is coincident with a footprint of DNA-RNA duplex, as previously observed.Additionally, it was previously shown that average nucleosome density around TAS correlates with average RNA-seq signals. To test how strong is that correlation, we performed gene clustering using k-means with either nucleosome occupancy or mRNA signals relative to TAS as predictor variables. From the MNase-seq clustering, we observed a homogenous distribution of average nucleosome density in the three organisms except for a subset of genes with unusually high nucleosome density in T. cruzi. As opposed, from RNA-seq analysis, we obtained well-defined gene clusters for the three organisms supported by high silhouette values. Particularly, we observed that there is a subset of genes with markedly high mRNA levels compared to the rest, but the correspondence between nucleosome density and mRNA signal is only partial. To have a better understanding of the role and conservation of those subsets of genes with unusual characteristics among TriTryps, we are currently performing GO and Metabolic pathway analysis.