Biology subjects

Ocampo, J.

Publications and source records attributed to Ocampo, J..

6 recordsLinked to original sources

bioRxiv2026

H3K4me3 exhibits length-dependent deposition patterns at transcription initiation regions in Trypanosoma cruzi and correlates with transcriptional activity

In trypanosmatids genes, transcribed by RNA polymerase II do not have canonical promoters and are organized into directional gene clusters that mature into monocistronic transcripts by a co-transcriptional process known as trans-splicing. Even though gene expression is regulated mainly post-transcriptionally, it is currently understood that chromatin and epigenetics are also involved in this regulation. In eukaryotes, specific signals are normally required for the occurrence of an appropriate transcription initiation. Among them, trimethylation of histone H3 in lysine 4 is the most conserved signal normally detected at transcription start sites of actively transcribed genes. Unlike many model organisms, trypanosomes do not have defined promoters. Instead, transcription initiates in a bidirectional manner from dispersed regions coincident with divergent strand switch regions located between directional gene clusters (DGCs). In T. cruzi, H3K4me3 was observed at the origins of transcription coincident with divergent strand switch regions (dSSRs) in epimastigotes, but it has not been mapped throughout the whole genome at base-pair resolution or in other life stages so far. Here, we set up the CUT&RUN technique for T. cruzi epimastigotes and trypomastigotes. Consistent with a predominant post-transcriptional regulation along the life cycle, we did not find significant differences between life stages. We corroborated that H3K4me3 is enriched at dSSR adjacent to actively expressed DGCs. Moreover, we noticed that this histone mark exhibits different patterns that correlate with the genomic span of the transcription initiation regions and with transcriptional activity. Furthermore, we unveiled that the most actively transcribed DGCs are associated with shorter dSSRs and are located within the core compartment of the genome displaying a more accessible chromatin.

genomics↗

bioRxiv2026

Histone H3 Ser10 phosphorylation occurs exclusively in replicative stages and peaks during mitosis in Trypanosoma cruzi

Protein phosphorylation is a central post-translational modification that regulates signaling pathways across all living organisms. Through the antagonistic activities of protein kinases and phosphatases, phosphorylation modulates protein function by inducing conformational changes that affect enzyme activity, protein-protein interactions, stability, and subcellular localization. These molecular events regulate diverse cellular processes, including cell cycle progression, differentiation, gene expression, and metabolism. In unicellular parasites such as Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp., specialized signaling pathways have evolved to enable adaptation to the fluctuating environments of insect vectors and mammalian hosts. In many eukaryotes, phosphorylation of histone H3 at serine 10 (H3Ser10p) is essential for proper chromosome condensation during mitosis and is catalyzed by Aurora kinase B. Although trypanosomatids possess an Aurora kinase B homolog and a conserved serine residue at position 10 of histone H3, this modification had not been previously detected in these organisms. Here, using a stage-specific approach, we report the first detection of H3Ser10p in T. cruzi and explore its association with cell cycle progression. Western blot analyses using a specific antibody revealed H3Ser10p in exponentially growing epimastigotes, both in total protein extracts and nucleosome-enriched fractions, indicating its incorporation into chromatin. Fluorescence microscopy showed that this histone mark is restricted to the nuclei of dividing cells. Furthermore, H3Ser10p was detected exclusively in replicative stages of the parasite. Analysis of cell cycle-associated structures and flow cytometry demonstrated that H3Ser10 phosphorylation is dynamically regulated, peaking in the G2/M phase. These findings identify H3Ser10p as a novel epigenetic mark in T. cruzi that is tightly regulated during the cell cycle.

cell biology↗

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↗

bioRxiv2019

DNA accessibility is not the primary determinant of chromatin-mediated gene regulation

DNA accessibility is thought to be of major importance in regulating gene expression. We test this hypothesis using a restriction enzyme as a probe of chromatin structure and as a proxy for transcription factors. We measured the digestion rate and the fraction of accessible DNA at all genomic AluI sites in budding yeast and mouse liver nuclei. Hepatocyte DNA is more accessible than yeast DNA, consistent with longer linkers between nucleosomes, and indicating that nucleosome spacing is a major determinant of accessibility. DNA accessibility varies from cell to cell, such that essentially no sites are accessible or inaccessible in every cell. AluI sites in inactive mouse promoters are accessible in some cells, implying that transcription factors could bind without activating the gene. Euchromatin and heterochromatin have very similar accessibilities, suggesting that transcription factors can penetrate heterochromatin. Thus, DNA accessibility is not likely to be the primary determinant of gene regulation.

genomics↗

bioRxiv2018

Transfer RNA Genes Affect Chromosome Structure and Function via Local Effects

The genome is packaged and organized in an ordered, non-random manner and specific chromatin segments contact nuclear substructures to mediate this organization. While transfer RNA genes (tDNAs) are essential for the generation of tRNAs, these loci are also binding sites for transcription factors and architectural proteins and are thought to play an important role in the organization of the genome. In this study, we investigate the role of tDNAs in genomic organization and chromosome function by editing a chromosome so that it lacks any tDNAs. Surprisingly our analyses of this tDNA-less chromosome show that loss of tDNAs does not grossly affect chromosome folding or chromosome tethering. However, loss of tDNAs affects local nucleosome positioning and the binding of SMC proteins at these loci. The absence of tDNAs also leads to changes in centromere clustering and a reduction in the frequency of long range HML-HMR heterochromatin clustering. We propose that the tDNAs primarily affect local chromatin structure that result in effects on long-range chromosome architecture.

molecular biology↗