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.

We developed OmicIntegrator, a broadly adaptable pipeline designed to standardize and integrate publicly available transcriptomic, proteomic, and phosphoproteomic datasets. We applied this workflow to Arabidopsis thaliana etiolated seedlings to identify protein kinases and phosphatases relevant to skotomorphogenic development, a phase during which seedlings rely on tightly regulated signaling networks to ensure survival in darkness. This meta-analysis provided a comprehensive view of gene and protein expression, revealing discrepancies between transcript and protein abundance, suggesting post-transcriptional and post-translational regulation. By integrating multiple datasets, OmicIntegrator reduces experimental bias and enables the detection of phosphorylation events that may be missed in single-condition studies. Distinct phosphorylation patterns were detected across different protein kinase families. Motif enrichment analysis showed a strong overrepresentation of RxxS motifs among phosphosites in protein phosphatases and microtubule-associated proteins, consistent with potential regulation by calcium-dependent protein kinases (CPKs). Across omics layers, CPK3 and CPK9 repeatedly emerged as prominent candidates, highlighting them as priorities for future functional studies in skotomorphogenesis. Overall, our results demonstrate the power of OmicIntegrator as a flexible framework to contextualize signaling landscapes and identify robust patterns and candidate genes and for generating testable hypotheses from integrated multi-omics data in plant developmental biology.

We developed a pipeline to standardize and integrate publicly available transcriptomic, proteomic, and phosphoproteomic datasets from Arabidopsis thaliana etiolated seedlings. The pipeline is broadly adaptable, as its database schema and processing scripts can be applied to datasets from any organism or experimental condition. We applied it to etiolated seedlings to uncover protein kinases and protein phosphatases relevant to early skotomorphogenic development, a stage in which seedlings grow underground and rely on precisely regulated signaling networks to ensure survival and successful emergence. This multi-omics approach revealed a comprehensive landscape of protein expression at this developmental stage; however, we observed widespread discrepancies between transcript and protein abundance, pointing to post-transcriptional and/or post-translational regulation. This cross-study minimizes experimental bias and allows the detection of phosphorylation events that may have been overlooked in single-condition studies. A search of kinase specific motifs revealed that RxxS motifs were significantly enriched among phosphorylated peptides in protein phosphatases and microtubule-associated proteins, suggesting regulation by calcium dependent protein kinases (CPKs). CPK3 and CPK9 were identified as the most relevant isoforms in the proteome and phosphokinome, supporting their central role in skotomorphogenesis. Our results underscore the importance of phosphorylation-mediated signaling in coordinating early seedling development and provide a resource for dissecting regulatory networks and candidate genes involved in skotomorphogenesis.