Long G4-rich regions (LG4s) are defined as DNA sequences containing a high density of guanine triplets capable of forming non-B DNA structures called G-quadruplexes (G4s). These regions frequently overlap with enhancers, which are regulatory DNA elements that modulate gene expression by interacting with DNA regions that dictate where transcription is initiated known as promoters. While LG4s have now been well-characterized in the human genome, neither LG4 occurrence, nor the ability of LG4s to function as enhancers, in other species has been described. To address this, we screened the genomes of 16 different species from various taxa to identify LG4s and then determined if they were conserved, and additionally, we show the ability of one of the conserved LG4s to interact with its cognate promoter in vitro is functionally maintained across species. Our analyses characterized a number of previously unreported LG4s in the human genome as well as LG4s in 13 additional species. Of note, we identified a highly conserved LG4 enhancer predicted to regulate over 40 genes. This LG4 is embedded in the MAZ (Myc-Associated Zinc finger protein) locus, and we find this LG4 possesses the ability to directly interact with the same target promoter in both human and mouse. In summary, this work describes LG4s in the genomes of both unicellular and multicellular species including vertebrates, invertebrates, plants, and fungi, and finds many of these LG4 sequences highly conserved.

Rumen flukes, particularly the trematode Calicophoron daubneyi, are emerging parasites of livestock in Europe, yet transcriptomic insights into their environmental and intermediate host stages remain limited. Here, we present a comprehensive transcriptomic analysis of eggs at three distinct developmental stages (freshly excreted, early developmental and eye-spot stages), as well as rediae and cercariae, of C. daubneyi. High-quality RNA-sequencing (RNA-seq) datasets revealed both shared and stage-specific transcriptional profiles with each developmental stage exhibiting its own distinct expression pattern. Subsequent GO-Term enrichment analyses revealed that fully embryonated eggs in eye-spot-stage especially upregulated genes related to cilia assembly, movement and motility, reflecting preparation for miracidial hatching and host-seeking behavior. Rediae showed enhanced transcription of genes involved in diverse metabolic and biosynthetic processes, supporting rapid asexual proliferation within the snail intermediate host. Cercariae exhibited predominant upregulation of genes associated with signal transduction and energy metabolism, indicating the adaptation to its changing environmental conditions. These findings provide the first transcriptomic insights into the biology of C. daubneyi outside the definitive host, reveal molecular mechanisms underlying development, transmission and adaptation to a changing environment and identify stage-specific genes as potential targets for interventions aimed at disrupting the parasites life cycle and controlling rumen flukes in the future.