Pericentromeric heterochromatin in Drosophila generally consists of repetitive DNA, forming the environment associated with gene silencing. Despite the expanding knowledge of the impact of transposable elements (TEs) on the host genome, little is known about the evolution of pericentromeric heterochromatin, its structural composition, and age. During the evolution of the Drosophilidae, hundreds of genes have become embedded within pericentromeric regions yet retained activity. We investigated a pericentromeric heterochromatin fragment found in D. virilis and related species, describing the evolution of genes in this region and the age of TE invasion. Regardless of the heterochromatic environment, the amino acid composition of the genes is under purifying selection. However, the selective pressure affects parts of genes in varying degrees, resulting in expansion of gene introns due to TEs invasion. According to the divergence of TEs, the pericentromeric heterochromatin of the species of virilis group began to form more than 20 million years ago by invasions of retroelements, miniature inverted repeat transposable elements (MITEs), and Helitrons. Importantly, invasions into the heterochromatin continue to occur by TEs that fall under the scope of piRNA silencing. Thus, the pericentromeric heterochromatin, in spite of its ability to induce silencing, has the means for being dynamic, incorporating the regions of active transcription.

Complete metamorphosis of holometabolous insects is a complex biological process characterized by profound morphological, physiological, and transcriptional changes. To reveal the temporal dynamics of gene expression during this critical developmental transition, we conducted a detailed analysis of the developmental transcriptomes of two Drosophila species, Drosophila melanogaster and Drosophila virilis. We confirmed partial recapitulation of the embryonic transcriptional program in pupae, but unlike the traditional hourglass model, suggesting maximal conservation at mid-embryonic stages, at different stages of pupae we observed a more complicated pattern of alternating low and high diversity, resembling an inverted hourglass, or “spindle”. This underscores the complexity of developmental processes during complete metamorphosis. Notably, recently formed genes (specific to insects) exhibit pronounced expression peaks during mid-pupal development, indicating their potential role in developmental transitions.

Transposable elements (TEs) drive genomic innovation, but their dynamics in non-model species remain unclear. Here, we integrated multi-omics data to explore TE dynamics in Drosophila virilis, an important model for repetitive DNA research. By combining computational predictions with manual curation, we identified 100 TE families and delineated three temporal waves of TE mobilization: recent activity, speciation-associated, and ancient invasions. TEs in D. virilis dynamically colonise both euchromatin and heterochromatin, suggesting heterochromatin is not solely a repository for degenerate repeats. While most TEs are widespread across strains, some exhibit strain-specific expansions, indicating varied activity and silencing. We found substantial evidence for horizontal transfer of TEs among close relatives, demonstrating that the D. virilis species group functions effectively as a TE “ecosystem”, allowing for recurrent invasion, loss, and re-invasion of TE lineages across the group. Epigenetic profiling revealed that H3K9me3 spreading from TEs represses adjacent genes in a distance-dependent manner, influenced by insertion length and genomic context, affecting developmental and metabolic genes. We also discovered the first spontaneous polymorphic inversion in D. virilis linked to retrotransposons. Our findings illuminate TEs as drivers of genomic innovation, influencing gene regulation and evolutionary trajectories, providing a framework for studying TE dynamics across animal species.

D. melanogaster has two paralogs, tth and dd4, related to the evolutionarily conserved d4 family genes. In mammals, the family consists of Dpf1-3, encoding transcription co-factors involved in the regulation of development and cell fate determination. The function of tth and dd4 in Drosophila remains unclear. The typical domain structure of the proteins encoded by the d4 family consists of an N-terminal 2/3 domain (Requiem_N), a central Kruppel-type zinc finger, and a C-terminal D4 domain of paired PHD zinc fingers (DPFs). In Drosophila, both paralogs lack the Kruppel-type ZF, and tth encodes a protein that contains only Requiem_N. In contrast, vertebrate Dpf1-3 paralogs encode all the domains, but some paralogs have specific splice isoforms. For example, the DPF3a isoform lacks the D4 domain necessary for histone reading. The occurrence of proteins without the D4 domain in mammals and flies implies functional significance and analogous roles across animal taxa. In this study, we reconstructed the evolutionary events that led to the emergence of Drosophila tth by analyzing the divergence of d4 paralogs across different evolutionary lineages. Our genomic and transcriptomic data analysis revealed duplications and gene copy loss events. Among insects, gene duplication was only observed in Diptera. In other lineages, we found the specialization of paralogs for producing isoforms and further specialization for coding proteins with specific domain organizations. We hypothesize that this pathway is a common mechanism for the emergence of paralogues lacking the D4 domain across different evolutionary lineages. We, thus, postulate that TTH may function as a splice isoform of the ancestral single-copy gene, possibly a DPF3a-like isoform characteristic of related insect species. Our analysis provides insights into the possible impact of paralogue divergence, emphasizing the functional significance of the 2/3 domain and the potential roles of isoforms lacking the D4 domain.

Piwi-interacting RNAs (piRNAs) play a crucial role in silencing transposable elements (TEs) in the germ cells of Metazoa by acting as sequence-specific guides. Originating from distinct genomic loci, called piRNA clusters, piRNA can trigger an epigenetic conversion of TE insertions into piRNA clusters by means of a paramutation-like process. However, the variability in piRNA clusters’ capacity to induce such conversions remains poorly understood. Here, we investigated two Drosophila virilis strains with differing capacities to produce piRNAs from the subtelomeric RhoGEF3 and Adar gene loci. We found that active piRNA generation correlates with high levels of the heterochromatic mark histone 3 lysine 9 trimethylation (H3K9me3) over genomic regions that give rise to piRNAs. Importantly, the maternal transmission of piRNAs drives their production in the progeny, even from homologous loci previously inactive in piRNA biogenesis. The RhoGEF3 locus, once epigenetically converted, maintained enhanced piRNA production in subsequent generations lacking the original allele carrying the active piRNA cluster. In contrast, piRNA expression from the converted Adar locus was lost in offspring lacking the inducer allele. The present findings suggest that the paramutation-like behavior of piRNA clusters may be influenced not only by piRNAs but also by structural features and the chromatin environment in the proximity to telomeres, providing new insights into the epigenetic regulation of the Drosophila genome.

Pericentric heterochromatin in Drosophila is generally composed of repetitive DNA forming a transcriptionally repressive environment. Nevertheless, dozens of genes were embedded into pericentric genome regions during evolution of Drosophilidae lineage and retained functional activity. However, factors that contribute to “immunity” of these gene loci to transcriptional silencing remain unknown. Here, we investigated molecular evolution of the essential Myb and Ranbp16 genes. These protein-coding genes reside in euchromatic loci of chromosome X in D. melanogaster and related species, while in other studied Drosophila species, including evolutionary distant ones, they are located in genomic regions highly enriched with the remnants of transposable elements (TEs), suggesting their heterochromatic nature and location. The promoter region of Myb exhibits a conserved structure throughout the Drosophila phylogeny and carries motifs for binding of chromatin remodeling factors, including insulator BEAF-32, regardless of eu- or heterochromatic surroundings. Importantly, BEAF-32 occupies not only the promoter region of Myb but is also found in the vicinity of transcriptional start sites (TSS) of Ranbp16 gene as well as in a wide range of genes located in the contrasting chromatin types in D. melanogaster and D. virilis, denoting the boundary of the nucleosome-free region available for RNA polymerase II recruitment and the surrounding heterochromatin. We also find that along with BEAF-32, insulators dCTCF and GAF are enriched at the TSS of heterochromatic genes in D. melanogaster . Thus, we propose that insulator proteins contribute to gene expression in the heterochromatic environment and, hence, facilitate the evolutionary repositioning of gene loci into heterochromatin. Author summary: Heterochromatin in Drosophila is generally associated with transcriptional silencing. Nevertheless, hundreds of essential genes have been identified in the pericentric heterochromatin of Drosophila melanogaster . Interestingly, genes embedded in pericentric heterochromatin of D. melanogaster may occupy different genomic loci, euchromatic or heterochromatic, due to repositioning in the course of evolution of Drosophila species. By surveying factors that contribute to the normal functioning of the relocated genes in distant Drosophila species, i.e. D. melanogaster and D. virilis , we identify certain insulator proteins (e.g.BEAF-32) that facilitate the expression of heterochromatic genes in spite of the repressive environment.