Telomeric small RNAs related to PIWI-interacting RNAs (piRNAs) have been described in various eukaryotes; however, their role in germline-specific telomere function remains poorly understood. Using a Drosophila model, we performed an in-depth study of the biogenesis of telomeric piRNAs and their function in telomere homeostasis in the germline. To fully characterize telomeric piRNA clusters, we integrated the data obtained from analysis of endogenous telomeric repeats, as well as transgenes inserted into different telomeric and subtelomeric regions. The small RNA-seq data from strains carrying telomeric transgenes demonstrated that all transgenes belong to a class of dual-strand piRNA clusters; however, their capacity to produce piRNAs varies significantly. Rhino, a paralog of heterochromatic protein 1 (HP1) expressed exclusively in the germline, is associated with all telomeric transgenes, but its enrichment correlates with the abundance of transgenic piRNAs. It is likely that this heterogeneity is determined by the sequence peculiarities of telomeric retrotransposons. In contrast to the heterochromatic non-telomeric germline piRNA clusters, piRNA loss leads to a dramatic decrease in HP1, Rhino, and trimethylated histone H3 lysine 9 in telomeric regions. Therefore, the presence of piRNAs is required for the maintenance of telomere chromatin in the germline. Moreover, piRNA loss causes telomere translocation from the nuclear periphery toward the nuclear interior but does not affect telomere end capping. Analysis of the telomere-associated sequences (TASs) chromatin revealed strong tissue specificity. In the germline, TASs are enriched with HP1 and Rhino, in contrast to somatic tissues, where they are repressed by Polycomb group proteins. piRNAs play an essential role in the assembly of telomeric chromatin, as well as in nuclear telomere positioning in the germline. Telomeric arrays and TASs belong to a unique type of Rhino-dependent piRNA clusters with transcripts that serve simultaneously as piRNA precursors and as their only targets. Telomeric chromatin is highly sensitive to piRNA loss, implying the existence of a novel developmental checkpoint that depends on telomere integrity in the germline.

Expression of transposable elements in the germline is controlled by Piwi-interacting (pi) RNAs produced by genomic loci termed piRNA clusters and associated with Rhino, a heterochromatin protein 1 (HP1) homolog. Previously, we have shown that transgenes containing a fragment of the I retrotransposon form de novo piRNA clusters in the Drosophila germline providing suppression of I-element activity. We noted that identical transgenes located in different genomic sites vary considerably in piRNA production and classified them as “strong” and “weak” piRNA clusters. Here, we investigated what chromatin and transcriptional changes occur at the transgene insertion sites after their conversion into piRNA clusters. We found that the formation of a transgenic piRNA cluster is accompanied by activation of transcription from both genomic strands that likely initiates at multiple random sites. The chromatin of all transgene-associated piRNA clusters contain high levels of trimethylated lysine 9 of histone H3 (H3K9me3) and HP1a, whereas Rhino binding is considerably higher at the strong clusters. None of these chromatin marks was revealed at the “empty” sites before transgene insertion. Finally, we have shown that in the nucleus of polyploid nurse cells, the formation of a piRNA cluster at a given transgenic genomic copy works according to an “all-or-nothing” model: either there is high Rhino enrichment or there is no association with Rhino at all. As a result, genomic copies of a weak piRNA transgenic cluster show a mosaic association with Rhino foci, while the majority of strong transgene copies associate with Rhino and are hence involved in piRNA production.

In the germ line and during early embryogenesis, DNA methylation (DNAme) undergoes global erasure and re-establishment to support germ cell and embryonic development. While DNAme acquisition during male germ cell development is essential for setting genomic DNA methylation imprints, other intergenerational roles for paternal DNAme in defining embryonic chromatin are unknown. Through conditional gene deletion of the de novo DNA methyltransferases Dnmt3a and/or Dnmt3b, we observe that DNMT3A primarily safeguards against DNA hypomethylation in undifferentiated spermatogonia, while DNMT3B catalyzes de novo DNAme during spermatogonial differentiation. Failing de novo DNAme in Dnmt3a/Dnmt3b double deficient spermatogonia is associated with increased nucleosome occupancy in mature sperm, preferentially at sites with higher CpG content, supporting the model that DNAme modulates nucleosome retention in sperm. To assess the impact of altered sperm chromatin in formatting embryonic chromatin, we measure H3K4me3 occupancy at paternal and maternal alleles in 2-cell embryos using a transposon-based tagging approach. Our data show that reduced DNAme in sperm renders paternal alleles permissive for H3K4me3 establishment in early embryos, independently of possible paternal inheritance of sperm born H3K4me3. Together, this study provides evidence that paternally inherited DNAme directs chromatin formation during early embryonic development.

The study of the telomeric complex in oogenesis and early development is important for understanding the mechanisms which maintain genome integrity. Telomeric transcripts are the key components of the telomeric complex and are essential for regulation of telomere function. We study the biogenesis of transcripts generated by the major Drosophila telomere repeat HeT-A in oogenesis and early development with disrupted telomeric repeat silencing. In wild type ovaries, HeT-A expression is downregulated by the Piwi-interacting RNAs (piRNAs). By repressing piRNA pathway, we show that overexpressed HeT-A transcripts interact with their product, RNA-binding protein Gag-HeT-A, forming ribonucleoprotein particles (RNPs) during oogenesis and early embryonic development. Moreover, during early stages of oogenesis, in the nuclei of dividing cystoblasts, HeT-A RNP form spherical structures, which supposedly represent the retrotransposition complexes participating in telomere elongation. During the later stages of oogenesis, abundant HeT-A RNP are detected in the cytoplasm and nuclei of the nurse cells, as well as in the cytoplasm of the oocyte. Further on, we demonstrate that HeT-A products co-localize with the transporter protein Egalitarian (Egl) both in wild type ovaries and upon piRNA loss. This finding suggests a role of Egl in the transportation of the HeT-A RNP to the oocyte using a dynein motor. Following germline piRNA depletion, abundant maternal HeT-A RNP interacts with Egl resulting in ectopic accumulation of Egl close to the centrosomes during the syncytial stage of embryogenesis. Given the essential role of Egl in the proper localization of numerous patterning mRNAs, we suggest that its abnormal localization likely leads to impaired embryonic axis specification typical for piRNA pathway mutants.

Piwi-interacting RNAs (piRNAs) control transposable element (TE) activity in the germline. piRNAs are produced from single-stranded precursors transcribed from distinct genomic loci, enriched by TE fragments and termed piRNA clusters. The specific chromatin organization and transcriptional regulation of Drosophila germline-specific piRNA clusters ensure transcription and processing of piRNA precursors. TEs harbour various regulatory elements that could affect piRNA cluster integrity. One of such elements is the suppressor-of-hairy-wing (Su(Hw))-mediated insulator, which is harboured in the retrotransposon gypsy. To understand how insulators contribute to piRNA cluster activity, we studied the effects of transgenes containing gypsy insulators on local organization of endogenous piRNA clusters. We show that transgene insertions interfere with piRNA precursor transcription, small RNA production and the formation of piRNA cluster-specific chromatin, a hallmark of which is Rhino, the germline homolog of the heterochromatin protein 1 (HP1). The mutations of Su(Hw) restored the integrity of piRNA clusters in transgenic strains. Surprisingly, Su(Hw) depletion enhanced the production of piRNAs by the domesticated telomeric retrotransposon TART, indicating that Su(Hw)-dependent elements protect TART transcripts from piRNA processing machinery in telomeres. A genome-wide analysis revealed that Su(Hw)-binding sites are depleted in endogenous germline piRNA clusters, suggesting that their functional integrity is under strict evolutionary constraints.

Insulators are architectural elements implicated in the organization of higher-order chromatin structures and transcriptional regulation. However, it is still unknown how insulators contribute to Drosophila telomere maintenance. Although the Drosophila telomeric retrotransposons HeT-A and TART occupy a common genomic niche, they are regulated independently. TART elements are believed to provide reverse transcriptase activity, whereas HeT-A transcripts serve as a template for telomere elongation. Here, we report that insulator complexes associate with TART and contribute to its transcriptional regulation in the Drosophila germline. Chromatin immunoprecipitation revealed that the insulator complex containing BEAF32, Chriz, and DREF proteins occupy the TART promoter. BEAF32 depletion causes derepression and chromatin changes at TART in ovaries. Moreover, an expansion of TART copy number was observed in the genome of the BEAF32 mutant strain. BEAF32 localizes between the TART enhancer and promoter, suggesting that it blocks enhancer–promoter interactions. Our study found that TART repression is released in the germ cysts as a result of the normal reduction of BEAF32 expression at this developmental stage. We suggest that coordinated expression of telomeric repeats during development underlies telomere elongation control.