Key role of piRNAs in telomeric chromatin maintenance and telomere nuclear positioning in Drosophila germline

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Chromatin; Drosophila; Germline; HP1; Retrotransposon; Rhino; Telomere; Transcription; Transgene; piRNA cluster.

Fig. 1

Generation of small RNAs by telomeric transgenes. a Schematic structure of telomeric elements is shown above. Insertion sites of transgenes are indicated as triangles situated above and below the schemes, which correspond to their genomic orientation. The profiles of small RNAs in ovaries of yw strain are shown along the canonical sequences of HeT-A, TAHRE, TART-A, TART-B, and TART-C telomeric retrotransposons. Normalized numbers of small RNAs (RPM, reads per million, 0–3 mismatches) in a 30-bp window were calculated. Length distribution of the telomeric element small RNAs is shown below. Percentages of reads having 1U are indicated for each strand (only 24–29-nt reads were considered). b Scheme of transgenic insertion sites in euchromatin and TAS of chromosome 2R. c Normalized numbers of small RNAs mapped to transgenic constructs (blue–sense; brown–antisense; no mismatches allowed). Mapping of piRNAs (24–29 nt) and siRNAs (21 nt) onto the transgenes is shown separately. Scheme of the P{EPgy2} transgene is shown above. Short names of telomeric insertions are indicated. d Length distribution of transgenic small RNAs. Percentage of reads having 1U are indicated for each strand (only 24–29-nt reads were considered). e Relative frequencies (Z-score) of 5′ overlap for sense and antisense 24–29-nt piRNAs (ping-pong signature). f Northern blot hybridization of the RNA isolated from the ovaries of EY08176, EY03383, EY00453, EY00802, EY09966, and EY03241 strains was done with the white riboprobe to detect antisense piRNAs. Lower panel represents hybridization to mir-13b1 microRNA. P³²-labeled RNA oligonucleotides were used as size markers

Fig. 2

Chromatin components of the telomeric regions. HP1, H3K9me3, and Rhi occupancies at P{EPgy2} transgenes were estimated by ChIP-qPCR using primers corresponding to 5′-P-element transgenic sequence. Primers corresponding to ORFs were used for the analysis of endogenous HeT-A, TART-A, and TAHRE. Two regions of the 42AB piRNA cluster are enriched in all studied chromatin components. rp49, metRS-m, and 60D regions are used as negative controls. Asterisks indicate statistically significant differences in Rhi enrichment relative to EY03241 (control) (*P < 0.05 to 0.01, **P < 0.01 to 0.001, ***P < 0.001, unpaired t test). The difference in the HP1 binding between transgenes is statistically insignificant

Fig. 3

Role of piRNA pathway in the deposition of HP1, Rhi, and H3K9me3 at telomeric transgenes and endogenous telomeric retrotransposons in ovaries. a ChIP-qPCR analysis of HP1, Rhi, and H3K9me3 enrichment at EY08176 transgene (insTAHRE), endogenous HeT-A, TART-A, TAHRE and a set of dual-strand piRNA clusters in ovaries of hetero- and trans-heterozygous (spn-E¹/spn-E^hls3987) spindle-E mutants. Asterisks indicate statistically significant differences in chromatin protein levels at the indicated regions between spnE/+ and spnE/spnE (*P < 0.05 to 0.01, **P < 0.01 to 0.001, ***P < 0.001, unpaired t test). b ChIP-qPCR analysis of HP1, Rhi, and H3K9me3 enrichment at EY00453 (ins2TART-B), endogenous HeT-A, TART-B, and dual-strand piRNA cluster 6 in ovaries upon spnE germline knockdown. Here, we used primers specific to the transgene insertion site instead of those to 5′P due to the presence of additional P-element-based constructs in the genome. The TART-B promoter was amplified using a primer pair surrounding the insTART-B insertion

Fig. 4

piRNAs are required for telomere localization at nuclear periphery. a DNA FISH with HeT-A (green) combined with Rhi staining (red) was performed on ovaries of the yw strain and of the spn-E¹/spn-E^hls3987 mutants. b Estimation of the positioning of clustered HeT-A signals relative to the nuclear surface of nurse cells by 3D quantitative confocal image analysis of HeT-A DNA FISH on ovaries of spnE/+, spn-E¹/spn-E^hls3987, piwi/+, and piwi²/piwi^Nt mutants. c piRNAs are dispensable for telomere capping and telomere clustering. DNA FISH with HeT-A probe combined with HOAP staining was performed on ovaries of the yw and spn-E¹/spn-E^hls3987 strains. d Double DNA FISH with HeT-A (red) and TART (green) probes was performed on ovaries of the yw strain. e, f piRNA pathway disruption causes loss of Rhi from TART but not from the 42AB piRNA cluster. DNA FISH (green) with TART-A (e) or 42AB (f) probes combined with Rhi staining (red) was performed on ovaries of the yw and spn-E¹/spn-E^hls3987 strains. DNA is stained with DAPI (blue). Nuclei of nurse cells from VIII to X stages of oogenesis are shown

Fig. 5

Comparison of subtelomeric chromatin structure in somatic and ovarian tissues. a HP1, H3K9me3, and Rhi occupancy at the EY03383 transgene located in the 2R TAS was estimated by ChIP-qPCR. rp49 and metRS-m regions are used as negative controls. b DNA FISH with TAS 2R-3R probe (green) combined with Rhi (red) or H3K27me3 (red) staining was done on ovaries of yw and spn-E¹/spn-E^hls3987 strains. Nuclei of nurse cells (stage VIII–X) are shown. c RT-qPCR analysis of the expression levels of transgenic mini-white in ovaries of transgenic strains. white-specific primers detect only transgenic transcripts because endogenous white is partially deleted

Fig. 6

Telomeres represent a distinct type of self-targeting dual-strand piRNA cluster. a Schematic representation of three types of dual-strand piRNA clusters. The chromatin structure of “canonical” dual-strand piRNA clusters is established by maternally inherited piRNAs but maintained by a piRNA-independent mechanism. On the contrary, piRNAs are strongly required for maintenance of the chromatin state of telomeric and euchromatic TE-associated piRNA clusters during oogenesis. Assembly of telomere protection capping complex is not affected by piRNAs. b Comparison of telomeric chromatin in somatic and germ cells. A schematic distribution of chromatin components along telomeric retrotransposon arrays and TAS is based on our study and previously published results [30, 31, 33]. In somatic tissues, TAS and HeT-A–TART–TAHRE arrays are subdivided into repressed and transcriptionally active domains, respectively. In the germline, both telomeric regions form piRNA cluster(s) enriched in HP1, H3K9me3, and Rhi