The insulator BEAF32 controls the spatial-temporal expression profile of the telomeric retrotransposon TART in the Drosophila germline.

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.

HiConfidence: a novel approach uncovering the biological signal in Hi-C data affected by technical biases.

The chromatin interaction assays, particularly Hi-C, enable detailed studies of genome architecture in multiple organisms and model systems, resulting in a deeper understanding of gene expression regulation mechanisms mediated by epigenetics. However, the analysis and interpretation of Hi-C data remain challenging due to technical biases, limiting direct comparisons of datasets obtained in different experiments and laboratories. As a result, removing biases from Hi-C-generated chromatin contact matrices is a critical data analysis step. Our novel approach, HiConfidence, eliminates biases from the Hi-C data by weighing chromatin contacts according to their consistency between replicates so that low-quality replicates do not substantially influence the result. The algorithm is effective for the analysis of global changes in chromatin structures such as compartments and topologically associating domains. We apply the HiConfidence approach to several Hi-C datasets with significant technical biases, that could not be analyzed effectively using existing methods, and obtain meaningful biological conclusions. In particular, HiConfidence aids in the study of how changes in histone acetylation pattern affect chromatin organization in Drosophila melanogaster S2 cells. The method is freely available at GitHub: https://github.com/victorykobets/HiConfidence.

Extended disordered regions of ribosome-associated NAC proteins paralogs belong only to the germline in Drosophila melanogaster.

The nascent polypeptide-associated complex (NAC) consisting of α- and ß-subunits is an essential ribosome-associated protein conserved in eukaryotes. NAC is a ubiquitously expressed co-translational regulator of nascent protein folding and sorting providing for homeostasis of cellular proteins. Here we report on discovering the germline-specific NACαß paralogs (gNACs), whose ß-subunits, non-distinguishable by ordinary immunodetection, are encoded by five highly homologous gene copies, while the α-subunit is encoded by a single αNAC gene. The gNAC expression is detected in the primordial embryonic and adult gonads via immunostaining. The germline-specific α and ß subunits differ from the ubiquitously expressed paralogs by the extended intrinsically disordered regions (IDRs) acquired at the N- and C-termini of the coding regions, predicted to be phosphorylated. The presence of distinct phosphorylated isoforms of gNAC-ß subunits is confirmed by comparing of their profiles by 2D-isoeletrofocusing resolution before and after phosphatase treatment of testis ribosomes. We revealed that the predicted S/T sites of phosphorylation in the individual orthologous IDRs of gNAC-ß sequences of Drosophila species are positionally conserved despite these disordered regions are drastically different. We propose the IDR-dependent molecular crowding and specific coordination of NAC and other proteostasis regulatory factors at the ribosomes of germinal cells. Our findings imply that there may be a functional crosstalk between the germinal and ubiquitous α- and ß-subunits based on assessing their depletion effects on the fly viability and gonad development.

Impaired function of rDNA transcription initiation machinery leads to derepression of ribosomal genes with insertions of R2 retrotransposon.

Eukaryotic genomes harbor hundreds of rRNA genes, many of which are transcriptionally silent. However, little is known about selective regulation of individual rDNA units. In Drosophila melanogaster, some rDNA repeats contain insertions of the R2 retrotransposon, which is capable to be transcribed only as part of pre-rRNA molecules. rDNA units with R2 insertions are usually inactivated, although R2 expression may be beneficial in cells with decreased rDNA copy number. Here we found that R2-inserted rDNA units are enriched with HP1a and H3K9me3 repressive mark, whereas disruption of the heterochromatin components slightly affects their silencing in ovarian germ cells. Surprisingly, we observed a dramatic upregulation of R2-inserted rRNA genes in ovaries lacking Udd (Under-developed) or other subunits (TAF1b and TAF1c-like) of the SL1-like complex, which is homologues to mammalian Selective factor 1 (SL1) involved in rDNA transcription initiation. Derepression of rRNA genes with R2 insertions was accompanied by a reduction of H3K9me3 and HP1a enrichment. We suggest that the impairment of the SL1-like complex affects a mechanism of selective activation of intact rDNA units which competes with heterochromatin formation. We also propose that R2 derepression may serve as an adaptive response to compromised rRNA synthesis.

Loss of telomere silencing is accompanied by dysfunction of Polo kinase and centrosomes during Drosophila oogenesis and early development.

Telomeres are nucleoprotein complexes that protect the ends of eukaryotic linear chromosomes from degradation and fusions. Telomere dysfunction leads to cell growth arrest, oncogenesis, and premature aging. Telomeric RNAs have been found in all studied species; however, their functions and biogenesis are not clearly understood. We studied the mechanisms of development disorders observed upon overexpression of telomeric repeats in Drosophila. In somatic cells, overexpression of telomeric retrotransposon HeT-A is cytotoxic and leads to the accumulation of HeT-A Gag near centrosomes. We found that RNA and RNA-binding protein Gag encoded by the telomeric retrotransposon HeT-A interact with Polo and Cdk1 mitotic kinases, which are conserved regulators of centrosome biogenesis and cell cycle. The depletion of proteins Spindle E, Ccr4 or Ars2 resulting in HeT-A overexpression in the germline was accompanied by mislocalization of Polo as well as its abnormal stabilization during oogenesis and severe deregulation of centrosome biogenesis leading to maternal-effect embryonic lethality. These data suggest a mechanistic link between telomeric HeT-A ribonucleoproteins and cell cycle regulators that ensures the cell response to telomere dysfunction.

Order and stochasticity in the folding of individual Drosophila genomes.

Mammalian and Drosophila genomes are partitioned into topologically associating domains (TADs). Although this partitioning has been reported to be functionally relevant, it is unclear whether TADs represent true physical units located at the same genomic positions in each cell nucleus or emerge as an average of numerous alternative chromatin folding patterns in a cell population. Here, we use a single-nucleus Hi-C technique to construct high-resolution Hi-C maps in individual Drosophila genomes. These maps demonstrate chromatin compartmentalization at the megabase scale and partitioning of the genome into non-hierarchical TADs at the scale of 100 kb, which closely resembles the TAD profile in the bulk in situ Hi-C data. Over 40% of TAD boundaries are conserved between individual nuclei and possess a high level of active epigenetic marks. Polymer simulations demonstrate that chromatin folding is best described by the random walk model within TADs and is most suitably approximated by a crumpled globule build of Gaussian blobs at longer distances. We observe prominent cell-to-cell variability in the long-range contacts between either active genome loci or between Polycomb-bound regions, suggesting an important contribution of stochastic processes to the formation of the Drosophila 3D genome.

Special vulnerability of somatic niche cells to transposable element activation in Drosophila larval ovaries.

In the Drosophila ovary, somatic escort cells (ECs) form a niche that promotes differentiation of germline stem cell (GSC) progeny. The piRNA (Piwi-interacting RNA) pathway, which represses transposable elements (TEs), is required in ECs to prevent the accumulation of undifferentiated germ cells (germline tumor phenotype). The soma-specific piRNA cluster flamenco (flam) produces a substantial part of somatic piRNAs. Here, we characterized the biological effects of somatic TE activation on germ cell differentiation in flam mutants. We revealed that the choice between normal and tumorous phenotypes of flam mutant ovaries depends on the number of persisting ECs, which is determined at the larval stage. Accordingly, we found much more frequent DNA breaks in somatic cells of flam larval ovaries than in adult ECs. The absence of Chk2 or ATM checkpoint kinases dramatically enhanced oogenesis defects of flam mutants, in contrast to the germline TE-induced defects that are known to be mostly suppressed by сhk2 mutation. These results demonstrate a crucial role of checkpoint kinases in protecting niche cells against deleterious TE activation and suggest substantial differences between DNA damage responses in ovarian somatic and germ cells.

Yb body assembly on the flamenco piRNA precursor transcripts reduces genic piRNA production.

In Drosophila ovarian somatic cells, PIWI-interacting small RNAs (piRNAs) against transposable elements are mainly produced from the ∼180-kb flamenco (flam) locus. flam transcripts are gathered into foci, located close to the nuclear envelope, and processed into piRNAs in the cytoplasmic Yb bodies. The mechanism of Yb body formation remains unknown. Using RNA fluorescence in situ hybridization, we found that in the follicle cells of ovaries the 5'-ends of flam transcripts are usually located in close proximity to the nuclear envelope and outside of Yb bodies, whereas their extended downstream regions mostly overlap with Yb bodies. In flamKG mutant ovaries, flam transcripts containing the first and, partially, second exons but lacking downstream regions are gathered into foci at the nuclear envelope, but Yb bodies are not assembled. Strikingly, piRNAs from the protein-coding gene transcripts accumulate at higher levels in flamKG ovaries indicating that piRNA biogenesis may occur without Yb bodies. We propose that normally in follicle cells, flam downstream transcript regions function not only as a substrate for generation of piRNAs but also as a scaffold for Yb body assembly, which competitively decreases piRNA production from the protein-coding gene transcripts. By contrast, in ovarian somatic cap and escort cells Yb body assembly does not require flam transcription.

Nuclear lamina integrity is required for proper spatial organization of chromatin in Drosophila.

How the nuclear lamina (NL) impacts on global chromatin architecture is poorly understood. Here, we show that NL disruption in Drosophila S2 cells leads to chromatin compaction and repositioning from the nuclear envelope. This increases the chromatin density in a fraction of topologically-associating domains (TADs) enriched in active chromatin and enhances interactions between active and inactive chromatin. Importantly, upon NL disruption the NL-associated TADs become more acetylated at histone H3 and less compact, while background transcription is derepressed. Two-colour FISH confirms that a TAD becomes less compact following its release from the NL. Finally, polymer simulations show that chromatin binding to the NL can per se compact attached TADs. Collectively, our findings demonstrate a dual function of the NL in shaping the 3D genome. Attachment of TADs to the NL makes them more condensed but decreases the overall chromatin density in the nucleus by stretching interphase chromosomes.

The nucleolar transcriptome regulates Piwi shuttling between the nucleolus and the nucleoplasm.

The nucleolus contains a lot of proteins unrelated to ribosome biogenesis. Some of these proteins shuttle between the nucleolus and the nucleoplasm regulating the cell cycle and stress response. The piRNA binding protein Piwi is involved in silencing of transposable elements (TEs) in the Drosophila gonads. Here we used cultured ovarian somatic cells (OSC) to characterize Piwi as a visitor to the nucleolus. Dynamic Piwi localization was shown to vary from its uniform distribution between the nucleoplasm and the nucleolus to pronounced nucleolar immobilization. We were intrigued by this localization behavior and revealed that nascent nucleolar transcripts recruit Piwi for nucleolar retention. Piwi eviction from the nucleolus was observed upon RNase treatment and after RNA polymerase (Pol) I inhibition, but not after Pol II inactivation. On the contrary, heat shock caused drastic Piwi redistribution from the nucleoplasm to the nucleolus, which occurred only in the presence of Pol I-mediated transcription. These results allow us to hypothesize that specific stress-induced transcripts made by Pol I promote the nucleolar sequestration of proteins in Drosophila, similar to previous observations in mammalian cells. We also found that in OSC, Piwi partially restricts expression of the rDNA copies containing R1 and R2 retrotransposon insertions especially upon heat shock-induced activation of these copies. Therefore, we suggest that Piwi intranuclear shuttling may have a functional role in ensuring a balance between silencing of rDNA-specific TEs under stress and the canonical Piwi function in non-nucleolar TE repression.

The large fraction of heterochromatin in Drosophila neurons is bound by both B-type lamin and HP1a.

BACKGROUND: In most mammalian cell lines, chromatin located at the nuclear periphery is represented by condensed heterochromatin, as evidenced by microscopy observations and DamID mapping of lamina-associated domains (LADs) enriched in dimethylated Lys9 of histone H3 (H3K9me2). However, in Kc167 cell culture, the only Drosophilla cell type where LADs have previously been mapped, they are neither H3K9me2-enriched nor overlapped with the domains of heterochromatin protein 1a (HP1a). RESULTS: Here, using cell type-specific DamID we mapped genome-wide LADs, HP1a and Polycomb (Pc) domains from the central brain, Repo-positive glia, Elav-positive neurons and the fat body of Drosophila third instar larvae. Strikingly, contrary to Kc167 cells of embryonic origin, in neurons and, to a lesser extent, in glia and the fat body, HP1a domains appear to overlap strongly with LADs in both the chromosome arms and pericentromeric regions. Accordingly, centromeres reside closer to the nuclear lamina in neurons than in Kc167 cells. As expected, active gene promoters are mostly not present in LADs, HP1a and Pc domains. These domains are occupied by silent or weakly expressed genes with genes residing in the HP1a-bound LADs expressed at the lowest level. CONCLUSIONS: In various differentiated Drosophila cell types, we discovered the existence of peripheral heterochromatin, similar to that observed in mammals. Our findings support the model that peripheral heterochromatin matures enhancing the repression of unwanted genes as cells terminally differentiate.

Piwi interacts with chromatin at nuclear pores and promiscuously binds nuclear transcripts in Drosophila ovarian somatic cells.

Piwi in a complex with Piwi-interacting RNAs (piRNAs) triggers transcriptional silencing of transposable elements (TEs) in Drosophila ovaries, thus ensuring genome stability. To do this, Piwi must scan the nascent transcripts of genes and TEs for complementarity to piRNAs. The mechanism of this scanning is currently unknown. Here we report the DamID-seq mapping of multiple Piwi-interacting chromosomal domains in somatic cells of Drosophila ovaries. These domains significantly overlap with genomic regions tethered to Nuclear Pore Complexes (NPCs). Accordingly, Piwi was coimmunoprecipitated with the component of NPCs Elys and with the Xmas-2 subunit of RNA transcription and export complex, known to interact with NPCs. However, only a small Piwi fraction has transient access to DNA at nuclear pores. Importantly, although 36% of the protein-coding genes overlap with Piwi-interacting domains and RNA-immunoprecipitation results demonstrate promiscuous Piwi binding to numerous genic and TE nuclear transcripts, according to available data Piwi does not silence these genes, likely due to the absence of perfect base-pairing between piRNAs and their transcripts.

Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains.

Recent advances enabled by the Hi-C technique have unraveled many principles of chromosomal folding that were subsequently linked to disease and gene regulation. In particular, Hi-C revealed that chromosomes of animals are organized into topologically associating domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. Mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principles of TAD folding in Drosophila melanogaster, we performed Hi-C and poly(A)(+) RNA-seq in four cell lines of various origins (S2, Kc167, DmBG3-c2, and OSC). Contrary to previous studies, we find that regions between TADs (i.e., the inter-TADs and TAD boundaries) in Drosophila are only weakly enriched with the insulator protein dCTCF, while another insulator protein Su(Hw) is preferentially present within TADs. However, Drosophila inter-TADs harbor active chromatin and constitutively transcribed (housekeeping) genes. Accordingly, we find that binding of insulator proteins dCTCF and Su(Hw) predicts TAD boundaries much worse than active chromatin marks do. Interestingly, inter-TADs correspond to decompacted inter-bands of polytene chromosomes, whereas TADs mostly correspond to densely packed bands. Collectively, our results suggest that TADs are condensed chromatin domains depleted in active chromatin marks, separated by regions of active chromatin. We propose the mechanism of TAD self-assembly based on the ability of nucleosomes from inactive chromatin to aggregate, and lack of this ability in acetylated nucleosomal arrays. Finally, we test this hypothesis by polymer simulations and find that TAD partitioning may be explained by different modes of inter-nucleosomal interactions for active and inactive chromatin.

Role of histone deacetylases in gene regulation at nuclear lamina.

Theoretical models suggest that gene silencing at the nuclear periphery may involve "closing" of chromatin by transcriptional repressors, such as histone deacetylases (HDACs). Here we provide experimental evidence confirming these predictions. Histone acetylation, chromatin compactness, and gene repression in lamina-interacting multigenic chromatin domains were analyzed in Drosophila S2 cells in which B-type lamin, diverse HDACs, and lamina-associated proteins were downregulated by dsRNA. Lamin depletion resulted in decreased compactness of the repressed multigenic domain associated with its detachment from the lamina and enhanced histone acetylation. Our data reveal the major role for HDAC1 in mediating deacetylation, chromatin compaction, and gene silencing in the multigenic domain, and an auxiliary role for HDAC3 that is required for retention of the domain at the lamina. These findings demonstrate the manifold and central involvement of class I HDACs in regulation of lamina-associated genes, illuminating a mechanism by which these enzymes can orchestrate normal and pathological development.

Separation of stem cell maintenance and transposon silencing functions of Piwi protein.

Piwi-interacting RNAs (piRNAs) and Piwi proteins have the evolutionarily conserved function of silencing of repetitive genetic elements in germ lines. The founder of the Piwi subfamily, Drosophila nuclear Piwi protein, was also shown to be required for the maintenance of germ-line stem cells (GSCs). Hence, null mutant piwi females exhibit two types of abnormalities, overexpression of transposons and severely underdeveloped ovaries. It remained unknown whether the failure of GSC maintenance is related to transposon derepression or if GSC self-renewal and piRNA silencing are two distinct functions of the Piwi protein. We have revealed a mutation, piwi(Nt), removing the nuclear localization signal of the Piwi protein. piwi(Nt) females retain the ability of GSC self-renewal and a near-normal number of egg chambers in the ovarioles but display a drastic transposable element derepression and nuclear accumulation of their transcripts in the germ line. piwi(Nt) mutants are sterile most likely because of the disturbance of piRNA-mediated transposon silencing. Analysis of chromatin modifications in the piwi(Nt) ovaries indicated that Piwi causes chromatin silencing only of certain types of transposons, whereas others are repressed in the nuclei without their chromatin modification. Thus, Piwi nuclear localization that is required for its silencing function is not essential for the maintenance of GSCs. We suggest that the Piwi function in GSC self-renewal is independent of transposon repression and is normally realized in the cytoplasm of GSC niche cells.