Evolutionary Dynamics of the Pericentromeric Heterochromatin in Drosophila virilis and Related Species

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

Expression of Drosophila virilis Retroelements and Role of Small RNAs in Their Intrastrain Transposition

Transposition of two retroelements (Ulysses and Penelope) mobilized in the course of hybrid dysgenesis in Drosophila virilis has been investigated by in situ hybridization on polytene chromosomes in two D. virilis strains of different cytotypes routinely used to get dysgenic progeny. The analysis has been repeatedly performed over the last two decades, and has revealed transpositions of Penelope in one of the strains, while, in the other strain, the LTR-containing element Ulysses was found to be transpositionally active. The gypsy retroelement, which has been previously shown to be transpositionally inactive in D. virilis strains, was also included in the analysis. Whole mount is situ hybridization with the ovaries revealed different subcellular distribution of the transposable elements transcripts in the strains studied. Ulysses transpositions occur only in the strain where antisense piRNAs homologous to this TE are virtually absent and the ping-pong amplification loop apparently does not take place. On the other hand small RNAs homologous to Penelope found in the other strain, belong predominantly to the siRNA category (21nt), and consist of sense and antisense species observed in approximately equal proportion. The number of Penelope copies in the latter strain has significantly increased during the last decades, probably because Penelope-derived siRNAs are not maternally inherited, while the low level of Penelope-piRNAs, which are faithfully transmitted from mother to the embryo, is not sufficient to silence this element completely. Therefore, we speculate that intrastrain transposition of the three retroelements studied is controlled predominantly at the post-transcriptional level

Maternal deposition and distribution levels of <i>Penelope</i>-derived small RNAs.

<p>siRNAs at (A, B) and piRNAs at (C, D) in strain 160 and its 0–2 h embryos. Sense small RNAs are indicated in red, antisense–in blue.</p

The pattern of piRNAs distribution along transposons in testes.

<p>Distribution of <i>Ulysses</i>-piRNAs in testes of strain 9 (A) and strain 160 (C). The distribution of piRNAs homologous to <i>gypsyDv</i> in testes of strain 9 (B) and strain 160 (D). Sense small RNAs are indicated in red, antisense – in blue.</p

Transcription levels of selected <i>D. virilis</i> TEs.

<p>(A) semiquantitative RT-PCR data for ovaries and carcasses; (B) Quantitative RT-PCR analysis of TE transcription levels in ovaries. Since RT-PCR failed to reveal any transcription of <i>Penelope</i> and <i>Helena</i> in strain 9, we do not include the results of comparative analysis of these TEs by qRT-PCR in the panel; (C) Northern blot detection of <i>Ulysses</i> and <i>gypsyDv</i> sense transcripts in strains 9 and 160. Poly-A RNAs isolated from strain 9, strain 160 and <i>D. melanogaster</i> yw^67c23 strain ovaries were used. The size of marker RNA is given in nt at the right. The filter was rehybridized with a fragment of constitutively expressed <i>D. melanogaster rp49</i> to monitor the level of loaded RNA.</p

Copy number of <i>Penelope</i>, <i>Ulysses</i> and <i>gypsyDv</i> in polytene chromosomes of <i>D. virilis</i> strains 9 and 160.

<p>Copy number was determined by <i>in situ</i> hybridization analysis within the last two decades (1991–2008). When performing <i>in situ</i> hybridization analysis in 2008, we excluded a few <i>Ulysses</i> sites that were polymorphic in 1991 (did not contain <i>Ulysses</i> in 100% of larvae). Asterisks indicate site 49F where all three TEs were found.</p

Small RNA-based silencing strategies for transposons in the process of invading Drosophila species

Colonization of a host by an active transposon can increase mutation rates or cause sterility, a phenotype termed hybrid dysgenesis. As an example, intercrosses of certain Drosophila virilis strains can produce dysgenic progeny. The Penelope element is present only in a subset of laboratory strains and has been implicated as a causative agent of the dysgenic phenotype. We have also introduced Penelope into Drosophila melanogaster, which are otherwise naive to the element. We have taken advantage of these natural and experimentally induced colonization processes to probe the evolution of small RNA pathways in response to transposon challenge. In both species, Penelope was predominantly targeted by endo-small-interfering RNAs (siRNAs) rather than by piwi-interacting RNAs (piRNAs). Although we do observe correlations between Penelope transcription and dysgenesis, we could not correlate differences in maternally deposited Penelope piRNAs with the sterility of progeny. Instead, we found that strains that produced dysgenic progeny differed in their production of piRNAs from clusters in subtelomeric regions, possibly indicating that changes in the overall piRNA repertoire underlie dysgenesis. Considered together, our data reveal unexpected plasticity in small RNA pathways in germ cells, both in the character of their responses to invading transposons and in the piRNA clusters that define their ability to respond to mobile elements

Activity of heat shock genes' promoters in thermally contrasting animal species.

Heat shock gene promoters represent a highly conserved and universal system for the rapid induction of transcription after various stressful stimuli. We chose pairs of mammalian and insect species that significantly differ in their thermoresistance and constitutive levels of Hsp70 to compare hsp promoter strength under normal conditions and after heat shock (HS). The first pair includes the HSPA1 gene promoter of camel (Camelus dromedarius) and humans. It was demonstrated that the camel HSPA1A and HSPA1L promoters function normally in vitro in human cell cultures and exceed the strength of orthologous human promoters under basal conditions. We used the same in vitro assay for Drosophila melanogaster Schneider-2 (S2) cells to compare the activity of the hsp70 and hsp83 promoters of the second species pair represented by Diptera, i.e., Stratiomys singularior and D. melanogaster, which dramatically differ in thermoresistance and the pattern of Hsp70 accumulation. Promoter strength was also monitored in vivo in D. melanogaster strains transformed with constructs containing the S. singularior hsp70 ORF driven either by its own promoter or an orthologous promoter from the D. melanogaster hsp70Aa gene. Analysis revealed low S. singularior hsp70 promoter activity in vitro and in vivo under basal conditions and after HS in comparison with the endogenous promoter in D. melanogaster cells, which correlates with the absence of canonical GAGA elements in the promoters of the former species. Indeed, the insertion of GAGA elements into the S. singularior hsp70 regulatory region resulted in a dramatic increase in promoter activity in vitro but only modestly enhanced the promoter strength in the larvae of the transformed strains. In contrast with hsp70 promoters, hsp83 promoters from both of the studied Diptera species demonstrated high conservation and universality

Spontaneous gain of susceptibility suggests a novel mechanism of resistance to hybrid dysgenesis in Drosophila virilis.

Syndromes of hybrid dysgenesis (HD) have been critical for our understanding of the transgenerational maintenance of genome stability by piRNA. HD in D. virilis represents a special case of HD since it includes simultaneous mobilization of a set of TEs that belong to different classes. The standard explanation for HD is that eggs of the responder strains lack an abundant pool of piRNAs corresponding to the asymmetric TE families transmitted solely by sperm. However, there are several strains of D. virilis that lack asymmetric TEs, but exhibit a "neutral" cytotype that confers resistance to HD. To characterize the mechanism of resistance to HD, we performed a comparative analysis of the landscape of ovarian small RNAs in strains that vary in their resistance to HD mediated sterility. We demonstrate that resistance to HD cannot be solely explained by a maternal piRNA pool that matches the assemblage of TEs that likely cause HD. In support of this, we have witnessed a cytotype shift from neutral (N) to susceptible (M) in a strain devoid of all major TEs implicated in HD. This shift occurred in the absence of significant change in TE copy number and expression of piRNAs homologous to asymmetric TEs. Instead, this shift is associated with a change in the chromatin profile of repeat sequences unlikely to be causative of paternal induction. Overall, our data suggest that resistance to TE-mediated sterility during HD may be achieved by mechanisms that are distinct from the canonical syndromes of HD

Adaptation of gene loci to heterochromatin in the course of Drosophila evolution is associated with insulator proteins

Pericentromeric heterochromatin is generally composed of repetitive DNA forming a transcriptionally repressive environment. Dozens of genes were embedded into pericentromeric heterochromatin during evolution of Drosophilidae lineage while retaining activity. However, factors that contribute to insusceptibility of gene loci to transcriptional silencing remain unknown. Here, we find that the promoter region of genes that can be embedded in both euchromatin and heterochromatin exhibits a conserved structure throughout the Drosophila phylogeny and carries motifs for binding of certain chromatin remodeling factors, including insulator proteins. Using ChIP-seq data, we demonstrate that evolutionary gene relocation between euchromatin and pericentric heterochromatin occurred with preservation of sites of insulation of BEAF-32 in evolutionarily distant species, i.e. D. melanogaster and D. virilis. Moreover, promoters of virtually all protein-coding genes located in heterochromatin in D. melanogaster are enriched with insulator proteins BEAF-32, GAF and dCTCF. Applying RNA-seq of a BEAF-32 mutant, we show that the impairment of BEAF-32 function has a complex effect on gene expression in D. melanogaster, affecting even those genes that lack BEAF-32 association in their promoters. We propose that conserved intrinsic properties of genes, such as sites of insulation near the promoter regions, may contribute to adaptation of genes to the heterochromatic environment and, hence, facilitate the evolutionary relocation of genes loci between euchromatin and heterochromatin.peerReviewe