Paramutation-like Epigenetic Conversion by piRNA at the Telomere of Drosophila virilis

First discovered in maize, paramutation is a phenomenon in which one allele can trigger an epigenetic conversion of an alternate allele. This conversion causes a genetically heterozygous individual to transmit alleles that are functionally the same, in apparent violation of Mendelian segregation. Studies over the past several decades have revealed a strong connection between mechanisms of genome defense against transposable elements by small RNA and the phenomenon of paramutation. For example, a system of paramutation in Drosophila melanogaster has been shown to be mediated by piRNAs, whose primary function is to silence transposable elements in the germline. In this paper, we characterize a second system of piRNA-mediated paramutation-like behavior at the telomere of Drosophila virilis. In Drosophila, telomeres are maintained by arrays of retrotransposons that are regulated by piRNAs. As a result, the telomere and sub-telomeric regions of the chromosome have unique regulatory and chromatin properties. Previous studies have shown that maternally deposited piRNAs derived from a sub-telomeric piRNA cluster can silence the sub-telomeric center divider gene of Drosophila virilis in trans. In this paper, we show that this silencing can also be maintained in the absence of the original silencing allele in a subsequent generation. The precise mechanism of this paramutation-like behavior may be explained by either the production of retrotransposon piRNAs that differ across strains or structural differences in the telomere. Altogether, these results show that the capacity for piRNAs to mediate paramutation in trans may depend on the local chromatin environment and proximity to the uniquely structured telomere regulated by piRNAs. This system promises to provide significant insights into the mechanisms of paramutation

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

Drosophila Model for the Analysis of Genesis of LIM-kinase 1-Dependent Williams-Beuren Syndrome Cognitive Phenotypes: INDELs, Transposable Elements of the Tc1/Mariner Superfamily and MicroRNAs

Genomic disorders, the syndromes with multiple manifestations, may occur sporadically due to unequal recombination in chromosomal regions with specific architecture. Therefore, each patient may carry an individual structural variant of DNA sequence (SV) with small insertions and deletions (INDELs) sometimes less than 10 bp. The transposable elements of the Tc1/mariner superfamily are often associated with hotspots for homologous recombination involved in human genetic disorders, such as Williams Beuren Syndromes (WBS) with LIM-kinase 1-dependent cognitive defects. The Drosophila melanogaster mutant agnts3 has unusual architecture of the agnostic locus harboring LIMK1: it is a hotspot of chromosome breaks, ectopic contacts, underreplication, and recombination. Here, we present the analysis of LIMK1-containing locus sequencing data in agnts3 and three D. melanogaster wild-type strains—Canton-S, Berlin, and Oregon-R. We found multiple strain-specific SVs, namely, single base changes and small INDEls. The specific feature of agnts3 is 28 bp A/T-rich insertion in intron 1 of LIMK1 and the insertion of mobile S-element from Tc1/mariner superfamily residing ~460 bp downstream LIMK1 3′UTR. Neither of SVs leads to amino acid substitutions in agnts3 LIMK1. However, they apparently affect the nucleosome distribution, non-canonical DNA structure formation and transcriptional factors binding. Interestingly, the overall expression of miRNAs including the biomarkers for human neurological diseases, is drastically reduced in agnts3 relative to the wild-type strains. Thus, LIMK1 DNA structure per se, as well as the pronounced changes in total miRNAs profile, probably lead to LIMK1 dysregulation and complex behavioral dysfunctions observed in agnts3 making this mutant a simple plausible Drosophila model for WBS

Comparative analysis of the ovarian piRNA profiles between <i>P</i>-like strain <i>160</i> and both <i>M</i>- and neutral <i>(N)</i> strains studied.

<p>A) and B) Scatter plots represent the result of pairwise comparison of normalized piRNAs (23–29 nt) in <i>P</i>-strain <i>160</i> versus <i>M</i>-like strains <i>9</i> and <i>13</i>, and in <i>P</i>-strain <i>160</i> versus <i>N</i>-strains <i>140</i>, <i>Argentina</i>, <i>Magarach</i> and <i>101</i>, respectively. Diagonal lines indicate 10-fold levels of difference. All the TEs that exceed 10-fold line are marked as gray dots. The red dots indicate TEs that are shared between <i>M</i>-strains <i>9</i> and <i>13</i> in terms of their low expression levels in comparison with <i>P</i>-strain <i>160</i>. Spearman’s correlation (R) is shown. C) Venn diagram depicting differences and similarities in a number of TEs exhibiting 10-fold lower piRNA expression level in <i>M</i>-strains <i>9</i> and <i>13</i> in comparison with <i>P</i>-strain <i>160</i>. 10 families show the same pattern of deficit in strains <i>9</i> and <i>13</i>, relative to strain 160. D) Venn diagram demonstrates distribution of piRNAs to eight essential elements distinguishing neutral strains from <i>M</i>-like in terms of piRNA-mediated silencing among studied <i>N</i>-strains.</p

The frequency (in %) of gonadal atrophy in the progeny of dysgenic and reciprocal involving <i>P</i>-like strain <i>160</i> and <i>Penelope</i>-transformed strains.

<p>The number of examined individuals was ~100 separately for females and males.</p

The alteration of the frequency (in %) of female and male gonadal atrophy in initially neutral strain <i>101</i>.

<p>The number of examined individuals was ~100 separately for females and males for each indicated time of monitoring.</p

Characterization of small RNA mediated silencing in substrains of <i>101</i> exhibiting different cytotype.

<p><b>A)</b> Scatter plot represent the result of pairwise comparison of normalized piRNAs (23–29 nt) in <i>M</i>-like strain <i>101</i> versus its neutral variant. Diagonal lines indicate 10-fold levels of difference. Gray dots indicate the repeats exhibiting more than 10-fold greater piRNA level. Spearman’s rank test was used for correlation (R) calculation. B) (1) The coverage of normalized <i>315</i>, <i>635</i>, <i>850</i>, <i>904</i> and <i>931</i>-derived piRNA reads (23–29 nt) from the <i>101(N)</i> strain on the entire body of correspondent elements. Sense reads are shown as [+], antisense as [–]. (2) The ping-pong signature of <i>315</i>, <i>635</i>, <i>850</i>, <i>904</i> and <i>931</i>-derived piRNAs. C) Expression levels of <i>315</i>, <i>635</i>, <i>850</i> elements in the ovaries of <i>160(P)</i>, <i>101(M)</i> and <i>101(N)</i> strains. <i>P</i>-values were calculated using <i>t</i>-test. D) Enrichment of H3K9me3 mark on the genomic loci carrying <i>315</i>, <i>635</i> and <i>850</i> using ChIP-qPCR on ovaries of substrains <i>101</i>. E) Heatmap of the ovarian expression of <i>315</i>, <i>635</i>, <i>850</i>, <i>904</i>, <i>931</i>-derived piRNAs in dysgenic and reciprocal hybrids. F) Expression levels of <i>315</i>, <i>635</i>, <i>850</i> elements in the progeny from dysgenic and reciprocal crosses relative to parental <i>101(N)</i> levels and G) to <i>160(P)</i> levels. H) Heatmap represents expression pattern of <i>315</i>, <i>635</i>, <i>850</i>, <i>904</i>, <i>931-</i>targeted piRNAs among the studied <i>P</i>-like, <i>M</i>-like and neutral strains.</p

Characterization of <i>Penelope</i> activity in the ovaries of <i>Penelope</i>-transformed strains.

<p>A) Expression levels of <i>Penelope</i> in <i>9(w3)</i>, <i>Tf1</i>, <i>Tf2</i> strains relative to <i>P</i>-strain <i>160</i>. B) (1) The coverage of normalized <i>Penelope</i>-piRNA reads (23–29 nt) on the entire body of the element, across transformed strains. Sense reads are shown as [+], antisense as [–]. (2) The ping-pong signature of <i>Penelope</i>-derived piRNAs. C) Mapping proportions of <i>Penelope</i>-piRNAs to canonical sequence of the element with the perfect match and with the assumption of up to 3 mismatches.</p

Genomic abundance and expression levels of putative HD-implicated TEs (<i>Penelope</i>, <i>Paris</i>, <i>Polyphemus</i> and <i>Helena</i>) in both cytotype variants of strain <i>101</i>.

<p>A) Southern blot analysis of genomic DNA of <i>160(P)</i>, <i>9(M)</i>, <i>101(M)</i> and <i>101(N)</i> strains. B) Expression levels of described <i>D</i>. <i>virilis</i> TEs in the ovaries of both variants of strain <i>101</i>. Spearman’s rank test was used to calculate the correlation (R) between the strains studied.</p

Comparative analysis of a set of HD-implicated TEs in the ovaries of dysgenic and reciprocal hybrids.

<p>A) Expression levels of <i>Penelope</i>, <i>Paris</i>, <i>Polyphemus</i>, <i>Helena</i> and <i>Ulysses</i> among the studied <i>P</i>-like, <i>M</i>-like and neutral strain. B) mRNA and piRNAs expression levels in the ovaries of the progeny from dysgenic and reciprocal hybrids. At the left—expression levels of indicated TEs relative to the level in <i>P</i>-strain <i>160</i>. At the right–normalized piRNAs expression levels. The dotted line indicates level in <i>P</i>-strain <i>160</i>. <i>P</i>-values were calculated using <i>t</i>-test. C) Expression level of <i>Penelope</i>, <i>Paris</i>, <i>Polyphemus</i>, <i>Helena</i> and <i>Ulysses</i> in the ovaries of dysgenic crosses compared to reciprocal ones. <i>P</i>-values were calculated using <i>t</i>-test.</p