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

Low level of expression of C-terminally truncated human FUS causes extensive changes in the spinal cord transcriptome of asymptomatic transgenic mice

A number of mutations in a gene encoding RNA-binding protein FUS have been linked to the development of a familial form of amyotrophic lateral sclerosis known as FUS-ALS. C-terminal truncations of FUS by either nonsense or frameshift mutations lead to the development of FUS-ALS with a particularly early onset and fast progression. However, even in patients bearing these highly pathogenic mutations the function of motor neurons is not noticeably compromised for at least a couple of decades, suggesting that until cytoplasmic levels of FUS lacking its C-terminal nuclear localisation signal reaches a critical threshold, motor neurons are able to tolerate its permanent production.In order to identify how the nervous system responds to low levels of pathogenic variants of FUS we produced and characterised a mouse line, L-FUS[1-359], with a low neuronal expression level of a highly aggregation-prone and pathogenic form of C-terminally truncated FUS. In contrast to mice that express substantially higher level of the same FUS variant and develop severe early onset motor neuron pathology, L-FUS[1-359] mice do not develop any clinical or histopathological signs of motor neuron deficiency even at old age. Nevertheless, we detected substantial changes in the spinal cord transcriptome of these mice compared to their wild type littermates. We suggest that at least some of these changes reflect activation of cellular mechanisms compensating for the potentially damaging effect of pathogenic FUS production. Further studies of these mechanism might reveal effective targets for therapy of FUS-ALS and possibly, other forms of ALS

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

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

Genes Responsible for H2S Production and Metabolism Are Involved in Learning and Memory in Drosophila melanogaster

The gasotransmitter hydrogen sulfide (H2S) produced by the transsulfuration pathway (TSP) is an important biological mediator, involved in many physiological and pathological processes in multiple higher organisms, including humans. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) enzymes play a central role in H2S production and metabolism. Here, we investigated the role of H2S in learning and memory processes by exploring several Drosophila melanogaster strains with single and double deletions of CBS and CSE developed by the CRISPR/Cas9 technique. We monitored the learning and memory parameters of these strains using the mating rejection courtship paradigm and demonstrated that the deletion of the CBS gene, which is expressed predominantly in the central nervous system, and double deletions completely block short- and long-term memory formation in fruit flies. On the other hand, the flies with CSE deletion preserve short- and long-term memory but fail to exhibit long-term memory retention. Transcriptome profiling of the heads of the males from the strains with deletions in Gene Ontology terms revealed a strong down-regulation of many genes involved in learning and memory, reproductive behavior, cognition, and the oxidation–reduction process in all strains with CBS deletion, indicating an important role of the hydrogen sulfide production in these vital processes

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

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

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

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