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

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

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

The Role of p53 Protein in the Realization of the Exogenous Heat Shock Protein 70 Anti-Apoptotic Effect during Axotomy

The search for effective neuroprotective agents for the treatment of neurotrauma has always been of great interest to researchers around the world. Extracellular heat shock protein 70 (eHsp70) is considered a promising agent to study, as it has been demonstrated to exert a significant neuroprotective activity against various neurodegenerative diseases. We showed that eHsp70 can penetrate neurons and glial cells when added to the incubation medium, and can accumulate in the nuclei of neurons and satellite glial cells after axotomy. eHsp70 reduces apoptosis and necrosis of the glial cells, but not the neurons. At the same time, co-localization of eHsp70 with p53 protein, one of the key regulators of apoptosis, was noted. eHsp70 reduces the level of the p53 protein apoptosis promoter both in glial cells and in the nuclei and cytoplasm of neurons, which indicates its neuroprotective effect. The ability of eHsp70 to reverse the proapoptotic effect of the p53 activator WR1065 may indicate its ability to regulate p53 activity or its proteosome-dependent degradation

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

Left panel: General arrangement of constructs used in the transformation experiments.

<p>The thick green arrows in I(S-GAGA) indicate the position of the inserted GAGA elements. HSE, GAGA elements and TATA boxes are indicated by square boxes of different color. Right panel: <i>in situ</i> hybridization of heat-shocked salivary gland chromosomes with the <i>white</i> gene fragment included in the constructs. The sites of the inserts are shown by arrows with puffs formed only in the strains (the bottom panel) containing constructs with <i>D</i>. <i>melanogaster hsp70</i> promoters. In all panels, the heat shock puffs formed in the locations of major <i>D</i>. <i>melanogaster hsp</i> genes (i.e., 63B, 61C and 95D) that represent an internal control are indicated. 3B is the <i>white</i> locus that hybridizes with the labeled probe and represents an internal control for hybridization efficiency. In each strain, at least ten larvae were used for puff detection after HS.</p

The results of RT-PCR experiments using RNA isolated from adult flies (A) or the third instar larvae (B) of <i>D</i>. <i>melanogaster</i> strains transformed with constructs containing different <i>hsp70</i> promoters.

<p>A significant dichotomy in the expression of the I(S-GAGA) construct in adults and larvae is evident, while all other constructs exhibit similar strengths at both stages. *** p ≤ 0.001.</p

A. EMSA experiments with protein extracts from S2 cells and adult <i>D</i>. <i>melanogaster</i> flies exploring labeled fragments of the <i>D</i>. <i>melanogaster hsp70Aa</i> and <i>S</i>. <i>singularior hsp70S3</i> and <i>S4</i> genes. 1—Control (25°C), 2—heat shock (37°C), 3—heat shock + preimmune serum, 4—super shift with anti-HSF. The arrow indicates the position of the HSF-HSE complex. B. Recombinant <i>D</i>. <i>melanogaster</i> GAF protein effectively binds to the <i>D</i>. <i>melanogaster hsp70</i> and <i>S</i>. <i>singularior hsp70S3</i> promoters with the experimental insertion of three GAGA elements (lanes 1 and 4) but not with the wild-type <i>hsp70S3</i> and <i>hsp70S4</i> promoters (lanes 2 and 3). The arrow indicates the position of the GAF-GAGA complexes.

<p>A. EMSA experiments with protein extracts from S2 cells and adult <i>D</i>. <i>melanogaster</i> flies exploring labeled fragments of the <i>D</i>. <i>melanogaster hsp70Aa</i> and <i>S</i>. <i>singularior hsp70S3</i> and <i>S4</i> genes. 1—Control (25°C), 2—heat shock (37°C), 3—heat shock + preimmune serum, 4—super shift with anti-HSF. The arrow indicates the position of the HSF-HSE complex. B. Recombinant <i>D</i>. <i>melanogaster</i> GAF protein effectively binds to the <i>D</i>. <i>melanogaster hsp70</i> and <i>S</i>. <i>singularior hsp70S3</i> promoters with the experimental insertion of three GAGA elements (lanes 1 and 4) but not with the wild-type <i>hsp70S3</i> and <i>hsp70S4</i> promoters (lanes 2 and 3). The arrow indicates the position of the GAF-GAGA complexes.</p