Michael B. Evgen’ev

Larvae of related Diptera species from thermally contrasting habitats exhibit continuous up-regulation of heat shock proteins and high thermotolerance.

A population of Stratiomys japonica, a species belonging to the family Stratiomyidae (Diptera), common name ‘soldier flies’, occurs in a hot volcanic spring, which is apparently among the most inhospitable environments for animals because of chemical and thermal conditions. Larvae of this species, which naturally often experience temperatures more than 40 °C, have constitutively high concentrations of the normally inducible heat‐shock protein Hsp70, but very low level of corresponding mRNA. Larvae of three other species of the same family, Stratiomys singularior, Nemotelus bipunctatus and Oxycera pardalina, are confined to different type semi‐aquatic habitats with contrasting thermal regime. However, all of them shared the same pattern of Hsp70 expression. Interestingly, heat‐shock treatment of S. japonica larvae activates heat‐shock factor and significantly induces Hsp70 synthesis, whereas larvae of O. pardalina, a species from constant cold environment, produce significantly less Hsp70 in response to heat shock. Adults of the four species also exhibit lower, but detectable levels of Hsp70 without heat shock. Larvae of all species studied have very high tolerance to temperature stress in comparison with other Diptera species investigated, probably representing an inherent adaptive feature of all Stratiomyidae enabling successful colonization of highly variable and extreme habitats.

Distribution and evolution of mobile elements in the virilis species group of Drosophila

Abstract.The distributions of Penelope and Ulysses, two transposable elements that can induce hybrid dysgenesis, were studied in several species groups of Drosophila. No significant hybridization to Penelope and Ulysses probes was detected by Southern blot analyses of species outside the virilis group. In contrast, both element families have had a long residence in all species of the virilis species group, as indicated by their strong presence in the heterochromatic chromocenter. Except for D. kanekoi, D. lummei, and some strains of D. virilis, species of the group carry full-sized, and at least potentially functional, copies of both element families. Consistent with the occurrence of recent transposition, Penelope and Ulysses elements are located at different chromosomal sites in different geographical strains of the same species. A total of 79 Penelope and 47 Ulysses euchromatic insertion sites were localized to chromosomal subsections in species of the virilis group. Highly significant deviations from independence of the distributions of Penelope and Ulysses and previously established inversion breakpoints were documented, suggesting that these transposable elements may have played an important role in genomic reorganization and evolution of the virilis species group, which is especially rich in karyotypic variation.

Evolution and Dynamics of Small RNA Response to a Retroelement Invasion in Drosophila

Although small RNAs efficiently control transposition activity of most transposons in the host genome, such an immune system is not always applicable against a new transposons invasions. Here, we explored a possibility to introduce potentially mobile copy of the Penelope retroelement previously implicated in hybrid dysgenesis syndrome in Drosophila virilis into the genomes of two distant Drosophila species. The consequences of such introduction were monitored at different phases after experimental colonization as well as in D. virilis species, which is apparently in the process of ongoing Penelope invasion. We investigated the expression of Penelope and biogenesis of Penelope-derived small RNAs in D. virilis and D. melanogaster strains originally lacking active copies of this element after experimental Penelope invasion. These strains were transformed by constructs containing intact Penelope copies. We show that immediately after transformation, which imitates the first stage of retroelement invasion, Penelope undergoes transposition predominantly in somatic tissues, and may produce siRNAs that are apparently unable to completely silence its activity. However, at the later stages of colonization Penelope copies may jump into one of the piRNA-clusters, which results in production of homologous piRNAs that are maternally deposited and can silence euchromatic transcriptionally active copies of Penelope in trans and, hence, prevent further amplification of the invader in the host genome. Intact Penelope copies and different classes of Penelope-derived small RNAs were found in most geographical strains of D. virilis collected throughout the world. Importantly, all strains of this species containing full-length Penelope tested do not produce gonadal sterility in dysgenic crosses and, hence, exhibit neutral cytotype. To understand whether RNA interference mechanism able to target Penelope operates in related species of the virilis group, we correlated the presence of full-length and potentially active Penelope with the occurrence of piRNAs homologous to this transposable element in the ovaries of species comprising the group. It was demonstrated that Penelope-derived piRNAs are present in all virilis group species containing full-length but transcriptionally silent copies of this element that probably represent the remnants of its previous invasions taking place in the course of the virilis species divergent evolution.

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.

What happens when Penelope comes?: An unusual retroelement invades a host species genome exploring different strategies

Transposable elements (TEs) are ubiquitous residents in eukaryotic genomes. They can cause dramatic changes in gene expression and lead to gross rearrangements of chromosome structure, providing the basis for rapid evolution. The virilis species group of Drosophila contains certain species that can be crossed under experimental conditions and their phylogeny is thoroughly investigated. We have shown that Drosophila virilis, the most primitive karyotypically and probably the ancestral species of the group, is in the process of colonization by a very unusual retroelement Penelope which apparently repeatedly invaded the species of the group in the past. However, the molecular mechanisms and evolutionary consequences of such invasions are poorly understood. In this commentary, we discuss the implications of our recent investigation into the response of the RNA silencing system to Penelope invasion of a new host genome which can be achieved in different ways.Transposable elements (TEs) are ubiquitous residents in eukaryotic genomes. They can cause dramatic changes in gene expression and lead to gross rearrangements of chromosome structure, providing the basis for rapid evolution. The virilis species group of Drosophila contains certain species that can be crossed under experimental conditions and their phylogeny is thoroughly investigated. We have shown that Drosophila virilis, the most primitive karyotypically and probably the ancestral species of the group, is in the process of colonization by a very unusual retroelement Penelope which apparently repeatedly invaded the species of the group in the past. However, the molecular mechanisms and evolutionary consequences of such invasions are poorly understood. In this commentary, we discuss the implications of our recent investigation into the response of the RNA silencing system to Penelope invasion of a new host genome which can be achieved in different ways.

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.

Intranasal Administration of Hsp70: Molecular and Therapeutic Consequences

Hsp70 and other molecular chaperones function as a complex neuroprotective system, which fails in the brains of aged people and Alzheimer’s disease (AD)-type neuropathologies. It was demonstrated that intranasally injected exogenous Hsp70 (eHsp70) effectively bypassed the blood-brain barrier and penetrates brain regions of the model animals. It was shown that chronic administration of eHsp70 decreases beta-amyloid level and the number of Aβ-plaques in two mouse models of AD. In both cases eHsp70 restored learning and memory parameters as well as functional state of neurons. Characteristically, eHsp70 treatment increased synaptophysin level and protects neurons in brain areas most affected in AD patients such as hippocampus and neocortex. It was also demonstrated that eHsp70 can promote longevity and life quality in male mice. The eHsp70 treatment decreased accumulation of aging marker lipofuscin and modulates the activity of UPS by increasing expression of several proteasome subunits including immunoproteasome subunit β5i. Deep sequencing studies exploring brain regions of AD-model – 5XFAD mice treated with eHsp70 revealed candidate genes and signal pathways probably underlying beneficial effects of eHsp70 treatment. Taken together, our findings establish intranasal administration of exogenous human Hsp70 as a practical therapeutic approach for the treatment of various neurodegenerative diseases and aging.

The Role of Mobile Elements in the Evolution and Function of HSPS Systems

The “selfish DNA” theory postulates that transposable elements (TEs) are intragenomic parasites, and that natural selection against deleterious effects associated with their presence is the main force preventing their genomic spread in natural populations (Orgel and Crick 1980).

Experimental Modulation of Heat Shock Response

Soon after the discovery of molecular mechanisms underlying the heat shock response and, specifically, the synthesis of a restricted set of proteins with different molecular weights (heat shock proteins) multiple experimental approaches were applied with the goal to somehow modify the HSR and monitor the biological consequences of such manipulations (see reviews of Feder and Hoffman 1999).

Fine Tuning of the HSR in Various Organisms

It is widely assumed that heat shock response system in eukaryotes is amazingly conserved. Thus, all described organisms possess one or several genes encoding transcription factors belonging to HSF family that recognize the same sequences (HSEs) in the promoters of various Hsps genes (Akerfelt et al. 2010; Morimoto 1998; Wu 1995). Furthermore, HSF family members in different organisms contain two highly similar domains responsible for DNA-binding and heat-induced trimerization as described in detail in Chap. 3. HSF recognizes practically the same simple sequences within promoters of Hsp genes (GAANNTTCNNGAA) that usually present in several copies at a regular distance from the transcription start. Various lines of evidence demonstrated that Hsp70 gene promoter is able to efficiently function in the cells of phylogenetically distant organisms even belonging to different phyla. Thus, reporter constructs under the control of Drosophila melanogaster Hsp70 gene promoter were readily expressed in the cells of mosquito Aedes aegypti, silkworm Bombix mori transgenic strains and in sea urchin embryos (Berger et al. 1985; McMahon et al. 1984; Uhlirova et al. 2002). Furthermore, constructs with D. melanogaster Hsp70 regulatory region were efficiently transcribed in Xenopus oocytes, rat fibroblasts and monkey COS cells (Bienz and Pelham 1982; Burke and Ish-Horowicz 1982; Mirault et al. 1982; Voellmy and Rungger 1982). Importantly, all such constructs exhibited clear-cut heat-inducible pattern of expression in the cells of the foreign hosts, thus corroborating the presumed high conservatism of HS response in unrelated organisms.