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

Medicine and improvement in the Scots Magazine; and Edinburgh Literary Miscellany, 1804-17

Megan Coyer’s chapter engages with periodical print as a vehicle for an improving medical culture in Scotland, concentrating on the second series of the Scots Magazine. Coyer demonstrates how the Scottish press often complemented improving civic initiatives like the Edinburgh Lunatic Asylum campaign. She focuses attention on the distinctive national dynamics associated with medical improvement efforts in early nineteenth-century Scotland, with the Scots Magazine ‘providing a public forum for the expression of a national medical identity’. This identity, as Coyer shows, had an ideology of improvement at its core. This work recovers the cultural significance of the Scots Magazine as ‘the third major player in popular periodical culture in Romantic-era Scotland’; a status overshadowed by the recent critical attention devoted to the second Edinburgh Review and Blackwood’s in Scottish Romantic studies. Coyer also shows how the efforts of public health reformers highlight the complexity of improvement as both a material and moral process. She argues that print efforts dedicated to improving public health represent a ‘discursive strand in the magazine identifying a lack of cleanliness … as a moral and material blight on an otherwise improving Scottish society’. This bringing together of moral and practical aspects of improvement in the Scots also finds expression in the magazine’s series of Scottish medical biographies, whose narratives, Coyer notes, provide ‘ideal exemplars of lives dedicated to a culture of improvement’

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

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

Supplementary Tables S1-S5 from Interplay between RNA interference and heat shock response systems in <i>Drosophila melanogaster</i>

The genome expression pattern is strongly modified during the heat shock response (HSR) to form adaptive state. This may be partly achieved by modulating microRNA levels that control the expression of a great number of genes that are embedded within the gene circuitry. Here, we investigated the cross-talk between two highly conserved and universal house-keeping systems, the HSR and microRNA machinery, in <i>Drosophila melanogaster</i>. We demonstrated that pronounced interstrain differences in the microRNA levels are alleviated after heat shock (HS) to form a uniform microRNA pattern. However, individual strains exhibit different patterns of microRNA expression during the course of recovery. Importantly, HS-regulated microRNAs may target functionally similar HS-responsive genes involved in the HSR. Despite the observed general downregulation of primary microRNA precursor expression as well as core microRNA pathway genes after HS, the levels of many mature microRNAs are upregulated. This indicates that the regulation of miRNA expression after HS occurs at transcriptional and post-transcriptional levels. It was also shown that deletion of all <i>hsp70</i> genes had no significant effect on microRNA biogenesis but might influence the dynamics of microRNA expression during the HSR

List of transgenic strains used in the investigation.

<p>^π–All localizations were performed using Lefevre’s map of <i>D</i>. <i>melanogaster</i> salivary glands polytene chromosomes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115536#pone.0115536.ref053" target="_blank">53</a>]. Ch.-chromosome.</p><p>List of transgenic strains used in the investigation.</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. General arrangement of the <i>HSPA1</i> cluster in mammals. B. The level of transcription activity of individual <i>hsp70</i> gene promoters comprising the cluster based on the measurement of transient luciferase luminescence driven by constructs carrying different human and camel <i>hspA1</i> promoters. (i)—<i>HSPA1A</i>, (ii)—<i>HSPA1B</i> and (iii)—<i>HSPA1L</i>. The signal levels are shown as the ratio of the intensity of the luminescence of firefly (pFF) and renilla (pRL) luciferase. C. EMSA experiments with human and camel <i>HSPA1L</i> promoters fragments: 1—<i>H</i>. <i>sapiens</i> promoter without 112 bp fragment specific for primates only, 2—isolated <i>H</i>. <i>sapiens</i> 112 bp fragment, 3—<i>C</i>. <i>dromedarius</i> promoter. *** and ### p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

<p>A. General arrangement of the <i>HSPA1</i> cluster in mammals. B. The level of transcription activity of individual <i>hsp70</i> gene promoters comprising the cluster based on the measurement of transient luciferase luminescence driven by constructs carrying different human and camel <i>hspA1</i> promoters. (i)—<i>HSPA1A</i>, (ii)—<i>HSPA1B</i> and (iii)—<i>HSPA1L</i>. The signal levels are shown as the ratio of the intensity of the luminescence of firefly (pFF) and renilla (pRL) luciferase. C. EMSA experiments with human and camel <i>HSPA1L</i> promoters fragments: 1—<i>H</i>. <i>sapiens</i> promoter without 112 bp fragment specific for primates only, 2—isolated <i>H</i>. <i>sapiens</i> 112 bp fragment, 3—<i>C</i>. <i>dromedarius</i> promoter. *** and ### p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.</p

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

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