Eu-heterochromatic Rearrangements Induce Replication of Heterochromatic Sequences Normally Underreplicated in Polytene Chromosomes of Drosophila melanogaster

In polytene chromosomes of D. melanogaster the heterochromatic pericentric regions are underreplicated (underrepresented). In this report, we analyze the effects of eu-heterochromatic rearrangements involving a cluster of the X-linked heterochromatic (Xh) Stellate repeats on the representation of these sequences in salivary gland polytene chromosomes. The discontinuous heterochromatic Stellate cluster contains specific restriction fragments that were mapped along the distal region of Xh. We found that transposition of a fragment of the Stellate cluster into euchromatin resulted in its replication in polytene chromosomes. Interestingly, only the Stellate repeats that remain within the pericentric Xh and are close to a new eu-heterochromatic boundary were replicated, strongly suggesting the existence of a spreading effect exerted by the adjacent euchromatin. Internal rearrangements of the distal Xh did not affect Stellate polytenization. We also demonstrated trans effects exerted by heterochromatic blocks on the replication of the rearranged heterochromatin; replication of transposed Stellate sequences was suppressed by a deletion of Xh and restored by addition of Y heterochromatin. This phenomenon is discussed in light of a possible role of heterochromatic proteins in the process of heterochromatin underrepresentation in polytene chromosomes

Determination of Main Spectral and Luminescent Characteristics of Winter Wheat Seeds Infected with Pathogenic Microflora

In connection with the constant growth of demand for high-quality food products, there is a need to develop effective methods for storing agricultural products, and the registration and predicting infection in the early stages. The studying of the physical properties of infected plants and seeds has fundamental importance for determining crop losses, conducting a survey of diseases, and assessing the effectiveness of their control (assessment of the resistance of crops and varieties, the effect of fungicides, etc.). Presently, photoluminescent methods for diagnosing seeds in the ultraviolet and visible ranges have not been studied. For research, seeds of winter wheat were selected, and were infected with one of the most common and dangerous diseases for plants—fusarium. The research of luminescence was carried out based on a hardware–software complex consisting of a multifunctional spectrofluorometer “Fluorat-02-Panorama”, a computer with software “Panorama Pro” installed, and an external camera for the samples under study. Spectra were obtained with a diagnostic range of winter wheat seeds of 220–400 nm. Based on the results obtained for winter wheat seeds, it is possible to further develop a method for determining the degree of fusarium infection

Key role of piRNAs in telomeric chromatin maintenance and telomere nuclear positioning in Drosophila germline

Abstract Background Telomeric small RNAs related to PIWI-interacting RNAs (piRNAs) have been described in various eukaryotes; however, their role in germline-specific telomere function remains poorly understood. Using a Drosophila model, we performed an in-depth study of the biogenesis of telomeric piRNAs and their function in telomere homeostasis in the germline. Results To fully characterize telomeric piRNA clusters, we integrated the data obtained from analysis of endogenous telomeric repeats, as well as transgenes inserted into different telomeric and subtelomeric regions. The small RNA-seq data from strains carrying telomeric transgenes demonstrated that all transgenes belong to a class of dual-strand piRNA clusters; however, their capacity to produce piRNAs varies significantly. Rhino, a paralog of heterochromatic protein 1 (HP1) expressed exclusively in the germline, is associated with all telomeric transgenes, but its enrichment correlates with the abundance of transgenic piRNAs. It is likely that this heterogeneity is determined by the sequence peculiarities of telomeric retrotransposons. In contrast to the heterochromatic non-telomeric germline piRNA clusters, piRNA loss leads to a dramatic decrease in HP1, Rhino, and trimethylated histone H3 lysine 9 in telomeric regions. Therefore, the presence of piRNAs is required for the maintenance of telomere chromatin in the germline. Moreover, piRNA loss causes telomere translocation from the nuclear periphery toward the nuclear interior but does not affect telomere end capping. Analysis of the telomere-associated sequences (TASs) chromatin revealed strong tissue specificity. In the germline, TASs are enriched with HP1 and Rhino, in contrast to somatic tissues, where they are repressed by Polycomb group proteins. Conclusions piRNAs play an essential role in the assembly of telomeric chromatin, as well as in nuclear telomere positioning in the germline. Telomeric arrays and TASs belong to a unique type of Rhino-dependent piRNA clusters with transcripts that serve simultaneously as piRNA precursors and as their only targets. Telomeric chromatin is highly sensitive to piRNA loss, implying the existence of a novel developmental checkpoint that depends on telomere integrity in the germline

Stellate Repeats: Targets of Silencing and Modules Causing cis-Inactivation and trans-Activation

The mechanism of silencing of testis expressed X-linked Stellate repeats by homologous Y-linked Suppressor of Stellate [Su(Ste)] repeats localized in the crystal locus was studied. The double stranded RNA as a product of symmetrical transcription of Su(Ste) repeat and small iinterfaceSu(Ste) siRNA were revealed suggesting the mechanism of RNA interference (RNAi) for Stellate silencing. The relief of Stellate silencing as a result of impaired complementarity between the sequences of putative target Stellate transcripts and Su(Ste) repeats was shown. The role of RNAi mechanism in the silencing of heterochromatic retrotransposon GATE inserted in Stellate cluster was revealed. The studies of cis-effects of Stellate tandem repeats causing variegated expression of juxtaposed reporter genes were extended and the lacZ variegation in imaginal disc was shown. The exceptional case of a non-variegated expression of mini-white gene juxtaposed to Stellate repeats in a construct inserted into the 39C region was shown to be accompanied by trans-activation in homozygous state. Trans-activation effect was retained after transposition of this construct into heterochromatic environment in spite of strong variegation of a mini-white gene

Role of Histone Deacetylases in Gene Regulation at Nuclear Lamina

<div><p>Theoretical models suggest that gene silencing at the nuclear periphery may involve “closing” of chromatin by transcriptional repressors, such as histone deacetylases (HDACs). Here we provide experimental evidence confirming these predictions. Histone acetylation, chromatin compactness, and gene repression in lamina-interacting multigenic chromatin domains were analyzed in Drosophila <em>S2</em> cells in which B-type lamin, diverse HDACs, and lamina-associated proteins were downregulated by dsRNA. Lamin depletion resulted in decreased compactness of the repressed multigenic domain associated with its detachment from the lamina and enhanced histone acetylation. Our data reveal the major role for HDAC1 in mediating deacetylation, chromatin compaction, and gene silencing in the multigenic domain, and an auxiliary role for HDAC3 that is required for retention of the domain at the lamina. These findings demonstrate the manifold and central involvement of class I HDACs in regulation of lamina-associated genes, illuminating a mechanism by which these enzymes can orchestrate normal and pathological development.</p> </div

Effect of LEM domain protein depletion on the expression of the <i>60D1</i> gene-cluster.

<p>Bars show increased expression of the cluster genes in <i>S2</i> cells determined with RT-qPCR after treatment with the mixture of <i>dMAN1</i>, <i>Bocksbeutel</i>, and <i>Otefin</i> dsRNAs. The control <i>LacZ</i> dsRNA-treated cells served as the reference. Gene symbols are shown on the X-axis; the <i>60D1</i> cluster is boxed. n = 6; error bars show SEM; *, p≤0.05 for comparison of individual transcript levels between <i>LacZ</i> RNAi and target RNAi; ††, p≤0.01 for comparison between the <i>60D1</i> cluster and control housekeeping genes. Inserts show the knockdown efficiency of the RNAi at the RNA levels.</p

Effects of Class I HDAC depletion on histone acetylation and chromatin compactness.

<p>(<b>A</b>) ChIP assay shows increased acetylation of histones H3 (left panel) and H4 (right panel) in cells treated with <i>HDAC1</i> dsRNA and <i>HDAC3</i> dsRNAs as compared to the <i>LacZ</i> dsRNA-treated control cells. n = 4; error bars represent SEM. (<b>B</b>) Decreased chromatin compactness revealed by the general sensitivity to DNase I assay in <i>HDAC1</i> dsRNA-treated cells as compared to the control <i>LacZ</i> dsRNA treatment. Gene positions are shown below the X-axis with the <i>60D1</i> cluster framed. n = 2 to 4; error bars show SEM. *, p≤0.05; **, p≤0.01; ***, p≤0.001 for comparisons to the control. Inserts show the knockdown efficiency of the RNAi at the RNA levels.</p

Effect of B-type Lamin depletion on chromatin compactness and histone acetylation.

<p>(<b>A</b>) Increase in general sensitivity to DNase I upon dsRNA-induced depletion of <i>LamDm_o</i>. Permeabilized cells were treated with DNase I and DNA damage was quantified by qPCR and normalized to the amplicons located at 37–39 kb (outside the <i>60D1</i> cluster), as shown for the <i>LamDm_o</i>-depleted cells in comparison to control <i>LacZ</i> dsRNA-treated cells. Horizontal axis shows positions of amplicons relative to the testis-specific <i>60D1</i> gene cluster outlined with a box, and its genes highlighted in black. (<b>B</b>) Increase in histone acetylation along the <i>60D1</i> cluster in <i>LamDm_o</i> dsRNA-treated cells as compared to control <i>LacZ</i> dsRNA-treated cells. Acetylation of histones H3 (left panel) and H4 (right panel) was detected by ChIP assay. Horizontal axis is same as in (A). n = 3 to 6; error bars show SEM; *, p≤0.05; **, p≤0.01 for comparisons to the control. Inserts show the knockdown efficiency of the dsRNA at the RNA levels.</p