Paucity and preferential suppression of transgenes in late replication domains of the D. melanogaster genome

<p>Abstract</p> <p>Background</p> <p>Eukaryotic genomes are organized in extended domains with distinct features intimately linking genome structure, replication pattern and chromatin state. Recently we identified a set of long late replicating euchromatic regions that are underreplicated in salivary gland polytene chromosomes of <it>D. melanogaster</it>.</p> <p>Results</p> <p>Here we demonstrate that these underreplicated regions (URs) have a low density of <it>P</it>-<it>element </it>and <it>piggyBac </it>insertions compared to the genome average or neighboring regions. In contrast, <it>Minos</it>-based transposons show no paucity in URs but have a strong bias to testis-specific genes. We estimated the suppression level in 2,852 stocks carrying a single <it>P</it>-<it>element </it>by analysis of eye color determined by the mini-<it>white </it>marker gene and demonstrate that the proportion of suppressed transgenes in URs is more than three times higher than in the flanking regions or the genomic average. The suppressed transgenes reside in intergenic, genic or promoter regions of the annotated genes. We speculate that the low insertion frequency of <it>P-elemen</it>ts and <it>piggyBac</it>s in URs partially results from suppression of transgenes that potentially could prevent identification of transgenes due to complete suppression of the marker gene. In a similar manner, the proportion of suppressed transgenes is higher in loci replicating late or very late in Kc cells and these loci have a lower density of <it>P-elements </it>and <it>piggyBac </it>insertions. In transgenes with two marker genes suppression of mini-<it>white </it>gene in eye coincides with suppression of <it>yellow </it>gene in bristles.</p> <p>Conclusions</p> <p>Our results suggest that the late replication domains have a high inactivation potential apparently linked to the silenced or closed chromatin state in these regions, and that such inactivation potential is largely maintained in different tissues.</p

Genome-wide analysis of gene regulation mechanisms during Drosophila spermatogenesis

Background During Drosophila spermatogenesis, testis-specific meiotic arrest complex (tMAC) and testis-specific TBP-associated factors (tTAF) contribute to activation of hundreds of genes required for meiosis and spermiogenesis. Intriguingly, tMAC is paralogous to the broadly expressed complex Myb-MuvB (MMB)/dREAM and Mip40 protein is shared by both complexes. tMAC acts as a gene activator in spermatocytes, while MMB/dREAM was shown to repress gene activity in many cell types. Results Our study addresses the intricate interplay between tMAC, tTAF, and MMB/dREAM during spermatogenesis. We used cell type-specific DamID to build the DNA-binding profiles of Cookie monster (tMAC), Cannonball (tTAF), and Mip40 (MMB/dREAM and tMAC) proteins in male germline cells. Incorporating the whole transcriptome analysis, we characterized the regulatory effects of these proteins and identified their gene targets. This analysis revealed that tTAFs complex is involved in activation of achi, vis, and topi meiosis arrest genes, implying that tTAFs may indirectly contribute to the regulation of Achi, Vis, and Topi targets. To understand the relationship between tMAC and MMB/dREAM, we performed Mip40 DamID in tTAF- and tMAC-deficient mutants demonstrating meiosis arrest phenotype. DamID profiles of Mip40 were highly dynamic across the stages of spermatogenesis and demonstrated a strong dependence on tMAC in spermatocytes. Integrative analysis of our data indicated that MMB/dREAM represses genes that are not expressed in spermatogenesis, whereas tMAC recruits Mip40 for subsequent gene activation in spermatocytes. Conclusions Discovered interdependencies allow to formulate a renewed model for tMAC and tTAFs action in Drosophila spermatogenesis demonstrating how tissue-specific genes are regulated

L Chromosome Behaviour and Chromosomal Imprinting in Sciara Coprophila

The retention of supernumerary chromosomes in the germ-line of Sciara coprophila is part of a highly-intricate pattern of chromosome behaviours that have fascinated cytogeneticists for over 80 years. Germ-line limited (termed L or &ldquo;limited&rdquo;) chromosomes are cytologically heterochromatic and late-replicating, with more recent studies confirming they possess epigenetic hallmarks characteristic of constitutive heterochromatin. Little is known about their genetic constitution although they have been found to undergo cycles of condensation and de-condensation at different stages of development. Unlike most supernumeraries, the L chromosomes in S. coprophila are thought to be indispensable, although in two closely related species Sciara ocellaris and Sciara reynoldsi the L chromosomes, have been lost during evolution. Here, we review what we know about L chromosomes in Sciara coprophila. We end by discussing how study of the L chromosome condensation cycle has provided insight into the site and timing of both the erasure of parental &ldquo;imprints&rdquo; and also the placement of a putative &ldquo;imprint&rdquo; that might be carried by the sperm into the egg

The Development Of Drosophila Melanogaster under Different Duration Space Flight and Subsequent Adaptation to Earth Gravity.

In prospective human exploration of outer space, the need to preserve a species over several generations under changed gravity conditions may arise. This paper demonstrates our results in the creation of the third generation of fruit fly Drosophila melanogaster (third-stage larvae) during the 44.5-day space flight (Foton-M4 satellite (2014, Russia)), then the fourth generation on Earth and the fifth generation again in conditions of the 12-day space flight (2014, in the Russian Segment of the ISS). The species preserves fertility despite a number of changes in the level of expression and content of cytoskeletal proteins, which are the key components of the cleavage spindle and the contractile ring of cells. The results of transcriptome screening and space analysis of cytoskeletal proteins show that the exposure to weightless conditions leads to the increased transcription of metabolic genes, cuticle components and the decreased transcription of genes involved in morphogenesis, cell differentiation, cytoskeletal organization and genes associated with the plasma membrane. "Subsequent" exposure to the microgravity for 12 days resulted in an even more significant increase/decrease in the transcription of the same genes. On the contrary, the transition from the microgravity conditions to the gravity of Earth leads to the increased transcription of genes whose products are involved in the morphogenesis, cytoskeletal organization, motility of cells and transcription regulation, and to the decreased transcription of cuticle genes and proteolytic processes

Maternal regulation of chromosomal imprinting in animals

Chromosomal imprinting requires an epigenetic system that imprints one of the two parental chromosomes such that it results in a heritable (cell-to-cell) change in behavior of the imprinted chromosome. Imprinting takes place when the parental genomes are separate, which occurs during gamete formation in the respective germ-lines and post-fertilization during the period when the parental pro-nuclei lie separately within the ooplasm of the zygote. In the mouse, chromosomal imprinting is regulated by germ-line specific DNA methylation. But the methylation machinery in the respective germ-lines does not discriminate between imprinted and non-imprinted regions. As a consequence, the mouse oocyte nucleus contains over a thousand oocyte-specific germ-line differentially methylated regions (gDMRs). Upon fertilization, the sperm provides a few hundred sperm-specific gDMRs of its own. Combined, there are around 1600 imprinted and non-imprinted gDMRs in the pro-nuclei of the newly fertilized zygote. It is a remarkable fact that beginning in the maternal ooplasm, there are mechanisms that manage to preserve DNA methylation at 26 known imprinted gDMRs in the face of the ongoing genome-wide DNA de-methylation that characterizes pre-implantation development. Specificity is achieved through the binding of KRAB-zinc finger proteins to their cognate recognition sequences within the gDMRs of imprinted genes. This in turn nucleates the assembly of localized heterochromatin-like complexes that preserve methylation at imprinted gDMRs through recruitment of the maintenance methyl transferase Dnmt1. These studies have shown that a germ-line imprint may cause parent-of-origin-specific behavior only if licensed by mechanisms that operate post-fertilization. Study of the germ-line and post-fertilization contributions to the imprinting of chromosomes in classical insect systems (Coccidae and Sciaridae) show that the ooplasm is the likely site where imprinting takes place. By comparing molecular and genetic studies across these three species, we suggest that mechanisms which operate post-fertilization play a key role in chromosomal imprinting phenomena in animals and conserved components of heterochromatin are shared by these mechanisms

BIOLOGY AND PHYSICS OF HETEROCHROMATIN-LIKE DOMAINS/COMPLEXESA DETAILED ASSESSMENT OF GROUNDWATER QUALITY IN THE KABUL BASIN, AFGHANISTAN, AND SUITABILITY FOR FUTURE DEVELOPMENT

The hallmarks of constitutive heterochromatin, HP1 and H3K9me2/3, assemble heterochromatin-like domains/complexes outside canonical constitutively heterochromatic territories where they regulate chromatin template-dependent processes. Domains are more than 100 kb in size; complexes less than 100 kb. They are present in the genomes of organisms ranging from fission yeast to human, with an expansion in size and number in mammals. Some of the likely functions of domains/complexes include silencing of the donor mating type region in fission yeast, preservation of DNA methylation at imprinted germline differentially methylated regions (gDMRs) and regulation of the phylotypic progression during vertebrate development. Far cis- and trans-contacts between micro-phase separated domains/complexes in mammalian nuclei contribute to the emergence of epigenetic compartmental domains (ECDs) detected in Hi-C maps. A thermodynamic description of micro-phase separation of heterochromatin-like domains/complexes may require a gestalt shift away from the monomer as the “unit of incompatibility” that determines the sign and magnitude of the Flory–Huggins parameter, χ. Instead, a more dynamic structure, the oligo-nucleosomal “clutch”, consisting of between 2 and 10 nucleosomes is both the long sought-after secondary structure of chromatin and its unit of incompatibility. Based on this assumption we present a simple theoretical framework that enables an estimation of χ for domains/complexes flanked by euchromatin and thereby an indication of their tendency to phase separate. The degree of phase separation is specified by χN, where N is the number of “clutches” in a domain/complex. Our approach could provide an additional tool for understanding the biophysics of the 3D genome

Relative content of actin in the membrane (MF) and cytoplasmic (CF) fractions.

<p>(A) and (B) Total actin. The total actin content in the MF from the 3LS and 3LS+12h groups exceeded the level in the MF from the 5LSS group by 45% and 51% (p<0.05) but was not significantly different in the CF. In the 3LS, 3LS+12h, 5LSF, and 5LFF groups, the total actin contents in the MF and CF were reduced by 24% and 46% (p<0.05), 27% and 58% (p<0.05), 19% and 36% (p<0.05), and 17% and 29% (p<0.05), respectively, compared with the 5LSS group. (C) and (D) Beta-actin. The beta-actin content in the MF from the 3LS and 3LS+12h groups exceeded the level of 5LSS group by 61% and 44% (p<0.05), respectively, but remained unchanged in the CF. The beta-actin content in the CF from the 3LF and 3LF+12h groups was reduced by 27% and 29% (p<0.05) compared with the CF of the 5LSS group but remained unchanged in the MF. In the 3LF+24h group, the beta-actin content in the MF exceeded the level in the MF of the 5LSS group by 28% (p<0.05) but remained unchanged in the CF.</p

Relative mRNA content of genes (qPCR data) that encode actin-binding proteins.

<p>(A) and (B) The <i>Arpc3A</i> (A) and <i>Tmod</i> (B) mRNA contents were the same in the different groups, with the exception that the contents were reduced by 76% and 73%, respectively, in the 5LFF group compared with the 5LSS group (p<0.05). (C) The <i>Svil</i> mRNA content in the 3LS, 3LS+12h, 3LS+24h, 5LSS, 5LFS, and 5LSF groups was the same. The <i>Svil</i> mRNA content was significantly reduced by 32% and 29% in the 3LF and 3LF+12h groups, respectively, compared with the 5LSS group (p<0.05), whereas the levels were restored to the control level (5LSS group) in the 3LF+24h group. In the 5LFF group, the <i>Svil</i> mRNA content was reduced by 62% compared with the 5LSS group (p<0.05). (D) The <i>Fim</i> mRNA content in the 3LS, 3LS+12h, 3LS+24h, 5LSS, 5LFS groups was the same. The <i>Fim</i> mRNA content was the same in the 3LF group and 5LSS group, it was decreased by 46% in the 3LF+12h group (p<0.05), and was restored to the control level (5LSS group) in the 3LF+24h group. In the 5LSF group, the <i>Fim</i> mRNA content was reduced by 57% compared with the 5LSS group (p<0.05) and by 79% compared with the 5LFF group (p<0.05). (E) The <i>Actn</i> mRNA content in the 3LS, 3LS+12h and 3LS+24h groups was increased by 80%, 91% and 82% (p<0.05), respectively, compared with the 5LSS group. The <i>Actn</i> mRNA content was significantly reduced by 29% and 27% in the 3LF and 3LF+12h groups, respectively, compared with the 5LSS group (p<0.05) and was increased by 90% in the 3LF+24h group (p<0.05). In the 5LSF and 5LFF groups, <i>Actn</i> mRNA content was reduced by 28% and 72% (p<0.05), respectively, compared with the 5LSS group.</p

Design and cyclogram of the experiment.

<p>The experimental groups are highlighted by the circles.</p

Relative mRNA content of genes (qPCR data) that encode actin isoforms.

<p>(A) The <i>Act57B</i> mRNA content was increased by 100% in the 3LS group (p<0.05), by 111% in the 3LS+12h group(p<0.05), and by 42% in the 3LS+24h group (p<0.05) compared with the 5LSS group. In 3LF group, <i>Act57B</i> mRNA was reduced by 24% (p<0.05), it was the same in the 3LF+12h group, and it exceeded the level of the 5LSS group by 130% in the 3LF+24h group (p<0.05). In the 5LSS, 5LFS, 5LSF, and 5LFF groups, the mRNA content was the same. (B) The <i>Act87E</i> mRNA content was the same in the 3LS group, was increased by 31% in the 3LS+12h group (p<0.05), and was decreased by 31% in the 3LS+24h group (p<0.05) compared with the 5LSS group. <i>Act87E</i> mRNA was reduced by 26% (p<0.05) in the 3LF group, was the same in the 3LF+12h group, and was increased by 54% in the 3LF+24h group (p<0.05) compared with the 5LSS group. Similar to <i>Act57B</i>, the mRNA content was the same in the 5LSS, 5LF, 5LSF, and 5LFF groups. (C) The <i>Act5C</i> mRNA content in the 3LS, 3LS+12h, 3LS+24h, 3LF, 3LF+12h and 3LF+24h groups, as well as the 5LFF group was reduced by 23%, 46%, 79%, 62%, 44%, 49% and 86% compared with the 5LSS group (p<0.05).</p