H3K9 and H4K20 methyltransferases are directly involved in the heterochromatinization of the paternal chromosomes in male Planococcus citri embryos.

Using a new method for bulk preparation of early stage embryos, we have investigated the role played by putative Planococcus citri H3K9 and H4K20 histone methyl transferases (HMTases) in regulating heterochromatinization of the imprinted paternal chromosomal set in male embryos. We found that H3K9 and H420 HMTases are required for heterochromatinization of the paternal chromosomes. We present evidence that both HMTases maintain the paternal "imprint" during the cleavage divisions when both parental chromosome sets are euchromatic. A testable model that accommodates our findings is proposed.

Dosage Compensation in Drosophila: Its Canonical and Non-Canonical Mechanisms.

Dosage compensation equalizes gene expression in a single male X chromosome with that in the pairs of autosomes and female X chromosomes. In the fruit fly Drosophila, canonical dosage compensation is implemented by the male-specific lethal (MSL) complex functioning in all male somatic cells. This complex contains acetyl transferase males absent on the first (MOF), which performs H4K16 hyperacetylation specifically in the male X chromosome, thus facilitating transcription of the X-linked genes. However, accumulating evidence points to an existence of additional, non-canonical dosage compensation mechanisms operating in somatic and germline cells. In this review, we discuss current advances in the understanding of both canonical and non-canonical mechanisms of dosage compensation in Drosophila.

ASIP Promoter Variants Predict the Sesame Coat Color in Shiba Inu Dogs.

Animals exhibit a wide variety of genetically determined coat colors and pigmentation patterns that serve important roles in adaptation and communication. Although the genetics of the main coat colors in dogs have been studied extensively, there are types of coat pigmentation that have not been explained yet. Recently, an association between the variants in the ASIP gene Ventral (VP) and Hair Cycle (HCP) promoters with different coat colors in dogs has been established. Here, we used the new findings as a basis to investigate the genetics of the red sesame coat color in Shiba Inu dogs. Our study revealed that red sesame dogs carry a specific heterozygous ASIP promoter diplotype, VP2-HCP1/VP2-HCP3, where VP2-HCP1 is responsible for the red coat with a dark overlay, and VP2-HCP3 for a tan point-like pattern. This finding explains the inheritance of this coat color pattern and can be used by breeders to produce dogs with this rare phenotype. A comparison of sesame dogs (VP2-HCP1/VP2-HCP3) to a dog homozygous for the VP2-HCP1 promoter haplotype suggests that the incomplete dominance between the ASIP alleles may be involved in the sesame coat formation. These results are in good agreement with the new model explaining how different levels of ASIP gene expression affect the regulation of pigment synthesis in melanocytes.

Comparison of genome architecture at two stages of male germline cell differentiation in Drosophila.

Eukaryotic chromosomes are spatially segregated into topologically associating domains (TADs). Some TADs are attached to the nuclear lamina (NL) through lamina-associated domains (LADs). Here, we identified LADs and TADs at two stages of Drosophila spermatogenesis - in bamΔ86 mutant testes which is the commonly used model of spermatogonia (SpG) and in larval testes mainly filled with spermatocytes (SpCs). We found that initiation of SpC-specific transcription correlates with promoters' detachment from the NL and with local spatial insulation of adjacent regions. However, this insulation does not result in the partitioning of inactive TADs into sub-TADs. We also revealed an increased contact frequency between SpC-specific genes in SpCs implying their de novo gathering into transcription factories. In addition, we uncovered the specific X chromosome organization in the male germline. In SpG and SpCs, a single X chromosome is stronger associated with the NL than autosomes. Nevertheless, active chromatin regions in the X chromosome interact with each other more frequently than in autosomes. Moreover, despite the absence of dosage compensation complex in the male germline, randomly inserted SpG-specific reporter is expressed higher in the X chromosome than in autosomes, thus evidencing that non-canonical dosage compensation operates in SpG.

Distemper, extinction, and vaccination of the Amur tiger.

Canine distemper virus (CDV) has recently emerged as an extinction threat for the endangered Amur tiger (Panthera tigris altaica). CDV is vaccine-preventable, and control strategies could require vaccination of domestic dogs and/or wildlife populations. However, vaccination of endangered wildlife remains controversial, which has led to a focus on interventions in domestic dogs, often assumed to be the source of infection. Effective decision making requires an understanding of the true reservoir dynamics, which poses substantial challenges in remote areas with diverse host communities. We carried out serological, demographic, and phylogenetic studies of dog and wildlife populations in the Russian Far East to show that a number of wildlife species are more important than dogs, both in maintaining CDV and as sources of infection for tigers. Critically, therefore, because CDV circulates among multiple wildlife sources, dog vaccination alone would not be effective at protecting tigers. We show, however, that low-coverage vaccination of tigers themselves is feasible and would produce substantive reductions in extinction risks. Vaccination of endangered wildlife provides a valuable component of conservation strategies for endangered species.

Biology and Physics of Heterochromatin-Like Domains/Complexes.

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.

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.

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 "limited") 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 "imprints" and also the placement of a putative "imprint" that might be carried by the sperm into the egg.

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.

Genome-wide analysis of SU(VAR)3-9 distribution in chromosomes of Drosophila melanogaster.

Histone modifications represent one of the key factors contributing to proper genome regulation. One of histone modifications involved in gene silencing is methylation of H3K9 residue. Present in the chromosomes across different eukaryotes, this epigenetic mark is controlled by SU(VAR)3-9 and its orthologs. Despite SU(VAR)3-9 was discovered over two decades ago, little is known about the details of its chromosomal distribution pattern. To fill in this gap, we used DamID-seq approach and obtained high-resolution genome-wide profiles for SU(VAR)3-9 in two somatic (salivary glands and brain ganglia) and two germline (ovarian nurse cells and testes) tissues of Drosophila melanogaster. Analysis of tissue and developmental expression of SU(VAR)3-9-bound genes indicates that in the somatic tissues tested, as well as in the ovarian nurse cells, SU(VAR)3-9 tends to associate with transcriptionally silent genes. In contrast, in the testes, SU(VAR)3-9 shows preferential association with testis-specific genes, and its binding appears dynamic during spermatogenesis. In somatic cells, the mere presence/absence of SU(VAR)3-9 binding correlates with lower/higher expression. No such correlation is found in the male germline. Interestingly, transcription units in piRNA clusters (particularly flanks thereof) are frequently targeted by SU(VAR)3-9, and Su(var)3-9 mutation affects the expression of select piRNA species. Our analyses suggest a context-dependent role of SU(VAR)3-9. In euchromatin, SU(VAR)3-9 may serve to fine-tune the expression of individual genes, whereas in heterochromatin, chromosome 4, and piRNA clusters, it may act more broadly over large chromatin domains.

Functional dissection of Drosophila melanogaster SUUR protein influence on H3K27me3 profile.

BACKGROUND: In eukaryotes, heterochromatin replicates late in S phase of the cell cycle and contains specific covalent modifications of histones. SuUR mutation found in Drosophila makes heterochromatin replicate earlier than in wild type and reduces the level of repressive histone modifications. SUUR protein was shown to be associated with moving replication forks, apparently through the interaction with PCNA. The biological process underlying the effects of SUUR on replication and composition of heterochromatin remains unknown. RESULTS: Here we performed a functional dissection of SUUR protein effects on H3K27me3 level. Using hidden Markow model-based algorithm we revealed SuUR-sensitive chromosomal regions that demonstrated unusual characteristics: They do not contain Polycomb and require SUUR function to sustain H3K27me3 level. We tested the role of SUUR protein in the mechanisms that could affect H3K27me3 histone levels in these regions. We found that SUUR does not affect the initial H3K27me3 pattern formation in embryogenesis or Polycomb distribution in the chromosomes. We also ruled out the possible effect of SUUR on histone genes expression and its involvement in DSB repair. CONCLUSIONS: Obtained results support the idea that SUUR protein contributes to the heterochromatin maintenance during the chromosome replication. A model that explains major SUUR-associated phenotypes is proposed.

Piwi interacts with chromatin at nuclear pores and promiscuously binds nuclear transcripts in Drosophila ovarian somatic cells.

Piwi in a complex with Piwi-interacting RNAs (piRNAs) triggers transcriptional silencing of transposable elements (TEs) in Drosophila ovaries, thus ensuring genome stability. To do this, Piwi must scan the nascent transcripts of genes and TEs for complementarity to piRNAs. The mechanism of this scanning is currently unknown. Here we report the DamID-seq mapping of multiple Piwi-interacting chromosomal domains in somatic cells of Drosophila ovaries. These domains significantly overlap with genomic regions tethered to Nuclear Pore Complexes (NPCs). Accordingly, Piwi was coimmunoprecipitated with the component of NPCs Elys and with the Xmas-2 subunit of RNA transcription and export complex, known to interact with NPCs. However, only a small Piwi fraction has transient access to DNA at nuclear pores. Importantly, although 36% of the protein-coding genes overlap with Piwi-interacting domains and RNA-immunoprecipitation results demonstrate promiscuous Piwi binding to numerous genic and TE nuclear transcripts, according to available data Piwi does not silence these genes, likely due to the absence of perfect base-pairing between piRNAs and their transcripts.

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.

Data analysis algorithm for DamID-seq profiling of chromatin proteins in Drosophila melanogaster.

Analysis of gene expression regulation typically requires identification of genomic sites bound by regulatory proteins. For this purpose, chromatin immunoprecipitation (ChIP) and Dam identification (DamID) methods can be applied to cell lines, whole organisms, or enriched cell populations. In this work, we present modifications to the experimental DamID protocol, as well as a custom data processing algorithm, that allow to confidently identify genomic sites enriched with the proteins of interest. This algorithm is implemented in Perl and is also available as executable files, thereby making DamID analysis relatively straightforward. Finally, we demonstrate how this pipeline performs when fed with real experimental data.

The effects of SUUR protein suggest its role in repressive chromatin renewal during replication in Drosophila.

Replication of chromosomes is central to heredity. To become available for replication machinery, DNA invariably needs to dissociate from chromatin proteins. Yet, chromatin landscape must be promptly re-established during or soon after replication. Although this process underlies the epigenetic inheritance, little is known about its molecular mechanisms. This mini-review is focused on Drosophila melanogaster SUppressor of UnderReplication (SUUR) protein, which is involved both in replication and chromatin maintenance in polytene tissues. Existing data suggest that it is involved in the regulation of chromatin renewal during replication. According to this model, SUUR protein moves along the chromosomes together with the replication complex. When the replication fork enters the repressed, H3K27me3- or H3K9me3-enriched, chromatin, SUUR-containing complex slows down the replisome until these histone modifications are properly placed on the newly-synthesized DNA strands. Suggested model provides an insight into the mechanism of epigenetic information inheritance. This hypothesis could be tested by further analysis of the interplay between local enrichment of repressive histone modifications and the replication fork progression rate.

Tethering of SUUR and HP1 proteins results in delayed replication of euchromatic regions in Drosophila melanogaster polytene chromosomes.

We analyze how artificial targeting of Suppressor of Under-Replication (SUUR) and HP1 proteins affects DNA replication in the "open," euchromatic regions. Normally these regions replicate early in the S phase and display no binding of either SUUR or HP1. These proteins were expressed as fusions with DNA-binding domain of GAL4 and recruited to multimerized UAS integrated in three euchromatic sites of the polytene X chromosome: 3B, 8D, and 18B. Using PCNA staining as a marker of ongoing replication, we showed that targeting of SUUR(GAL4DBD) and HP1(GAL4DBD) results in delayed replication of appropriate euchromatic regions. Specifically, replication at these regions starts early, much like in the absence of the fusion proteins; however, replication completion is significantly delayed. Notably, delayed replication was insufficient to induce underreplication. Recruitment of SUUR(GAL4DBD) and HP1(GAL4DBD) had distinct effects on expression of a mini-white reporter, found near UAS. Whereas SUUR(GAL4DBD) had no measurable influence on mini-white expression, HP1(GAL4DBD) targeting silenced mini-white, even in the absence of functional SU(VAR)3-9. Furthermore, recruitment of SUUR(GAL4DBD) and HP1(GAL4DBD) had distinct effects on the protein composition of target regions. HP1(GAL4DBD) but not SUUR(GAL4DBD) could displace an open chromatin marker, CHRIZ, from the tethering sites.

Developmental variation of the SUUR protein binding correlates with gene regulation and specific chromatin types in D. melanogaster.

Eukaryotic genomes are organized in large chromatin domains that maintain proper gene activity in the cell. These domains may be permissive or repressive to the transcription of underlying genes. Based on its protein makeup, chromatin in Drosophila cell culture has been recently categorized into five color-coded states. Suppressor of Under-Replication (SUUR) protein was found to be the major component present in all three repressive chromatin states named BLACK, BLUE, and GREEN and to be depleted from the active YELLOW and RED chromatin types. Here, we addressed the question of developmental dynamics of SUUR binding as a marker of repressed chromatin types. We established genomewide SUUR binding profiles in larval salivary gland, brain, and embryos using DNA adenine methyltransferase identification (DamID) technique, performed their pairwise comparisons and comparisons with the published data from Drosophila Kc cells. SUUR binding pattern was found to vary between the samples. Increase in SUUR binding predominantly correlated with local gene repression suggesting heterochromatin formation. Reduction in SUUR binding often coincided with activation of tissue-specific genes probably reflecting the transition to permissive chromatin state and increase in accessibility to specific transcription factors. SUUR binding plasticity accompanied by the regulation of the underlying genes was mainly observed in BLACK, BLUE, and RED chromatin types. Our results provide novel insight into the developmental dynamics of repressive chromatin and reveal a link to the chromatin-guided regulation of gene expression.

Comparison of ultra-conserved elements in drosophilids and vertebrates.

Metazoan genomes contain many ultra-conserved elements (UCEs), long sequences identical between distant species. In this study we identified UCEs in drosophilid and vertebrate species with a similar level of phylogenetic divergence measured at protein-coding regions, and demonstrated that both the length and number of UCEs are larger in vertebrates. The proportion of non-exonic UCEs declines in distant drosophilids whilst an opposite trend was observed in vertebrates. We generated a set of 2,126 Sophophora UCEs by merging elements identified in several drosophila species and compared these to the eutherian UCEs identified in placental mammals. In contrast to vertebrates, the Sophophora UCEs are depleted around transcription start sites. Analysis of 52,954 P-element, piggyBac and Minos insertions in the D. melanogaster genome revealed depletion of the P-element and piggyBac insertions in and around the Sophophora UCEs. We examined eleven fly strains with transposon insertions into the intergenic UCEs and identified associated phenotypes in five strains. Four insertions behave as recessive lethals, and in one case we observed a suppression of the marker gene within the transgene, presumably by silenced chromatin around the integration site. To confirm the lethality is caused by integration of transposons we performed a phenotype rescue experiment for two stocks and demonstrated that the excision of the transposons from the intergenic UCEs restores viability. Sequencing of DNA after the transposon excision in one fly strain with the restored viability revealed a 47 bp insertion at the original transposon integration site suggesting that the nature of the mutation is important for the appearance of the phenotype. Our results suggest that the UCEs in flies and vertebrates have both common and distinct features, and demonstrate that a significant proportion of intergenic drosophila UCEs are sensitive to disruption.

Induced transcription results in local changes in chromatin structure, replication timing, and DNA polytenization in a site of intercalary heterochromatin.

In salivary gland polytene chromosomes of Drosophila melanogaster, the regions of intercalary heterochromatin are characterized by late replication, under-replication, and genetic silencing. Using Gal4/UAS system, we induced transcription of sequences adjacent to transgene insertions in the band 11A6-9. This activation resulted in a loss of "silent" and appearance of "active" epigenetic marks, recruitment of RNA polymerase II, and formation of a puff. The activated region is now early replicating and shows increased level of DNA polytenization. Notably, all these changes are restricted to the area around the inserts, whereas the rest of the band remains inactive and late replicating. Although only a short area near the insertion site is transcribed, it results in an "open" chromatin conformation in a much broader region. We conclude that regions of intercalary heterochromatin do not form stand-alone units of late replication and under-replication. Every part of such regions can be activated and polytenized independently of other parts.

Gene density profile reveals the marking of late replicated domains in the Drosophila melanogaster genome.

Regulation of replication timing has been a focus of many studies. It has been shown that numerous chromosomal regions switch their replication timing on cell differentiation in Drosophila and mice. However, it is not clear which features of these regions are essential for such regulation. In this study, we examined the organization of late underreplicated regions (URs) of the Drosophila melanogaster genome. When compared with their flanks, these regions showed decreased gene density. A detailed view revealed that these regions originate from unusual combination of short genes and long intergenic spacers. Furthermore, gene expression study showed that this pattern is mostly contributed by short testis-specific genes abundant in the URs. Based on these observations, we developed a genome scanning algorithm and identified 110 regions possessing similar gene density and transcriptional profiles. According to the published data, replication of these regions has been significantly shifted towards late S-phase in two Drosophila cell lines and in polytene chromosomes. Our results suggest that genomic organization of the underreplicated areas of Drosophila polytene chromosomes may be associated with the regulation of their replication timing.