Chromosome-nuclear envelope attachments affect interphase chromosome territories and entanglement.

BACKGROUND: It is well recognized that the interphase chromatin of higher eukaryotes folds into non-random configurations forming territories within the nucleus. Chromosome territories have biologically significant properties, and understanding how these properties change with time during lifetime of the cell is important. Chromosome-nuclear envelope (Chr-NE) interactions play a role in epigenetic regulation of DNA replication, repair, and transcription. However, their role in maintaining chromosome territories remains unclear. RESULTS: We use coarse-grained molecular dynamics simulations to study the effects of Chr-NE interactions on the dynamics of chromosomes within a model of the Drosophila melanogaster regular (non-polytene) interphase nucleus, on timescales comparable to the duration of interphase. The model simulates the dynamics of chromosomes bounded by the NE. Initially, the chromosomes in the model are prearranged in fractal-like configurations with physical parameters such as nucleus size and chromosome persistence length taken directly from experiment. Time evolution of several key observables that characterize the chromosomes is quantified during each simulation: chromosome territories, chromosome entanglement, compactness, and presence of the Rabl (polarized) chromosome arrangement. We find that Chr-NE interactions help maintain chromosome territories by slowing down and limiting, but not eliminating, chromosome entanglement on biologically relevant timescales. At the same time, Chr-NE interactions have little effect on the Rabl chromosome arrangement as well as on how chromosome compactness changes with time. These results are rationalized by simple dimensionality arguments, robust to model details. All results are robust to the simulated activity of topoisomerase, which may be present in the interphase cell nucleus. CONCLUSIONS: Our study demonstrates that Chr-NE attachments may help maintain chromosome territories, while slowing down and limiting chromosome entanglement on biologically relevant timescales. However, Chr-NE attachments have little effect on chromosome compactness or the Rabl chromosome arrangement.

Thiazole Orange as an Alternative to Antibody Binding for Detecting Triple-helical DNA in Heterochromatin of Drosophila and Rhynchosciara.

The standard method for detecting triple-stranded DNA over the last 1.5 decades has been immune detection using antibodies raised against non-canonical nucleic acid structures. Many fluorescent dyes bind differentially to nucleic acids and often exhibit distinctive staining patterns along metaphase chromosomes dependent upon features, including binding to the major and minor DNA grooves, level of chromatin compaction, nucleotide specificity, and level of dye stacking. Relatively recently, the fluorochrome Thiazole Orange (TO) was shown to preferentially bind to triplex DNA in gels. Here, we demonstrate that TO also detects triplex DNA in salivary gland chromosomes of Drosophila melanogaster and Rhynchosciara americana identical in location and specificity to observations using antibodies. This finding may enable triple-stranded DNA investigations to be carried out on a much broader and reproducible scale than hitherto possible using antibodies, where a frequently encountered problem is the difference in detection specificity and sensitivity between one antibody and another.

Non-farnesylated B-type lamin can tether chromatin inside the nucleus and its chromatin interaction requires the Ig-fold region.

Lamins are thought to direct heterochromatin to the nuclear lamina (NL); however, this function of lamin has not been clearly demonstrated in vivo. To address this, we analyzed polytene chromosome morphology when artificial lamin variants were expressed in Drosophila endoreplicating cells. We found that the CaaX-motif-deleted B-type lamin Dm0, but not A-type lamin C, was able to form a nuclear envelope-independent layer that was closely associated with chromatin. Other nuclear envelope proteins were not detected in this "ectopic lamina," and the associated chromatin showed a repressive histone modification maker but not a permissive histone modification marker nor RNA polymerase II proteins. Furthermore, deletion of the C-terminal lamin-Ig-fold domain prevents chromatin association with this ectopic lamina. Thus, non-farnesylated B-type lamin Dm0 can form an ectopic lamina and induce changes to chromatin structure and status inside the interphase nucleus.

Exon junction complex proteins bind nascent transcripts independently of pre-mRNA splicing in Drosophila melanogaster.

Although it is currently understood that the exon junction complex (EJC) is recruited on spliced mRNA by a specific interaction between its central protein, eIF4AIII, and splicing factor CWC22, we found that eIF4AIII and the other EJC core proteins Y14 and MAGO bind the nascent transcripts of not only intron-containing but also intronless genes on Drosophila polytene chromosomes. Additionally, Y14 ChIP-seq demonstrates that association with transcribed genes is also splicing-independent in Drosophila S2 cells. The association of the EJC proteins with nascent transcripts does not require CWC22 and that of Y14 and MAGO is independent of eIF4AIII. We also show that eIF4AIII associates with both polysomal and monosomal RNA in S2 cell extracts, whereas Y14 and MAGO fractionate separately. Cumulatively, our data indicate a global role of eIF4AIII in gene expression, which would be independent of Y14 and MAGO, splicing, and of the EJC, as currently understood.

Guanine quadruplex structures localize to heterochromatin.

Increasing amounts of data support a role for guanine quadruplex (G4) DNA and RNA structures in various cellular processes. We stained different organisms with monoclonal antibody 1H6 specific for G4 DNA. Strikingly, immuno-electron microscopy showed exquisite specificity for heterochromatin. Polytene chromosomes from Drosophila salivary glands showed bands that co-localized with heterochromatin proteins HP1 and the SNF2 domain-containing protein SUUR. Staining was retained in SUUR knock-out mutants but lost upon overexpression of SUUR. Somatic cells in Macrostomum lignano were strongly labeled, but pluripotent stem cells labeled weakly. Similarly, germline stem cells in Drosophila ovaries were weakly labeled compared to most other cells. The unexpected presence of G4 structures in heterochromatin and the difference in G4 staining between somatic cells and stem cells with germline DNA in ciliates, flatworms, flies and mammals point to a conserved role for G4 structures in nuclear organization and cellular differentiation.

Stable Chromosome Condensation Revealed by Chromosome Conformation Capture.

Chemical cross-linking and DNA sequencing have revealed regions of intra-chromosomal interaction, referred to as topologically associating domains (TADs), interspersed with regions of little or no interaction, in interphase nuclei. We find that TADs and the regions between them correspond with the bands and interbands of polytene chromosomes of Drosophila. We further establish the conservation of TADs between polytene and diploid cells of Drosophila. From direct measurements on light micrographs of polytene chromosomes, we then deduce the states of chromatin folding in the diploid cell nucleus. Two states of folding, fully extended fibers containing regulatory regions and promoters, and fibers condensed up to 10-fold containing coding regions of active genes, constitute the euchromatin of the nuclear interior. Chromatin fibers condensed up to 30-fold, containing coding regions of inactive genes, represent the heterochromatin of the nuclear periphery. A convergence of molecular analysis with direct observation thus reveals the architecture of interphase chromosomes.

A standard cytogenetic map of Culex quinquefasciatus polytene chromosomes in application for fine-scale physical mapping.

BACKGROUND: Southern house mosquito Culex quinquefasciatus belongs to the C. pipiens cryptic species complex, with global distribution and unclear taxonomy. Mosquitoes of the complex can transmit human and animal pathogens, such as filarial worm, West Nile virus and avian malarial Plasmodium. Physical gene mapping is crucial to understanding genome organization, function, and systematic relationships of cryptic species, and is a basis for developing new vector control strategies. However, physical mapping was not established previously for Culex due to the lack of well-structured polytene chromosomes. METHODS: Inbreeding was used to diminish inversion polymorphism and asynapsis of chromosomal homologs. Identification of larvae of the same developmental stage using the shape of imaginal discs allowed achievement of uniformity in chromosomal banding pattern. This together with high-resolution phase-contrast photography enabled the development of a cytogenetic map. Fluorescent in situ hybridization was used for gene mapping. RESULTS: A detailed cytogenetic map of C. quinquefasciatus polytene chromosomes was produced. Landmarks for chromosome recognition and cytological boundaries for two inversions were identified. Locations of 23 genes belonging to 16 genomic supercontigs, and 2 cDNA were established. Six supercontigs were oriented and one was found putatively misassembled. The cytogenetic map was linked to the previously developed genetic linkage groups by corresponding positions of 2 genetic markers and 10 supercontigs carrying genetic markers. Polytene chromosomes were numbered according to the genetic linkage groups. CONCLUSIONS: This study developed a new standard cytogenetic photomap of the polytene chromosomes for C. quinquefasciatus and was applied for the fine-scale physical mapping. It allowed us to infer chromosomal position of 1333 of annotated genes belonging to 16 genomic supercontigs and find orientation of 6 of these supercontigs; the new cytogenetic and previously developed genetic linkage maps were integrated based on 12 matches. The map will further assist in finding chromosomal position of the medically important and other genes, contributing into improvement of the genome assembly. Better assembled C. quinquefasciatus genome can serve as a reference for studying other vector species of C. pipiens complex and will help to resolve their taxonomic relationships. This, in turn, will contribute into future development of vector and disease control strategies.

Quantified effects of chromosome-nuclear envelope attachments on 3D organization of chromosomes.

We use a combined experimental and computational approach to study the effects of chromosome-nuclear envelope (Chr-NE) attachments on the 3D genome organization of Drosophila melanogaster (fruit fly) salivary gland nuclei. We consider 3 distinct models: a Null model - without specific Chr-NE attachments, a 15-attachment model - with 15 previously known Chr-NE attachments, and a 48-attachment model - with 15 original and 33 recently identified Chr-NE attachments. The radial densities of chromosomes in the models are compared to the densities observed in 100 experimental images of optically sectioned salivary gland nuclei forming "z-stacks." Most of the experimental z-stacks support the Chr-NE 48-attachment model suggesting that as many as 48 chromosome loci with appreciable affinity for the NE are necessary to reproduce the experimentally observed distribution of chromosome density in fruit fly nuclei. Next, we investigate if and how the presence and the number of Chr-NE attachments affect several key characteristics of 3D genome organization: chromosome territories and gene-gene contacts. This analysis leads to novel insight about the possible role of Chr-NE attachments in regulating the genome architecture. Specifically, we find that model nuclei with more numerous Chr-NE attachments form more distinct chromosome territories and their chromosomes intertwine less frequently. Intra-chromosome and intra-arm contacts are more common in model nuclei with Chr-NE attachments compared to the Null model (no specific attachments), while inter-chromosome and inter-arm contacts are less common in nuclei with Chr-NE attachments. We demonstrate that Chr-NE attachments increase the specificity of long-range inter-chromosome and inter-arm contacts. The predicted effects of Chr-NE attachments are rationalized by intuitive volume vs. surface accessibility arguments.

In vivo analysis of a fluorescent SUMO fusion in transgenic Drosophila.

Sumoylation, the covalent attachment of SUMO, a 90 amino acid peptide related to ubiquitin, is a major modulator of protein functions. Fluorescent SUMO protein fusions have been used in cell cultures to visualize SUMO in vivo but not in multicellular organisms. We generated a transgenic line of Drosophila expressing an mCherry-SUMO fusion. We analyzed its pattern in vivo in salivary gland nuclei expressing Venus-HP1 to recognize the different chromatin components (Chromocenter, chromosome IV). We compared it to SUMO immunostaining on squashed polytene chromosomes and observed similar patterns. In addition to the previously reported SUMO localizations (chromosome arms and chromocenter), we identify 2 intense binding sites: the fourth chromosome telomere and the DAPI-bright band in the region 81F.

A simple approach for multicolor immunofluorescence staining in different Drosophila cell types.

Multicolor immunostaining analysis is often a desirable tool in cell biology for most researchers. Nonetheless, this is not an easy task and often not affordable by many laboratories as it might require expensive instrumentation and sophisticated analysis software. Here, we describe a simple protocol for performing sequential immunostainings on two different Drosophila specimens. Our strategy relies on an efficient and reproducible method for removal primary antibodies and/or fluorophore-conjugated secondary antibodies that does not affect antigene integrity. We show that alternation of multiple rounds of antibody incubation and removal on the same slide, followed by registration of the same DAPI-stained image, provides a simple framework for the sequential detection of several antigens in the same cell. Given that the sample fixation procedures used for Drosophila tissues are compatible with most specimen processing protocols, we can envisage that the multicolor immunostaining strategy presented here can be also adapted to different samples including mammalian tissues and/or cells.

Macrogenomic evidence for the origin of the black fly Simulium suzukii (Diptera: Simuliidae) on Okinawa Island, Japan.

To determine the geographic origin of the black fly Simulium suzukii on Okinawa Island, Japan, macrogenomic profiles derived from its polytene chromosomes were compared with those of mainland and other insular populations of S. suzukii and of the isomorphic Simulium tani species complex. The Okinawan population is a chromosomally unique cytoform, designated 'D,' which is essentially monomorphic and differs by about 27 fixed rearrangements from the chromosomal standard sequence for the subgenus Simulium and by two fixed differences from its nearest known relative, representing the type of S. suzukii, on the main islands of Japan. Chromosomal band sequences revealed two additional, sympatric cytoforms of S. suzukii, designated 'A' and 'B,' each with species status, in Korea, and a third cytoform, designated 'C,' on Hokkaido, Japan. A new cytoform, 'K,' of S. tani from Malaysia, representing the type of S. tani, is more closely related to cytoforms in Thailand, as are populations from Taiwan previously treated as S. suzukii but more closely aligned with S. tani and newly recognized as cytoform 'L' of the latter nominal species. Rooting of chromosomal band sequences by outgroup comparisons allowed directionality of chromosomal rearrangements to be established, enabling phylogenetic inference of cytoforms. Of 41 macrogenomic rearrangements discovered in the five new cytoforms, four provide evidence for a stepwise origin of the Okinawan population from populations characteristic of the main islands of Japan. The macrogenomic approach applied to black flies on Okinawa Island illustrates its potential utility in defining source areas for other species of flies including those that might pose medical and veterinary risks.

Maintenance of interphase chromosome compaction and homolog pairing in Drosophila is regulated by the condensin cap-h2 and its partner Mrg15.

Dynamic regulation of chromosome structure and organization is critical for fundamental cellular processes such as gene expression and chromosome segregation. Condensins are conserved chromosome-associated proteins that regulate a variety of chromosome dynamics, including axial shortening, lateral compaction, and homolog pairing. However, how the in vivo activities of condensins are regulated and how functional interactors target condensins to chromatin are not well understood. To better understand how Drosophila melanogaster condensin is regulated, we performed a yeast two-hybrid screen and identified the chromo-barrel domain protein Mrg15 to interact with the Cap-H2 condensin subunit. Genetic interactions demonstrate that Mrg15 function is required for Cap-H2-mediated unpairing of polytene chromosomes in ovarian nurse cells and salivary gland cells. In diploid tissues, transvection assays demonstrate that Mrg15 inhibits transvection at Ubx and cooperates with Cap-H2 to antagonize transvection at yellow. In cultured cells, we show that levels of chromatin-bound Cap-H2 protein are partially dependent on Mrg15 and that Cap-H2-mediated homolog unpairing is suppressed by RNA interference depletion of Mrg15. Thus, maintenance of interphase chromosome compaction and homolog pairing status requires both Mrg15 and Cap-H2. We propose a model where the Mrg15 and Cap-H2 protein-protein interaction may serve to recruit Cap-H2 to chromatin and facilitates compaction of interphase chromatin.

[Karyotypes of four species of chironomids (Diptera, Chironomidae) from Northern Italy].

Karyotypes of 4 chironomid species were studied: Cryptochironomus obreptans Walker, Criptochironomus sp., Chironomus plumosus Linnaeus and Stictochironomus rosenscholdi Zetterstedt. All these species belong to the subfamily Chironominae. Each species is characterized by the specific karyotype structure. The first species in the list has 2n = 4, while the other 3 species have 2n = 8.

[Localization of the Dras1 sequence to polytene chromosomes in sibling species of the Drosophila virilis group].

Drasl gene was mapped by in situ hybridization to polytene chromosomes of several sibling species of the Drosophila virilis group and hybrids between them. A 1037 bp fragment of the Drasl gene of the D. virilis genome was used as a probe. The gene sequence is localized to the region of the disk 25 A-B on the chromosome 2 of the polytene chromosome map of D. virilis.

[Patterns of transposable elements distribution on the Drosophila melanogaster polytene chromosomes before and after selection for a quantitative trait].

The effect of selection for radius vein length on the distribution of hybridization sites of the P and hobo transposons and the mdgl and mdg2 retrotransposons on polytene chromosomes of Drosophila melanogaster salivary glands was studied. The patterns of these transposable elements (TEs) distribution were polymorphic in both the parental strain and selected strains. The similarity in mdg1 and mdg2 patterns between strains selected in one direction was closer than between strains selected in opposite directions, but the selected strains were closer to each other than to the parental strain regardless of selection direction. No mdg2 hybridization sites that would be absent in the control were found in the selected strains. There were more mdg2 and hobo hybridization sites in the strains selected in the (+) direction than in the (-) direction. The mobility of hobo copies in the strains studied correlated with the presence of its full-sized copy in the genome. The polymorphism of all TEs studied except for mdgl was greater for strains selected in the (+) direction that in the (-) direction. These facts suggest that some TEs migrate over the genome independently of selection, and others are markers of evolutionary events rather than their causes.

An integrated chromosome map of microsatellite markers and inversion breakpoints for an Asian malaria mosquito, Anopheles stephensi.

Anopheles stephensi is one of the major vectors of malaria in the Middle East and Indo-Pakistan subcontinent. Understanding the population genetic structure of malaria mosquitoes is important for developing adequate and successful vector control strategies. Commonly used markers for inferring anopheline taxonomic and population status include microsatellites and chromosomal inversions. Knowledge about chromosomal locations of microsatellite markers with respect to polymorphic inversions could be useful for better understanding a genetic structure of natural populations. However, fragments with microsatellites used in population genetic studies are usually too short for successful labeling and hybridization with chromosomes. We designed new primers for amplification of microsatellite loci identified in the A. stephensi genome sequenced with next-generation technologies. Twelve microsatellites were mapped to polytene chromosomes from ovarian nurse cells of A. stephensi using fluorescent in situ hybridization. All microsatellites hybridized to unique locations on autosomes, and 7 of them localized to the largest arm 2R. Ten microsatellites were mapped inside the previously described polymorphic chromosomal inversions, including 4 loci located inside the widespread inversion 2Rb. We analyzed microsatellite-based population genetic data available for A. stephensi in light of our mapping results. This study demonstrates that the chromosomal position of microsatellites may affect estimates of population genetic parameters and highlights the importance of developing physical maps for nonmodel organisms.

Molecular and cytological characterization of repetitive DNA sequences from the centromeric heterochromatin of Sciara coprophila.

Sciara coprophila (Diptera, Nematocera) constitutes a classic model to analyze unusual chromosome behavior such as the somatic elimination of paternal X chromosomes, the elimination of the whole paternal, plus non-disjunction of the maternal X chromosome at male meiosis. The molecular organization of the heterochromatin in S. coprophila is mostly unknown except for the ribosomal DNA located in the X chromosome pericentromeric heterochromatin. The characterization of the centromeric regions, thus, is an essential and required step for the establishment of S. coprophila as a model system to study fundamental mechanisms of chromosome segregation. To accomplish such a study, heterochromatic sections of the X chromosome centromeric region from salivary glands polytene chromosomes were microdissected and microcloned. Here, we report the identification and characterization of two tandem repeated DNA sequences from the pericentromeric region of the X chromosome, a pericentromeric RTE element and an AT-rich centromeric satellite. These sequences will be important tools for the cloning of S. coprophila centromeric heterochromatin using libraries of large genomic clones.

Sex-biased transcription enhancement by a 5' tethered Gal4-MOF histone acetyltransferase fusion protein in Drosophila.

BACKGROUND: In male Drosophila melanogaster, the male specific lethal (MSL) complex is somehow responsible for a two-fold increase in transcription of most X-linked genes, which are enriched for histone H4 acetylated at lysine 16 (H4K16ac). This acetylation requires MOF, a histone acetyltransferase that is a component of the MSL complex. MOF also associates with the non-specific lethal or NSL complex. The MSL complex is bound within active genes on the male X chromosome with a 3' bias. In contrast, the NSL complex is enriched at promoter regions of many autosomal and X-linked genes in both sexes. In this study we have investigated the role of MOF as a transcriptional activator. RESULTS: MOF was fused to the DNA binding domain of Gal4 and targeted to the promoter region of UAS-reporter genes in Drosophila. We found that expression of a UAS-red fluorescent protein (DsRed) reporter gene was strongly induced by Gal4-MOF. However, DsRed RNA levels were about seven times higher in female than male larvae. Immunostaining of polytene chromosomes showed that Gal4-MOF co-localized with MSL1 to many sites on the X chromosome in male but not female nuclei. However, in female nuclei that express MSL2, Gal4-MOF co-localized with MSL1 to many sites on polytene chromosomes but DsRed expression was reduced. Mutation of conserved active site residues in MOF (Glu714 and Cys680) reduced HAT activity in vitro and UAS-DsRed activation in Drosophila. In the presence of Gal4-MOF, H4K16ac levels were enriched over UAS-lacZ and UAS-arm-lacZ reporter genes. The latter utilizes the constitutive promoter from the arm gene to drive lacZ expression. In contrast to the strong induction of UAS-DsRed expression, UAS-arm-lacZ expression increased by about 2-fold in both sexes. CONCLUSIONS: Targeting MOF to reporter genes led to transcription enhancement and acetylation of histone H4 at lysine 16. Histone acetyltransferase activity was required for the full transcriptional response. Incorporation of Gal4-MOF into the MSL complex in males led to a lower transcription enhancement of UAS-DsRed but not UAS-arm-lacZ genes. We discuss how association of Gal4-MOF with the MSL or NSL proteins could explain our results.