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ELife Dec 2022Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early...
Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Intercalary heterochromatin domains replicate very late in both diploid chromosomes of dividing cells and in endoreplicating polytene chromosomes where they are also underreplicated. SNF2-related factor SUUR imparts locus-specific underreplication of polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. Here, we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, or ) mutations reverse underreplication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. Our findings implicate a SNF2 family ATP-dependent motor protein SUUR in the insulator function, reveal that DNA replication can be delayed by a chromatin barrier, and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for the establishment of late replication that does not depend on an asynchronous firing of late replication origins.
Topics: Animals; Drosophila; DNA-Binding Proteins; Heterochromatin; Drosophila melanogaster; Drosophila Proteins; Euchromatin; Chromatin; DNA Replication
PubMed: 36458689
DOI: 10.7554/eLife.81828 -
Nucleus (Austin, Tex.) 2015We use a combined experimental and computational approach to study the effects of chromosome-nuclear envelope (Chr-NE) attachments on the 3D genome organization of...
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.
Topics: Animals; Chromatin; Drosophila melanogaster; Genome, Insect; Models, Biological; Nuclear Envelope; Polytene Chromosomes; Salivary Glands; User-Computer Interface
PubMed: 26068134
DOI: 10.1080/19491034.2015.1056441 -
Chromosome Research : An International... Dec 2022Studying the probability distribution of replication initiation along a chromosome is a huge challenge. Drosophila polytene chromosomes in combination with...
Studying the probability distribution of replication initiation along a chromosome is a huge challenge. Drosophila polytene chromosomes in combination with super-resolution microscopy provide a unique opportunity for analyzing the probabilistic nature of replication initiation at the ultrastructural level. Here, we developed a method for synchronizing S-phase induction among salivary gland cells. An analysis of the replication label distribution in the first minutes of S phase and in the following hours after the induction revealed the dynamics of replication initiation. Spatial super-resolution structured illumination microscopy allowed identifying multiple discrete replication signals and to investigate the behavior of replication signals in the first minutes of the S phase at the ultrastructural level. We identified replication initiation zones where initiation occurs stochastically. These zones differ significantly in the probability of replication initiation per time unit. There are zones in which initiation occurs on most strands of the polytene chromosome in a few minutes. In other zones, the initiation on all strands takes several hours. Compact bands are free of replication initiation events, and the replication runs from outer edges to the middle, where band shapes may alter.
Topics: Animals; Drosophila; Polytene Chromosomes; Microscopy; DNA Replication; Chromosomes; Drosophila melanogaster
PubMed: 35226231
DOI: 10.1007/s10577-021-09679-w -
Proceedings of the National Academy of... Jun 2001Salivary gland cells in the larvae of the dipteran Chironomus tentans offer unique possibilities to visualize the assembly and nucleocytoplasmic transport of a specific... (Review)
Review
Salivary gland cells in the larvae of the dipteran Chironomus tentans offer unique possibilities to visualize the assembly and nucleocytoplasmic transport of a specific transcription product. Each nucleus harbors four giant polytene chromosomes, whose transcription sites are expanded, or puffed. On chromosome IV, there are two puffs of exceptional size, Balbiani ring (BR) 1 and BR 2. A BR gene is 35-40 kb, contains four short introns, and encodes a 1-MDa salivary polypeptide. The BR transcript is packed with proteins into a ribonucleoprotein (RNP) fibril that is folded into a compact ring-like structure. The completed RNP particle is released into the nucleoplasm and transported to the nuclear pore, where the RNP fibril is gradually unfolded and passes through the pore. On the cytoplasmic side, the exiting extended RNP fibril becomes engaged in protein synthesis and the ensuing polysome is anchored to the endoplasmic reticulum. Several of the BR particle proteins have been characterized, and their fate during the assembly and transport of the BR particle has been elucidated. The proteins studied are all added cotranscriptionally to the pre-mRNA molecule. The various proteins behave differently during RNA transport, and the flow pattern of each protein is related to the particular function of the protein. Because the cotranscriptional assembly of the pre-mRNP particle involves proteins functioning in the nucleus as well as proteins functioning in the cytoplasm, it is concluded that the fate of the mRNA molecule is determined to a considerable extent already at the gene level.
Topics: Animals; Cell Nucleus; Chironomidae; Chromosome Mapping; Larva; Protein Transport; RNA-Binding Proteins; Ribonucleoproteins; Salivary Proteins and Peptides; Spliceosomes
PubMed: 11416180
DOI: 10.1073/pnas.111145498 -
PloS One 2017Spatial organization of chromosome territories is important for maintenance of genomic stability and regulation of gene expression. Recent studies have shown...
Spatial organization of chromosome territories is important for maintenance of genomic stability and regulation of gene expression. Recent studies have shown tissue-specific features of chromosome attachments to the nuclear envelope in various organisms including malaria mosquitoes. However, other spatial characteristics of nucleus organization, like volume and shape of chromosome territories, have not been studied in Anopheles. We conducted a thorough analysis of tissue-specific features of the X chromosome and nucleolus volume and shape in follicular epithelium and nurse cells of the Anopheles atroparvus ovaries using a modern open-source software. DNA of the polytene X chromosome from ovarian nurse cells was obtained by microdissection and was used as a template for amplification with degenerate oligo primers. A fluorescently labeled X chromosome painting probe was hybridized with formaldehyde-fixed ovaries of mosquitoes using a 3D-FISH method. The nucleolus was stained by immunostaining with an anti-fibrillarin antibody. The analysis was conducted with TANGO-a software for a chromosome spatial organization analysis. We show that the volume and position of the X chromosome have tissue-specific characteristics. Unlike nurse cell nuclei, the growth of follicular epithelium nuclei is not accompanied with the proportional growth of the X chromosome. However, the shape of the X chromosome does not differ between the tissues. The dynamics of the X chromosome attachment regions location is tissue-specific and it is correlated with the process of nucleus growth in follicular epithelium and nurse cells.
Topics: Animals; Anopheles; Cell Nucleolus; Female; In Situ Hybridization, Fluorescence; Malaria; Organ Specificity; Polytene Chromosomes; X Chromosome
PubMed: 28158219
DOI: 10.1371/journal.pone.0171290 -
Cells Oct 2021Although heterochromatin makes up 40% of the genome, its organization remains little explored, especially in polytene chromosomes, as it is virtually not represented in...
Although heterochromatin makes up 40% of the genome, its organization remains little explored, especially in polytene chromosomes, as it is virtually not represented in them due to underreplication. Two all-new approaches were used in this work: (i) with the use of a newly synthesized line that carries three mutations, , and , suppressing the underreplication of heterochromatic regions, we obtained their fullest representation in polytene chromosomes and described their structure; (ii) 20 DNA fragments with known positions on the physical map as well as molecular genetic features of the genome (gene density, histone marks, heterochromatin proteins, origin recognition complex proteins, replication timing sites and satellite DNAs) were mapped in the newly polytenized heterochromatin using FISH and bioinformatics data. The borders of the heterochromatic regions and variations in their positions on arm 3L have been determined for the first time. The newly polytenized heterochromatic material exhibits two main types of morphology: a banding pattern (locations of genes and short satellites) and reticular chromatin (locations of large blocks of satellite DNA). The locations of the banding and reticular polytene heterochromatin was determined on the physical map.
Topics: Animals; Antibodies; Carrier Proteins; Chromosomal Proteins, Non-Histone; DNA Replication; DNA-Binding Proteins; Drosophila Proteins; Drosophila melanogaster; Genes, Essential; Genome, Insect; Guanine Nucleotide Exchange Factors; Heterochromatin; Histones; Introns; Models, Biological; Mutation; Nuclear Proteins; Polytene Chromosomes; Protein Processing, Post-Translational; Repetitive Sequences, Nucleic Acid; Replication Origin; Repressor Proteins
PubMed: 34831030
DOI: 10.3390/cells10112809 -
G3 (Bethesda, Md.) Mar 2015The spatial organization of the genome within the eukaryotic nucleus is a dynamic process that plays a central role in cellular processes such as gene expression, DNA...
The spatial organization of the genome within the eukaryotic nucleus is a dynamic process that plays a central role in cellular processes such as gene expression, DNA replication, and chromosome segregation. Condensins are conserved multi-subunit protein complexes that contribute to chromosome organization by regulating chromosome compaction and homolog pairing. Previous work in our laboratory has shown that the Cap-H2 subunit of condensin II physically and genetically interacts with the Drosophila homolog of human MORF4-related gene on chromosome 15 (MRG15). Like Cap-H2, Mrg15 is required for interphase chromosome compaction and homolog pairing. However, the mechanism by which Mrg15 and Cap-H2 cooperate to maintain interphase chromatin organization remains unclear. Here, we show that Cap-H2 localizes to interband regions on polytene chromosomes and co-localizes with Mrg15 at regions of active transcription across the genome. We show that co-localization of Cap-H2 on polytene chromosomes is partially dependent on Mrg15. We have identified a binding motif within Cap-H2 that is essential for its interaction with Mrg15, and have found that mutation of this motif results in loss of localization of Cap-H2 on polytene chromosomes and results in partial suppression of Cap-H2-mediated compaction and homolog unpairing. Our data are consistent with a model in which Mrg15 acts as a loading factor to facilitate Cap-H2 binding to chromatin and mediate changes in chromatin organization.
Topics: Adenosine Triphosphatases; Amino Acid Motifs; Animals; Cell Line; Cells, Cultured; Chromatin; Chromosomal Proteins, Non-Histone; DNA-Binding Proteins; Drosophila; Drosophila Proteins; Gene Expression; Genes, Reporter; Genome; Interphase; Multiprotein Complexes; Mutation; Polytene Chromosomes; Protein Binding; Protein Interaction Domains and Motifs; Protein Transport; Transcription, Genetic; Transcriptional Activation
PubMed: 25758823
DOI: 10.1534/g3.115.016634 -
ELife May 2016Duplicating chromosomes once each cell cycle produces sister chromatid pairs, which separate accurately at anaphase. In contrast, reduplicating chromosomes without...
Duplicating chromosomes once each cell cycle produces sister chromatid pairs, which separate accurately at anaphase. In contrast, reduplicating chromosomes without separation frequently produces polytene chromosomes, a barrier to accurate mitosis. Chromosome reduplication occurs in many contexts, including: polytene tissue development, polytene tumors, and following treatment with mitosis-blocking chemotherapeutics. However, mechanisms responding to or resolving polyteny during mitosis are poorly understood. Here, using Drosophila, we uncover two distinct reduplicated chromosome responses. First, when reduplicated polytene chromosomes persist into metaphase, an anaphase delay prevents tissue malformation and apoptosis. Second, reduplicated polytene chromosomes can also separate prior to metaphase through a spindle-independent mechanism termed Separation-Into-Recent-Sisters (SIRS). Both reduplication responses require the spindle assembly checkpoint protein Mad2. While Mad2 delays anaphase separation of metaphase polytene chromosomes, Mad2's control of overall mitotic timing ensures efficient SIRS. Our results pinpoint mechanisms enabling continued proliferation after genome reduplication, a finding with implications for cancer progression and prevention.
Topics: Animals; Cell Cycle; Drosophila; Drosophila Proteins; Endoreduplication; Mad2 Proteins; Polytene Chromosomes
PubMed: 27159240
DOI: 10.7554/eLife.15204 -
Nucleic Acids Research Jan 2016Increasing amounts of data support a role for guanine quadruplex (G4) DNA and RNA structures in various cellular processes. We stained different organisms with...
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.
Topics: Animals; Ciliophora; Drosophila; G-Quadruplexes; Germ Cells; Guanine; Heterochromatin; Histones; Islets of Langerhans; Platyhelminths; Polytene Chromosomes; Rats
PubMed: 26384414
DOI: 10.1093/nar/gkv900 -
PloS One 2018Morphologically, polytene chromosomes of Drosophila melanogaster consist of compact "black" bands alternating with less compact "grey" bands and interbands. We developed...
Morphologically, polytene chromosomes of Drosophila melanogaster consist of compact "black" bands alternating with less compact "grey" bands and interbands. We developed a comprehensive approach that combines cytological mapping data of FlyBase-annotated genes and novel tools for predicting cytogenetic features of chromosomes on the basis of their protein composition and determined the genomic coordinates for all black bands of polytene chromosome 2R. By a PCNA immunostaining assay, we obtained the replication timetable for all the bands mapped. The results allowed us to compare replication timing between polytene chromosomes in salivary glands and chromosomes from cultured diploid cell lines and to observe a substantial similarity in the global replication patterns at the band resolution level. In both kinds of chromosomes, the intervals between black bands correspond to early replication initiation zones. Black bands are depleted of replication initiation events and are characterized by a gradient of replication timing; therefore, the time of replication completion correlates with the band length. The bands are characterized by low gene density, contain predominantly tissue-specific genes, and are represented by silent chromatin types in various tissues. The borders of black bands correspond well to the borders of topological domains as well as to the borders of the zones showing H3K27me3, SUUR, and LAMIN enrichment. In conclusion, the characteristic pattern of polytene chromosomes reflects partitioning of the Drosophila genome into two global types of domains with contrasting properties. This partitioning is conserved in different tissues and determines replication timing in Drosophila.
Topics: Animals; Chromosomes, Insect; DNA Replication; DNA Replication Timing; Diploidy; Drosophila melanogaster; Genome, Insect; Physical Chromosome Mapping; Polytene Chromosomes; Salivary Glands
PubMed: 29659604
DOI: 10.1371/journal.pone.0195207