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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 -
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 -
Genome Research Mar 2015Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial...
Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.
Topics: Animals; Chromosome Mapping; Chromosomes, Artificial, Bacterial; Computational Biology; Contig Mapping; Drosophila melanogaster; Genome; High-Throughput Nucleotide Sequencing; In Situ Hybridization, Fluorescence; Molecular Sequence Data; Polytene Chromosomes; Restriction Mapping
PubMed: 25589440
DOI: 10.1101/gr.185579.114 -
Insects Sep 2018Anopheline mosquitoes are important vectors of human malaria. Next-generation sequencing opens new opportunities for studies of mosquito genomes to uncover the genetic...
Anopheline mosquitoes are important vectors of human malaria. Next-generation sequencing opens new opportunities for studies of mosquito genomes to uncover the genetic basis of a transmission. Physical mapping of genome sequences to polytene chromosomes significantly improves reference assemblies. High-resolution cytogenetic maps are essential for anchoring genome sequences to chromosomes as well as for studying breakpoints of chromosome rearrangements and chromatin protein localization. Here we describe a detailed pipeline for the development of high-resolution cytogenetic maps using polytene chromosomes of malaria mosquitoes. We apply this workflow to the refinement of the cytogenetic map developed for
PubMed: 30227611
DOI: 10.3390/insects9030121 -
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 -
Cells Mar 2023Nrf2 is the dominant cellular stress response factor that protects cells through transcriptional responses to xenobiotic and oxidative stimuli. Nrf2 malfunction is...
Nrf2 is the dominant cellular stress response factor that protects cells through transcriptional responses to xenobiotic and oxidative stimuli. Nrf2 malfunction is highly correlated with many human diseases, but the underlying molecular mechanisms remain to be fully uncovered. GATA4 is a conserved GATA family transcription factor that is essential for cardiac and dorsal epidermal development. Here, we describe a novel interaction between Nrf2 and GATA4 proteins, i.e., cap'n'collar C (CncC) and Pannier (Pnr), respectively. Using the bimolecular fluorescence complementation (BiFC) assay-a unique imaging tool for probing protein complexes in living cells-we detected CncC-Pnr complexes in the nuclei of embryonic and salivary gland cells. Visualization of CncC-Pnr BiFC signals on the polytene chromosome revealed that CncC and Pnr tend to form complexes in euchromatic regions, with a preference for loci that are not highly occupied by CncC or Pnr alone. Most genes within these loci are activated by the CncC-Pnr BiFC, but not by individually expressed CncC or Pnr fusion proteins, indicating a novel mechanism whereby CncC and Pnr interact at specific genomic loci and coactivate genes at these loci. Finally, CncC-induced early lethality can be rescued by Pnr depletion, suggesting that CncC and Pnr function in the same genetic pathway during the early development of . Taken together, these results elucidate a novel crosstalk between the Nrf2 xenobiotic/oxidative response factor and GATA factors in the transcriptional regulation of development. This study also demonstrates that the polytene chromosome BiFC assay is a valuable tool for mapping genes that are targeted by specific transcription factor complexes.
Topics: Animals; Chromatin; Drosophila; Drosophila Proteins; GATA4 Transcription Factor; NF-E2-Related Factor 2; Polytene Chromosomes; Xenobiotics; Transcriptional Activation
PubMed: 36980279
DOI: 10.3390/cells12060938 -
Cell Nov 2015Chemical cross-linking and DNA sequencing have revealed regions of intra-chromosomal interaction, referred to as topologically associating domains (TADs), interspersed...
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.
Topics: Animals; Cell Nucleus; Chromosomal Puffs; Diploidy; Drosophila melanogaster; Genetic Techniques; Larva; Polytene Chromosomes
PubMed: 26544940
DOI: 10.1016/j.cell.2015.10.026 -
Scientific Reports Nov 2018Establishment and maintenance of histone acetylation levels are critical for metazoan development and viability. Disruption of the balance between acetylation and...
Establishment and maintenance of histone acetylation levels are critical for metazoan development and viability. Disruption of the balance between acetylation and deacetylation by treatment with chemical histone deacetylase (HDAC) inhibitors results in loss of cell proliferation, differentiation and/or apoptosis. Histone deacetylation by the SIN3 complex is essential in Drosophila and mice, as loss of the scaffolding factor SIN3 or the associated HDAC results in lethality. The objective of this study is to elucidate contributions of SIN3 complex components to these essential processes. We used the Drosophila model organism to carry out a systematic functional analysis of the SIN3 complex. We find that SIN3 associated proteins are essential for viability and cell proliferation during development. Additionally, tissue specific reduction of SIN3 complex components results in abnormal wing development. Interestingly, while knockdown of each factor resulted in similar phenotypes, their individual effects on recruitment of SIN3 to polytene chromosomes are distinct. Reduction of some factors leads to large changes in the morphology of the chromosome and/or greatly reduced SIN3 binding. These findings suggest that while individual SIN3 complex components work through distinct molecular mechanisms, they each make a substantial contribution to the overall function of this highly conserved histone deacetylase complex.
Topics: Animals; Cell Proliferation; Cell Survival; Drosophila Proteins; Drosophila melanogaster; Female; Histones; Male; Sin3 Histone Deacetylase and Corepressor Complex
PubMed: 30451916
DOI: 10.1038/s41598-018-35093-0 -
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 -
G3 (Bethesda, Md.) Aug 2017Eukaryote genomes are replete with repetitive DNAs. This class includes tandemly repeated satellite DNAs (satDNA) which are among the most abundant, fast evolving (yet...
Eukaryote genomes are replete with repetitive DNAs. This class includes tandemly repeated satellite DNAs (satDNA) which are among the most abundant, fast evolving (yet poorly studied) genomic components. Here, we used high-throughput sequencing data from three cactophilic species, , , and , to access and study their whole satDNA landscape. In total, the software identified five satDNAs, three previously described (, and ) and two novel ones ( and ). Only is shared among all three species. The satDNA repeat length falls within only two classes, between 130 and 200 bp or between 340 and 390 bp. FISH on metaphase and polytene chromosomes revealed the presence of satDNA arrays in at least one of the following genomic compartments: centromeric, telomeric, subtelomeric, or dispersed along euchromatin. The chromosomal distribution ranges from a single chromosome to almost all chromosomes of the complement. Fiber-FISH and sequence analysis of contigs revealed interspersion between and in the microchromosomes of Phylogenetic analyses showed that the pBuM satDNA underwent concerted evolution at both interspecific and intraspecific levels. Based on RNA-seq data, we found transcription activity for (in ) and (in ) in all five analyzed developmental stages, most notably in pupae and adult males. Our data revealed that cactophilic present the lowest amount of satDNAs (1.9-2.9%) within the genus reported so far. We discuss how our findings on the satDNA location, abundance, organization, and transcription activity may be related to functional aspects.
Topics: Animals; Cactaceae; Centromere; DNA Probes; DNA Transposable Elements; DNA, Satellite; Drosophila; Evolution, Molecular; Genome, Insect; In Situ Hybridization, Fluorescence; Phylogeny; Polytene Chromosomes; Repetitive Sequences, Nucleic Acid; Sequence Analysis, DNA; Species Specificity; Telomere; Transcription, Genetic
PubMed: 28659292
DOI: 10.1534/g3.117.042093