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Experimental Biology and Medicine... Nov 2019Repairing DNA double-strand breaks is particularly challenging in pericentromeric heterochromatin, where the abundance of repeated sequences exacerbates the risk of...
UNLABELLED
Repairing DNA double-strand breaks is particularly challenging in pericentromeric heterochromatin, where the abundance of repeated sequences exacerbates the risk of ectopic recombination. In Kc cells, accurate homologous recombination repair of heterochromatic double-strand breaks relies on the relocalization of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. This movement is driven by Arp2/3-dependent nuclear actin filaments and myosins’ ability to walk along them. Conserved mechanisms enable the relocalization of heterochromatic repair sites in mouse cells, and defects in these pathways lead to massive ectopic recombination in heterochromatin and chromosome rearrangements. In polytene chromosomes, extensive DNA movement is blocked by a stiff structure of chromosome bundles. Repair pathways in this context are poorly characterized, and whether heterochromatic double-strand breaks relocalize in these cells is unknown. Here, we show that damage in heterochromatin results in relaxation of the heterochromatic chromocenter, consistent with a dynamic response. Arp2/3, the Arp2/3 activator Scar, and the myosin activator Unc45, are required for heterochromatin stability in polytene cells, suggesting that relocalization enables heterochromatin repair also in this tissue. Together, these studies reveal critical roles for actin polymerization and myosin motors in heterochromatin repair and genome stability across different organisms and tissue types.
IMPACT STATEMENT
Heterochromatin relies on dedicated pathways for ‘safe’ recombinational repair. In mouse and fly cultured cells, DNA double-strand break repair requires the movement of damaged sites away from the heterochromatin ‘domain’ nuclear actin filaments and myosins. Here, we explore the importance of these pathways in salivary gland cells, which feature a stiff bundle of endoreduplicated polytene chromosomes. Repair pathways in polytene chromosomes are largely obscure and how nuclear dynamics operate in this context is unknown. We show that heterochromatin relaxes in response to damage, and relocalization pathways are necessary to prevent abnormal repair and promote the stability of heterochromatic sequences. These results deepen our understanding of DNA damage response mechanisms in polytene chromosomes, revealing unexpected dynamics. It also provides a first understanding of nuclear dynamics responding to replication damage and rDNA breaks, providing a new understanding of the importance of nuclear architecture in genome stability. We expect these discoveries will shed light on tumorigenic processes, including therapy-induced cancer relapses.
Topics: Actin Cytoskeleton; Actin-Related Protein 2-3 Complex; Animals; DNA Breaks, Double-Stranded; DNA Repair; Drosophila Proteins; Drosophila melanogaster; Genomic Instability; Heterochromatin; Molecular Chaperones; Myosins; Polytene Chromosomes
PubMed: 31364400
DOI: 10.1177/1535370219862282 -
Heredity Jul 2019
Review
Topics: Animals; Caenorhabditis elegans; Drosophila; Halobacterium; Models, Genetic; Phycomyces; Polytene Chromosomes; RNA Interference; T-Phages; Tetrahymena
PubMed: 31189909
DOI: 10.1038/s41437-019-0191-5 -
Chromosoma Jun 2019The fourth chromosome smallest in the genome of Drosophila melanogaster differs from other chromosomes in many ways. It has high repeat density in conditions of a large...
The fourth chromosome smallest in the genome of Drosophila melanogaster differs from other chromosomes in many ways. It has high repeat density in conditions of a large number of active genes. Gray bands represent a significant part of this polytene chromosome. Specific proteins including HP1a, POF, and dSETDB1 establish the epigenetic state of this unique chromatin domain. In order to compare maps of localization of genes, bands, and chromatin types of the fourth chromosome, we performed FISH analysis of 38 probes chosen according to the model of four chromatin types. It allowed clarifying the dot chromosome cytological map consisting of 16 loose gray bands, 11 dense black bands, and 26 interbands. We described the relation between chromatin states and bands. Open aquamarine chromatin mostly corresponds to interbands and it contains 5'UTRs of housekeeping genes. Their coding parts are embedded in gray bands substantially composed of lazurite chromatin of intermediate compaction. Polygenic black bands contain most of dense ruby chromatin, and also some malachite and lazurite. Having an accurate map of the fourth chromosome bands and its correspondence to physical map, we found that DNase I hypersensitivity sites, ORC2 protein, and P-elements are mainly located in open aquamarine chromatin, while element 1360, characteristic of the fourth chromosome, occupies band chromatin types. POF and HP1a proteins providing special organization of this chromosome are mostly located in aquamarine and lazurite chromatin. In general, band organization of the fourth chromosome shares the features of the whole Drosophila genome.
Topics: Animals; Chromosome Banding; Chromosomes, Insect; Drosophila Proteins; Drosophila melanogaster; Female; Genome, Insect; Male; Polytene Chromosomes
PubMed: 31041520
DOI: 10.1007/s00412-019-00703-x -
ELife Mar 2019UPF1 is an RNA helicase that is required for nonsense-mediated mRNA decay (NMD) in eukaryotes, and the predominant view is that UPF1 mainly operates on the 3'UTRs of...
UPF1 is an RNA helicase that is required for nonsense-mediated mRNA decay (NMD) in eukaryotes, and the predominant view is that UPF1 mainly operates on the 3'UTRs of mRNAs that are directed for NMD in the cytoplasm. Here we offer evidence, obtained from , that UPF1 constantly moves between the nucleus and cytoplasm by a mechanism that requires its RNA helicase activity. UPF1 is associated, genome-wide, with nascent RNAs at most of the active Pol II transcription sites and at some Pol III-transcribed genes, as demonstrated microscopically on the polytene chromosomes of salivary glands and by ChIP-seq analysis in S2 cells. Intron recognition seems to interfere with association and translocation of UPF1 on nascent pre-mRNAs, and cells depleted of UPF1 show defects in the release of mRNAs from transcription sites and their export from the nucleus.
Topics: Animals; Drosophila; Drosophila Proteins; RNA Helicases; RNA, Messenger; Transcription, Genetic
PubMed: 30907728
DOI: 10.7554/eLife.41444 -
BMC Genetics Mar 2019Expression of the CNDP2 gene is frequently up- or down-regulated in different types of human cancers. However, how the product of this gene is involved in cell growth...
BACKGROUND
Expression of the CNDP2 gene is frequently up- or down-regulated in different types of human cancers. However, how the product of this gene is involved in cell growth and proliferation is poorly understood. Moreover, our knowledge of the functions of the CNDP2 orthologs in well-established model organisms is scarce. In particular, the function of the D. melanogaster ortholog of CNDP2, encoded by the CG17337 gene (hereafter referred to as dCNDP2), is still unknown.
RESULTS
This study was aimed at developing a set of genetic and molecular tools to study the roles of dCNDP2. We generated a dCNDP2 null mutation (hereafter ∆dCNDP2) using CRISPR/Cas9-mediated homologous recombination (HR) and found that the ∆dCNDP2 mutants are homozygous viable, morphologically normal and fertile. We also generated transgenic fly lines expressing eGFP-tagged and non-tagged dCNDP2 protein, all under the control of the UAS promoter, as well as polyclonal antibodies specific to dCNDP2. Using these tools, we demonstrate that only one of the two predicted dCNDP2 isoforms is expressed throughout the different tissues tested. dCNDP2 was detected in both the cytoplasm and the nucleus, and was found to be associated with multiple sites in the salivary gland polytene chromosomes.
CONCLUSIONS
The dCNDP2 gene is not essential for fly viability under standard laboratory conditions. The subcellular localization pattern of dCNDP2 suggests that this protein might have roles in both the cytoplasm and the nucleus. The genetic and molecular tools developed in this study will allow further functional characterization of the conserved CNDP2 protein using D. melanogaster as a model system.
Topics: Animals; Animals, Genetically Modified; Cell Line; Cell Proliferation; Drosophila melanogaster; Protein Isoforms
PubMed: 30885138
DOI: 10.1186/s12863-019-0726-z -
Genes Jan 2019Chromatin structure and its organization contributes to the proper regulation and timing of DNA replication. Yet, the precise mechanism by which chromatin contributes to...
Chromatin structure and its organization contributes to the proper regulation and timing of DNA replication. Yet, the precise mechanism by which chromatin contributes to DNA replication remains incompletely understood. This is particularly true for cell types that rely on polyploidization as a developmental strategy for growth and high biosynthetic capacity. During larval development, cells of the salivary gland undergo endoreplication, repetitive rounds of DNA synthesis without intervening cell division, resulting in ploidy values of ~1350C. S phase of these endocycles displays a reproducible pattern of early and late replicating regions of the genome resulting from the activity of the same replication initiation factors that are used in diploid cells. However, unlike diploid cells, the latest replicating regions of polyploid salivary gland genomes, composed primarily of pericentric heterochromatic enriched in H3K9 methylation, are not replicated each endocycle, resulting in under-replicated domains with reduced ploidy. Here, we employ a histone gene replacement strategy in to demonstrate that mutation of a histone residue important for heterochromatin organization and function (H3K9) but not mutation of a histone residue important for euchromatin function (H4K16), disrupts proper endoreplication in salivary gland polyploid genomes thereby leading to DNA copy gain in pericentric heterochromatin. These findings reveal that H3K9 is necessary for normal levels of under-replication of pericentric heterochromatin and suggest that under-replication at pericentric heterochromatin is mediated through H3K9 methylation.
Topics: Animals; Centromere; DNA Replication; Drosophila melanogaster; Heterochromatin; Histones; Methylation; Polytene Chromosomes; Protein Processing, Post-Translational; Salivary Glands
PubMed: 30700014
DOI: 10.3390/genes10020093 -
Epigenetics & Chromatin Jan 2019Boundaries in the Drosophila bithorax complex delimit autonomous regulatory domains that activate the parasegment (PS)-specific expression of homeotic genes. The Fab-7...
BACKGROUND
Boundaries in the Drosophila bithorax complex delimit autonomous regulatory domains that activate the parasegment (PS)-specific expression of homeotic genes. The Fab-7 boundary separates the iab-6 and iab-7 regulatory domains that control Abd-B expression in PS11 and PS12. This boundary is composed of multiple functionally redundant elements and has two key activities: it blocks crosstalk between iab-6 and iab-7 and facilitates boundary bypass.
RESULTS
Here, we have used a structure-function approach to elucidate the biochemical properties and the in vivo activities of a conserved BEN domain protein, Insensitive, that is associated with Fab-7. Our biochemical studies indicate that in addition to the C-terminal BEN DNA-binding domain, Insv has two domains that mediate multimerization: one is a coiled-coil domain in the N-terminus, and the other is next to the BEN domain. These multimerization domains enable Insv to bind simultaneously to two canonical 8-bp recognition motifs, as well as to a ~ 100-bp non-canonical recognition sequence. They also mediate the assembly of higher-order multimers in the presence of DNA. Transgenic proteins lacking the N-terminal coiled-coil domain are compromised for boundary function in vivo. We also show that Insv interacts directly with CP190, a protein previously implicated in the boundary functions of several DNA-binding proteins, including Su(Hw) and dCTCF. While CP190 interaction is required for Insv binding to a subset of sites on polytene chromosomes, it has only a minor role in the boundary activity of Insv in the context of Fab-7.
CONCLUSIONS
The subdivision of eukaryotic chromosomes into discrete topological domains depends upon the pairing of boundary elements. In flies, pairing interactions are specific and typically orientation dependent. They occur in cis between neighboring heterologous boundaries, and in trans between homologous boundaries. One potential mechanism for ensuring pairing-interaction specificity is the use of sequence-specific DNA-binding proteins that can bind simultaneously with two or more recognition sequences. Our studies indicate that Insv can assemble into a multivalent DNA-binding complex and that the N-terminal Insv multimerization domain is critical for boundary function.
Topics: Animals; Binding Sites; Co-Repressor Proteins; Drosophila; Drosophila Proteins; Insulator Elements; Microtubule-Associated Proteins; Nuclear Proteins; Protein Binding; Protein Multimerization
PubMed: 30602385
DOI: 10.1186/s13072-018-0249-2 -
Cells Nov 2018Polynucleotide chains obeying Watson-Crick pairing are apt to form non-canonical complexes such as triple-helical nucleic acids. From early characterization in vitro,...
Polynucleotide chains obeying Watson-Crick pairing are apt to form non-canonical complexes such as triple-helical nucleic acids. From early characterization in vitro, their occurrence in vivo has been strengthened by increasing evidence, although most remain circumstantial particularly for triplex DNA. Here, different approaches were employed to specify triple-stranded DNA sequences in the chromosomes. Antibodies to triplex nucleic acids, previously characterized, bind to centromeric regions of mitotic chromosomes and also to the polytene section 59E of mutant strains carrying the allele, indicating that AAGAG tandem satellite repeats are triplex-forming sequences. The satellite probe hybridized to AAGAG-containing regions omitting chromosomal DNA denaturation, as expected, for the intra-molecular triplex DNA formation model in which single-stranded DNA coexists with triplexes. In addition, Thiazole Orange, previously described as capable of reproducing results obtained by antibodies to triple-helical DNA, binds to AAGAG repeats in situ thus validating both detection methods. Unusual phenotype and nuclear structure exhibited by correlate with the non-canonical conformation of tandem satellite arrays. From the approaches that lead to the identification of triple-helical DNA in chromosomes, facilities particularly provided by Thiazole Orange use may broaden the investigation on the occurrence of triplex DNA in eukaryotic genomes.
PubMed: 30477098
DOI: 10.3390/cells7120227 -
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 -
Nature Communications Oct 2018TDP-43 is the major disease protein associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-TDP)....
TDP-43 is the major disease protein associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-TDP). Here we identify the transcriptional elongation factor Ell-a shared component of little elongation complex (LEC) and super elongation complex (SEC)-as a strong modifier of TDP-43-mediated neurodegeneration. Our data indicate select targets of LEC and SEC become upregulated in the fly ALS/FTLD-TDP model. Among them, U12 snRNA and a stress-induced long non-coding RNA Hsrω, functionally contribute to TDP-43-mediated degeneration. We extend the findings of Hsrω, which we identify as a chromosomal target of TDP-43, to show that the human orthologue Sat III is elevated in a human cellular disease model and FTLD-TDP patient tissue. We further demonstrate an interaction between TDP-43 and human ELL2 by co-immunoprecipitation from human cells. These findings reveal important roles of Ell-complexes LEC and SEC in TDP-43-associated toxicity, providing potential therapeutic insight for TDP-43-associated neurodegeneration.
Topics: Animals; DNA-Binding Proteins; Drosophila Proteins; Drosophila melanogaster; Female; Genetic Loci; HEK293 Cells; Humans; Male; Models, Biological; Nuclear Proteins; Polytene Chromosomes; Protein Binding; RNA, Small Nuclear; RNA, Untranslated; Transcription Elongation, Genetic; Transcription Factors; Transcriptional Elongation Factors
PubMed: 30353006
DOI: 10.1038/s41467-018-06543-0