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Olga I. Podgornaya - One of the best experts on this subject based on the ideXlab platform.
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Mouse chromocenters DNA content: sequencing and in silico analysis
BMC Genomics, 2018Co-Authors: Dmitrii I. Ostromyshenskii, Ekaterina N. Chernyaeva, Inna S. Kuznetsova, Olga I. PodgornayaAbstract:BackgroundChromocenters are defined as a punctate condensed blocks of chromatin in the interphase cell nuclei of certain cell types with unknown biological significance. In recent years a progress in revealing of chromocenters protein content has been made although the details of DNA content within constitutive Heterochromatin still remain unclear. It is known that these regions are enriched in tandem repeats (TR) and transposable elements. Quick improvement of genome sequencing does not help to assemble the heterochromatic regions due to lack of appropriate bioinformatics techniques.ResultsChromocenters DNA have been isolated by a biochemical approach from mouse liver cells nuclei and sequenced on the Illumina MiSeq resulting in ChrmC dataset. Analysis of ChrmC dataset by the bioinformatics tools available revealed that the major component of chromocenter DNA are TRs: ~ 66% MaSat and ~ 4% MiSat. Other previously classified TR families constitute ~ 1% of ChrmC dataset. About 6% of chromocenters DNA are mostly unannotated sequences. In the contigs assembled with IDBA_UD there are many fragments of heterochromatic Y-chromosome, rDNA and other pseudo-genes and non-coding DNA. A protein coding sfi1 homolog gene fragment was also found in contigs. The Sfi1 homolog gene is located on the chromosome 11 in the reference genome very close to the Golden Pass Gap (a ~ 3 Mb empty region reserved to the pericentromeric region) and proves the purity of chromocenters isolation. The second major fraction are non-LTR retroposons (SINE and LINE) with overwhelming majority of LINE - ~ 11% of ChrmC. Most of the LINE fragments are from the ~ 2 kb region at the end of the 2nd ORF and its’ flanking region. The precise LINEs’ segment of ~ 2 kb is the necessary mouse constitutive heterohromatin component together with TR. The third most abundant fraction are ERVs. The ERV distribution in chromocenters differs from the whole genome: IAP (ERV2 class) is the most numerous in ChrmC while MaLR (ERV3 class) prevails in the reference genome. IAP and its LTR also prevail in TR containing contigs extracted from the WGS dataset. In silico prediction of IAP and LINE fragments in chromocenters was confirmed by direct fluorescent in situ hybridization (FISH).ConclusionOur data of chromocenters’ DNA (ChrmC) sequencing demonstrate that IAP with LTR and a precise ~ 2 kb fragment of LINE represent a substantial fraction of mouse chromocenters (constitutive heteroсhromatin) along with TRs.
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mouse chromocenters dna content sequencing and in silico analysis
BMC Genomics, 2018Co-Authors: Dmitrii I. Ostromyshenskii, Ekaterina N. Chernyaeva, Inna S. Kuznetsova, Olga I. PodgornayaAbstract:Chromocenters are defined as a punctate condensed blocks of chromatin in the interphase cell nuclei of certain cell types with unknown biological significance. In recent years a progress in revealing of chromocenters protein content has been made although the details of DNA content within constitutive Heterochromatin still remain unclear. It is known that these regions are enriched in tandem repeats (TR) and transposable elements. Quick improvement of genome sequencing does not help to assemble the heterochromatic regions due to lack of appropriate bioinformatics techniques. Chromocenters DNA have been isolated by a biochemical approach from mouse liver cells nuclei and sequenced on the Illumina MiSeq resulting in ChrmC dataset. Analysis of ChrmC dataset by the bioinformatics tools available revealed that the major component of chromocenter DNA are TRs: ~ 66% MaSat and ~ 4% MiSat. Other previously classified TR families constitute ~ 1% of ChrmC dataset. About 6% of chromocenters DNA are mostly unannotated sequences. In the contigs assembled with IDBA_UD there are many fragments of heterochromatic Y-chromosome, rDNA and other pseudo-genes and non-coding DNA. A protein coding sfi1 homolog gene fragment was also found in contigs. The Sfi1 homolog gene is located on the chromosome 11 in the reference genome very close to the Golden Pass Gap (a ~ 3 Mb empty region reserved to the pericentromeric region) and proves the purity of chromocenters isolation. The second major fraction are non-LTR retroposons (SINE and LINE) with overwhelming majority of LINE - ~ 11% of ChrmC. Most of the LINE fragments are from the ~ 2 kb region at the end of the 2nd ORF and its’ flanking region. The precise LINEs’ segment of ~ 2 kb is the necessary mouse constitutive heterohromatin component together with TR. The third most abundant fraction are ERVs. The ERV distribution in chromocenters differs from the whole genome: IAP (ERV2 class) is the most numerous in ChrmC while MaLR (ERV3 class) prevails in the reference genome. IAP and its LTR also prevail in TR containing contigs extracted from the WGS dataset. In silico prediction of IAP and LINE fragments in chromocenters was confirmed by direct fluorescent in situ hybridization (FISH). Our data of chromocenters’ DNA (ChrmC) sequencing demonstrate that IAP with LTR and a precise ~ 2 kb fragment of LINE represent a substantial fraction of mouse chromocenters (constitutive heteroсhromatin) along with TRs.
Shiv I S Grewal - One of the best experts on this subject based on the ideXlab platform.
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asf1 hira facilitate global histone deacetylation and associate with hp1 to promote nucleosome occupancy at heterochromatic loci
Molecular Cell, 2011Co-Authors: Kenichi Yamane, Kenichi Noma, Martin Zofall, Takeshi Mizuguchi, Bowen Cui, Shiv I S GrewalAbstract:Heterochromatin impacts various nuclear processes by providing a recruiting platform for diverse chromosomal proteins. In fission yeast, HP1 proteins Chp2 and Swi6, which bind to methylated histone H3 lysine 9, associate with SHREC (Snf2/HDAC repressor complex) and Clr6 histone deacetylases (HDACs) involved in heterochromatic silencing. However, heterochromatic silencing machinery is not fully defined. We describe a histone chaperone complex containing Asf1 and HIRA that spreads across silenced domains via its association with Swi6 to enforce transcriptional silencing. Asf1 functions in concert with a Clr6 HDAC complex to silence heterochromatic repeats, and it suppresses antisense transcription by promoting histone deacetylation. Furthermore, we demonstrate that Asf1 and SHREC facilitate nucleosome occupancy at heterochromatic regions but TFIIIC transcription factor binding sites within boundary elements are refractory to these factors. These analyses uncover a role for Asf1 in global histone deacetylation and suggest that HP1-associated histone chaperone promotes nucleosome occupancy to assemble repressive Heterochromatin.
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hulc a histone h2b ubiquitinating complex modulates Heterochromatin independent of histone methylation in fission yeast
Journal of Biological Chemistry, 2007Co-Authors: Martin Zofall, Shiv I S GrewalAbstract:Heterochromatin in fission yeast is targeted dynamically by opposing chromatin-modifying activities capable of alleviating or promoting transcriptional gene silencing. In this study, we report the biochemical and genetic characterization of a ubiquitin-conjugating enzyme Rhp6 (a homolog of budding yeast Rad6), which has been shown to negatively affect stability of heterochromatic structures. We show that Rhp6 is a component of the multisubunit protein complex (termed HULC) that also contains two RING finger proteins Rfp1 and Rfp2, sharing homology with budding yeast Bre1 protein and a unique serine-rich protein Shf1. HULC is required for ubiquitination of histone H2B at lysine 119 (H2B-K119), and it localizes to heterochromatic sequences. Moreover, our analyses suggest that Rhp6-induced changes in heterochromatic silencing are mediated predominantly through H2B ubiquitination (ubH2B), and they correlate with increased RNA polymerase II levels at repeat elements embedded within Heterochromatin domains. Interestingly, heterochromatic derepression caused by Rhp6 occurs independently of the involvement of HULC subunits and ubH2B in methylation of histone H3 at lysine 4 (H3K4me). These analyses implicate ubH2B in modulation of Heterochromatin, which has important implications for dynamics and many functions associated with heterochromatic structures.
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rits acts in cis to promote rna interference mediated transcriptional and post transcriptional silencing
Nature Genetics, 2004Co-Authors: Kenichi Noma, Martin Zofall, Tomoyasu Sugiyama, Hugh P Cam, Andre Verdel, Songtao Jia, Danesh Moazed, Shiv I S GrewalAbstract:RNA interference is a conserved mechanism by which double-stranded RNA is processed into short interfering RNAs (siRNAs) that can trigger both post-transcriptional and transcriptional gene silencing. In fission yeast, the RNA-induced initiation of transcriptional gene silencing (RITS) complex contains Dicer-generated siRNAs and is required for heterochromatic silencing. Here we show that RITS components, including Argonaute protein, bind to all known heterochromatic loci. At the mating-type region, RITS is recruited to the centromere-homologous repeat cenH in a Dicer-dependent manner, whereas the spreading of RITS across the entire 20-kb silenced domain, as well as its subsequent maintenance, requires Heterochromatin machinery including Swi6 and occurs even in the absence of Dicer. Furthermore, our analyses suggest that RNA interference machinery operates in cis as a stable component of heterochromatic domains with RITS tethered to silenced loci by methylation of histone H3 at Lys9. This tethering promotes the processing of transcripts and generation of additional siRNAs for Heterochromatin maintenance.
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recruitment of cohesin to heterochromatic regions by swi6 hp1 in fission yeast
Nature Cell Biology, 2002Co-Authors: Nobuhiro Nonaka, Shiv I S Grewal, Tomoya S Kitajima, Shihori Yokobayashi, Guoping Xiao, Masayuki Yamamoto, Yoshinori WatanabeAbstract:Fission yeast centromeres, like those of higher eukaryotes, are composed of repeated DNA structures and associated Heterochromatin protein complexes, that have a critical function in the faithful segregation of chromosomes during cell division1,2,3. Cohesin protein complexes, which are essential for sister-chromatid cohesion and proper chromosome segregation, are enriched at centromeric repeats4,5. We have identified a functional and physical link between Heterochromatin and cohesin. We find that the preferential localization of cohesins at the centromeric repeats is dependent on Swi6, a conserved Heterochromatin protein that is required for proper kinetochore function. Cohesin is also enriched at the mating-type heterochromatic region in a manner that depends on Swi6 and is required to preserve the genomic integrity of this locus. We provide evidence that a cohesin subunit Psc3 interacts with Swi6 and its mouse homologue HP1. These data define a conserved function of Swi6/HP1 in recruitment of cohesin to heterochromatic regions, promoting the proper segregation of chromosomes.
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transitions in distinct histone h3 methylation patterns at the Heterochromatin domain boundaries
Science, 2001Co-Authors: Kenichi Noma, David C Allis, Shiv I S GrewalAbstract:Eukaryotic genomes are organized into discrete structural and functional chromatin domains. Here, we show that distinct site-specific histone H3 methylation patterns define euchromatic and heterochromatic chromosomal domains within a 47-kilobase region of the mating-type locus in fission yeast. H3 methylated at lysine 9 (H3 Lys9), and its interacting Swi6 protein, are strictly localized to a 20-kilobase silent heterochromatic interval. In contrast, H3 methylated at lysine 4 (H3 Lys4) is specific to the surrounding euchromatic regions. Two inverted repeats flanking the silent interval serve as boundary elements to mark the borders between Heterochromatin and euchromatin. Deletions of these boundary elements lead to spreading of H3 Lys9 methylation and Swi6 into neighboring sequences. Furthermore, the H3 Lys9methylation and corresponding Heterochromatin-associated complexes prevent H3 Lys4 methylation in the silent domain.
Dmitrii I. Ostromyshenskii - One of the best experts on this subject based on the ideXlab platform.
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Mouse chromocenters DNA content: sequencing and in silico analysis
BMC Genomics, 2018Co-Authors: Dmitrii I. Ostromyshenskii, Ekaterina N. Chernyaeva, Inna S. Kuznetsova, Olga I. PodgornayaAbstract:BackgroundChromocenters are defined as a punctate condensed blocks of chromatin in the interphase cell nuclei of certain cell types with unknown biological significance. In recent years a progress in revealing of chromocenters protein content has been made although the details of DNA content within constitutive Heterochromatin still remain unclear. It is known that these regions are enriched in tandem repeats (TR) and transposable elements. Quick improvement of genome sequencing does not help to assemble the heterochromatic regions due to lack of appropriate bioinformatics techniques.ResultsChromocenters DNA have been isolated by a biochemical approach from mouse liver cells nuclei and sequenced on the Illumina MiSeq resulting in ChrmC dataset. Analysis of ChrmC dataset by the bioinformatics tools available revealed that the major component of chromocenter DNA are TRs: ~ 66% MaSat and ~ 4% MiSat. Other previously classified TR families constitute ~ 1% of ChrmC dataset. About 6% of chromocenters DNA are mostly unannotated sequences. In the contigs assembled with IDBA_UD there are many fragments of heterochromatic Y-chromosome, rDNA and other pseudo-genes and non-coding DNA. A protein coding sfi1 homolog gene fragment was also found in contigs. The Sfi1 homolog gene is located on the chromosome 11 in the reference genome very close to the Golden Pass Gap (a ~ 3 Mb empty region reserved to the pericentromeric region) and proves the purity of chromocenters isolation. The second major fraction are non-LTR retroposons (SINE and LINE) with overwhelming majority of LINE - ~ 11% of ChrmC. Most of the LINE fragments are from the ~ 2 kb region at the end of the 2nd ORF and its’ flanking region. The precise LINEs’ segment of ~ 2 kb is the necessary mouse constitutive heterohromatin component together with TR. The third most abundant fraction are ERVs. The ERV distribution in chromocenters differs from the whole genome: IAP (ERV2 class) is the most numerous in ChrmC while MaLR (ERV3 class) prevails in the reference genome. IAP and its LTR also prevail in TR containing contigs extracted from the WGS dataset. In silico prediction of IAP and LINE fragments in chromocenters was confirmed by direct fluorescent in situ hybridization (FISH).ConclusionOur data of chromocenters’ DNA (ChrmC) sequencing demonstrate that IAP with LTR and a precise ~ 2 kb fragment of LINE represent a substantial fraction of mouse chromocenters (constitutive heteroсhromatin) along with TRs.
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mouse chromocenters dna content sequencing and in silico analysis
BMC Genomics, 2018Co-Authors: Dmitrii I. Ostromyshenskii, Ekaterina N. Chernyaeva, Inna S. Kuznetsova, Olga I. PodgornayaAbstract:Chromocenters are defined as a punctate condensed blocks of chromatin in the interphase cell nuclei of certain cell types with unknown biological significance. In recent years a progress in revealing of chromocenters protein content has been made although the details of DNA content within constitutive Heterochromatin still remain unclear. It is known that these regions are enriched in tandem repeats (TR) and transposable elements. Quick improvement of genome sequencing does not help to assemble the heterochromatic regions due to lack of appropriate bioinformatics techniques. Chromocenters DNA have been isolated by a biochemical approach from mouse liver cells nuclei and sequenced on the Illumina MiSeq resulting in ChrmC dataset. Analysis of ChrmC dataset by the bioinformatics tools available revealed that the major component of chromocenter DNA are TRs: ~ 66% MaSat and ~ 4% MiSat. Other previously classified TR families constitute ~ 1% of ChrmC dataset. About 6% of chromocenters DNA are mostly unannotated sequences. In the contigs assembled with IDBA_UD there are many fragments of heterochromatic Y-chromosome, rDNA and other pseudo-genes and non-coding DNA. A protein coding sfi1 homolog gene fragment was also found in contigs. The Sfi1 homolog gene is located on the chromosome 11 in the reference genome very close to the Golden Pass Gap (a ~ 3 Mb empty region reserved to the pericentromeric region) and proves the purity of chromocenters isolation. The second major fraction are non-LTR retroposons (SINE and LINE) with overwhelming majority of LINE - ~ 11% of ChrmC. Most of the LINE fragments are from the ~ 2 kb region at the end of the 2nd ORF and its’ flanking region. The precise LINEs’ segment of ~ 2 kb is the necessary mouse constitutive heterohromatin component together with TR. The third most abundant fraction are ERVs. The ERV distribution in chromocenters differs from the whole genome: IAP (ERV2 class) is the most numerous in ChrmC while MaLR (ERV3 class) prevails in the reference genome. IAP and its LTR also prevail in TR containing contigs extracted from the WGS dataset. In silico prediction of IAP and LINE fragments in chromocenters was confirmed by direct fluorescent in situ hybridization (FISH). Our data of chromocenters’ DNA (ChrmC) sequencing demonstrate that IAP with LTR and a precise ~ 2 kb fragment of LINE represent a substantial fraction of mouse chromocenters (constitutive heteroсhromatin) along with TRs.
Joel C. Eissenberg - One of the best experts on this subject based on the ideXlab platform.
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Heterochromatin and euchromatin
eLS, 2014Co-Authors: Joel C. Eissenberg, Sarah C R ElginAbstract:Eukaryotes are characterised by the extensive packaging of their genomes, initially in a nucleosomal array, and further into higher order domains. Differential packaging is used as a mechanism of gene regulation, with stable silencing of large domains achieved by packaging the deoxyribonucleic acid (DNA) into a heterochromatic structure. Chromosome rearrangements and transgene insertions that misplace euchromatic genes near or within the Heterochromatin result in silencing of the euchromatic genes, testifying to a distinct Heterochromatin assembly that can antagonise transcription. Most heterochromatic regions are rich in repetitious sequences, frequently derived from transposable elements, and such packaging helps to silence such elements. Domains of Heterochromatin and euchromatin are defined by specific covalent modifications of histones and, in some cases, DNA, as well as by associations with a specific subset of nonhistone chromosomal proteins. Chromosomal domains may be targeted for Heterochromatin formation by specific noncoding ribonucleic acids (RNAs). Key Concepts: The chromatin of eukaryotes is differentially packaged into domains of euchromatin and Heterochromatin. Displacing a euchromatic gene to near or within the Heterochromatin often results in silencing of the euchromatic gene in some of the cells in which it should be active, resulting in a variegating phenotype. Euchromatin and Heterochromatin are distinguishable biochemically by different covalent modifications of histones (and in some cases DNA) and by distinct nonhistone proteins. Members of the HP1a chromo domain protein family bind methylated histone H3 and interact with the H3K9 histone methyltransferase to organise transcriptionally repressive Heterochromatin. The piRNA pathway is implicated in targeting transposon silencing through local Heterochromatin formation. Keywords: euchromatin; Heterochromatin; histone acetylation; HP1a; position-effect variegation; SIR2
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Heterochromatin protein 1 is required for the normal expression of two Heterochromatin genes in drosophila
Genetics, 2000Co-Authors: Peter Emtage, Brenda J Duyf, Arthur J Hilliker, Joel C. EissenbergAbstract:The Su(var)2-5 locus, an essential gene in Drosophila, encodes the Heterochromatin-associated protein HP1. Here, we show that the Su(var)2-5 lethal period is late third instar. Maternal HP1 is still detectable in first instar larvae, but disappears by third instar, suggesting that developmentally late lethality is probably the result of depletion of maternal protein. We demonstrate that heterochromatic silencing of a normally euchromatic reporter gene is completely lost by third instar in zygotically HP1 mutant larvae, implying a defect in Heterochromatin-mediated transcriptional regulation in these larvae. However, expression of the essential heterochromatic genes rolled and light is reduced in Su(var)2-5 mutant larvae, suggesting that reduced expression of essential heterochromatic genes could underlie the recessive lethality of Su(var)2-5 mutations. These results also show that HP1, initially recognized as a transcriptional silencer, is required for the normal transcriptional activation of heterochromatic genes.
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functional analysis of the chromo domain of hp1
The EMBO Journal, 1995Co-Authors: J S Platero, T. Hartnett, Joel C. EissenbergAbstract:Abstract Heterochromatin protein 1 (HP1) is a non-histone chromosomal protein in Drosophila with dosage-dependent effects on Heterochromatin-mediated gene silencing. An evolutionarily conserved amino acid sequence in the N-terminal half of HP1 (the 'chromo domain') shares > 60% sequence identity with a motif found in the Polycomb protein, a silencer of homeotic genes. We report here that point mutations in the HP1 chromo domain abolish the ability of HP1 to promote gene silencing. We show that the HP1 chromo domain, like the Polycomb chromo domain, has chromosome binding activity, but to distinct chromosomal sites. We constructed a chimeric HP1-Polycomb protein, consisting of the chromo domain of Polycomb in the context of HP1, and show that it binds to both Heterochromatin and Polycomb binding sites in polytene chromosomes. In flies expressing chimeric HP1-Polycomb protein, endogenous HP1 is mislocalized to Polycomb binding sites, and endogenous polycomb is misdirected to the heterochromatic chromocenter, suggesting that both proteins are recruited to their distinct chromosomal binding sites through protein-protein contacts. Chimeric HP1-Polycomb protein expression in transgenic flies promotes Heterochromatin-mediated gene silencing, supporting the view that the chromo domain homology reflects a common mechanistic basis for homeotic and heterochromatic silencing.
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The Heterochromatin-associated protein HP-1 is an essential protein in Drosophila with dosage-dependent effects on position-effect variegation.
Genetics, 1992Co-Authors: Joel C. Eissenberg, Gunter Reuter, G D Morris, T. HartnettAbstract:Chromosome rearrangements which place euchromatic genes adjacent to a heterochromatic breakpoint frequently result in gene repression (position-effect variegation). This repression is thought to reflect the spreading of a heterochromatic structure into neighboring euchromatin. Two allelic dominant suppressors of position-effect variegation were found to contain mutations within the gene encoding the Heterochromatin-specific chromosomal protein HP-1. The site of mutation for each allele is given: one converts Lys169 into a nonsense (ochre) codon, while the other is a frameshift after Ser10. In flies heterozygous for one of the mutant alleles (Su(var)2-504), a truncated HP-1 protein was detectable by Western blot analysis. An HP-1 minigene, consisting of HP-1 cDNA under the control of an Hsp70 heat-inducible promoter, was transduced into flies by P element-mediated germ line transformation. Heat-shock driven expression of this minigene results in elevated HP-1 protein level and enhancement of position-effect variegation. Levels of variegating gene expression thus appear to depend upon the level of expression of a Heterochromatin-specific protein. The implications of these observations for mechanism of heterochromatic position effects and Heterochromatin function are discussed.
Irene Chiolo - One of the best experts on this subject based on the ideXlab platform.
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arp2 3 and unc45 maintain Heterochromatin stability in drosophila polytene chromosomes
bioRxiv, 2019Co-Authors: George Dialynas, Laetitia Delabaere, Irene ChioloAbstract:Repairing DNA double-strand breaks (DSBs) is particularly challenging in pericentromeric Heterochromatin, where the abundance of repeated sequences exacerbates the risk of ectopic recombination. In Drosophila Kc cells, accurate homologous recombination (HR) repair of heterochromatic DSBs 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 myosins9 ability to walk along them. Conserved mechanisms enable the relocalization of heterochromatic DSBs in mouse cells, and their defects lead to massive ectopic recombination in Heterochromatin and chromosome rearrangements. In Drosophila 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 DSBs 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 in this structure. 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 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.
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nuclear f actin and myosins drive relocalization of heterochromatic breaks
Nature, 2018Co-Authors: Christophe P Caridi, Laetitia Delabaere, Nuno Amaral, Carla Dagostino, Taehyu Ryu, Grzegorz Zapotoczny, Varand Y Khodaverdia, Alesandra R Rau, Irene ChioloAbstract:Heterochromatin mainly comprises repeated DNA sequences that are prone to ectopic recombination. In Drosophila cells, ‘safe’ repair of heterochromatic double-strand breaks by homologous recombination relies on the relocalization of repair sites to the nuclear periphery before strand invasion. The mechanisms responsible for this movement were unknown. Here we show that relocalization occurs by directed motion along nuclear actin filaments assembled at repair sites by the Arp2/3 complex. Relocalization requires nuclear myosins associated with the Heterochromatin repair complex Smc5/6 and the myosin activator Unc45, which is recruited to repair sites by Smc5/6. ARP2/3, actin nucleation and myosins also relocalize heterochromatic double-strand breaks in mouse cells. Defects in this pathway result in impaired Heterochromatin repair and chromosome rearrangements. These findings identify de novo nuclear actin filaments and myosins as effectors of chromatin dynamics for Heterochromatin repair and stability in multicellular eukaryotes. Relocalization of heterochromatic double-strand breaks to the nuclear periphery in Drosophila cells occurs via directed motions driven by nuclear actin filaments and myosins activated by the Smc5/6 complex.
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Heterochromatic breaks move to the nuclear periphery to continue recombinational repair
Nature Cell Biology, 2015Co-Authors: Taehyun Ryu, Laetitia Delabaere, Brett N. Spatola, Katherine Bowlin, Hannah Hopp, Ryan Kunitake, Gary H. Karpen, Irene ChioloAbstract:Heterochromatin mostly comprises repeated sequences prone to harmful ectopic recombination during double-strand break (DSB) repair. In Drosophila cells, 'safe' homologous recombination (HR) repair of heterochromatic breaks relies on a specialized pathway that relocalizes damaged sequences away from the Heterochromatin domain before strand invasion. Here we show that heterochromatic DSBs move to the nuclear periphery to continue HR repair. Relocalization depends on nuclear pores and inner nuclear membrane proteins (INMPs) that anchor repair sites to the nuclear periphery through the Smc5/6-interacting proteins STUbL/RENi. Both the initial block to HR progression inside the Heterochromatin domain, and the targeting of repair sites to the nuclear periphery, rely on SUMO and SUMO E3 ligases. This study reveals a critical role for SUMOylation in the spatial and temporal regulation of HR repair in Heterochromatin, and identifies the nuclear periphery as a specialized site for Heterochromatin repair in a multicellular eukaryote.
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double strand breaks in Heterochromatin move outside of a dynamic hp1a domain to complete recombinational repair
Cell, 2011Co-Authors: Irene Chiolo, Gary H. Karpen, Aki Minoda, Serafin U Colmenares, Aris Polyzos, Sylvain V CostesAbstract:Double-strand breaks (DSBs) in heterochromatic repetitive DNAs pose significant threats to genome integrity, but information about how such lesions are processed and repaired is sparse. We observe dramatic expansion and dynamic protrusions of the Heterochromatin domain in response to ionizing radiation (IR) in Drosophila cells. We also find that heterochromatic DSBs are repaired by homologous recombination (HR) but with striking differences from euchromatin. Proteins involved in early HR events (resection) are rapidly recruited to DSBs within Heterochromatin. In contrast, Rad51, which mediates strand invasion, only associates with DSBs that relocalize outside of the domain. Heterochromatin expansion and relocalization of foci require checkpoint and resection proteins. Finally, the Smc5/6 complex is enriched in Heterochromatin and is required to exclude Rad51 from the domain and prevent abnormal recombination. We propose that the spatial and temporal control of DSB repair in Heterochromatin safeguards genome stability by preventing aberrant exchanges between repeats.