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1.
Sci Rep ; 14(1): 1324, 2024 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-38225252

RESUMO

Despite surging interest in space travel in recent decades, the impacts of prolonged, elevated exposure to galactic cosmic radiation (GCR) on human health remain poorly understood. This form of ionizing radiation causes significant changes to biological systems including damage to DNA structure by altering epigenetic phenotype with emphasis on DNA methylation. Building on previous work by Kennedy et al. (Sci Rep 8(1): 6709. 10.1038/S41598-018-24755-8), we evaluated spatial DNA methylation patterns triggered by high-LET (56Fe, 28Si) and low-LET (X-ray) radiation and the influence of chromosome positioning and epigenetic architecture in distinct radial layers of cell nucleus. Next, we validated our results using gene expression data of mice irradiated with simulated GCR and JAXA astronauts. We showed that primarily 56Fe induces a persistent DNA methylation increase whereas 28Si and X-ray induce a decrease DNA methylation which is not persistent with time. Moreover, we highlighted the role of nuclear chromatin architecture in cell response to external radiation. In summary, our study provides novel insights towards epigenetic and transcriptomic response as well as chromatin multidimensional structure influence on galactic cosmic radiation damage.


Assuntos
Radiação Cósmica , Humanos , Camundongos , Animais , Radiação Cósmica/efeitos adversos , Metilação de DNA , Posicionamento Cromossômico , Epigênese Genética , Cromatina/genética
2.
Nature ; 620(7972): 209-217, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37438531

RESUMO

The human genome functions as a three-dimensional chromatin polymer, driven by a complex collection of chromosome interactions1-3. Although the molecular rules governing these interactions are being quickly elucidated, relatively few proteins regulating this process have been identified. Here, to address this gap, we developed high-throughput DNA or RNA labelling with optimized Oligopaints (HiDRO)-an automated imaging pipeline that enables the quantitative measurement of chromatin interactions in single cells across thousands of samples. By screening the human druggable genome, we identified more than 300 factors that influence genome folding during interphase. Among these, 43 genes were validated as either increasing or decreasing interactions between topologically associating domains. Our findings show that genetic or chemical inhibition of the ubiquitous kinase GSK3A leads to increased long-range chromatin looping interactions in a genome-wide and cohesin-dependent manner. These results demonstrate the importance of GSK3A signalling in nuclear architecture and the use of HiDRO for identifying mechanisms of spatial genome organization.


Assuntos
Cromatina , Posicionamento Cromossômico , Cromossomos Humanos , Genoma Humano , Quinases da Glicogênio Sintase , Ensaios de Triagem em Larga Escala , Análise de Célula Única , Humanos , Cromatina/efeitos dos fármacos , Cromatina/genética , Cromatina/metabolismo , Posicionamento Cromossômico/efeitos dos fármacos , Cromossomos Humanos/efeitos dos fármacos , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , DNA/análise , DNA/metabolismo , Genoma Humano/efeitos dos fármacos , Genoma Humano/genética , Quinases da Glicogênio Sintase/antagonistas & inibidores , Quinases da Glicogênio Sintase/deficiência , Quinases da Glicogênio Sintase/genética , Ensaios de Triagem em Larga Escala/métodos , Interfase , Reprodutibilidade dos Testes , RNA/análise , RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Célula Única/métodos , Coesinas
3.
Nature ; 607(7919): 604-609, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35831506

RESUMO

Chromosome segregation errors during cell divisions generate aneuploidies and micronuclei, which can undergo extensive chromosomal rearrangements such as chromothripsis1-5. Selective pressures then shape distinct aneuploidy and rearrangement patterns-for example, in cancer6,7-but it is unknown whether initial biases in segregation errors and micronucleation exist for particular chromosomes. Using single-cell DNA sequencing8 after an error-prone mitosis in untransformed, diploid cell lines and organoids, we show that chromosomes have different segregation error frequencies that result in non-random aneuploidy landscapes. Isolation and sequencing of single micronuclei from these cells showed that mis-segregating chromosomes frequently also preferentially become entrapped in micronuclei. A similar bias was found in naturally occurring micronuclei of two cancer cell lines. We find that segregation error frequencies of individual chromosomes correlate with their location in the interphase nucleus, and show that this is highest for peripheral chromosomes behind spindle poles. Randomization of chromosome positions, Cas9-mediated live tracking and forced repositioning of individual chromosomes showed that a greater distance from the nuclear centre directly increases the propensity to mis-segregate. Accordingly, chromothripsis in cancer genomes9 and aneuploidies in early development10 occur more frequently for larger chromosomes, which are preferentially located near the nuclear periphery. Our findings reveal a direct link between nuclear chromosome positions, segregation error frequencies and micronucleus content, with implications for our understanding of tumour genome evolution and the origins of specific aneuploidies during development.


Assuntos
Aneuploidia , Posicionamento Cromossômico , Segregação de Cromossomos , Cromossomos , Proteína 9 Associada à CRISPR , Linhagem Celular , Linhagem Celular Tumoral , Segregação de Cromossomos/genética , Cromossomos/genética , Cromossomos/metabolismo , Cromotripsia , Crescimento e Desenvolvimento/genética , Humanos , Interfase , Micronúcleos com Defeito Cromossômico , Mitose , Neoplasias/genética , Neoplasias/patologia , Organoides/citologia , Organoides/metabolismo , Análise de Sequência de DNA , Análise de Célula Única
4.
Proc Natl Acad Sci U S A ; 118(34)2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34417301

RESUMO

Canonical Wnt signaling plays critical roles in development and tissue renewal by regulating ß-catenin target genes. Recent evidence showed that ß-catenin-independent Wnt signaling is also required for faithful execution of mitosis. However, the targets and specific functions of mitotic Wnt signaling still remain uncharacterized. Using phosphoproteomics, we identified that Wnt signaling regulates the microtubule depolymerase KIF2A during mitosis. We found that Dishevelled recruits KIF2A via its N-terminal and motor domains, which is further promoted upon LRP6 signalosome formation during cell division. We show that Wnt signaling modulates KIF2A interaction with PLK1, which is critical for KIF2A localization at the spindle. Accordingly, inhibition of basal Wnt signaling leads to chromosome misalignment in somatic cells and pluripotent stem cells. We propose that Wnt signaling monitors KIF2A activity at the spindle poles during mitosis to ensure timely chromosome alignment. Our findings highlight a function of Wnt signaling during cell division, which could have important implications for genome maintenance, notably in stem cells.


Assuntos
Segregação de Cromossomos , Cromossomos Humanos/genética , Cinesinas/metabolismo , Mitose , Fuso Acromático/fisiologia , Via de Sinalização Wnt , Posicionamento Cromossômico , Humanos , Cinesinas/genética
5.
Chromosoma ; 130(2-3): 163-175, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34231035

RESUMO

Chromosome territoriality is not random along the cell cycle and it is mainly governed by intrinsic chromosome factors and gene expression patterns. Conversely, very few studies have explored the factors that determine chromosome territoriality and its influencing factors during meiosis. In this study, we analysed chromosome positioning in murine spermatogenic cells using three-dimensionally fluorescence in situ hybridization-based methodology, which allows the analysis of the entire karyotype. The main objective of the study was to decipher chromosome positioning in a radial axis (all analysed germ-cell nuclei) and longitudinal axis (only spermatozoa) and to identify the chromosomal factors that regulate such an arrangement. Results demonstrated that the radial positioning of chromosomes during spermatogenesis was cell-type specific and influenced by chromosomal factors associated to gene activity. Chromosomes with specific features that enhance transcription (high GC content, high gene density and high numbers of predicted expressed genes) were preferentially observed in the inner part of the nucleus in virtually all cell types. Moreover, the position of the sex chromosomes was influenced by their transcriptional status, from the periphery of the nucleus when its activity was repressed (pachytene) to a more internal position when it is partially activated (spermatid). At pachytene, chromosome positioning was also influenced by chromosome size due to the bouquet formation. Longitudinal chromosome positioning in the sperm nucleus was not random either, suggesting the importance of ordered longitudinal positioning for the release and activation of the paternal genome after fertilisation.


Assuntos
Posicionamento Cromossômico , Meiose , Animais , Hibridização in Situ Fluorescente , Masculino , Camundongos , Cromossomos Sexuais , Espermatogênese/genética
6.
Mol Reprod Dev ; 88(5): 349-361, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33843103

RESUMO

This study investigated the effect of the antioxidant dieckol, a component of Ecklonia cava, on maturation and developmental competence of porcine oocytes exposed to oxidative stress in vitro. Oocytes were matured in in vitro maturation (IVM) medium containing various concentrations of dieckol. The blastocyst formation rate was highest in the 0.5 µM dieckol-treated (0.5 DEK) group. The reactive oxygen species level was decreased, and the level of glutathione and expression of antioxidant genes (NFE2L, SOD1, and SOD2) at metaphase II were increased in the 0.5 DEK group. Abnormal spindle organization and chromosome misalignment were prevented in the 0.5 DEK group. Expression of maternal markers (CCNB1 and MOS) and activity of p44/42 mitogen-activated protein kinase were increased in the 0.5 DEK group. After parthenogenetic activation, the total number of cells per blastocyst was increased and the percentage of apoptotic cells was decreased in the 0.5 DEK group. Expression of development-related genes (CX45, CDX2, POU5F1, and NANOG), antiapoptotic genes (BCL2L1 and BIRC5), and a proapoptotic gene (CASP3) were altered in the 0.5 DEK group. These results indicate that the antioxidant dieckol improves IVM and subsequent development of porcine oocytes and can be used to improve the quality of oocytes under peroxidation experimental conditions.


Assuntos
Antioxidantes/farmacologia , Benzofuranos/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Animais , Antioxidantes/administração & dosagem , Benzofuranos/administração & dosagem , Blastocisto/citologia , Posicionamento Cromossômico/efeitos dos fármacos , Relação Dose-Resposta a Droga , Técnicas de Cultura Embrionária , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glutationa/metabolismo , Técnicas de Maturação in Vitro de Oócitos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Meiose , Oócitos/metabolismo , Phaeophyceae/química , Espécies Reativas de Oxigênio/metabolismo , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/ultraestrutura , Suínos
7.
Nature ; 593(7858): 289-293, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33854237

RESUMO

Fundamental features of 3D genome organization are established de novo in the early embryo, including clustering of pericentromeric regions, the folding of chromosome arms and the segregation of chromosomes into active (A-) and inactive (B-) compartments. However, the molecular mechanisms that drive de novo organization remain unknown1,2. Here, by combining chromosome conformation capture (Hi-C), chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq), 3D DNA fluorescence in situ hybridization (3D DNA FISH) and polymer simulations, we show that heterochromatin protein 1a (HP1a) is essential for de novo 3D genome organization during Drosophila early development. The binding of HP1a at pericentromeric heterochromatin is required to establish clustering of pericentromeric regions. Moreover, HP1a binding within chromosome arms is responsible for overall chromosome folding and has an important role in the formation of B-compartment regions. However, depletion of HP1a does not affect the A-compartment, which suggests that a different molecular mechanism segregates active chromosome regions. Our work identifies HP1a as an epigenetic regulator that is involved in establishing the global structure of the genome in the early embryo.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Posicionamento Cromossômico , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Genoma de Inseto/genética , Conformação Molecular , Animais , Imunoprecipitação da Cromatina , Cromossomos de Insetos/química , Cromossomos de Insetos/genética , Cromossomos de Insetos/metabolismo , Drosophila melanogaster/citologia , Embrião não Mamífero/citologia , Desenvolvimento Embrionário/genética , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , Hibridização in Situ Fluorescente
8.
Science ; 371(6532)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33384301

RESUMO

Understanding genome organization requires integration of DNA sequence and three-dimensional spatial context; however, existing genome-wide methods lack either base pair sequence resolution or direct spatial localization. Here, we describe in situ genome sequencing (IGS), a method for simultaneously sequencing and imaging genomes within intact biological samples. We applied IGS to human fibroblasts and early mouse embryos, spatially localizing thousands of genomic loci in individual nuclei. Using these data, we characterized parent-specific changes in genome structure across embryonic stages, revealed single-cell chromatin domains in zygotes, and uncovered epigenetic memory of global chromosome positioning within individual embryos. These results demonstrate how IGS can directly connect sequence and structure across length scales from single base pairs to whole organisms.


Assuntos
Genoma Humano , Genoma , Análise de Sequência de DNA , Animais , Sequência de Bases , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Cromatina/química , Cromatina/ultraestrutura , Posicionamento Cromossômico , Cromossomos Humanos/ultraestrutura , Cromossomos de Mamíferos/ultraestrutura , Embrião de Mamíferos , Desenvolvimento Embrionário , Epigênese Genética , Fibroblastos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Análise de Célula Única , Análise Espacial
9.
Methods Mol Biol ; 2198: 255-268, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32822037

RESUMO

Male infertility is associated with several causes affecting the paternal nucleus such as DNA lesions (breaks, deletions, mutations, ...) or numerical chromosome anomalies. More recently, male infertility has also been associated with changes in the sperm epigenome, including modification in the topology of chromatin (Olszewska et al., Chromosome Research 16:875-890, 2008; Alladin et al., Syst Biol Reprod Med 59: 146-152, 2013) ref with number 1, 2. Indeed, the positioning of chromosomes in the sperm nucleus is nonrandom and defines chromosome territories (Champroux et al., Genes (Basel) 9:501, 2018) ref with number 3 whose optimal organization determines the success of embryonic development. In this context, the study of the spatial distribution of chromosomes in sperm cells could be relevant for clinical diagnosis. We describe here a in situ fluorescence hybridization (FISH) strategy coupled with a fluorescent immunocytochemistry approach followed by confocal analysis and reconstruction (2D/3D) as a powerful tool to analyze the location of chromosomes in the sperm nucleus using the mouse sperm as a model. Already, the two-dimensional (2D) analysis of FISH and immunofluorescence data reveal the location of chromosomes as well as the different markings on the spermatic nucleus. In addition, a good 3D rendering after Imaris software processing was obtained when Z-stacks of images were acquired over a defined volume (10 µm × 13 µm × 15 µm) with a sequential scanning mode to minimize bleed-through effects and avoid overlapping wavelengths.


Assuntos
Posicionamento Cromossômico/imunologia , Microscopia Confocal/métodos , Espermatozoides/imunologia , Aneuploidia , Animais , Núcleo Celular/imunologia , Cromatina , Aberrações Cromossômicas , Posicionamento Cromossômico/genética , Cromossomos/imunologia , Modelos Animais de Doenças , Imunofluorescência/métodos , Hibridização in Situ Fluorescente/métodos , Infertilidade Masculina/imunologia , Masculino , Camundongos , Espermatozoides/citologia
10.
PLoS Genet ; 16(9): e1009001, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886661

RESUMO

During meiosis, diploid organisms reduce their chromosome number by half to generate haploid gametes. This process depends on the repair of double strand DNA breaks as crossover recombination events between homologous chromosomes, which hold homologs together to ensure their proper segregation to opposite spindle poles during the first meiotic division. Although most organisms are limited in the number of crossovers between homologs by a phenomenon called crossover interference, the consequences of excess interfering crossovers on meiotic chromosome segregation are not well known. Here we show that extra interfering crossovers lead to a range of meiotic defects and we uncover mechanisms that counteract these errors. Using chromosomes that exhibit a high frequency of supernumerary crossovers in Caenorhabditis elegans, we find that essential chromosomal structures are mispatterned in the presence of multiple crossovers, subjecting chromosomes to improper spindle forces and leading to defects in metaphase alignment. Additionally, the chromosomes with extra interfering crossovers often exhibited segregation defects in anaphase I, with a high incidence of chromatin bridges that sometimes created a tether between the chromosome and the first polar body. However, these anaphase I bridges were often able to resolve in a LEM-3 nuclease dependent manner, and chromosome tethers that persisted were frequently resolved during Meiosis II by a second mechanism that preferentially segregates the tethered sister chromatid into the polar body. Altogether these findings demonstrate that excess interfering crossovers can severely impact chromosome patterning and segregation, highlighting the importance of limiting the number of recombination events between homologous chromosomes for the proper execution of meiosis.


Assuntos
Segregação de Cromossomos/genética , Troca Genética/genética , Meiose/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Cromátides/genética , Cromatina/genética , Posicionamento Cromossômico/genética , Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/genética , Recombinação Genética
11.
Adv Exp Med Biol ; 1267: 45-58, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32894476

RESUMO

In this chapter, we will focus on ParABS: an apparently simple, three-component system, required for the segregation of bacterial chromosomes and plasmids. We will specifically describe how biophysical measurements combined with physical modeling advanced our understanding of the mechanism of ParABS-mediated complex assembly, segregation and positioning.


Assuntos
Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos/metabolismo , Posicionamento Cromossômico , DNA Bacteriano/metabolismo , Plasmídeos/metabolismo
12.
Nature ; 587(7833): 285-290, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32879492

RESUMO

Gene expression in eukaryotes requires the effective separation of nuclear transcription and RNA processing from cytosolic translation1. This separation is achieved by the nuclear envelope, which controls the exchange of macromolecules through nuclear pores2. During mitosis, however, the nuclear envelope in animal and plant cells disassembles, allowing cytoplasmic and nuclear components to intermix3. When the nuclear envelope is reformed, cytoplasmic components are removed from the nucleus by receptor-mediated transport through nuclear pores2. These pores have a size limit of 39 nanometres4-7, which raises the question of how larger cytoplasmic molecules are cleared from the nucleus. Here we show in HeLa cells that large cytoplasmic components are displaced before nuclear envelope assembly by the movement of chromosomes to a dense cluster. This clustering occurs when chromosomes approach the poles of anaphase spindles, and is mediated by a microtubule-independent mechanism that involves the surfactant-like protein Ki-67. Ki-67 forms repulsive molecular brushes during the early stages of mitosis8, but during mitotic exit the brushes collapse and Ki-67 promotes chromosome clustering. We show that the exclusion of mature ribosomes from the nucleus after mitosis depends on Ki-67-regulated chromosome clustering. Thus, our study reveals that chromosome mechanics help to re-establish the compartmentalization of eukaryotic cells after open mitosis.


Assuntos
Posicionamento Cromossômico , Cromossomos Humanos/metabolismo , Citoplasma/metabolismo , Antígeno Ki-67/metabolismo , Membrana Nuclear/metabolismo , Transporte Biológico , Células HeLa , Humanos , Mitose , Ribossomos/metabolismo , Fuso Acromático
13.
J Exp Bot ; 71(20): 6262-6272, 2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-32805034

RESUMO

Despite much recent progress, our understanding of the principles of plant genome organization and its dynamics in three-dimensional space of interphase nuclei remains surprisingly limited. Notably, it is not clear how these processes could be affected by the size of a plant's nuclear genome. In this study, DNA replication timing and interphase chromosome positioning were analyzed in seven Poaceae species that differ in their genome size. To provide a comprehensive picture, a suite of advanced, complementary methods was used: labeling of newly replicated DNA by ethynyl-2'-deoxyuridine, isolation of nuclei at particular cell cycle phases by flow cytometric sorting, three-dimensional immunofluorescence in situ hybridization, and confocal microscopy. Our results revealed conserved dynamics of DNA replication in all species, and a similar replication timing order for telomeres and centromeres, as well as for euchromatin and heterochromatin regions, irrespective of genome size. Moreover, stable chromosome positioning was observed while transitioning through different stages of interphase. These findings expand upon earlier studies in suggesting that a more complex interplay exists between genome size, organization of repetitive DNA sequences along chromosomes, and higher order chromatin structure and its maintenance in interphase, albeit controlled by currently unknown factors.


Assuntos
Núcleo Celular , Posicionamento Cromossômico , Núcleo Celular/genética , Centrômero/genética , Replicação do DNA , Genoma de Planta , Interfase
14.
Biochemistry (Mosc) ; 85(6): 643-650, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32586227

RESUMO

Functional compartmentalization of the cell nucleus plays an important role in the regulation of genome activity by providing accumulation of enzymes and auxiliary factors in the reaction centers, such as transcription factories, Cajal bodies, speckles, etc. The mechanisms behind the nucleus functional compartmentalization are still poorly understood. There are reasons to believe that the key role in the nucleus compartmentalization belongs to the process of liquid-liquid phase separation. In this brief review, we analyze results of experimental studies demonstrating that liquid-liquid phase separation not only governs functional compartmentalization of the cell nucleus but also contributes to the formation of the 3D genomic architecture.


Assuntos
Compartimento Celular , Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Cromossomos Humanos/genética , Genoma Humano , Extração Líquido-Líquido/métodos , Transcrição Gênica , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Posicionamento Cromossômico , Humanos , Proteínas Nucleares/genética
15.
Nucleus ; 11(1): 99-110, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32453974

RESUMO

Nuclear lamins form an elastic meshwork underlying the inner nuclear membrane and provide mechanical rigidity to the nucleus and maintain shape. Lamins also maintain chromosome positioning and play important roles in several nuclear processes like replication, DNA damage repair, transcription, and epigenetic modifications. LMNA mutations affect cardiac tissue, muscle tissues, adipose tissues to precipitate several diseases collectively termed as laminopathies. However, the rationale behind LMNA mutations and laminopathies continues to elude scientists. During interphase, several chromosomes form inter/intrachromosomal contacts inside nucleoplasm and several chromosomal loops also stretch out to make a 'loop-cluster' which are key players to regulate gene expressions. In this perspective, we have proposed that the lamin network in tandem with nuclear actin and myosin provide mechanical rigidity to the chromosomal contacts and facilitate loop-clusters movements. LMNA mutations thus might perturb the landscape of chromosomal contacts or loop-clusters positioning which can impair gene expression profile.


Assuntos
Núcleo Celular/genética , Posicionamento Cromossômico , Regulação da Expressão Gênica , Lamina Tipo A/metabolismo , Lâmina Nuclear/metabolismo , Animais , Núcleo Celular/metabolismo , Posicionamento Cromossômico/genética , Humanos , Mutação
16.
Nature ; 580(7801): 142-146, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32238933

RESUMO

Paternal and maternal epigenomes undergo marked changes after fertilization1. Recent epigenomic studies have revealed the unusual chromatin landscapes that are present in oocytes, sperm and early preimplantation embryos, including atypical patterns of histone modifications2-4 and differences in chromosome organization and accessibility, both in gametes5-8 and after fertilization5,8-10. However, these studies have led to very different conclusions: the global absence of local topological-associated domains (TADs) in gametes and their appearance in the embryo8,9 versus the pre-existence of TADs and loops in the zygote5,11. The questions of whether parental structures can be inherited in the newly formed embryo and how these structures might relate to allele-specific gene regulation remain open. Here we map genomic interactions for each parental genome (including the X chromosome), using an optimized single-cell high-throughput chromosome conformation capture (HiC) protocol12,13, during preimplantation in the mouse. We integrate chromosome organization with allelic expression states and chromatin marks, and reveal that higher-order chromatin structure after fertilization coincides with an allele-specific enrichment of methylation of histone H3 at lysine 27. These early parental-specific domains correlate with gene repression and participate in parentally biased gene expression-including in recently described, transiently imprinted loci14. We also find TADs that arise in a non-parental-specific manner during a second wave of genome assembly. These de novo domains are associated with active chromatin. Finally, we obtain insights into the relationship between TADs and gene expression by investigating structural changes to the paternal X chromosome before and during X chromosome inactivation in preimplantation female embryos15. We find that TADs are lost as genes become silenced on the paternal X chromosome but linger in regions that escape X chromosome inactivation. These findings demonstrate the complex dynamics of three-dimensional genome organization and gene expression during early development.


Assuntos
Blastocisto/citologia , Blastocisto/metabolismo , Cromatina/metabolismo , Desenvolvimento Embrionário/genética , Fertilização/genética , Células Germinativas/citologia , Pais , Alelos , Animais , Cromatina/química , Cromatina/genética , Posicionamento Cromossômico , Cromossomos de Mamíferos/química , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genoma/genética , Impressão Genômica , Células Germinativas/metabolismo , Histonas/química , Histonas/metabolismo , Masculino , Metilação , Camundongos , Proteínas do Grupo Polycomb/metabolismo , Análise de Célula Única , Inativação do Cromossomo X/genética
17.
BMC Genomics ; 21(1): 175, 2020 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-32087673

RESUMO

BACKGROUND: Proximity ligation based techniques, like Hi-C, involve restriction digestion followed by ligation of formaldehyde cross-linked chromatin. Distinct chromatin states can impact the restriction digestion, and hence the visibility in the contact maps, of engaged loci. Yet, the extent and the potential impact of digestion bias remain obscure and under-appreciated in the literature. RESULTS: Through analysis of 45 Hi-C datasets, lamina-associated domains (LADs), inactive X-chromosome in mammals, and polytene bands in fly, we first established that the DNA in condensed chromatin had lesser accessibility to restriction endonucleases used in Hi-C as compared to that in decondensed chromatin. The observed bias was independent of known systematic biases, was not appropriately corrected by existing computational methods, and needed an additional optimization step. We then repurposed this bias to identify novel condensed domains outside LADs, which were bordered by insulators and were dynamically associated with the polycomb mediated epigenetic and transcriptional states during development. CONCLUSIONS: Our observations suggest that the corrected one-dimensional read counts of existing Hi-C datasets can be reliably repurposed to study the gene-regulatory dynamics associated with chromatin condensation and decondensation, and that the existing Hi-C datasets should be interpreted with cautions.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Posicionamento Cromossômico , Genômica/métodos , Cromossomos Politênicos , Cromossomo X , Animais , Imunoprecipitação da Cromatina , Drosophila/genética , Epigenômica , Humanos , Camundongos , Análise de Sequência de DNA
18.
Mol Biol (Mosk) ; 53(6): 933-953, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31876274

RESUMO

Chromatin packing in eukaryotic chromosomes has been traditionally viewed as a hierarchical process, in which nucleosome chains fold into helical chromatin fibers. These fibers would then fold into more complex regular structures. However, recent chromatin imaging studies and analyses of chromosomal DNA contacts within the 3D space of the cell nucleus have necessitated a radical revision of the hierarchical chromatin packing model. According to the new studies, the nucleosome chain has a free spatial configuration without regular helical fibers in most cell types. The overall 3D organization of DNA in the cell nucleus includes chromatin loops and contact domains of up to several million base pairs in size. During cell differentiation, individual structure-functional chromatin domains marked by similar types of histone modifications and functional states can merge together and form chromosomal subcompartments suited for local gene activation or repression. This "attraction of likenesses" may be mediated by direct self-association of nucleosome chains as well as by architectural chromatin proteins making oligomeric protein "bridges" between nucleosomes as well as larger dynamic condensates leading to liquid-liquid phase separation inside the cell nucleus. Future studies of mechanisms of chromatin self-association and compartmentalization will require a combination of molecular, imaging, and computational approaches capable of revealing the 3D organization of the eukaryotic genome with nucleosomal resolution.


Assuntos
Cromatina/química , Cromatina/metabolismo , Posicionamento Cromossômico , Eucariotos/genética , DNA/química , DNA/metabolismo , Eucariotos/citologia , Histonas/química , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo
19.
J Cell Biol ; 219(2)2019 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31881080

RESUMO

Aurora kinases create phosphorylation gradients within the spindle during prometaphase and anaphase, thereby locally regulating factors that promote spindle organization, chromosome condensation and movement, and cytokinesis. We show that one such factor is the kinesin KIF4A, which is present along the chromosome axes throughout mitosis and the central spindle in anaphase. These two pools of KIF4A depend on condensin I and PRC1, respectively. Previous work has shown KIF4A is activated by Aurora B at the anaphase central spindle. However, whether or not chromosome-associated KIF4A bound to condensin I is regulated by Aurora kinases remain unclear. To determine the roles of the two different pools of KIF4A, we generated specific point mutants that are unable to interact with either condensin I or PRC1 or are deficient for Aurora kinase regulation. By analyzing these mutants, we show that Aurora A phosphorylates the condensin I-dependent pool of KIF4A and thus actively promotes chromosome congression from the spindle poles to the metaphase plate.


Assuntos
Adenosina Trifosfatases/metabolismo , Aurora Quinase A/metabolismo , Segregação de Cromossomos/fisiologia , Cromossomos/metabolismo , Cromossomos/fisiologia , Proteínas de Ligação a DNA/metabolismo , Cinesinas/metabolismo , Complexos Multiproteicos/metabolismo , Anáfase/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Posicionamento Cromossômico/fisiologia , Células HEK293 , Células HeLa , Humanos , Microtúbulos/metabolismo , Mitose/fisiologia , Fosforilação/fisiologia , Fuso Acromático/metabolismo , Fuso Acromático/fisiologia
20.
Nature ; 569(7756): 345-354, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31092938

RESUMO

How cells adopt different identities has long fascinated biologists. Signal transduction in response to environmental cues results in the activation of transcription factors that determine the gene-expression program characteristic of each cell type. Technological advances in the study of 3D chromatin folding are bringing the role of genome conformation in transcriptional regulation to the fore. Characterizing this role of genome architecture has profound implications, not only for differentiation and development but also for diseases including developmental malformations and cancer. Here we review recent studies indicating that the interplay between transcription and genome conformation is a driving force for cell-fate decisions.


Assuntos
Diferenciação Celular/genética , Células/citologia , Células/metabolismo , Genoma , Fatores de Transcrição/metabolismo , Animais , Montagem e Desmontagem da Cromatina/genética , Posicionamento Cromossômico , Regulação da Expressão Gênica , Genoma/genética , Humanos , Especificidade de Órgãos/genética
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