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1.
BMC Biol ; 20(1): 99, 2022 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-35524220

RESUMO

BACKGROUND: The three-dimensional (3D) architecture of the genome has a highly ordered and hierarchical nature, which influences the regulation of essential nuclear processes at the basis of gene expression, such as gene transcription. While the hierarchical organization of heterochromatin and euchromatin can underlie differences in gene expression that determine evolutionary differences among species, the way 3D genome architecture is affected by evolutionary forces within major lineages remains unclear. Here, we report a comprehensive comparison of 3D genomes, using high resolution Hi-C data in fibroblast cells of fish, chickens, and 10 mammalian species. RESULTS: This analysis shows a correlation between genome size and chromosome length that affects chromosome territory (CT) organization in the upper hierarchy of genome architecture, whereas lower hierarchical features, including local transcriptional availability of DNA, are selected through the evolution of vertebrates. Furthermore, conservation of topologically associating domains (TADs) appears strongly associated with the modularity of expression profiles across species. Additionally, LINE and SINE transposable elements likely contribute to heterochromatin and euchromatin organization, respectively, during the evolution of genome architecture. CONCLUSIONS: Our analysis uncovers organizational features that appear to determine the conservation and transcriptional regulation of functional genes across species. These findings can guide ongoing investigations of genome evolution by extending our understanding of the mechanisms shaping genome architecture.


Assuntos
Cromatina , Heterocromatina , Animais , Galinhas/genética , Elementos de DNA Transponíveis , Eucromatina/genética , Heterocromatina/genética , Mamíferos/genética , Vertebrados/genética
2.
Genome Biol ; 23(1): 91, 2022 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-35410387

RESUMO

BACKGROUND: Interactions of chromatin with the nuclear lamina via lamina-associated domains (LADs) confer structural stability to the genome. The dynamics of positioning of LADs during differentiation, and how LADs impinge on developmental gene expression, remains, however, elusive. RESULTS: We examined changes in the association of lamin B1 with the genome in the first 72 h of differentiation of adipose stem cells into adipocytes. We demonstrate a repositioning of entire stand-alone LADs and of LAD edges as a prominent nuclear structural feature of early adipogenesis. Whereas adipogenic genes are released from LADs, LADs sequester downregulated or repressed genes irrelevant for the adipose lineage. However, LAD repositioning only partly concurs with gene expression changes. Differentially expressed genes in LADs, including LADs conserved throughout differentiation, reside in local euchromatic and lamin-depleted sub-domains. In these sub-domains, pre-differentiation histone modification profiles correlate with the LAD versus inter-LAD outcome of these genes during adipogenic commitment. Lastly, we link differentially expressed genes in LADs to short-range enhancers which overall co-partition with these genes in LADs versus inter-LADs during differentiation. CONCLUSIONS: We conclude that LADs are predictable structural features of adipose nuclear architecture that restrain non-adipogenic genes in a repressive environment.


Assuntos
Adipogenia , Eucromatina , Cromatina/metabolismo , Eucromatina/metabolismo , Lâmina Nuclear/genética
3.
Eur Phys J E Soft Matter ; 45(4): 33, 2022 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-35403917

RESUMO

No systematic method exists to derive inter-nucleosomal potentials between nucleosomes along a chromosome consistently across a given genome. Such potentials can yield information on nucleosomal ordering, thermal as well as mechanical properties of chromosomes. Thus, indirectly, they shed light on a possible mechanical genomic code along a chromosome. To develop a method yielding effective inter-nucleosomal potentials between nucleosomes, a generalized Lennard-Jones potential for the parameterization is developed based on nucleosomal positioning data. This approach eliminates some of the problems that the underlying nucleosomal positioning data have, rendering the extraction difficult on the individual nucleosomal level. Furthermore, patterns on which to base a classification along a chromosome appear on larger domains, such as hetero- and euchromatin. An intuitive selection strategy for the noisy optimization problem is employed to derive effective exponents for the generalized potential. The method is tested on the Candida albicans genome. Applying k-means clustering based on potential parameters and thermodynamic compressibilities, a genome-wide clustering of nucleosome sequences is obtained for C. albicans. This clustering shows that a chromosome beyond the classical dichotomic categories of hetero- and euchromatin is more feature-rich.


Assuntos
Eucromatina , Nucleossomos , Nucleossomos/genética , Termodinâmica
4.
Int J Mol Sci ; 23(7)2022 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-35409128

RESUMO

Double-strand breaks (DSBs) in nuclear DNA represents radiation-induced damage that has been identified as particularly deleterious. Calculating this damage using Monte Carlo track structure modeling could be a suitable indicator to better assess and anticipate the side-effects of radiation therapy. However, as already demonstrated in previous work, the geometrical description of the nucleus and the DNA content used in the simulation significantly influence damage calculations. Therefore, in order to obtain accurate results, this geometry must be as realistic as possible. In this study, a new geometrical model of an endothelial cell nucleus and DNA distribution according to the isochore theory are presented and used in a Monte Carlo simulation chain based on the Geant4-DNA toolkit. In this theory, heterochromatin and euchromatin compaction are distributed along the genome according to five different families (L1, L2, H1, H2, and H3). Each of these families is associated with a different hetero/euchromatin rate related to its compaction level. In order to compare the results with those obtained using a previous nuclear geometry, simulations were performed for protons with linear energy transfers (LETs) of 4.29 keV/µm, 19.51 keV/µm, and 43.25 keV/µm. The organization of the chromatin fibers at different compaction levels linked to isochore families increased the DSB yield by 6-10%, and it allowed the most affected part of the genome to be identified. These new results indicate that the genome core is more radiosensitive than the genome desert, with a 3-8% increase in damage depending on the LET. This work highlights the importance of using realistic distributions of chromatin compaction levels to calculate radio-induced damage using Monte Carlo simulation methods.


Assuntos
Eucromatina , Isocoros , Cromatina , DNA/química , Dano ao DNA , Eucromatina/genética , Humanos , Método de Monte Carlo
5.
Genes (Basel) ; 13(3)2022 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-35328082

RESUMO

Pericentromeric heterochromatin is mostly composed of repetitive DNA sequences prone to aberrant recombination. Cells have developed highly specialized mechanisms to enable 'safe' homologous recombination (HR) repair while preventing aberrant recombination in this domain. Understanding heterochromatin repair responses is essential to understanding the critical mechanisms responsible for genome integrity and tumor suppression. Here, we review the tools, approaches, and methods currently available to investigate double-strand break (DSB) repair in pericentromeric regions, and also suggest how technologies recently developed for euchromatin repair studies can be adapted to characterize responses in heterochromatin. With this ever-growing toolkit, we are witnessing exciting progress in our understanding of how the 'dark matter' of the genome is repaired, greatly improving our understanding of genome stability mechanisms.


Assuntos
Quebras de DNA de Cadeia Dupla , Heterocromatina , Reparo do DNA/genética , Eucromatina , Heterocromatina/genética , Reparo de DNA por Recombinação
6.
Nucleic Acids Res ; 50(7): 3852-3866, 2022 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-35333354

RESUMO

Crucial mechanisms are required to restrict self-propagating heterochromatin spreading within defined boundaries and prevent euchromatic gene silencing. In the filamentous fungus Neurospora crassa, the JmjC domain protein DNA METHYLATION MODULATOR-1 (DMM-1) prevents aberrant spreading of heterochromatin, but the molecular details remain unknown. Here, we revealed that DMM-1 is highly enriched in a well-defined 5-kb heterochromatin domain upstream of the cat-3 gene, hereby called 5H-cat-3 domain, to constrain aberrant heterochromatin spreading. Interestingly, aberrant spreading of the 5H-cat-3 domain observed in the dmm-1KO strain is accompanied by robust deposition of histone variant H2A.Z, and deletion of H2A.Z abolishes aberrant spreading of the 5H-cat-3 domain into adjacent euchromatin. Furthermore, lysine 56 of histone H3 is deacetylated at the expanded heterochromatin regions, and mimicking H3K56 acetylation with an H3K56Q mutation effectively blocks H2A.Z-mediated aberrant spreading of the 5H-cat-3 domain. Importantly, genome-wide analyses demonstrated the general roles of H3K56 deacetylation and H2A.Z deposition in aberrant spreading of heterochromatin. Together, our results illustrate a previously unappreciated regulatory process that mediates aberrant heterochromatin spreading.


Assuntos
Heterocromatina , Histonas , Neurospora crassa/metabolismo , Metilação de DNA , Eucromatina/genética , Estudo de Associação Genômica Ampla , Heterocromatina/genética , Histonas/genética , Histonas/metabolismo
7.
G3 (Bethesda) ; 12(5)2022 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-35244156

RESUMO

The eukaryotic genome must be precisely organized for its proper function, as genome topology impacts transcriptional regulation, cell division, replication, and repair, among other essential processes. Disruptions to human genome topology can lead to diseases, including cancer. The advent of chromosome conformation capture with high-throughput sequencing (Hi-C) to assess genome organization has revolutionized the study of nuclear genome topology; Hi-C has elucidated numerous genomic structures, including chromosomal territories, active/silent chromatin compartments, Topologically Associated Domains, and chromatin loops. While low-resolution heatmaps can provide important insights into chromosomal level contacts, high-resolution Hi-C datasets are required to reveal folding principles of individual genes. Of particular interest are high-resolution chromosome conformation datasets of organisms modeling the human genome. Here, we report the genome topology of the fungal model organism Neurospora crassa at a high resolution. Our composite Hi-C dataset, which merges 2 independent datasets generated with restriction enzymes that monitor euchromatin (DpnII) and heterochromatin (MseI), along with our DpnII/MseI double digest dataset, provide exquisite detail for both the conformation of entire chromosomes and the folding of chromatin at the resolution of individual genes. Within constitutive heterochromatin, we observe strong yet stochastic internal contacts, while euchromatin enriched with either activating or repressive histone post-translational modifications associates with constitutive heterochromatic regions, suggesting intercompartment contacts form to regulate transcription. Consistent with this, a strain with compromised heterochromatin experiences numerous changes in gene expression. Our high-resolution Neurospora Hi-C datasets are outstanding resources to the fungal community and provide valuable insights into higher organism genome topology.


Assuntos
Neurospora crassa , Cromatina/metabolismo , Cromossomos Fúngicos/genética , Eucromatina , Heterocromatina/metabolismo , Humanos , Neurospora crassa/genética , Neurospora crassa/metabolismo
8.
Cells ; 11(3)2022 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-35159258

RESUMO

The zeppelin (zep) locus is known for its essential role in the development of the embryonic cuticle of Drosophila melanogaster. We show here that zep encodes Gfat1 (Glutamine: Fructose-6-Phosphate Aminotransferase 1; CG12449), the enzyme that catalyzes the rate-limiting step in the hexosamine biosynthesis pathway (HBP). This conserved pathway diverts 2%-5% of cellular glucose from glycolysis and is a nexus of sugar (fructose-6-phosphate), amino acid (glutamine), fatty acid [acetyl-coenzymeA (CoA)], and nucleotide/energy (UDP) metabolism. We also describe the isolation and characterization of lethal mutants in the euchromatic paralog, Gfat2 (CG1345), and demonstrate that ubiquitous expression of Gfat1+ or Gfat2+ transgenes can rescue lethal mutations in either gene. Gfat1 and Gfat2 show differences in mRNA and protein expression during embryogenesis and in essential tissue-specific requirements for Gfat1 and Gfat2, suggesting a degree of functional evolutionary divergence. An evolutionary, cytogenetic analysis of the two genes in six Drosophila species revealed Gfat2 to be located within euchromatin in all six species. Gfat1 localizes to heterochromatin in three melanogaster-group species, and to euchromatin in the more distantly related species. We have also found that the pattern of flanking-gene microsynteny is highly conserved for Gfat1 and somewhat less conserved for Gfat2.


Assuntos
Drosophila melanogaster , Hexosaminas , Animais , Vias Biossintéticas/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Eucromatina , Glutamina/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo
9.
BMC Plant Biol ; 22(1): 77, 2022 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-35193491

RESUMO

BACKGROUND: Structural variants (SVs) constitute a large proportion of the genomic variation that results in phenotypic variation in plants. However, they are still a largely unexplored feature in most plant genomes. Here, we present the whole-genome landscape of SVs between two model legume Medicago truncatula ecotypes-Jemalong A17 and R108- that have been extensively used in various legume biology studies. RESULTS: To catalogue SVs, we first resolved the previously published R108 genome assembly (R108 v1.0) to chromosome-scale using 124 × Hi-C data, resulting in a high-quality genome assembly. The inter-chromosomal reciprocal translocations between chromosomes 4 and 8 were confirmed by performing syntenic analysis between the two genomes. Combined with the Hi-C data, it appears that these translocation events had a significant effect on chromatin organization. Using both whole-genome and short-read alignments, we identified the genomic landscape of SVs between the two genomes, some of which may account for several phenotypic differences, including their differential responses to aluminum toxicity and iron deficiency, and the development of different anthocyanin leaf markings. We also found extensive SVs within the nodule-specific cysteine-rich gene family which encodes antimicrobial peptides essential for terminal bacteroid differentiation during nitrogen-fixing symbiosis. CONCLUSIONS: Our results provide a near-complete R108 genome assembly and the first genomic landscape of SVs obtained by comparing two M. truncatula ecotypes. This may provide valuable genomic resources for the functional and molecular research of legume biology in the future.


Assuntos
Cromatina/genética , Genoma de Planta , Medicago truncatula/genética , Cromossomos de Plantas , Elementos de DNA Transponíveis , Ecótipo , Eucromatina/química , Eucromatina/genética , Genes de Plantas , Heterocromatina/química , Heterocromatina/genética , Medicago truncatula/fisiologia , Fixação de Nitrogênio/genética , Filogenia , Sequenciamento Completo do Genoma
10.
Nat Biotechnol ; 40(2): 235-244, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34635836

RESUMO

Recent efforts have succeeded in surveying open chromatin at the single-cell level, but high-throughput, single-cell assessment of heterochromatin and its underlying genomic determinants remains challenging. We engineered a hybrid transposase including the chromodomain (CD) of the heterochromatin protein-1α (HP-1α), which is involved in heterochromatin assembly and maintenance through its binding to trimethylation of the lysine 9 on histone 3 (H3K9me3), and developed a single-cell method, single-cell genome and epigenome by transposases sequencing (scGET-seq), that, unlike single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq), comprehensively probes both open and closed chromatin and concomitantly records the underlying genomic sequences. We tested scGET-seq in cancer-derived organoids and human-derived xenograft (PDX) models and identified genetic events and plasticity-driven mechanisms contributing to cancer drug resistance. Next, building upon the differential enrichment of closed and open chromatin, we devised a method, Chromatin Velocity, that identifies the trajectories of epigenetic modifications at the single-cell level. Chromatin Velocity uncovered paths of epigenetic reorganization during stem cell reprogramming and identified key transcription factors driving these developmental processes. scGET-seq reveals the dynamics of genomic and epigenetic landscapes underlying any cellular processes.


Assuntos
Eucromatina , Heterocromatina , Cromatina/genética , Epigênese Genética/genética , Eucromatina/genética , Heterocromatina/genética , Humanos , Transposases/genética
11.
Dev Biol ; 482: 7-16, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34822846

RESUMO

Regeneration is a response mechanism aiming to reconstruct lost or damaged structures. To achieve this, the cells repopulating the lost tissue often have to change their original identity, a process that involves chromatin remodelling.We have analysed the issue of chromatin remodelling during regeneration in the wing disc of Drosophila . In this disc the ablation of the central region (the pouch) induces the regenerative response of the cells from the lateral region (the hinge), which reconstitute the wing pouch. We have examined euchromatin and heterochromatin histone marks during the process and find that heterochromatin marks disappear but are recovered when regeneration is complete. Euchromatin marks are not modified. We also describe the transcription of two retrotransposons, Roo and F-element in the regenerating cells. We have established a temporal correlation between the alterations of heterochromatin marks and the levels of transcription of two retrotransposons, Roo and F-element, both during embryonic development and in the regeneration process.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Drosophila melanogaster/embriologia , Discos Imaginais/crescimento & desenvolvimento , Regeneração/fisiologia , Retroelementos/genética , Asas de Animais/embriologia , Acetilação , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Eucromatina/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , Metilação , Asas de Animais/crescimento & desenvolvimento
12.
Chromosome Res ; 30(1): 5-24, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34665365

RESUMO

Functional changes of cells upon developmental switches and in response to environmental cues are often reflected in nuclear phenotypes, showing distinctive chromatin states corresponding to transcriptional changes. Such characteristic nuclear shapes have been microscopically monitored and can be quantified after differential staining of euchromatin and heterochromatin domains. Here, we examined several nuclear parameters (size, DNA content, DNA density, chromatin compaction, relative heterochromatin fraction (RHF), and number of chromocenters) in relation to spatial distribution of genes and transposon elements (TEs), using standard 2D fluorescence microscopy. We provide nuclear profiles for different cell types and different accessions of Arabidopsis thaliana. A variable, yet significant, fraction of TEs was found outside chromocenters in all cell types, except for guard cells. The latter cell type features nuclei with the highest level of chromatin compaction, while their chromocenters seem to contain gene-rich regions. The highest number of parameter correlations was found in the accession Cvi, whereas Ler showed only few correlations. This may point at differences in phenotype robustness between accessions. The significantly high association of NOR chromocenters in accessions Ws and Cvi corresponds to their low RHF level.


Assuntos
Arabidopsis , Arabidopsis/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Eucromatina/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo
13.
Genome Biol ; 23(1): 5, 2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34980211

RESUMO

BACKGROUND: Direct analogs of chemically modified bases that carry important epigenetic information, such as 5-methylcytosine (m5C)/5-methyldeoxycytosine (5mC), 5-hydroxymethylcytosine (hm5C)/5-hydroxymethyldeoxycytosine (5hmC), and N6-methyladenosine (m6A)/N6-methyldeoxyadenosine (6mA), are detected in both RNA and DNA, respectively. The modified base N4-acetylcytosine (ac4C) is well studied in RNAs, but its presence and epigenetic roles in cellular DNA have not been explored. RESULTS: Here, we demonstrate the existence of N4-acetyldeoxycytosine (4acC) in genomic DNA of Arabidopsis with multiple detection methods. Genome-wide profiling of 4acC modification reveals that 4acC peaks are mostly distributed in euchromatin regions and present in nearly half of the expressed protein-coding genes in Arabidopsis. 4acC is mainly located around transcription start sites and positively correlates with gene expression levels. Imbalance of 5mC does not directly affect 4acC modification. We also characterize the associations of 4acC with 5mC and histone modifications that cooperatively regulate gene expression. Moreover, 4acC is also detected in genomic DNA of rice, maize, mouse, and human by mass spectrometry. CONCLUSIONS: Our findings reveal 4acC as a hitherto unknown DNA modification in higher eukaryotes. We identify potential interactions of this mark with other epigenetic marks in gene expression regulation.


Assuntos
Arabidopsis , 5-Metilcitosina , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Citosina/metabolismo , DNA/metabolismo , Metilação de DNA , Epigenômica , Eucromatina , Camundongos
14.
Cell Rep ; 37(10): 110089, 2021 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-34879283

RESUMO

Deleterious genetic variants in POGZ, which encodes the chromatin regulator Pogo Transposable Element with ZNF Domain protein, are strongly associated with autism spectrum disorder (ASD). Although it is a high-confidence ASD risk gene, the neurodevelopmental functions of POGZ remain unclear. Here we reveal the genomic binding of POGZ in the developing forebrain at euchromatic loci and gene regulatory elements (REs). We profile chromatin accessibility and gene expression in Pogz-/- mice and show that POGZ promotes the active chromatin state and transcription of clustered synaptic genes. We further demonstrate that POGZ forms a nuclear complex and co-occupies loci with ADNP, another high-confidence ASD risk gene, and provide evidence that POGZ regulates other neurodevelopmental disorder risk genes as well. Our results reveal a neurodevelopmental function of an ASD risk gene and identify molecular targets that may elucidate its function in ASD.


Assuntos
Transtorno Autístico/enzimologia , Encéfalo/enzimologia , Montagem e Desmontagem da Cromatina , Eucromatina/metabolismo , Sinapses/enzimologia , Transposases/metabolismo , Animais , Transtorno Autístico/genética , Transtorno Autístico/fisiopatologia , Sítios de Ligação , Encéfalo/crescimento & desenvolvimento , Elementos de DNA Transponíveis , Elementos Facilitadores Genéticos , Eucromatina/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese , Regiões Promotoras Genéticas , Sinapses/genética , Transposases/genética
15.
Proc Natl Acad Sci U S A ; 118(48)2021 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-34819378

RESUMO

We have studied the three-dimensional (3D) cytoarchitecture of the human hippocampus in neuropathologically healthy and Alzheimer's disease (AD) individuals, based on phase-contrast X-ray computed tomography of postmortem human tissue punch biopsies. In view of recent findings suggesting a nuclear origin of AD, we target in particular the nuclear structure of the dentate gyrus (DG) granule cells. Tissue samples of 20 individuals were scanned and evaluated using a highly automated approach of measurement and analysis, combining multiscale recordings, optimized phase retrieval, segmentation by machine learning, representation of structural properties in a feature space, and classification based on the theory of optimal transport. Accordingly, we find that the prototypical transformation between a structure representing healthy granule cells and the pathological state involves a decrease in the volume of granule cell nuclei, as well as an increase in the electron density and its spatial heterogeneity. The latter can be explained by a higher ratio of heterochromatin to euchromatin. Similarly, many other structural properties can be derived from the data, reflecting both the natural polydispersity of the hippocampal cytoarchitecture between different individuals in the physiological context and the structural effects associated with AD pathology.


Assuntos
Mapeamento Encefálico/métodos , Hipocampo/diagnóstico por imagem , Imageamento Tridimensional/métodos , Tomografia Computadorizada por Raios X/métodos , Núcleo Celular/metabolismo , Meios de Contraste , Giro Denteado/diagnóstico por imagem , Eucromatina/química , Substância Cinzenta/diagnóstico por imagem , Heterocromatina/química , Humanos , Aprendizado de Máquina , Distribuição Normal , Reconhecimento Automatizado de Padrão , Análise de Componente Principal , Reprodutibilidade dos Testes , Substância Branca/diagnóstico por imagem
16.
Int J Mol Sci ; 22(22)2021 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-34829981

RESUMO

Homologous recombination (HR) typically occurs during meiosis between homologs, at a few unplanned locations along the chromosomes. In this study, we tested whether targeted recombination between homologous chromosomes can be achieved via Clustered Regulatory Interspaced Short Palindromic Repeat associated protein Cas9 (CRISPR-Cas9)-induced DNA double-strand break (DSB) repair in Arabidopsis thaliana. Our experimental system includes targets for DSB induction in euchromatic and heterochromatic genomic regions of hybrid F1 plants, in one or both parental chromosomes, using phenotypic and molecular markers to measure Non-Homologous End Joining and HR repair. We present a series of evidence showing that targeted DSBs can be repaired via HR using a homologous chromosome as the template in various chromatin contexts including in pericentric regions. Targeted crossover was rare, but gene conversion events were the most frequent outcome of HR and were found in both "hot and cold" regions. The length of the conversion tracts was variable, ranging from 5 to 7505 bp. In addition, a typical feature of these tracks was that they often were interrupted. Our findings pave the way for the use of targeted gene-conversion for precise breeding.


Assuntos
Arabidopsis/genética , Eucromatina/genética , Heterocromatina/genética , Recombinação Homóloga/genética , Arabidopsis/crescimento & desenvolvimento , Sistemas CRISPR-Cas/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Genoma de Planta/genética , Reparo de DNA por Recombinação/genética
17.
Molecules ; 26(21)2021 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-34771102

RESUMO

Chromatin is a dynamic structure comprising of DNA and proteins. Its unique nature not only help to pack the DNA tightly within the cell but also is pivotal in regulating gene expression DNA replication. Furthermore it also protects the DNA from being damaged. Various proteins are involved in making a specific complex within a chromatin and the knowledge about these interacting partners is helpful to enhance our understanding about the pathophysiology of various chromatin associated diseases. Moreover, it could also help us to identify new drug targets and design more effective remedies. Due to the existence of chromatin in different forms under various physiological conditions it is hard to develop a single strategy to study chromatin associated proteins under all conditions. In our current review, we tried to provide an overview and comparative analysis of the strategies currently adopted to capture the DNA bounded protein complexes and their mass spectrometric identification and quantification. Precise information about the protein partners and their function in the DNA-protein complexes is crucial to design new and more effective therapeutic molecules against chromatin associated diseases.


Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteoma , Proteômica/métodos , Sítios de Ligação , Imunoprecipitação da Cromatina/métodos , Sequenciamento de Cromatina por Imunoprecipitação , Eucromatina/metabolismo , Heterocromatina/metabolismo , Humanos , Proteínas de Ligação a Telômeros
18.
Nat Commun ; 12(1): 6958, 2021 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-34845222

RESUMO

Epialleles are meiotically heritable variations in expression states that are independent from changes in DNA sequence. Although they are common in plant genomes, their molecular origins are unknown. Here we show, using mutant and experimental populations, that epialleles in Arabidopsis thaliana that result from ectopic hypermethylation are due to feedback regulation of pathways that primarily function to maintain DNA methylation at heterochromatin. Perturbations to maintenance of heterochromatin methylation leads to feedback regulation of DNA methylation in genes. Using single base resolution methylomes from epigenetic recombinant inbred lines (epiRIL), we show that epiallelic variation is abundant in euchromatin, yet, associates with QTL primarily in heterochromatin regions. Mapping three-dimensional chromatin contacts shows that genes that are hotspots for ectopic hypermethylation have increases in contact frequencies with regions possessing H3K9me2. Altogether, these data show that feedback regulation of pathways that have evolved to maintain heterochromatin silencing leads to the origins of spontaneous hypermethylated epialleles.


Assuntos
Alelos , Arabidopsis/genética , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Heterocromatina/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mapeamento Cromossômico , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eucromatina/química , Eucromatina/metabolismo , Retroalimentação Fisiológica , Frequência do Gene , Haplótipos , Heterocromatina/química , Histonas/genética , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Plantas Geneticamente Modificadas , Processamento de Proteína Pós-Traducional , Locos de Características Quantitativas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
Biophys J ; 120(22): 5005-5017, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34653387

RESUMO

The genetic material of eukaryotes is segregated into transcriptionally active euchromatin and silent heterochromatin compartments. The spatial arrangement of chromatin compartments evolves over the course of cellular life in a process that remains poorly understood. The latest nuclear imaging experiments reveal a number of dynamical signatures of chromatin that are reminiscent of active multiphase liquids. This includes the observations of viscoelastic response, coherent motions, Ostwald ripening, and coalescence of chromatin compartments. There is also growing evidence that liquid-liquid phase separation of protein and nucleic acid components is the underlying mechanism for the dynamical behavior of chromatin. To dissect the organizational and dynamical implications of chromatin's liquid behavior, we have devised a phenomenological field-theoretic model of the nucleus as a multiphase condensate of liquid chromatin types. Employing the liquid chromatin model of the Drosophila nucleus, we have carried out an extensive set of simulations with an objective to shed light on the dynamics and chromatin patterning observed in the latest nuclear imaging experiments. Our simulations reveal the emergence of experimentally detected mesoscale chromatin channels and spheroidal droplets which arise from the dynamic interplay of chromatin type to type interactions and intermingling of chromosomal territories. We also quantitatively reproduce coherent motions of chromatin domains observed in displacement correlation spectroscopy measurements which are explained within the framework of our model by phase separation of chromatin types operating within constrained intrachromosomal and interchromosomal boundaries. Finally, we illuminate the role of heterochromatin-lamina interactions in the nuclear organization by showing that these interactions enhance the mobility of euchromatin and indirectly introduce correlated motions of heterochromatin droplets.


Assuntos
Cromatina , Eucromatina , Animais , Núcleo Celular , Drosophila , Heterocromatina
20.
Prog Mol Subcell Biol ; 60: 145-167, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34386875

RESUMO

Satellite DNAs are tandemly repeated sequences organized in large clusters within (peri)centromeric and/or subtelomeric heterochromatin. However, in many species, satellite DNAs are not restricted to heterochromatin but are also dispersed as short arrays within euchromatin. Such genomic organization together with transcriptional activity seems to be a prerequisite for the gene-modulatory effect of satellite DNAs which was first demonstrated in the beetle Tribolium castaneum upon heat stress. Namely, enrichment of a silent histone mark at euchromatic repeats of a major beetle satellite DNA results in epigenetic silencing of neighboring genes. In addition, human satellite III transcripts induced by heat shock contribute to genome-wide gene silencing, providing protection against stress-induced cell death. Gene silencing mediated by satellite RNA was also shown to be fundamental for the early embryonic development of the mosquito Aedes aegypti. Apart from a physiological role during embryogenesis and heat stress response, activation of satellite DNAs in terms of transcription and proliferation can have an evolutionary impact. Spreading of satellite repeats throughout euchromatin promotes the variation of epigenetic landscapes and gene expression diversity, contributing to the evolution of gene regulatory networks and to genome adaptation in fluctuating environmental conditions.


Assuntos
DNA Satélite , Tribolium , Animais , DNA Satélite/genética , Eucromatina , Regulação da Expressão Gênica , Heterocromatina , Humanos , Tribolium/genética
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