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1.
Nat Commun ; 12(1): 5016, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408138

RESUMO

DNA damage prompts a diverse range of alterations to the chromatin landscape. The RNF168 E3 ubiquitin ligase catalyzes the mono-ubiquitination of histone H2A at lysine (K)13/15 (mUb-H2A), forming a binding module for DNA repair proteins. BRCA1 promotes homologous recombination (HR), in part, through its interaction with PALB2, and the formation of a larger BRCA1-PALB2-BRCA2-RAD51 (BRCA1-P) complex. The mechanism by which BRCA1-P is recruited to chromatin surrounding DNA breaks is unclear. In this study, we reveal that an RNF168-governed signaling pathway is responsible for localizing the BRCA1-P complex to DNA damage. Using mice harboring a Brca1CC (coiled coil) mutation that blocks the Brca1-Palb2 interaction, we uncovered an epistatic relationship between Rnf168- and Brca1CC alleles, which disrupted development, and reduced the efficiency of Palb2-Rad51 localization. Mechanistically, we show that RNF168-generated mUb-H2A recruits BARD1 through a BRCT domain ubiquitin-dependent recruitment motif (BUDR). Subsequently, BARD1-BRCA1 accumulate PALB2-RAD51 at DNA breaks via the CC domain-mediated BRCA1-PALB2 interaction. Together, these findings establish a series of molecular interactions that connect the DNA damage signaling and HR repair machinery.


Assuntos
Proteína BRCA1/metabolismo , Dano ao DNA , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteína BRCA1/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , DNA/genética , DNA/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Ligação Proteica , Transporte Proteico , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
2.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445207

RESUMO

Recent studies show a crucial role of post-transcriptional processes in the regulation of gene expression. Our research has shown that mRNA retention in the nucleus plays a significant role in such regulation. We studied larch microsporocytes during meiotic prophase, characterized by pulsatile transcriptional activity. After each pulse, the transcriptional activity is silenced, but the transcripts synthesized at this time are not exported immediately to the cytoplasm but are retained in the cell nucleus and especially in Cajal bodies, where non-fully-spliced transcripts with retained introns are accumulated. Analysis of the transcriptome of these cells and detailed analysis of the nuclear retention and transport dynamics of several mRNAs revealed two main patterns of nuclear accumulation and transport. The majority of studied transcripts followed the first one, consisting of a more extended retention period and slow release to the cytoplasm. We have shown this in detail for the pre-mRNA and mRNA encoding RNA pol II subunit 10. In this pre-mRNA, a second (retained) intron is posttranscriptionally spliced at a precisely defined time. Fully mature mRNA is then released into the cytoplasm, where the RNA pol II complexes are produced. These proteins are necessary for transcription in the next pulse to occur.mRNAs encoding translation factors and SERRATE followed the second pattern, in which the retention period was shorter and transcripts were rapidly transferred to the cytoplasm. The presence of such a mechanism in various cell types from a diverse range of organisms suggests that it is an evolutionarily conserved mechanism of gene regulation.


Assuntos
Núcleo Celular/metabolismo , Larix/metabolismo , Pólen/metabolismo , Prófase , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Núcleo Celular/genética , Larix/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pólen/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA de Plantas/genética
3.
Int J Mol Sci ; 22(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361059

RESUMO

In vertebrates, nucleostemin (NS) is an important marker of proliferation in several types of stem and cancer cells, and it can also interact with the tumor-suppressing transcription factor p53. In the present study, the intra-nuclear diffusional dynamics of native NS tagged with GFP and two GFP-tagged NS mutants with deleted guanosine triphosphate (GTP)-binding domains were analyzed by fluorescence correlation spectroscopy. Free and slow binding diffusion coefficients were evaluated, either under normal culture conditions or under treatment with specific cellular proliferation inhibitors actinomycin D (ActD), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), or trichostatin A (TSA). When treated with ActD, the fractional ratio of the slow diffusion was significantly decreased in the nucleoplasm. The decrease was proportional to ActD treatment duration. In contrast, DRB or TSA treatment did not affect NS diffusion. Interestingly, it was also found that the rate of diffusion of two NS mutants increased significantly even under normal conditions. These results suggest that the mobility of NS in the nucleoplasm is related to the initiation of DNA or RNA replication, and that the GTP-binding motif is also related to the large change of mobility.


Assuntos
Núcleo Celular/metabolismo , Dactinomicina/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Nucleares/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Transcrição Genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/genética , Células HeLa , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética
4.
Nat Commun ; 12(1): 4657, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34341359

RESUMO

Correlative imaging and quantification of intracellular nanoparticles with the underlying ultrastructure is crucial for understanding cell-nanoparticle interactions in biological research. However, correlative nanoscale imaging of whole cells still remains a daunting challenge. Here, we report a straightforward nanoscopic approach for whole-cell correlative imaging, by simultaneous ionoluminescence and ultrastructure mapping implemented with a highly focused beam of alpha particles. We demonstrate that fluorescent nanodiamonds exhibit fast, ultrabright and stable emission upon excitation by alpha particles. Thus, by using fluorescent nanodiamonds as imaging probes, our approach enables quantification and correlative localization of single nanodiamonds within a whole cell at sub-30 nm resolution. As an application example, we show that our approach, together with Monte Carlo simulations and radiobiological experiments, can be employed to provide unique insights into the mechanisms of nanodiamond radiosensitization at the single whole-cell level. These findings may benefit clinical studies of radio-enhancement effects by nanoparticles in charged-particle cancer therapy.


Assuntos
Partículas alfa , Núcleo Celular/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Histonas/metabolismo , Nanodiamantes/efeitos da radiação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células HeLa , Células Hep G2 , Humanos , Microscopia Confocal/métodos , Microscopia Eletrônica de Varredura/métodos , Nanodiamantes/química , Nanodiamantes/ultraestrutura , Fosforilação/efeitos da radiação
5.
Nat Commun ; 12(1): 4908, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389711

RESUMO

C9ORF72 hexanucleotide GGGGCC repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat-containing RNA mediates toxicity through nuclear granules and dipeptide repeat (DPR) proteins produced by repeat-associated non-AUG translation. However, it remains unclear how the intron-localized repeats are exported and translated in the cytoplasm. We use single molecule imaging approach to examine the molecular identity and spatiotemporal dynamics of the repeat RNA. We demonstrate that the spliced intron with G-rich repeats is stabilized in a circular form due to defective lariat debranching. The spliced circular intron, instead of pre-mRNA, serves as the translation template. The NXF1-NXT1 pathway plays an important role in the nuclear export of the circular intron and modulates toxic DPR production. This study reveals an uncharacterized disease-causing RNA species mediated by repeat expansion and demonstrates the importance of RNA spatial localization to understand disease etiology.


Assuntos
Proteína C9orf72/genética , Núcleo Celular/metabolismo , Íntrons/genética , Biossíntese de Proteínas/genética , RNA/genética , Transporte Ativo do Núcleo Celular/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Proteína C9orf72/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/genética , Expansão das Repetições de DNA/genética , Dipeptídeos/genética , Dipeptídeos/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Predisposição Genética para Doença/genética , Células HEK293 , Humanos , Microscopia de Fluorescência , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética
6.
BMC Plant Biol ; 21(1): 313, 2021 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215178

RESUMO

BACKGROUND: Harnessing heterosis is one of the major approaches to increase rice yield and has made a great contribution to food security. The identification and selection of outstanding parental genotypes especially among male sterile lines is a key step for exploiting heterosis. Two-line hybrid system is based on the discovery and application of photoperiod- and thermo-sensitive genic sensitive male sterile (PTGMS) materials. The development of wide-range of male sterile lines from a common gene pool leads to a narrower genetic diversity, which is vulnerable to biotic and abiotic stress. Hence, it is valuable to ascertain the genetic background of PTGMS lines and to understand their relationships in order to select and design a future breeding strategy. RESULTS: A collection of 118 male sterile rice lines and 13 conventional breeding lines from the major rice growing regions of China was evaluated and screened against the photosensitive (pms3) and temperature sensitive male sterility (tms5) genes. The total gene pool was divided into four major populations as P1 possessing the pms3, P2 possessing tms5, P3 possessing both pms3 and tms5 genes, and P4 containing conventional breeding lines without any male sterility allele. The high genetic purity was revealed by homozygous alleles in all populations. The population admixture, principle components and the phylogenetic analysis revealed the close relations of P2 and P3 with P4. The population differentiation analysis showed that P1 has the highest differentiation coefficient. The lines from P1 were observed as the ancestors of other three populations in a phylogenetic tree, while the lines in P2 and P3 showed a close genetic relation with conventional lines. A core collection of top 10% lines with maximum within and among populations genetic diversity was constructed for future research and breeding efforts. CONCLUSION: The low genetic diversity and close genetic relationship among PTGMS lines in P2, P3 and P4 populations suggest a selection sweep and they might result from a backcrossing with common ancestors including the pure lines of P1. The core collection from PTGMS panel updated with new diverse germplasm will serve best for further two-line hybrid breeding.


Assuntos
Oryza/genética , Fotoperíodo , Infertilidade das Plantas/genética , Sementes/genética , Temperatura , Núcleo Celular/genética , Núcleo Celular/efeitos da radiação , Análise por Conglomerados , Ontologia Genética , Estudos de Associação Genética , Marcadores Genéticos , Luz , Nucleotídeos/genética , Oryza/efeitos da radiação , Filogenia , Infertilidade das Plantas/efeitos da radiação , Polimorfismo de Nucleotídeo Único/genética , Análise de Componente Principal , Reprodutibilidade dos Testes , Sementes/efeitos da radiação
7.
Mol Cell ; 81(15): 3065-3081.e12, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34297911

RESUMO

The chromatin fiber folds into loops, but the mechanisms controlling loop extrusion are still poorly understood. Using super-resolution microscopy, we visualize that loops in intact nuclei are formed by a scaffold of cohesin complexes from which the DNA protrudes. RNA polymerase II decorates the top of the loops and is physically segregated from cohesin. Augmented looping upon increased loading of cohesin on chromosomes causes disruption of Lamin at the nuclear rim and chromatin blending, a homogeneous distribution of chromatin within the nucleus. Altering supercoiling via either transcription or topoisomerase inhibition counteracts chromatin blending, increases chromatin condensation, disrupts loop formation, and leads to altered cohesin distribution and mobility on chromatin. Overall, negative supercoiling generated by transcription is an important regulator of loop formation in vivo.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Transcrição Genética/fisiologia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular , Núcleo Celular/genética , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Laminas/genética , Laminas/metabolismo , RNA Polimerase II/metabolismo , Imagem Individual de Molécula/métodos
8.
Nat Commun ; 12(1): 4279, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257313

RESUMO

Divergent mitonuclear coadaptation could facilitate speciation. We investigate this possibility in two hybridizing species of warblers, Setophaga occidentalis and S. townsendi, in western North America. Inland S. townsendi harbor distinct mitochondrial DNA haplotypes from those of S. occidentalis. These populations also differ in several nuclear DNA regions. Coastal S. townsendi demonstrate mixed mitonuclear ancestry from S. occidentalis and inland S. townsendi. Of the few highly-differentiated chromosomal regions between inland S. townsendi and S. occidentalis, a 1.2 Mb gene block on chromosome 5 is also differentiated between coastal and inland S. townsendi. Genes in this block are associated with fatty acid oxidation and energy-related signaling transduction, thus linked to mitochondrial functions. Genetic variation within this candidate gene block covaries with mitochondrial DNA and shows signatures of divergent selection. Spatial variation in mitonuclear ancestries is correlated with climatic conditions. Together, these observations suggest divergent mitonuclear coadaptation underpins cryptic differentiation in this species complex.


Assuntos
Núcleo Celular/genética , DNA Mitocondrial/genética , Animais , Variação Genética/genética , Haplótipos/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Aves Canoras/genética
9.
Int J Mol Sci ; 22(14)2021 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-34299047

RESUMO

p57Kip2 protein is a member of the CIP/Kip family, mainly localized in the nucleus where it exerts its Cyclin/CDKs inhibitory function. In addition, the protein plays key roles in embryogenesis, differentiation, and carcinogenesis depending on its cellular localization and interactors. Mutations of CDKN1C, the gene encoding human p57Kip2, result in the development of different genetic diseases, including Beckwith-Wiedemann, IMAGe and Silver-Russell syndromes. We investigated a specific Beckwith-Wiedemann associated CDKN1C change (c.946 C>T) that results in the substitution of the C-terminal amino acid (arginine 316) with a tryptophan (R316W-p57Kip2). We found a clear redistribution of R316W-p57Kip2, in that while the wild-type p57Kip2 mostly occurs in the nucleus, the mutant form is also distributed in the cytoplasm. Transfection of two expression constructs encoding the p57Kip2 N- and C-terminal domain, respectively, allows the mapping of the nuclear localization signal(s) (NLSs) between residues 220-316. Moreover, by removing the basic RKRLR sequence at the protein C-terminus (from 312 to 316 residue), p57Kip2 was confined in the cytosol, implying that this sequence is absolutely required for nuclear entry. In conclusion, we identified an unreported p57Kip2 NLS and suggest that its absence or mutation might be of relevance in CDKN1C-associated human diseases determining significant changes of p57Kip2 localization/regulatory roles.


Assuntos
Síndrome de Beckwith-Wiedemann/genética , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/genética , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Mutação , Sinais de Localização Nuclear , Síndrome de Beckwith-Wiedemann/patologia , Ciclo Celular , Núcleo Celular/genética , Proliferação de Células , Células HEK293 , Células Hep G2 , Humanos
10.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201585

RESUMO

Forkhead box O3 (Foxo3) is a member of the FOXO subfamily within the forkhead box (FOX) family, which has been shown to be essential for ovarian follicular development and maturation. Previous studies have shown the abundant expression of miR-195-5p in the nuclei of porcine granulosa cells (GCs), suggesting its potential role during ovarian follicle growth. In this study, a conditional immortalized porcine granulosa cell (CIPGC) line was used to determine whether the expression of Foxo3 could be regulated by the nuclear-enriched miR-195-5p. Through silico target prediction, we identified a potential binding site of miR-195-5p within the Foxo3 promoter. The over-expression of miR-195-5p increased Foxo3 expression at both mRNA and protein levels, while the knockdown of miR-195-5p decreased the expression of Foxo3. Furthermore, driven by the Foxo3 promoter, luciferase reporter activity was increased in response to miR-195-5p, while the mutation of the miR-195-5p binding site in the promoter region abolished this effect. In addition, the siRNA knockdown of Argonaute (AGO) 2, but not AGO1, significantly decreased Foxo3 transcript level. However, miR-195-5p failed to upregulate Foxo3 expression when AGO2 was knocked down. Moreover, chromatin immunoprecipitation (CHIP) assay showed that anti-AGO2 antibody pulled down both AGO2 and the Foxo3 promoter sequence, suggesting that AGO2 may be required for miR-195-5p to regulate Foxo3 expression in the nucleus. Additionally, Foxo3 expression was significantly increased by valproic acid (VPA), the inhibitor of deacetylase, as well as by methyltransferase inhibitor BIX-01294, indicating the involvement of histone modification. These effects were further enhanced in the presence of miR-195-5p and were decreased when miR-195-5p was knocked down. Overall, our results suggest that nuclear-enriched miR-195-5p regulates Foxo3 expression, which may be associated with AGO2 recruitment, as well as histone demethylation and acetylation in ovarian granulosa cells.


Assuntos
Proteína Forkhead Box O3/genética , Células da Granulosa/fisiologia , MicroRNAs/genética , Animais , Proteínas Argonauta/genética , Sítios de Ligação , Linhagem Celular , Núcleo Celular/genética , Imunoprecipitação da Cromatina , Epigênese Genética , Feminino , Proteína Forkhead Box O3/metabolismo , Regulação da Expressão Gênica , Células da Granulosa/citologia , Células da Granulosa/efeitos dos fármacos , Histonas/genética , Histonas/metabolismo , Regiões Promotoras Genéticas , Suínos , Ácido Valproico/farmacologia
11.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201896

RESUMO

miR-29b2 and miR-29c play a suppressive role in breast cancer progression. C1orf132 (also named MIR29B2CHG) is the host gene for generating both microRNAs. However, the region also expresses longer transcripts with unknown functions. We employed bioinformatics and experimental approaches to decipher C1orf132 expression and function in breast cancer tissues. We also used the CRISPR/Cas9 technique to excise a predicted C1orf132 distal promoter and followed the behavior of the edited cells by real-time PCR, flow cytometry, migration assay, and RNA-seq techniques. We observed that C1orf132 long transcript is significantly downregulated in triple-negative breast cancer. We also identified a promoter for the longer transcripts of C1orf132 whose functionality was demonstrated by transfecting MCF7 cells with a C1orf132 promoter-GFP construct. Knocking-out the promoter by means of CRISPR/Cas9 revealed no alterations in the expression of the neighboring genes CD46 and CD34, while the expression of miR-29c was reduced by half. Furthermore, the promoter knockout elevated the migration ability of the edited cells. RNA sequencing revealed many up- and downregulated genes involved in various cellular pathways, including epithelial to mesenchymal transition and mammary gland development pathways. Altogether, we are reporting here the existence of an additional/distal promoter with an enhancer effect on miR-29 generation and an inhibitory effect on cell migration.


Assuntos
RNA Longo não Codificante/genética , Neoplasias de Mama Triplo Negativas/genética , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Regulação para Baixo , Transição Epitelial-Mesenquimal/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo
12.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34204945

RESUMO

A lesser known but crucially important downstream effect of Rho family GTPases is the regulation of gene expression. This major role is mediated via the cytoskeleton, the organization of which dictates the nucleocytoplasmic shuttling of a set of transcription factors. Central among these is myocardin-related transcription factor (MRTF), which upon actin polymerization translocates to the nucleus and binds to its cognate partner, serum response factor (SRF). The MRTF/SRF complex then drives a large cohort of genes involved in cytoskeleton remodeling, contractility, extracellular matrix organization and many other processes. Accordingly, MRTF, activated by a variety of mechanical and chemical stimuli, affects a plethora of functions with physiological and pathological relevance. These include cell motility, development, metabolism and thus metastasis formation, inflammatory responses and-predominantly-organ fibrosis. The aim of this review is twofold: to provide an up-to-date summary about the basic biology and regulation of this versatile transcriptional coactivator; and to highlight its principal involvement in the pathobiology of kidney disease. Acting through both direct transcriptional and epigenetic mechanisms, MRTF plays a key (yet not fully appreciated) role in the induction of a profibrotic epithelial phenotype (PEP) as well as in fibroblast-myofibroblast transition, prime pathomechanisms in chronic kidney disease and renal fibrosis.


Assuntos
Nefropatias/genética , Complexos Multiproteicos/genética , Fator de Resposta Sérica/genética , Transativadores/genética , Movimento Celular/genética , Núcleo Celular/genética , Citoesqueleto/genética , Regulação da Expressão Gênica/genética , Humanos , Nefropatias/patologia , Regiões Promotoras Genéticas/genética
13.
Int J Mol Sci ; 22(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34206020

RESUMO

Three dimensional (3D) ultra-structural imaging is an important tool for unraveling the organizational structure of individual chromosomes at various stages of the cell cycle. Performing hitherto uninvestigated ultra-structural analysis of the human genome at prophase, we used serial block-face scanning electron microscopy (SBFSEM) to understand chromosomal architectural organization within 3D nuclear space. Acquired images allowed us to segment, reconstruct, and extract quantitative 3D structural information about the prophase nucleus and the preserved, intact individual chromosomes within it. Our data demonstrate that each chromosome can be identified with its homolog and classified into respective cytogenetic groups. Thereby, we present the first 3D karyotype built from the compact axial structure seen on the core of all prophase chromosomes. The chromosomes display parallel-aligned sister chromatids with familiar chromosome morphologies with no crossovers. Furthermore, the spatial positions of all 46 chromosomes revealed a pattern showing a gene density-based correlation and a neighborhood map of individual chromosomes based on their relative spatial positioning. A comprehensive picture of 3D chromosomal organization at the nanometer level in a single human lymphocyte cell is presented.


Assuntos
Cromossomos/genética , Linfócitos/citologia , Mitose/genética , Troca de Cromátide Irmã/genética , Núcleo Celular/genética , Cromossomos/ultraestrutura , Humanos , Cariotipagem , Linfócitos/ultraestrutura , Microscopia Eletrônica de Varredura
14.
J Cell Sci ; 134(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34297126

RESUMO

Skeletal muscle myofibers are large and elongated cells with multiple and evenly distributed nuclei. Nuclear distribution suggests that each nucleus influences a specific compartment within the myofiber and implies a functional role for nuclear positioning. Compartmentalization of specific mRNAs and proteins has been reported at the neuromuscular and myotendinous junctions, but mRNA distribution in non-specialized regions of the myofibers remains largely unexplored. We report that the bulk of mRNAs are enriched around the nucleus of origin and that this perinuclear accumulation depends on recently transcribed mRNAs. Surprisingly, mRNAs encoding large proteins - giant mRNAs - are spread throughout the cell and do not exhibit perinuclear accumulation. Furthermore, by expressing exogenous transcripts with different sizes we found that size contributes to mRNA spreading independently of mRNA sequence. Both these mRNA distribution patterns depend on microtubules and are independent of nuclear dispersion, mRNA expression level and stability, and the characteristics of the encoded protein. Thus, we propose that mRNA distribution in non-specialized regions of skeletal muscle is size selective to ensure cellular compartmentalization and simultaneous long-range distribution of giant mRNAs.


Assuntos
Fibras Musculares Esqueléticas , Músculo Esquelético , Núcleo Celular/genética , RNA Mensageiro/genética , Tendões
15.
Am J Bot ; 108(7): 1166-1180, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34250591

RESUMO

PREMISE: The inference of evolutionary relationships in the species-rich family Orchidaceae has hitherto relied heavily on plastid DNA sequences and limited taxon sampling. Previous studies have provided a robust plastid phylogenetic framework, which was used to classify orchids and investigate the drivers of orchid diversification. However, the extent to which phylogenetic inference based on the plastid genome is congruent with the nuclear genome has been only poorly assessed. METHODS: We inferred higher-level phylogenetic relationships of orchids based on likelihood and ASTRAL analyses of 294 low-copy nuclear genes sequenced using the Angiosperms353 universal probe set for 75 species (representing 69 genera, 16 tribes, 24 subtribes) and a concatenated analysis of 78 plastid genes for 264 species (117 genera, 18 tribes, 28 subtribes). We compared phylogenetic informativeness and support for the nuclear and plastid phylogenetic hypotheses. RESULTS: Phylogenetic inference using nuclear data sets provides well-supported orchid relationships that are highly congruent between analyses. Comparisons of nuclear gene trees and a plastid supermatrix tree showed that the trees are mostly congruent, but revealed instances of strongly supported phylogenetic incongruence in both shallow and deep time. The phylogenetic informativeness of individual Angiosperms353 genes is in general better than that of most plastid genes. CONCLUSIONS: Our study provides the first robust nuclear phylogenomic framework for Orchidaceae and an assessment of intragenomic nuclear discordance, plastid-nuclear tree incongruence, and phylogenetic informativeness across the family. Our results also demonstrate what has long been known but rarely thoroughly documented: nuclear and plastid phylogenetic trees can contain strongly supported discordances, and this incongruence must be reconciled prior to interpretation in evolutionary studies, such as taxonomy, biogeography, and character evolution.


Assuntos
Genomas de Plastídeos , Orchidaceae , Núcleo Celular/genética , Orchidaceae/genética , Filogenia , Plastídeos/genética
16.
Am J Bot ; 108(7): 1252-1269, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34287829

RESUMO

PREMISE: The carrot family (Apiaceae) comprises 466 genera, which include many well-known crops (e.g., aniseed, caraway, carrots, celery, coriander, cumin, dill, fennel, parsley, and parsnips). Higher-level phylogenetic relationships among subfamilies, tribes, and other major clades of Apiaceae are not fully resolved. This study aims to address this important knowledge gap. METHODS: Target sequence capture with the universal Angiosperms353 probe set was used to examine phylogenetic relationships in 234 genera of Apiaceae, representing all four currently recognized subfamilies (Apioideae, Azorelloideae, Mackinlayoideae, and Saniculoideae). Recovered nuclear genes were analyzed using both multispecies coalescent and concatenation approaches. RESULTS: We recovered hundreds of nuclear genes even from old and poor-quality herbarium specimens. Of particular note, we placed with strong support three incertae sedis genera (Platysace, Klotzchia, and Hermas); all three occupy isolated positions, with Platysace resolved as sister to all remaining Apiaceae. We placed nine genera (Apodicarpum, Bonannia, Grafia, Haplosciadium, Microsciadium, Physotrichia, Ptychotis, Tricholaser, Xatardia) that have never previously been included in any molecular phylogenetic study. CONCLUSIONS: We provide support for the maintenance of the four existing subfamilies of Apiaceae, while recognizing that Hermas, Klotzschia, and the Platysace clade may each need to be accommodated in additional subfamilies (pending improved sampling). The placement of the currently apioid genus Phlyctidocarpa can be accommodated by the expansion of subfamily Saniculoideae, although adequate morphological synapomorphies for this grouping are yet to be defined. This is the first phylogenetic study of the Apiaceae using high-throughput sequencing methods and represents an unprecedented evolutionary framework for the group.


Assuntos
Apiaceae , Daucus carota , Apiaceae/genética , Evolução Biológica , Núcleo Celular/genética , Daucus carota/genética , Filogenia
17.
Front Immunol ; 12: 628564, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34211456

RESUMO

Neutrophil extracellular traps (NETs) and mitochondrial DNA (mtDNA) are inflammatory mediators involved in the development of type 1 diabetes (T1D). Pancreas-infiltrating neutrophils can release NETs, contributing to the inflammatory process. Levels of NETs are increased in serum from patients with T1D and mtDNA is increased in adult T1D patients. Our aim was to investigate extracellular DNA (NETs, mtDNA and nuclear DNA) in children with newly diagnosed T1D and in children at high risk of the disease. We also elucidated if extracellular DNA short after diagnosis could predict loss of endogenous insulin production. Samples were analysed for mtDNA and nuclear DNA using droplet digital PCR and NETs were assessed by a NET-remnants ELISA. In addition, in vitro assays for induction and degradation of NETs, as well as analyses of neutrophil elastase, HLA genotypes, levels of c-peptide, IL-1beta, IFN and autoantibodies (GADA, IA-2A, IAA and ZnT8A) were performed. In serum from children 10 days after T1D onset there was an increase in NETs (p=0.007), mtDNA (p<0.001) and nuclear DNA (p<0.001) compared to healthy children. The elevated levels were found only in younger children. In addition, mtDNA increased in consecutive samples short after onset (p=0.017). However, levels of extracellular DNA short after onset did not reflect future loss of endogenous insulin production. T1D serum induced NETs in vitro and did not deviate in the ability to degrade NETs. HLA genotypes and autoantibodies, except for ZnT8A, were not associated with extracellular DNA in T1D children. Serum from children with high risk of T1D showed fluctuating levels of extracellular DNA, sometimes increased compared to healthy children. Therefore, extracellular DNA in serum from autoantibody positive high-risk children does not seem to be a suitable biomarker candidate for prediction of T1D. In conclusion, we found increased levels of extracellular DNA in children with newly diagnosed T1D, which might be explained by an ongoing systemic inflammation.


Assuntos
Núcleo Celular/genética , DNA Mitocondrial/sangue , DNA/sangue , Diabetes Mellitus Tipo 1/sangue , Armadilhas Extracelulares/metabolismo , Adolescente , Fatores Etários , Autoanticorpos/sangue , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Feminino , Humanos , Estudos Longitudinais , Masculino , Medição de Risco , Fatores de Risco , Regulação para Cima
18.
Gene ; 801: 145841, 2021 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-34274481

RESUMO

Mitochondrial sequences were among the first molecular data collected for phylogenetic studies and they are plentiful in DNA sequence archives. However, the future value of mitogenomic data in phylogenetics is uncertain, because its phylogenetic signal sometimes conflicts with that of the nuclear genome. A thorough understanding of the causes and prevalence of cyto-nuclear discordance would aid in reconciling different results owing to sequence data type, and provide a framework for interpreting megaphylogenies when taxa which lack substantial nuclear data are placed using mitochondrial data. Here, we examine the prevalence and possible causes of cyto-nuclear discordance in the landfowl (Aves: Galliformes), leveraging 47 new mitogenomes assembled from off-target reads recovered as part of a target-capture study. We evaluated two hypotheses, that cyto-nuclear discordance is "genuine" and a result of biological processes such as incomplete lineage sorting or introgression, and that cyto-nuclear discordance is an artifact of inaccurate mitochondrial tree estimation (the "inaccurate estimation" hypothesis). We identified seven well-supported topological differences between the mitogenomic tree and trees based on nuclear data. These well-supported topological differences were robust to model selection. An examination of sites suggests these differences were driven by small number of sites, particularly from third-codon positions, suggesting that they were not confounded by convergent directional selection. Hence, the hypothesis of genuine discordance was supported.


Assuntos
Galliformes/genética , Genoma Mitocondrial/genética , Filogenia , Animais , Núcleo Celular/genética
19.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299219

RESUMO

Infertility is a problem affecting an increasing number of couples worldwide. Currently, marker tests for male factor infertility are complex, highly technical and relatively subjective. Up to 40% of cases of male factor infertility are currently diagnosed as idiopathic therefore, there is a clear need for further research into better ways of diagnosing it. Changes in sperm telomere length have been associated with infertility and closely linked to DNA damage and fragmentation, which are also known to be related to infertility. However, telomere distribution is a parameter thus far underexplored as an infertility marker. Here, we assessed morphological parameters of sperm nuclei in fertile control and male factor infertile cohorts. In addition, we used 2D and 3D fluorescence in situ hybridization (FISH) to compare telomere distribution between these two groups. Our findings indicate that the infertile cohort sperm nuclei were, on average, 2.9% larger in area and showed subtle differences in sperm head height and width. Telomeres were mainly distributed towards the periphery of the nuclei in the control cohort, with diminishing telomere signals towards the center of the nuclei. Sperm nuclei of infertile males, however, had more telomere signals towards the center of the nuclei, a finding supported by 3D imaging. We conclude that, with further development, both morphology and telomere distribution may prove useful investigative tools in the fertility clinic.


Assuntos
Dano ao DNA , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Cabeça do Espermatozoide/patologia , Telômero/metabolismo , Biomarcadores/metabolismo , Estudos de Casos e Controles , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Estudos de Coortes , Humanos , Infertilidade Masculina/genética , Masculino , Cabeça do Espermatozoide/metabolismo , Telômero/genética
20.
Nat Genet ; 53(8): 1143-1155, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34239132

RESUMO

The gene-regulatory landscape of the brain is highly dynamic in health and disease, coordinating a menagerie of biological processes across distinct cell types. Here, we present a multi-omic single-nucleus study of 191,890 nuclei in late-stage Alzheimer's disease (AD), accessible through our web portal, profiling chromatin accessibility and gene expression in the same biological samples and uncovering vast cellular heterogeneity. We identified cell-type-specific, disease-associated candidate cis-regulatory elements and their candidate target genes, including an oligodendrocyte-associated regulatory module containing links to APOE and CLU. We describe cis-regulatory relationships in specific cell types at a subset of AD risk loci defined by genome-wide association studies, demonstrating the utility of this multi-omic single-nucleus approach. Trajectory analysis of glial populations identified disease-relevant transcription factors, such as SREBF1, and their regulatory targets. Finally, we introduce single-nucleus consensus weighted gene coexpression analysis, a coexpression network analysis strategy robust to sparse single-cell data, and perform a systems-level analysis of the AD transcriptome.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Cromatina/genética , Córtex Pré-Frontal/patologia , Sequências Reguladoras de Ácido Nucleico , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Núcleo Celular/genética , Cromatina/metabolismo , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Humanos , Masculino , Neuroglia/patologia , Oligodendroglia/patologia , Oligodendroglia/fisiologia , Córtex Pré-Frontal/fisiologia , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Fatores de Transcrição/genética
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