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1.
Mol Cell ; 78(2): 236-249.e7, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32101700

RESUMO

The formation of silenced and condensed heterochromatin foci involves enrichment of heterochromatin protein 1 (HP1). HP1 can bridge chromatin segments and form liquid droplets, but the biophysical principles underlying heterochromatin compartmentalization in the cell nucleus are elusive. Here, we assess mechanistically relevant features of pericentric heterochromatin compaction in mouse fibroblasts. We find that (1) HP1 has only a weak capacity to form liquid droplets in living cells; (2) the size, global accessibility, and compaction of heterochromatin foci are independent of HP1; (3) heterochromatin foci lack a separated liquid HP1 pool; and (4) heterochromatin compaction can toggle between two "digital" states depending on the presence of a strong transcriptional activator. These findings indicate that heterochromatin foci resemble collapsed polymer globules that are percolated with the same nucleoplasmic liquid as the surrounding euchromatin, which has implications for our understanding of chromatin compartmentalization and its functional consequences.


Assuntos
Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Eucromatina/genética , Heterocromatina/genética , Animais , Fibroblastos , Camundongos
2.
PLoS Genet ; 15(5): e1008157, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31136569

RESUMO

Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.


Assuntos
Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , RNA Polimerase I/genética , DNA Ribossômico/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Precursores de RNA/genética , RNA Ribossômico , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição Genética
3.
Nucleic Acids Res ; 47(12): 6195-6207, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31114898

RESUMO

DNA folding and dynamics along with major nuclear functions are determined by chromosome structural properties, which remain, thus far, elusive in vivo. Here, we combine polymer modeling and single particle tracking experiments to determine the physico-chemical parameters of chromatin in vitro and in living yeast. We find that the motion of reconstituted chromatin fibers can be recapitulated by the Rouse model using mechanical parameters of nucleosome arrays deduced from structural simulations. Conversely, we report that the Rouse model shows some inconsistencies to analyze the motion and structural properties inferred from yeast chromosomes determined with chromosome conformation capture techniques (specifically, Hi-C). We hence introduce the Rouse model with Transient Internal Contacts (RouseTIC), in which random association and dissociation occurs along the chromosome contour. The parametrization of this model by fitting motion and Hi-C data allows us to measure the kinetic parameters of the contact formation reaction. Chromosome contacts appear to be transient; associated to a lifetime of seconds and characterized by an attractive energy of -0.3 to -0.5 kBT. We suggest attributing this energy to the occurrence of histone tail-DNA contacts and notice that its amplitude sets chromosomes in 'theta' conditions, in which they are poised for compartmentalization and phase separation.


Assuntos
Cromossomos Fúngicos/química , Modelos Genéticos , Cromatina/química , DNA Fúngico/química , Cinética , Movimento (Física) , Nucleossomos/química
4.
PLoS One ; 14(3): e0214122, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30913280

RESUMO

Branched chain amino acids (BCAA) are essential elements of the human diet, which display increased plasma levels in obesity and regained particular interest as potential biomarkers for development of diabetes. To define determinants of insulin resistance (IR) we investigated 73 genes involved in BCAA metabolism or regulation by fine-scale haplotype mapping in two European populations with metabolic syndrome. French and Romanians (n = 465) were genotyped for SNPs (Affymetrix) and enriched by imputation (BEAGLE 4.1) at 1000 genome project density. Initial association hits detected by sliding window were refined (HAPLOVIEW 3.1 and PHASE 2.1) and correlated to homeostasis model assessment (HOMAIR) index, in vivo insulin sensitivity (SI) and BCAA plasma levels (ANOVA). Four genomic regions were associated with IR located downstream of MUT, AACS, SLC6A15 and PRKCA genes (P between 9.3 and 3.7 x 10-5). Inferred haplotypes up to 13 SNPs length were associated with IR (e.g. MUT gene with P < 4.9 x 10-5; Bonferroni 1.3 x 10-3) and synergistic to HOMAIR. SNPs in the same regions were also associated with one order of magnitude lower P values (e.g. rs20167284 in the MUT gene with P < 1.27 x 10-4) and replicated in Mediterranean samples (n = 832). In French, influential haplotypes (OR > 2.0) were correlated with in vivo insulin sensitivity (1/SI) except for SLC6A15 gene. Association of these genes with BCAA levels was variable, but influential haplotypes confirmed implication of MUT from BCAA metabolism as well as a role of regulatory genes (AACS and PRKCA) and suggested potential changes in transcriptional activity. These data drive attention towards new regulatory regions involved in IR in relation with BCAA and show the ability of haplotypes in phased DNA to detect signals complimentary to SNPs, which may be useful in designing genetic markers for clinical applications in ethnic populations.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/genética , Aminoácidos de Cadeia Ramificada/genética , Haplótipos , Resistência à Insulina/genética , Síndrome Metabólica/genética , Proteínas do Tecido Nervoso/genética , Polimorfismo de Nucleotídeo Único , Proteína Quinase C-alfa/genética , Adulto , Aminoácidos de Cadeia Ramificada/metabolismo , Feminino , Humanos , Masculino , Síndrome Metabólica/sangue , Pessoa de Meia-Idade
5.
Nutrients ; 10(10)2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30275383

RESUMO

Branched-chained amino acids (BCAA) are essential dietary components for humans and can act as potential biomarkers for diabetes development. To efficiently estimate dietary intake, we developed a BCAA database for 1331 food items found in the French Centre d'Information sur la Qualité des Aliments (CIQUAL) food table by compiling BCAA content from international tables, published measurements, or by food similarity as well as by calculating 267 items from Greek, Turkish, Romanian, and Moroccan mixed dishes. The database embedded in MEDIPAD software capable of registering 24 h of dietary recalls (24HDR) with clinical and genetic data was evaluated based on archived 24HDR of the Saint Pierre Institute (France) from 2957 subjects, which indicated a BCAA content up to 4.2 g/100 g of food and differences among normal weight and obese subjects across BCAA quartiles. We also evaluated the database of 119 interviews of Romanians, Turkish and Albanians in Greece (27⁻65 years) during the MEDIGENE program, which indicated mean BCAA intake of 13.84 and 12.91 g/day in males and females, respectively, comparable to other studies. The MEDIPAD is user-friendly, multilingual, and secure software and with the BCAA database is suitable for conducting nutritional assessment in the Mediterranean area with particular facilities for food administration.


Assuntos
Aminoácidos de Cadeia Ramificada/análise , Bases de Dados Factuais/estatística & dados numéricos , Análise de Alimentos/métodos , Avaliação Nutricional , Software , Adulto , Idoso , Feminino , França , Grécia , Humanos , Masculino , Região do Mediterrâneo , Pessoa de Meia-Idade , Inquéritos Nutricionais
6.
Ann Endocrinol (Paris) ; 78(3): 146-155, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28624160

RESUMO

AIM OF THE STUDY: APOA5 has been linked to metabolic syndrome (MetS) or its traits in several populations. In North Africa, only the Moroccan population was investigated. Our aim is to assess the association between APOA5 gene polymorphisms with the susceptibility to MetS and its components in the Tunisian population. MATERIALS AND METHODS: A total of 594 participants from the Tunisian population were genotyped for two polymorphisms rs3135506 and rs651821 located in APOA5 gene using KASPar technology. Statistical analyses were performed using R software. RESULTS: The SNP rs651821 increased the risk of MetS under the dominant model (OR=1.91 [1.17-3.12], P=0.008) whereas the variant rs3135506 was not associated with MetS. After stratification of the cohort following the sex, only the variant rs651821 showed a significant association with MetS among the women group. The influence of the geographic origin of the studied population on the genotype distribution of APOA5 variants showed that the variant rs651821 was significantly associated with MetS only for the Northern population. The association analyses of the variants rs651821 and rs3135506 with different quantitative traits of MetS showed a significant association only between the variant rs3135506 and triglycerides levels. CONCLUSION: This is the first study reporting the association of APOA5 gene variants with MetS in Tunisia. Our study emphasizes the role of APOA5 variants in the regulation of the triglycerides blood levels. Further studies are needed to confirm the clinical relevance of these associations and to better understand the physiopathology of the MetS.


Assuntos
Apolipoproteína A-V/genética , Síndrome Metabólica/genética , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Metabolismo dos Lipídeos/genética , Masculino , Síndrome Metabólica/sangue , Síndrome Metabólica/epidemiologia , Pessoa de Meia-Idade , Fenótipo , Triglicerídeos/sangue , Tunísia/epidemiologia
7.
J Cell Sci ; 129(24): 4480-4495, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27831493

RESUMO

How spatial organization of the genome depends on nuclear shape is unknown, mostly because accurate nuclear size and shape measurement is technically challenging. In large cell populations of the yeast Saccharomyces cerevisiae, we assessed the geometry (size and shape) of nuclei in three dimensions with a resolution of 30 nm. We improved an automated fluorescence localization method by implementing a post-acquisition correction of the spherical microscopic aberration along the z-axis, to detect the three dimensional (3D) positions of nuclear pore complexes (NPCs) in the nuclear envelope. Here, we used a method called NucQuant to accurately estimate the geometry of nuclei in 3D throughout the cell cycle. To increase the robustness of the statistics, we aggregated thousands of detected NPCs from a cell population in a single representation using the nucleolus or the spindle pole body (SPB) as references to align nuclei along the same axis. We could detect asymmetric changes of the nucleus associated with modification of nucleolar size. Stereotypical modification of the nucleus toward the nucleolus further confirmed the asymmetric properties of the nuclear envelope.


Assuntos
Ciclo Celular , Forma do Núcleo Celular , Microscopia Confocal/métodos , Saccharomycetales/citologia , Carbono/farmacologia , Ciclo Celular/efeitos dos fármacos , Forma do Núcleo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Imageamento Tridimensional , Interfase/efeitos dos fármacos , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Saccharomycetales/efeitos dos fármacos , Saccharomycetales/metabolismo
8.
Methods Mol Biol ; 1455: 29-40, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27576708

RESUMO

Nucleoli form around RNA polymerase I transcribed ribosomal RNA (rRNA) genes. The direct electron microscopy observation of rRNA genes after nucleolar chromatin spreading (Miller's spreads) constitutes to date the only system to quantitatively assess transcription at a single molecule level. However, the spreading procedure is likely generating artifact and despite being informative, these spread rRNA genes are far from their in vivo situation. The integration of the structural characterization of spread rRNA genes in the three-dimensional (3D) organization of the nucleolus would represent an important scientific achievement. Here, we describe a correlative light and electron microscopy (CLEM) protocol allowing detection of tagged-Pol I by fluorescent microscopy and high-resolution imaging of the nucleolar ultrastructural context. This protocol can be implemented in laboratories equipped with conventional fluorescence and electron microscopes and does not require sophisticated "pipeline" for imaging.


Assuntos
Nucléolo Celular/genética , Nucléolo Celular/ultraestrutura , Microscopia Eletrônica , Microscopia de Fluorescência , Saccharomyces cerevisiae/genética , Transcrição Genética , Microscopia Eletrônica de Transmissão , RNA Polimerase I/genética , RNA Ribossômico/genética
9.
Methods Mol Biol ; 1455: 41-57, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27576709

RESUMO

Spatial organization of the genome has important impacts on all aspects of chromosome biology, including transcription, replication, and DNA repair. Frequent interactions of some chromosome domains with specific nuclear compartments, such as the nucleolus, are now well documented using genome-scale methods. However, direct measurement of distance and interaction frequency between loci requires microscopic observation of specific genomic domains and the nucleolus, followed by image analysis to allow quantification. The fluorescent repressor operator system (FROS) is an invaluable method to fluorescently tag DNA sequences and investigate chromosome position and dynamics in living cells. This chapter describes a combination of methods to define motion and region of confinement of a locus relative to the nucleolus in cell's nucleus, from fluorescence acquisition to automated image analysis using two dedicated pipelines.


Assuntos
Nucléolo Celular/genética , Cromossomos Fúngicos , Ensaios de Triagem em Larga Escala , Microscopia de Fluorescência , Saccharomyces cerevisiae/genética , Genes Reporter , Loci Gênicos , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência/métodos , Reprodutibilidade dos Testes , Razão Sinal-Ruído , Imagem com Lapso de Tempo
10.
Mol Biol Cell ; 27(20): 3164-3177, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27559135

RESUMO

The association of RNA polymerase III (Pol III)-transcribed genes with nucleoli seems to be an evolutionarily conserved property of the spatial organization of eukaryotic genomes. However, recent studies of global chromosome architecture in budding yeast have challenged this view. We used live-cell imaging to determine the intranuclear positions of 13 Pol III-transcribed genes. The frequency of association with nucleolus and nuclear periphery depends on linear genomic distance from the tethering elements-centromeres or telomeres. Releasing the hold of the tethering elements by inactivating centromere attachment to the spindle pole body or changing the position of ribosomal DNA arrays resulted in the association of Pol III-transcribed genes with nucleoli. Conversely, ectopic insertion of a Pol III-transcribed gene in the vicinity of a centromere prevented its association with nucleolus. Pol III-dependent transcription was independent of the intranuclear position of the gene, but the nucleolar recruitment of Pol III-transcribed genes required active transcription. We conclude that the association of Pol III-transcribed genes with the nucleolus, when permitted by global chromosome architecture, provides nucleolar and/or nuclear peripheral anchoring points contributing locally to intranuclear chromosome organization.


Assuntos
Nucléolo Celular/metabolismo , Centrômero/metabolismo , RNA Polimerase III/genética , Nucléolo Celular/fisiologia , Núcleo Celular/metabolismo , Centrômero/fisiologia , Cromossomos/fisiologia , Imagem Óptica/métodos , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , RNA Polimerase III/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Telômero/fisiologia , Transcrição Genética
11.
Coll Antropol ; 40(3): 195-8, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-29139639

RESUMO

The aim of this preliminary study is to analyze genetic specificity of Kosovo Albanians comparing with neighboring populations using new genetic tool - MEDISCOPE gene chip, to investigate the feasibility of this approach. We collected 37 DNA samples (9 Croats, 17 Albanians from Croatia and 11 Albanians from Kosovo) from unrelated males born in Croatia and Kosovo. Additionally, samples were expanded with female individuals and mtDNA analysis included a total of 61 samples (15 Croats, 23 Albanians from Croatia and 23 Albanians from Kosovo). This pilot study suggests that the usage of the MEDISCOPE chip could be recognized as an efficient tool within recognition of the population genetic specificity even within extremely small sample size.


Assuntos
Variação Genética/genética , Genética Populacional/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Cromossomos Humanos Y/genética , Croácia , DNA Mitocondrial/genética , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Marcadores Genéticos/genética , Humanos , Kosovo , Masculino , Projetos Piloto
12.
Endokrynol Pol ; 66(3): 198-206, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26136127

RESUMO

INTRODUCTION: Insulin gene VNTR was associated with polycystic ovary syndrome (PCOS) in some studies but not in others. This couldb be due to the heterogeneity of the definition of PCOS and/or the use of inappropriate gene mapping strategies. MATERIAL AND METHODS: In this investigation, the association of VNTR with PCOS was explored in a population of women from Central Europe (377 cases and 105 controls) in whom PCOS was diagnosed according to Rotterdam criteria. Seven SNPs: rs3842756 (G/A), rs3842755 (G/T), rs3842754 (C/T), rs3842753 (A/C), rs3842752 (C/T), rs3842748 (G/C), and rs689 (T/A) were genotyped in a portion of the population (160 cases and 95 controls) by sequencing or by SSO-PCR. Analysis of linkage disequilibrium (LD) pattern allowed selecting three tagSNPs (rs3842754, rs3842748, and rs689), which were genotyped in the rest of the population by KASPar. RESULTS: Six haplotypes were reconstructed, among which three (h1, h2 and h6) were more frequent. Statistical analysis allowed observation of the association of the SNP rs3842748, through its GC genotype, with obesity in PCOS (P = 0.049; OR CI95% 1,59 [1.00-2.51]) and in classical PCOS (YPCOS) (P = 0.010), as well as the correlation of the SNP rs689 and the pair of haplotypes h1/h1 with higher levels of testosteronaemia in the PCOS group, although this was at the limit of significance (P = 0.054) CONCLUSION: These results are in accordance with some studies in literature and highlight the role of insulin gene VNTR in complex metabolic disorders.


Assuntos
Insulina/genética , Repetições Minissatélites , Síndrome do Ovário Policístico/metabolismo , Polimorfismo de Nucleotídeo Único , Adulto , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Haplótipos , Humanos , Obesidade/metabolismo , Síndrome do Ovário Policístico/genética , Romênia , Análise de Sequência de DNA , Adulto Jovem
13.
Gene ; 565(2): 166-70, 2015 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-25498334

RESUMO

The FTO (fat mass and obesity associated) gene was associated with different metabolic disorders in populations from different origins but with great difference between African and non-African populations. North-African populations combine many genetic backgrounds, among which African, Berber and Caucasian components, which makes North-Africans a good model for studying the genetic association of FTO. In the present investigation we explored the association of FTO gene with polycystic ovary syndrome (PCOS) in a population from Tunisia (n=278). Single nucleotide polymorphisms (SNPs) used in this study were previously associated in non-African populations: rs8050136 (A/C), rs9939609 (A/T), rs9930506 (G/A), or in both African and non-African populations: rs8057044 (A/G). Genotyping was performed by allelic discrimination method on StepOne real-time PCR system or KASPar technology. Linkage disequilibrium (LD) pattern was assessed by HAPLOVIEW and reconstruction of haplotypes was performed by PHASE, while statistical analyses were performed using StatView and GoldenHelix programs. Among the 13 haplotypes in the population, three (h1, h7 and h13) were strongly associated with PCOS notably h13 (P<0.0001, OR95%CI=0.040 [0.005-0.294]) while SNPs display weaker association. Moreover the LD pattern in FTO in the Tunisian population (r(2) index) was intermediary between those of Caucasian and Africans. This highlights the need for studying the genetics of complex disorders in the North-African populations taking into-account the haplotype structure of candidate loci more than SNPs taken alone.


Assuntos
Predisposição Genética para Doença/genética , Haplótipos/genética , Síndrome do Ovário Policístico/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas/genética , Adulto , Afro-Americanos/genética , Alelos , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Humanos , Desequilíbrio de Ligação/genética , Tunísia
14.
Nucleic Acids Res ; 41(22): 10135-49, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24021628

RESUMO

Ribosome biogenesis is a major metabolic effort for growing cells. In Saccharomyces cerevisiae, Hmo1, an abundant high-mobility group box protein (HMGB) binds to the coding region of the RNA polymerase I transcribed ribosomal RNAs genes and the promoters of ∼70% of ribosomal protein genes. In this study, we have demonstrated the functional conservation of eukaryotic HMGB proteins involved in ribosomal DNA (rDNA) transcription. We have shown that when expressed in budding yeast, human UBF1 and a newly identified Sp-Hmo1 (Schizosaccharomyces pombe) localize to the nucleolus and suppress growth defect of the RNA polymerase I mutant rpa49-Δ. Owing to the multiple functions of both proteins, Hmo1 and UBF1 are not fully interchangeable. By deletion and domains swapping in Hmo1, we identified essential domains that stimulate rDNA transcription but are not fully required for stimulation of ribosomal protein genes expression. Hmo1 is organized in four functional domains: a dimerization module, a canonical HMGB motif followed by a conserved domain and a C-terminal nucleolar localization signal. We propose that Hmo1 has acquired species-specific functions and shares with UBF1 and Sp-Hmo1 an ancestral function to stimulate rDNA transcription.


Assuntos
DNA Ribossômico/metabolismo , Proteínas HMGB/química , Proteínas HMGB/metabolismo , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Nucléolo Celular/metabolismo , Sequência Conservada , Proteínas HMGB/genética , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Proteínas Pol1 do Complexo de Iniciação de Transcrição/química , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Estrutura Terciária de Proteína , RNA Polimerase I/metabolismo , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Relação Estrutura-Atividade
15.
J Cell Biol ; 202(2): 201-10, 2013 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-23878273

RESUMO

Chromosomes architecture is viewed as a key component of gene regulation, but principles of chromosomal folding remain elusive. Here we used high-throughput live cell microscopy to characterize the conformation and dynamics of the longest chromosome of Saccharomyces cerevisiae (XII). Chromosome XII carries the ribosomal DNA (rDNA) that defines the nucleolus, a major hallmark of nuclear organization. We determined intranuclear positions of 15 loci distributed every ~100 kb along the chromosome, and investigated their motion over broad time scales (0.2-400 s). Loci positions and motions, except for the rDNA, were consistent with a computational model of chromosomes based on tethered polymers and with the Rouse model from polymer physics, respectively. Furthermore, rapamycin-dependent transcriptional reprogramming of the genome only marginally affected the chromosome XII internal large-scale organization. Our comprehensive investigation of chromosome XII is thus in agreement with recent studies and models in which long-range architecture is largely determined by the physical principles of tethered polymers and volume exclusion.


Assuntos
Cromatina/genética , Cromossomos Fúngicos/genética , Genoma Fúngico , Saccharomyces cerevisiae/genética , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Mapeamento Cromossômico , Cromossomos Fúngicos/metabolismo , Simulação por Computador , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Loci Gênicos , Ensaios de Triagem em Larga Escala , Saccharomyces cerevisiae/metabolismo , Sirolimo , Telômero/genética , Telômero/metabolismo , Imagem com Lapso de Tempo , Transcrição Genética
16.
J Biol Chem ; 288(7): 4567-82, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23293027

RESUMO

Transcription by RNA polymerase I (Pol-I) is the main driving force behind ribosome biogenesis, a fundamental cellular process that requires the coordinated transcription of all three nuclear polymerases. Increased Pol-I transcription and the concurrent increase in ribosome biogenesis has been linked to the high rates of proliferation in cancers. The ellipticine family contains a number of potent anticancer therapeutic agents, some having progressed to stage I and II clinical trials; however, the mechanism by which many of the compounds work remains unclear. It has long been thought that inhibition of Top2 is the main reason behind the drugs antiproliferative effects. Here we report that a number of the ellipticines, including 9-hydroxyellipticine, are potent and specific inhibitors of Pol-I transcription, with IC(50) in vitro and in cells in the nanomolar range. Essentially, the drugs did not affect Pol-II and Pol-III transcription, demonstrating a high selectivity. We have shown that Pol-I inhibition occurs by a p53-, ATM/ATR-, and Top2-independent mechanism. We discovered that the drug influences the assembly and stability of preinitiation complexes by targeting the interaction between promoter recognition factor SL1 and the rRNA promoter. Our findings will have an impact on the design and development of novel therapeutic agents specifically targeting ribosome biogenesis.


Assuntos
Elipticinas/farmacologia , Inibidores Enzimáticos/farmacologia , RNA Polimerase I/metabolismo , Transcrição Genética , Bromodesoxiuridina/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Química Farmacêutica/métodos , Desenho de Fármacos , Humanos , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Ribossômico/metabolismo
17.
Biochim Biophys Acta ; 1819(6): 468-81, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22245105

RESUMO

Over the past decade, tremendous progress has been made in understanding the spatial organization of genes and chromosomes. Nuclear organization can be thought of as information that is not encoded in DNA, but which nevertheless impacts gene expression. Nuclear organizational influences can be cell-specific and are potentially heritable. Thus, nuclear organization fulfills all the criteria necessary for it to be considered an authentic level of epigenetic information. Chromosomal nuclear organization is primarily dictated by the biophysical properties of chromatin. Diffusion models of polymers confined in the crowded nuclear space accurately recapitulate experimental observation. Diffusion is a Brownian process, which implies that the positions of chromosomes and genes are not defined deterministically but are likely to be dictated by the laws of probability. Despite the small size of their nuclei, budding yeast have been instrumental in discovering how epigenetic information is encoded in the spatial organization of the genome. The relatively simple organization of the yeast nucleus and the very high number of genetically identical cells that can be observed under fluorescent microscopy allow statistically robust definitions of the gene and chromosome positions in the nuclear space to be constructed. In this review, we will focus on how the spatial organization of the chromatin in the yeast nucleus might impact transcription. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.


Assuntos
Fenômenos Biofísicos , Núcleo Celular/genética , Cromatina/genética , Saccharomyces cerevisiae/genética , Montagem e Desmontagem da Cromatina/genética , Cromossomos/genética , Difusão , Regulação Fúngica da Expressão Gênica , Genoma , Modelos Teóricos
18.
J Cell Biol ; 192(2): 277-93, 2011 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-21263028

RESUMO

RNA polymerase I (Pol I) produces large ribosomal RNAs (rRNAs). In this study, we show that the Rpa49 and Rpa34 Pol I subunits, which do not have counterparts in Pol II and Pol III complexes, are functionally conserved using heterospecific complementation of the human and Schizosaccharomyces pombe orthologues in Saccharomyces cerevisiae. Deletion of RPA49 leads to the disappearance of nucleolar structure, but nucleolar assembly can be restored by decreasing ribosomal gene copy number from 190 to 25. Statistical analysis of Miller spreads in the absence of Rpa49 demonstrates a fourfold decrease in Pol I loading rate per gene and decreased contact between adjacent Pol I complexes. Therefore, the Rpa34 and Rpa49 Pol I-specific subunits are essential for nucleolar assembly and for the high polymerase loading rate associated with frequent contact between adjacent enzymes. Together our data suggest that localized rRNA production results in spatially constrained rRNA production, which is instrumental for nucleolar assembly.


Assuntos
Regulação Fúngica da Expressão Gênica/genética , Genes de RNAr/genética , Subunidades Proteicas/metabolismo , RNA Polimerase I/metabolismo , Transcrição Genética/genética , Nucléolo Celular , Forma do Núcleo Celular , Sequência Conservada , Teste de Complementação Genética , Humanos , Sinais de Localização Nuclear , Multimerização Proteica , Subunidades Proteicas/química , RNA Polimerase I/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Fatores de Transcrição/metabolismo
19.
Nucleic Acids Res ; 38(6): 2044-56, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20044348

RESUMO

In DNA site-specific recombination catalysed by tyrosine recombinases, two pairs of DNA strands are sequentially exchanged between separate duplexes and the mechanisms that confer directionality to this theoretically reversible reaction remain unclear. The tyrosine recombinase TnpI acts at the internal resolution site (IRS) of the transposon Tn4430 to resolve intermolecular transposition products. Recombination is catalysed at the IRS core sites (IR1-IR2) and is regulated by adjacent TnpI-binding motifs (DR1 and DR2). These are dispensable accessory sequences that confer resolution selectivity to the reaction by stimulating synapsis between directly repeated IRSs. Here, we show that formation of the DR1-DR2-containing synapse imposes a specific order of activation of the TnpI catalytic subunits in the complex so that the IR1-bound subunits catalyse the first strand exchange and the IR2-bound subunits the second strand exchange. This ordered pathway was demonstrated for a complete recombination reaction using a TnpI catalytic mutant (TnpI-H234L) partially defective in DNA rejoining. The presence of the DR1- and DR2-bound TnpI subunits was also found to stabilize transient recombination intermediates, further displacing the reaction equilibrium towards product formation. Implication of TnpI/IRS accessory elements in the initial architecture of the synapse and subsequent conformational changes taking place during strand exchange is discussed.


Assuntos
DNA Nucleotidiltransferases/química , Recombinases/química , Motivos de Aminoácidos , Biocatálise , Clivagem do DNA , DNA Nucleotidiltransferases/metabolismo , Modelos Moleculares , Recombinases/metabolismo , Recombinação Genética , Sequências Repetitivas de Aminoácidos
20.
RNA ; 12(10): 1868-82, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16931875

RESUMO

During ribosome biogenesis, the RNA precursor to mature rRNAs undergoes numerous post-transcriptional chemical modifications of bases, including conversions of uridines to pseudouridines. In archaea and eukaryotes, these conversions are performed by box H/ACA small ribonucleoprotein particles (box H/ACA RNPs), which contain a small guide RNA responsible for the selection of substrate uridines and four proteins, including the pseudouridine synthase, Cbf5p. So far, no in vitro reconstitution of eukaryotic box H/ACA RNPs from purified components has been achieved, principally due to difficulties in purifying recombinant eukaryotic Cbf5p. In this study, we present the purification of a truncated derivative of yeast Cbf5p (Cbf5(Delta)p) that retains the highly conserved TRUB and PUA domains. We have used band retardation assays to show that Cbf5(Delta)p on its own binds to box H/ACA small nucleolar (sno)RNAs. We demonstrate that the conserved H and ACA boxes enhance the affinity of the protein for the snoRNA. Furthermore, like its archaeal homologs, Cbf5(Delta)p can bind to a single stem-loop-box ACA RNA. Finally, we report the first enzymatic footprinting analysis of a Cbf5-RNA complex. Our results are compatible with the view that two molecules of Cbf5p interact with a binding platform constituted by the 5' end of the RNA, the single-stranded hinge domain containing the conserved H box, and the 3' end of the molecule, including the conserved ACA box.


Assuntos
Hidroliases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Fúngico/metabolismo , RNA Nucleolar Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Sequência Conservada , Hidroliases/química , Hidroliases/genética , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Pegadas de Proteínas , Estrutura Terciária de Proteína , RNA Fúngico/química , RNA Fúngico/genética , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleases , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos
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