Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 55
Filtrar
1.
Mol Biol Evol ; 39(8)2022 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-35881460

RESUMO

Centromeres are epigenetically specified by the histone H3 variant CENP-A and typically associated with highly repetitive satellite DNA. We previously discovered natural satellite-free neocentromeres in Equus caballus and Equus asinus. Here, through ChIP-seq with an anti-CENP-A antibody, we found an extraordinarily high number of centromeres lacking satellite DNA in the zebras Equus burchelli (15 of 22) and Equus grevyi (13 of 23), demonstrating that the absence of satellite DNA at the majority of centromeres is compatible with genome stability and species survival and challenging the role of satellite DNA in centromere function. Nine satellite-free centromeres are shared between the two species in agreement with their recent separation. We assembled all centromeric regions and improved the reference genome of E. burchelli. Sequence analysis of the CENP-A binding domains revealed that they are LINE-1 and AT-rich with four of them showing DNA amplification. In the two zebras, satellite-free centromeres emerged from centromere repositioning or following Robertsonian fusion. In five chromosomes, the centromeric function arose near the fusion points, which are located within regions marked by traces of ancestral pericentromeric sequences. Therefore, besides centromere repositioning, Robertsonian fusions are an important source of satellite-free centromeres during evolution. Finally, in one case, a satellite-free centromere was seeded on an inversion breakpoint. At 11 chromosomes, whose primary constrictions seemed to be associated with satellite repeats by cytogenetic analysis, satellite-free neocentromeres were instead located near the ancestral inactivated satellite-based centromeres; therefore, the centromeric function has shifted away from a satellite repeat containing locus to a satellite-free new position.


Assuntos
Centrômero , DNA Satélite , Animais , Centrômero/genética , Centrômero/metabolismo , Proteína Centromérica A/genética , DNA Satélite/genética , Histonas/metabolismo , Cavalos/genética
2.
Int J Mol Sci ; 24(4)2023 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-36835543

RESUMO

In mammals, centromeres are epigenetically specified by the histone H3 variant CENP-A and are typically associated with satellite DNA. We previously described the first example of a natural satellite-free centromere on Equus caballus chromosome 11 (ECA11) and, subsequently, on several chromosomes in other species of the genus Equus. We discovered that these satellite-free neocentromeres arose recently during evolution through centromere repositioning and/or chromosomal fusion, after inactivation of the ancestral centromere, where, in many cases, blocks of satellite sequences were maintained. Here, we investigated by FISH the chromosomal distribution of satellite DNA families in Equus przewalskii (EPR), demonstrating a good degree of conservation of the localization of the major horse satellite families 37cen and 2PI with the domestic horse. Moreover, we demonstrated, by ChIP-seq, that 37cen is the satellite bound by CENP-A and that the centromere of EPR10, the ortholog of ECA11, is devoid of satellite sequences. Our results confirm that these two species are closely related and that the event of centromere repositioning which gave rise to EPR10/ECA11 centromeres occurred in the common ancestor, before the separation of the two horse lineages.


Assuntos
Proteína Centromérica A , Centrômero , DNA Satélite , Cavalos , Animais , Centrômero/metabolismo , Proteína Centromérica A/metabolismo , Cavalos/genética
3.
Int J Mol Sci ; 23(8)2022 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-35457002

RESUMO

The centromere is the chromosomal locus essential for proper chromosome segregation. While the centromeric function is well conserved and epigenetically specified, centromeric DNA sequences are typically composed of satellite DNA and represent the most rapidly evolving sequences in eukaryotic genomes. The presence of satellite sequences at centromeres hampered the comprehensive molecular analysis of these enigmatic loci. The discovery of functional centromeres completely devoid of satellite repetitions and fixed in some animal and plant species represented a turning point in centromere biology, definitively proving the epigenetic nature of the centromere. The first satellite-free centromere, fixed in a vertebrate species, was discovered in the horse. Later, an extraordinary number of satellite-free neocentromeres had been discovered in other species of the genus Equus, which remains the only mammalian genus with numerous satellite-free centromeres described thus far. These neocentromeres arose recently during evolution and are caught in a stage of incomplete maturation. Their presence made the equids a unique model for investigating, at molecular level, the minimal requirements for centromere seeding and evolution. This model system provided new insights on how centromeres are established and transmitted to the progeny and on the role of satellite DNA in different aspects of centromere biology.


Assuntos
DNA Satélite , Simulação de Dinâmica Molecular , Animais , Centrômero/genética , Segregação de Cromossomos , DNA Satélite/genética , Evolução Molecular , Cavalos/genética , Mamíferos/genética
4.
Genome Res ; 28(6): 789-799, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29712753

RESUMO

Mammalian centromeres are associated with highly repetitive DNA (satellite DNA), which has so far hindered molecular analysis of this chromatin domain. Centromeres are epigenetically specified, and binding of the CENPA protein is their main determinant. In previous work, we described the first example of a natural satellite-free centromere on Equus caballus Chromosome 11. Here, we investigated the satellite-free centromeres of Equus asinus by using ChIP-seq with anti-CENPA antibodies. We identified an extraordinarily high number of centromeres lacking satellite DNA (16 of 31). All of them lay in LINE- and AT-rich regions. A subset of these centromeres is associated with DNA amplification. The location of CENPA binding domains can vary in different individuals, giving rise to epialleles. The analysis of epiallele transmission in hybrids (three mules and one hinny) showed that centromeric domains are inherited as Mendelian traits, but their position can slide in one generation. Conversely, centromere location is stable during mitotic propagation of cultured cells. Our results demonstrate that the presence of more than half of centromeres void of satellite DNA is compatible with genome stability and species survival. The presence of amplified DNA at some centromeres suggests that these arrays may represent an intermediate stage toward satellite DNA formation during evolution. The fact that CENPA binding domains can move within relatively restricted regions (a few hundred kilobases) suggests that the centromeric function is physically limited by epigenetic boundaries.


Assuntos
Proteína Centromérica A/genética , Centrômero/genética , DNA Satélite/genética , Evolução Molecular , Animais , Autoantígenos/genética , Cromatina/genética , Instabilidade Genômica/genética , Cavalos , Mamíferos
5.
Int J Mol Sci ; 22(20)2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34681704

RESUMO

Interstitial telomeric sequences (ITSs) are stretches of telomeric-like repeats located at internal chromosomal sites. We previously demonstrated that ITSs have been inserted during the repair of DNA double-strand breaks in the course of evolution and that some rodent ITSs, called TERC-ITSs, are flanked by fragments retrotranscribed from the telomerase RNA component (TERC). In this work, we carried out an extensive search of TERC-ITSs in 30 vertebrate genomes and identified 41 such loci in 22 species, including in humans and other primates. The fragment retrotranscribed from the TERC RNA varies in different lineages and its sequence seems to be related to the organization of TERC. Through comparative analysis of TERC-ITSs with orthologous empty loci, we demonstrated that, at each locus, the TERC-like sequence and the ITS have been inserted in one step in the course of evolution. Our findings suggest that telomerase participated in a peculiar pathway of DNA double-strand break repair involving retrotranscription of its RNA component and that this mechanism may be active in all vertebrate species. These results add new evidence to the hypothesis that RNA-templated DNA repair mechanisms are active in vertebrate cells.


Assuntos
Evolução Molecular , RNA/metabolismo , Telomerase/metabolismo , Telômero/genética , Vertebrados/genética , Animais , Sequência de Bases , Quebras de DNA de Cadeia Dupla , Loci Gênicos , Genoma , Humanos , Filogenia , Alinhamento de Sequência , Telômero/química , Telômero/classificação
6.
Int J Mol Sci ; 21(8)2020 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-32325780

RESUMO

Interstitial telomeric sequences (ITSs) are short stretches of telomeric-like repeats (TTAGGG)n at nonterminal chromosomal sites. We previously demonstrated that, in the genomes of primates and rodents, ITSs were inserted during the repair of DNA double-strand breaks. These conclusions were derived from sequence comparisons of ITS-containing loci and ITS-less orthologous loci in different species. To our knowledge, insertion polymorphism of ITSs, i.e., the presence of an ITS-containing allele and an ITS-less allele in the same species, has not been described. In this work, we carried out a genome-wide analysis of 2504 human genomic sequences retrieved from the 1000 Genomes Project and a PCR-based analysis of 209 human DNA samples. In spite of the large number of individual genomes analyzed we did not find any evidence of insertion polymorphism in the human population. On the contrary, the analysis of ITS loci in the genome of a single horse individual, the reference genome, allowed us to identify five heterozygous ITS loci, suggesting that insertion polymorphism of ITSs is an important source of genetic variability in this species. Finally, following a comparative sequence analysis of horse ITSs and of their orthologous empty loci in other Perissodactyla, we propose models for the mechanism of ITS insertion during the evolution of this order.


Assuntos
Cromossomos/genética , Cavalos/genética , Telômero/genética , Alelos , Animais , Células Cultivadas , Evolução Molecular , Fibroblastos/citologia , Fibroblastos/metabolismo , Genoma Humano , Estudo de Associação Genômica Ampla , Heterozigoto , Humanos , Hibridização in Situ Fluorescente , Polimorfismo Genético , Sequências Repetitivas de Ácido Nucleico/genética
7.
Int J Mol Sci ; 19(1)2018 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-29342094

RESUMO

Telomeres are transcribed into noncoding telomeric repeat-containing RNAs (TERRA), which are essential for telomere maintenance. Deregulation of TERRA transcription impairs telomere metabolism and a role in tumorigenesis has been proposed. Head and neck cancer (HNC) is one of the most frequent cancers worldwide, with head and neck squamous cell carcinoma (HNSCC) being the predominant type. Since HNSCC patients are characterized by altered telomere maintenance, a dysfunction in telomere transcription can be hypothesized. In this prospective study, we compared TERRA levels in the tumor and matched normal tissue from 23 HNSCC patients. We then classified patients in two categories according to the level of TERRA expression in the tumor compared to the normal tissue: (1) lower expression in the tumor, (2) higher or similar expression in tumor. A significant proportion of patients in the first group died of the disease within less than 34 months postsurgery, while the majority of patients in the second group were alive and disease-free. Our results highlight a striking correlation between TERRA expression and tumor aggressiveness in HNSCC suggesting that TERRA levels may be proposed as a novel molecular prognostic marker for HNSCC.


Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidade , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/mortalidade , RNA Longo não Codificante/genética , Telômero/genética , Idoso , Carcinoma de Células Escamosas/patologia , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Avaliação de Resultados da Assistência ao Paciente , Prognóstico , Estudos Prospectivos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Homeostase do Telômero
8.
Prog Mol Subcell Biol ; 56: 337-354, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28840244

RESUMO

Centromeres are highly distinctive genetic loci whose function is specified largely by epigenetic mechanisms. Understanding the role of DNA sequences in centromere function has been a daunting task due to the highly repetitive nature of centromeres in animal chromosomes. The discovery of a centromere devoid of satellite DNA in the domestic horse consolidated observations on the epigenetic nature of centromere identity, showing that entirely natural chromosomes could function without satellite DNA cues. Horses belong to the genus Equus which exhibits a very high degree of evolutionary plasticity in centromere position and DNA sequence composition. Examination of horses has revealed that the position of the satellite-free centromere is variable among individuals. Analysis of centromere location and composition in other Equus species, including domestic donkey and zebras, confirms that the satellite-less configuration of centromeres is common in this group which has undergone particularly rapid karyotype evolution. These features have established the equids as a new mammalian system in which to investigate the molecular organization, dynamics and evolutionary behaviour of centromeres.


Assuntos
Centrômero/genética , DNA/genética , Equidae/genética , Animais , DNA Satélite , Cavalos/genética
9.
Mol Ecol ; 26(21): 6100-6109, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28851004

RESUMO

Individuals differ in realized fitness but the genetic/phenotypic traits that underpin such variation are often unknown. Telomere dynamics may be a major source of variation in fitness traits because physiological telomere shortening depends on environmental and genetic factors and may impair individual performance. Here, we showed that, in a population of a socially monogamous, biparental passerine bird, the barn swallow (Hirundo rustica), breeding in northern Italy, telomere length (TL) of both adult males and females positively correlated with seasonal reproductive and fledging success, as expected because long telomeres are supposed to boost performance. Telomere length was correlated with sexually dimorphic coloration in both sexes, showing for the first time in any species that coloration reliably reflects TL and may mediate mutual mate choice, leading to the observed positive assortative mating for TL in the barn swallow. Thus, TL appears to be associated with variation in a major fitness trait and may be an ultimate target of mate choice, as individuals of both sexes can use coloration to adaptively choose high-quality mates that possess long telomeres.


Assuntos
Plumas , Reprodução/fisiologia , Andorinhas/fisiologia , Telômero/ultraestrutura , Animais , Feminino , Aptidão Genética , Itália , Modelos Lineares , Masculino , Pigmentação , Estações do Ano , Andorinhas/genética , Encurtamento do Telômero
10.
Chromosoma ; 124(2): 277-87, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25413176

RESUMO

The centromere directs the segregation of chromosomes during mitosis and meiosis. It is a distinct genetic locus whose identity is established through epigenetic mechanisms that depend on the deposition of centromere-specific centromere protein A (CENP-A) nucleosomes. This important chromatin domain has so far escaped comprehensive molecular analysis due to its typical association with highly repetitive satellite DNA. In previous work, we discovered that the centromere of horse chromosome 11 is completely devoid of satellite DNA; this peculiar feature makes it a unique model to dissect the molecular architecture of mammalian centromeres. Here, we exploited this native satellite-free centromere to determine the precise localization of its functional domains in five individuals: We hybridized DNA purified from chromatin immunoprecipitated with an anti CENP-A antibody to a high resolution array (ChIP-on-chip) of the region containing the primary constriction of horse chromosome 11. Strikingly, each individual exhibited a different arrangement of CENP-A binding domains. We then analysed the organization of each domain using a single nucleotide polymorphism (SNP)-based approach and single molecule analysis on chromatin fibres. Examination of the ten instances of chromosome 11 in the five individuals revealed seven distinct 'positional alleles', each one extending for about 80-160 kb, were found across a region of about 500 kb. Our results demonstrate that CENP-A binding domains are autonomous relative to the underlying DNA sequence and are characterized by positional instability causing the sliding of centromere position. We propose that this dynamic behaviour may be common in mammalian centromeres and may determine the establishment of epigenetic alleles.


Assuntos
Centrômero/genética , Cromossomos de Mamíferos/genética , Cavalos/genética , Alelos , Animais , Autoantígenos/genética , Linhagem Celular , Proteína Centromérica A , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Clonagem Molecular , DNA Satélite , Epigênese Genética , Feminino , Masculino , Meiose , Procedimentos Analíticos em Microchip , Mitose , Nucleossomos/genética , Polimorfismo de Nucleotídeo Único
11.
BMC Genet ; 16: 126, 2015 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-26503543

RESUMO

BACKGROUND: In mammals, an important source of genomic variation is insertion polymorphism of retrotransposons. These may acquire a functional role when inserted inside genes or in their proximity. The aim of this work was to carry out a genome wide analysis of ERE1 retrotransposons in the horse and to analyze insertion polymorphism in relation to evolution and function. The effect of an ERE1 insertion in the promoter of the myostatin gene, which is involved in muscle development, was also investigated. RESULTS: In the horse population, the fraction of ERE1 polymorphic loci is related to the degree of similarity to their consensus sequence. Through the analysis of ERE1 conservation in seven equid species, we established that the level of identity to their consensus is indicative of evolutionary age of insertion. The position of ERE1s relative to genes suggests that some elements have acquired a functional role. Reporter gene assays showed that the ERE1 insertion within the horse myostatin promoter affects gene expression. The frequency of this variant promoter correlates with sport aptitude and racing performance. CONCLUSIONS: Sequence conservation and insertion polymorphism of ERE1 elements are related to the time of their appearance in the horse lineage, therefore, ERE1s are a useful tool for evolutionary and population studies. Our results suggest that the ERE1 insertion at the myostatin locus has been unwittingly selected by breeders to obtain horses with specific racing abilities. Although a complex combination of environmental and genetic factors contributes to athletic performance, breeding schemes may take into account ERE1 insertion polymorphism at the myostatin promoter.


Assuntos
Evolução Molecular , Regulação da Expressão Gênica , Genoma , Cavalos/genética , Mutagênese Insercional/genética , Miostatina/genética , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico/genética , Animais , Sequência de Bases , Sequência Conservada/genética , Genes Reporter , Loci Gênicos , Genótipo , Dados de Sequência Molecular , Filogenia , Polimorfismo de Nucleotídeo Único , Retroelementos/genética
12.
Proc Natl Acad Sci U S A ; 109(7): 2449-54, 2012 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-22308342

RESUMO

Archaeological and genetic evidence concerning the time and mode of wild horse (Equus ferus) domestication is still debated. High levels of genetic diversity in horse mtDNA have been detected when analyzing the control region; recurrent mutations, however, tend to blur the structure of the phylogenetic tree. Here, we brought the horse mtDNA phylogeny to the highest level of molecular resolution by analyzing 83 mitochondrial genomes from modern horses across Asia, Europe, the Middle East, and the Americas. Our data reveal 18 major haplogroups (A-R) with radiation times that are mostly confined to the Neolithic and later periods and place the root of the phylogeny corresponding to the Ancestral Mare Mitogenome at ~130-160 thousand years ago. All haplogroups were detected in modern horses from Asia, but F was only found in E. przewalskii--the only remaining wild horse. Therefore, a wide range of matrilineal lineages from the extinct E. ferus underwent domestication in the Eurasian steppes during the Eneolithic period and were transmitted to modern E. caballus breeds. Importantly, now that the major horse haplogroups have been defined, each with diagnostic mutational motifs (in both the coding and control regions), these haplotypes could be easily used to (i) classify well-preserved ancient remains, (ii) (re)assess the haplogroup variation of modern breeds, including Thoroughbreds, and (iii) evaluate the possible role of mtDNA backgrounds in racehorse performance.


Assuntos
Animais Domésticos/genética , DNA Mitocondrial/genética , Genoma , Haplótipos , Cavalos/genética , Animais , Cavalos/classificação , Filogenia
13.
Biochim Biophys Acta ; 1833(8): 1885-93, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23570868

RESUMO

Telomere length maintenance is critical for organisms' long-term survival and cancer cell proliferation. Telomeres are kept within species-specific length ranges by the interplay between telomerase activity and telomeric chromatin organization. In this paper, we exploited telomerase immortalized human fibroblasts (cen3tel) that gradually underwent neoplastic transformation during culture propagation to study telomere composition and length regulation during the transformation process. Just after telomerase catalytic subunit (hTERT) expression, cen3tel telomeres shortened despite the presence of telomerase activity. At a later stage and concomitantly with transformation, cells started elongating telomeres, which reached a mean length greater than 100kb in about 900 population doublings. Super-telomeres were stable and compatible with cell growth and tumorigenesis. Telomere extension was associated with increasing levels of telomerase activity that were linked to the deregulation of endogenous telomerase RNA (hTERC) and exogenous telomerase reverse transcriptase (hTERT) expression. Notably, the increase in hTERC levels paralleled the increase in telomerase activity, suggesting that this subunit plays a role in regulating enzyme activity. Telomeres ranging in length between 10 and more than 100kb were maintained in an extendible state although TRF1 and TRF2 binding increased with telomere length. Super-telomeres neither influenced subtelomeric region global methylation nor the expression of the subtelomeric gene FRG1, attesting the lack of a clear-cut relationship between telomere length, subtelomeric DNA methylation and expression in human cells. The cellular levels of the telomeric proteins hTERT, TRF1, TRF2 and Hsp90 rose with transformation and were independent of telomere length, pointing to a role of these proteins in tumorigenesis.


Assuntos
Transformação Celular Neoplásica/genética , Fibroblastos/fisiologia , Homeostase do Telômero/genética , Telômero/genética , Telômero/metabolismo , Linhagem Celular Transformada , Transformação Celular Neoplásica/metabolismo , Metilação de DNA , Fibroblastos/metabolismo , Humanos , Proteínas dos Microfilamentos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA/genética , Proteínas de Ligação a RNA , Telomerase/genética , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo
14.
Cytogenet Genome Res ; 144(2): 114-23, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25342230

RESUMO

Centromeres are the sites of kinetochore assembly and spindle fiber attachment and consist of protein-DNA complexes in which the DNA component is typically characterized by the presence of extended arrays of tandem repeats called satellite DNA. Here, we describe the isolation and characterization of a 137-bp-long new satellite DNA sequence from the horse genome (EC137), which is also present, even if less abundant, in the domestic donkey, the Grevy's zebra and the Burchelli's zebra. We investigated the chromosomal distribution of the EC137 sequence in these 4 species. Moreover, we analyzed its architectural organization by high-resolution FISH. The position of this sequence with respect to the primary constriction and in relation to the 2 major horse satellite tandem repeats (37 cen and 2PI) on horse chromosomes suggests that the new centromeric equine satellite is an accessory DNA element, presumably contributing to the organization of pericentromeric chromatin. FISH on combed DNA fibers reveals that the EC137 satellite is organized in relatively short stretches (2-8 kb) which are strictly intermingled within 37 cen or 2PI arrays. This arrangement suggests that interchanges between satellite families are a frequent occurrence in the horse genome.


Assuntos
DNA Satélite/genética , Animais , Sequência de Bases , Linhagem Celular , Centrômero/ultraestrutura , Cromossomos/ultraestrutura , DNA/genética , Equidae , Fibroblastos/citologia , Vetores Genéticos , Cavalos , Cinetocoros/ultraestrutura , Metáfase , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico
15.
Biol Reprod ; 90(5): 103, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24719256

RESUMO

Telomeres are ribonucleoprotein structures at the end of chromosomes composed of telomeric DNA, specific-binding proteins, and noncoding RNA (TERRA). Despite their importance in preventing chromosome instability, little is known about the cross talk between these three elements during the formation of the germ line. Here, we provide evidence that both TERRA and the telomerase enzymatic subunit (TERT) are components of telomeres in mammalian germ cells. We found that TERRA colocalizes with telomeres during mammalian meiosis and that its expression progressively increases during spermatogenesis until the beginning of spermiogenesis. While both TERRA levels and distribution would be regulated in a gender-specific manner, telomere-TERT colocalization appears to be regulated based on species-specific characteristics of the telomeric structure. Moreover, we found that TERT localization at telomeres is maintained throughout spermatogenesis as a structural component without affecting telomere elongation. Our results represent the first evidence of colocalization between telomerase and telomeres during mammalian gametogenesis.


Assuntos
Gametogênese/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Meiose/fisiologia , RNA não Traduzido/metabolismo , Telomerase/metabolismo , Telômero/metabolismo , Animais , Feminino , Citometria de Fluxo , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Masculino , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , RNA/química , RNA/genética , RNA não Traduzido/genética , Reação em Cadeia da Polimerase em Tempo Real , Telomerase/genética , Telômero/enzimologia , Telômero/genética
16.
Chromosoma ; 121(5): 475-88, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22797876

RESUMO

Many human primary somatic cells can be immortalized by inducing telomerase activity through the exogenous expression of the human telomerase catalytic subunit (hTERT). This approach has been extended to the immortalization of cell lines from several mammals. Here, we show that hTERT expression is not sufficient to immortalize primary fibroblasts from three equid species, namely donkey, Burchelli's zebra and Grevy's zebra. In vitro analysis of a reconstituted telomerase composed by hTERT and an equid RNA component of telomerase (TERC) revealed a low activity of this enzyme compared to human telomerase, suggesting a low compatibility of equid and human telomerase subunits. This conclusion was also strengthened by comparison of human and equid TERC sequences, which revealed nucleotide differences in key regions for TERC and TERT interaction. We then succeeded in immortalizing equid fibroblasts by expressing hTERT and hTERC concomitantly. Expression of both human telomerase subunits led to telomerase activity and telomere elongation, indicating that human telomerase is compatible with the other equid telomerase subunits and proteins involved in telomere metabolism. The immortalization procedure described herein could be extended to primary cells from other mammals. The availability of immortal cells from endangered species could be particularly useful for obtaining new information on the organization and function of their genomes, which is relevant for their preservation.


Assuntos
Fibroblastos/citologia , RNA/metabolismo , Telomerase/metabolismo , Animais , Sequência de Bases , Domínio Catalítico , Células Cultivadas , Equidae , Fibroblastos/metabolismo , Cavalos , Humanos , Camundongos , Dados de Sequência Molecular , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA/química , RNA/genética , Telomerase/química , Telomerase/genética , Telômero/genética , Telômero/metabolismo , Transfecção
17.
Hum Reprod ; 28(2): 414-22, 2013 02.
Artigo em Inglês | MEDLINE | ID: mdl-23049077

RESUMO

STUDY QUESTION: What is the distribution of telomeric repeat-containing RNA (TERRA) and of telomerase in human fetal oocytes? SUMMARY ANSWER: TERRA forms discrete foci at telomeres of human fetal oocytes and it co-localizes with both the shelterin component telomeric repeat-binding factor 2 (TRF2) and the catalytic subunit of human telomerase at the telomeres of meiotic chromosomes. WHAT IS KNOWN ALREADY: TERRA is a structural element of the telomeric chromatin that has been described in somatic cells of many different eukaryote species. The telomerase enzyme is inactive in adult somatic cells but is active in germ cells, stem cells and in the majority of tumors; however, its distribution in oocytes is still unknown. STUDY DESIGN, SIZE, DURATION: For this study, ovarian samples from four euploid fetuses of 22 gestational weeks were used. These samples were obtained with the consent of the parents and of the Ethics Committee of Hospital de la Vall d'Hebron. PARTICIPANTS/MATERIALS, SETTING, METHODS: We analyzed the distribution of TERRA and telomerase in cells derived from human fetal ovaries. The co-localization of TERRA, telomerase and telomeres was performed by optimizing a combination of immunofluorescence (IF) and RNA-fluorescent in situ hybridization (RNA-FISH) techniques. The synaptonemal complex protein 3 (SYCP3), TRF2 and protein component of telomerase [telomerase reverse transcriptase (TERT)] were detected by IF, whereas TERRA was revealed by RNA-FISH using a (CCCTAA)(3) oligonucleotide. SYCP3 signals allowed us to identify oocytes that had entered meiosis and classify them into the different stages of prophase I, whereas TRF2 indicated the telomeric regions of chromosomes. MAIN RESULTS AND THE ROLE OF CHANCE: We show for the first time the presence of TERRA and the intracellular distribution of telomerase in human fetal ovarian cells. TERRA is present, forming discrete foci, in 75% of the ovarian tissue cells and most of TERRA molecules (≈ 83%) are at telomeres (TRF2 co-localization). TERRA levels are higher in oocytes than in ovarian tissue cells (P = 0.00), and do not change along the progression of the prophase I stage (P = 0.37). TERRA is present on ≈ 23% of the telomeres in all cell types derived from human fetal ovaries. Moreover, ≈ 22% of TERRA foci co-localize with the protein component of telomerase (TERT). LIMITATIONS, REASONS FOR CAUTION: We present a descriptive/qualitative study of TERRA in human fetal ovarian tissue. Given the difficult access and manipulation of fetal samples, the number of fetal ovaries used in this study was limited. WIDER IMPLICATIONS OF THE FINDINGS: This is the first report on TERRA expression in oocytes from human fetal ovaries. The presence of TERRA at the telomeres of oocytes from the leptotene to pachytene stages and its co-localization with the telomerase protein component suggests that this RNA might participate in the maintenance of the telomere structure, at least through the processes that take place during the female meiotic prophase I. Since telomeres in oocytes have been mainly studied regarding the bouquet structure, our results introduce a new viewpoint of the telomeric structure during meiosis.


Assuntos
Feto/citologia , Oócitos/enzimologia , RNA/metabolismo , Telomerase/metabolismo , Células Cultivadas , Feminino , Feto/enzimologia , Células HeLa , Humanos
18.
PLoS Genet ; 6(2): e1000845, 2010 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-20169180

RESUMO

In a previous study, we showed that centromere repositioning, that is the shift along the chromosome of the centromeric function without DNA sequence rearrangement, has occurred frequently during the evolution of the genus Equus. In this work, the analysis of the chromosomal distribution of satellite tandem repeats in Equus caballus, E. asinus, E. grevyi, and E. burchelli highlighted two atypical features: 1) several centromeres, including the previously described evolutionary new centromeres (ENCs), seem to be devoid of satellite DNA, and 2) satellite repeats are often present at non-centromeric termini, probably corresponding to relics of ancestral now inactive centromeres. Immuno-FISH experiments using satellite DNA and antibodies against the kinetochore protein CENP-A demonstrated that satellite-less primary constrictions are actually endowed with centromeric function. The phylogenetic reconstruction of centromere repositioning events demonstrates that the acquisition of satellite DNA occurs after the formation of the centromere during evolution and that centromeres can function over millions of years and many generations without detectable satellite DNA. The rapidly evolving Equus species gave us the opportunity to identify different intermediate steps along the full maturation of ENCs.


Assuntos
Centrômero/metabolismo , DNA Satélite/genética , Equidae/genética , Animais , Autoantígenos/metabolismo , Sequência de Bases , Linhagem Celular , Proteína Centromérica A , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos de Mamíferos/genética , Evolução Molecular , Feminino , Hibridização in Situ Fluorescente , Masculino , Filogenia , Transporte Proteico
19.
Commun Biol ; 6(1): 963, 2023 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-37735603

RESUMO

Centromeres are epigenetically specified by the histone H3 variant CENP-A. Although mammalian centromeres are typically associated with satellite DNA, we previously demonstrated that the centromere of horse chromosome 11 (ECA11) is completely devoid of satellite DNA. We also showed that the localization of its CENP-A binding domain is not fixed but slides within an about 500 kb region in different individuals, giving rise to positional alleles. These epialleles are inherited as Mendelian traits but their position can move in one generation. It is still unknown whether centromere sliding occurs during meiosis or during development. Here, we first improve the sequence of the ECA11 centromeric region in the EquCab3.0 assembly. Then, to test whether centromere sliding may occur during development, we map the CENP-A binding domains of ECA11 using ChIP-seq in five tissues of different embryonic origin from the four horses of the equine FAANG (Functional Annotation of ANimal Genomes) consortium. Our results demonstrate that the centromere is localized in the same region in all tissues, suggesting that the position of the centromeric domain is maintained during development.


Assuntos
Centrômero , DNA Satélite , Humanos , Animais , Cavalos , Proteína Centromérica A/genética , Centrômero/genética , Histonas , Meiose , Mamíferos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA