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1.
EMBO J ; 39(21): e99520, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-32935369

RESUMO

Vertebrate genomes replicate according to a precise temporal program strongly correlated with their organization into A/B compartments. Until now, the molecular mechanisms underlying the establishment of early-replicating domains remain largely unknown. We defined two minimal cis-element modules containing a strong replication origin and chromatin modifier binding sites capable of shifting a targeted mid-late-replicating region for earlier replication. The two origins overlap with a constitutive or a silent tissue-specific promoter. When inserted side-by-side, these modules advance replication timing over a 250 kb region through the cooperation with one endogenous origin located 30 kb away. Moreover, when inserted at two chromosomal sites separated by 30 kb, these two modules come into close physical proximity and form an early-replicating domain establishing more contacts with active A compartments. The synergy depends on the presence of the active promoter/origin. Our results show that clustering of strong origins located at active promoters can establish early-replicating domains.


Assuntos
Período de Replicação do DNA , Replicação do DNA , Regiões Promotoras Genéticas , Actinas/genética , Sítios de Ligação , Cromatina , Cromossomos , Análise por Conglomerados , Epigenômica , Humanos , Origem de Replicação , Globinas beta/genética
2.
Bioessays ; 43(10): e2100141, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34319621

RESUMO

In vertebrates, single cell analyses of replication timing patterns brought to light a very well controlled program suggesting a tight regulation on initiation sites. Mapping of replication origins with different methods has revealed discrete preferential sites, enriched in promoters and potential G-quadruplex motifs, which can aggregate into initiation zones spanning several tens of kilobases (kb). Another characteristic of replication origins is a nucleosome-free region (NFR). A modified yeast strain containing a humanized origin recognition complex (ORC) fires new origins at NFRs revealing their regulatory role. In cooperation with NFRs, the histone variant H2A.Z facilitates ORC loading through di-methylation of lysine 20 of histone H4. Recent studies using genome editing methods show that efficient initiation sites associated with transcriptional activity can synergize over several tens of kb by establishing physical contacts and lead to the formation of early domains of DNA replication demonstrating a co-regulation between replication initiation and transcription.


Assuntos
Complexo de Reconhecimento de Origem , Origem de Replicação , Animais , Cromatina , Replicação do DNA/genética , Nucleossomos , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Origem de Replicação/genética , Vertebrados/genética , Vertebrados/metabolismo
3.
Nucleic Acids Res ; 47(10): 5155-5169, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-30926993

RESUMO

Eukaryotic genomes are replicated under the control of a highly sophisticated program during the restricted time period corresponding to S phase. The most widely used replication timing assays, which are performed on populations of millions of cells, suggest that most of the genome is synchronously replicated on homologous chromosomes. We investigated the stochastic nature of this temporal program, by comparing the precise replication times of allelic loci within single vertebrate cells progressing through S phase at six loci replicated from very early to very late. We show that replication timing is strictly controlled for the three loci replicated in the first half of S phase. Out of the three loci replicated in the second part of S phase, two present a significantly more stochastic pattern. Surprisingly, we find that the locus replicated at the very end of S phase, presents stochasticity similar to those replicated in early S phase. We suggest that the richness of loci in efficient origins of replication, which decreases from early- to late-replicating regions, and the strength of interaction with the nuclear lamina may underlie the variation of timing control during S phase.


Assuntos
Período de Replicação do DNA , Processos Estocásticos , Alelos , Animais , Linhagem Celular , Galinhas , Eletroporação , Corantes Fluorescentes , Processamento de Imagem Assistida por Computador , Lamina Tipo B/química , Proteínas Luminescentes/química , Microscopia de Fluorescência , Fase S
5.
Eur J Hum Genet ; 20(9): 990-4, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22378281

RESUMO

Waardenburg syndrome type 4 (WS4) is a rare neural crest disorder defined by the combination of Waardenburg syndrome (sensorineural hearing loss and pigmentation defects) and Hirschsprung disease (intestinal aganglionosis). Three genes are known to be involved in this syndrome, that is, EDN3 (endothelin-3), EDNRB (endothelin receptor type B), and SOX10. However, 15-35% of WS4 remains unexplained at the molecular level, suggesting that other genes could be involved and/or that mutations within known genes may have escaped previous screenings. Here, we searched for deletions within recently identified SOX10 regulatory sequences and describe the first characterization of a WS4 patient presenting with a large deletion encompassing three of these enhancers. Analysis of the breakpoint region suggests a complex rearrangement involving three Alu sequences that could be mediated by a FosTes/MMBIR replication mechanism. Taken together with recent reports, our results demonstrate that the disruption of highly conserved non-coding elements located within or at a long distance from the coding sequences of key genes can result in several neurocristopathies. This opens up new routes to the molecular dissection of neural crest disorders.


Assuntos
Elementos Alu , Elementos Facilitadores Genéticos , Fatores de Transcrição SOXE/genética , Deleção de Sequência , Síndrome de Waardenburg/genética , Pré-Escolar , Pontos de Quebra do Cromossomo , Cromossomos Humanos Par 22 , Endotelina-3/genética , Doença de Hirschsprung , Humanos , Masculino , Dados de Sequência Molecular , Crista Neural/metabolismo , Crista Neural/patologia , Receptor de Endotelina B/genética , Síndrome de Waardenburg/patologia
6.
Proc Natl Acad Sci U S A ; 104(9): 3336-41, 2007 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-17360648

RESUMO

Thioredoxins belong to a large family of enzymatic proteins that function as general protein disulfide reductases, therefore participating in several cellular processes via redox-mediated reactions. So far, none of the 18 members of this family has been involved in human pathology. Here we identified TXNDC3, which encodes a thioredoxin-nucleoside diphosphate kinase, as a gene implicated in primary ciliary dyskinesia (PCD), a genetic condition characterized by chronic respiratory tract infections, left-right asymmetry randomization, and male infertility. We show that the disease, which segregates as a recessive trait, results from the unusual combination of the following two transallelic defects: a nonsense mutation and a common intronic variant found in 1% of control chromosomes. This variant affects the ratio of two physiological TXNDC3 transcripts: the full-length isoform and a novel isoform, TXNDC3d7, carrying an in-frame deletion of exon 7. In vivo and in vitro expression data unveiled the physiological importance of TXNDC3d7 (whose expression was reduced in the patient) and the corresponding protein that was shown to bind microtubules. PCD is known to result from defects of the axoneme, an organelle common to respiratory cilia, embryonic nodal cilia, and sperm flagella, containing dynein arms, with, to date, the implication of genes encoding dynein proteins. Our findings, which identify a another class of molecules involved in PCD, disclose the key role of TXNDC3 in ciliary function; they also point to an unusual mechanism underlying a Mendelian disorder, which is an SNP-induced modification of the ratio of two physiological isoforms generated by alternative splicing.


Assuntos
Códon sem Sentido/genética , Síndrome de Kartagener/genética , Polimorfismo de Nucleotídeo Único/genética , Tiorredoxinas/genética , Primers do DNA , Componentes do Gene , Células HeLa , Humanos , Íntrons/genética , Síndrome de Kartagener/patologia , Masculino , Isoformas de Proteínas/genética , Mucosa Respiratória/metabolismo , Mucosa Respiratória/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Testículo/metabolismo
7.
Biol Reprod ; 77(5): 864-71, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17699735

RESUMO

The SPAG16 gene encodes two major transcripts, one for the 71-kDa SPAG16L, which is the orthologue of the Chlamydomonas rheinhardtii central apparatus protein PF20, and a smaller transcript, which codes for the 35-kDa SPAG16S nuclear protein that represents the C-terminus (exons 11-16) of SPAG16L. We have previously reported that a targeted mutation in exon 11 of the Spag16 gene impairs spermatogenesis and prevents transmission of the mutant allele in chimeric mice. In the present report, we describe a heterozygous mutation in exon 13 of the SPAG16 gene, which causes a frame shift and premature stop codon, affording the opportunity to compare mutations with similar impacts on SPAG16L and SPAG16S for male reproductive function in mice and men. We studied two male heterozygotes for the SPAG16 mutation, both of which were fertile. Freezing-boiling of isolated sperm from both affected males resulted in the loss of the SPAG16L protein, SPAG6, another central apparatus protein that interacts with SPAG16L, and the 28-kDa fragment of SPAG17, which associates with SPAG6. These proteins were also lost after freezing-boiling cycles of sperm extracts from mice that were heterozygous for an inactivating mutation (exons 2 and 3) in Spag16. Our findings suggest that a heterozygous mutation that affects both SPAG16L and SPAG16S does not cause male infertility in man, but is associated with reduced stability of the interacting proteins of the central apparatus in response to a thermal challenge, a phenotype shared by the sperm of mice heterozygous for a mutation that affects SPAG16L.


Assuntos
Axonema/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Espermatozoides/ultraestrutura , Sequência de Aminoácidos , Animais , Axonema/ultraestrutura , Análise Mutacional de DNA , Heterozigoto , Humanos , Masculino , Camundongos , Proteínas dos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Motilidade dos Espermatozoides/genética , Espermatozoides/metabolismo
8.
Am J Respir Crit Care Med ; 174(8): 858-66, 2006 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16858015

RESUMO

RATIONALE: Primary ciliary dyskinesia (PCD) is a rare, usually autosomal recessive, genetic disorder characterized by ciliary dysfunction, sino-pulmonary disease, and situs inversus. Disease-causing mutations have been reported in DNAI1 and DNAH5 encoding outer dynein arm (ODA) proteins of cilia. OBJECTIVES: We analyzed DNAI1 to identify disease-causing mutations in PCD and to determine if the previously reported IVS1+2_3insT (219+3insT) mutation represents a "founder" or "hot spot" mutation. METHODS: Patients with PCD from 179 unrelated families were studied. Exclusion mapping showed no linkage to DNAI1 for 13 families; the entire coding region was sequenced in a patient from the remaining 166 families. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on nasal epithelial RNA in 14 families. RESULTS: Mutations in DNAI1 including 12 novel mutations were identified in 16 of 179 (9%) families; 14 harbored biallelic mutations. Deep intronic splice mutations were not identified by reverse transcriptase-polymerase chain reaction. The prevalence of mutations in families with defined ODA defect was 13%; no mutations were found in patients without a defined ODA defect. The previously reported IVS1+2_3insT mutation accounted for 57% (17/30) of mutant alleles, and marker analysis indicates a common founder for this mutation. Seven mutations occurred in three exons (13, 16, and 17); taken together with previous reports, these three exons are emerging as mutation clusters harboring 29% (12/42) of mutant alleles. CONCLUSIONS: A total of 10% of patients with PCD are estimated to harbor mutations in DNAI1; most occur as a common founder IVS1+2_3insT or in exons 13, 16, and 17. This information is useful for establishing a clinical molecular genetic test for PCD.


Assuntos
DNA/genética , Dineínas/genética , Síndrome de Kartagener/genética , Mutação , Adolescente , Adulto , Idoso , Dineínas do Axonema , Criança , Pré-Escolar , Éxons , Feminino , Efeito Fundador , Frequência do Gene , Genótipo , Humanos , Lactente , Masculino , Linhagem , Fenótipo , Reação em Cadeia da Polimerase
9.
Am J Respir Cell Mol Biol ; 26(3): 362-70, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11867345

RESUMO

Primary ciliary dyskinesia (PCD) is a heterogeneous congenital disorder characterized by bronchiectasis and chronic sinusitis, sometimes associated with situs inversus (i.e., Kartagener's syndrome) and male infertility. At the cell level, the disease phenotype includes various axonemal abnormalities of respiratory cilia and sperm flagella. We have previously isolated DNAI1, the first gene involved in these diseases in patients lacking outer dynein arms. In this study, designed to find additional genes for other axonemal defects, we report the isolation of a novel human gene, hPF20, which is orthologous to Chlamydomonas pf20. The hPF20 gene is expressed as two major transcripts: one is expressed in testis only, whereas the second is weakly expressed in many other tissues. As flagella of Chlamydomonas strains carrying pf20 mutations lack the axonemal central complexes, we tested the involvement of the hPF20 gene in the disease phenotype of five patients in whom cilia or flagella display abnormal central complexes. Five intragenic polymorphisms were identified and used to exclude hPF20 in two consanguineous patients, while no mutation was found in the remaining patients. However, given the genetic heterogeneity of PCD, we consider that this gene remains a good candidate to be investigated in patients with abnormal central complexes.


Assuntos
Chlamydomonas/genética , Cromossomos Humanos Par 2 , Genoma Humano , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Protozoários , Adulto , Sequência de Aminoácidos , Animais , Criança , Pré-Escolar , Mapeamento Cromossômico , Feminino , Humanos , Síndrome de Kartagener/etiologia , Síndrome de Kartagener/genética , Síndrome de Kartagener/patologia , Masculino , Dados de Sequência Molecular , Mutação , Alinhamento de Sequência , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/fisiologia
10.
Am J Respir Crit Care Med ; 166(9): 1257-62, 2002 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-12403696

RESUMO

The diagnosis of primary ciliary dyskinesia is based on demonstration of ciliary defects, mainly concerning dynein arms. Whereas the absence of outer dynein arms can be easily distinguished, the absence of inner dynein arms is difficult to confirm because of their low contrast on electron microscopy. Ciliary ultrastructure was studied in 40 patients suffering from respiratory tract infections. Conventional transmission electron microscopy showed normal cilia in 6 patients, confirmed a diagnosis of primary ciliary dyskinesia in 26 patients, and was inconclusive in 8 patients. All doubtful cases were related to inner dynein arm determination. Conventional electron microscopic analysis was able to define the ultrastructural phenotype of inner dynein arms in 40.5% of cases (6 presence of inner dynein arms, 13 absence of inner dynein arms). We developed computer-assisted analysis of electron microscopic micrographs to improve inner dynein arm visualization. Computer-assisted analysis consisted of image transformations designed to enhance the signal/noise ratio, based on the symmetry of ciliary axonemes. The sensitivity and specificity of computer-assisted analysis were 100 and 98%, respectively. The efficiency of computer-assisted analysis to visualize inner dynein arms, evaluated in the patients with undetermined phenotype after electron microscopy, was 86% (three normal cilia, seven primary ciliary dyskinesia with absence of outer dynein arms, three primary ciliary dyskinesia with absence of inner dynein arms, five partial absence of inner dynein arms). Computer-assisted analysis of ciliary micrographs improves the characterization of inherited axonemal defects.


Assuntos
Transtornos da Motilidade Ciliar/patologia , Dineínas/ultraestrutura , Aumento da Imagem/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia Eletrônica/métodos , Infecções Respiratórias/patologia , Transtornos da Motilidade Ciliar/etiologia , Humanos , Técnicas In Vitro , Infecções Respiratórias/complicações , Sensibilidade e Especificidade
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