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1.
Genome Res ; 32(4): 699-709, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35264448

RESUMO

Eukaryotic genes are interrupted by introns that must be accurately spliced from mRNA precursors. With an average length of 25 nt, the more than 90,000 introns of Paramecium tetraurelia stand among the shortest introns reported in eukaryotes. The mechanisms specifying the correct recognition of these tiny introns remain poorly understood. Splicing can occur cotranscriptionally, and it has been proposed that chromatin structure might influence splice site recognition. To investigate the roles of nucleosome positioning in intron recognition, we determined the nucleosome occupancy along the P. tetraurelia genome. We show that P. tetraurelia displays a regular nucleosome array with a nucleosome repeat length of ∼151 bp, among the smallest periodicities reported. Our analysis has revealed that introns are frequently associated with inter-nucleosomal DNA, pointing to an evolutionary constraint favoring introns at the AT-rich nucleosome edge sequences. Using accurate splicing efficiency data from cells depleted for nonsense-mediated decay effectors, we show that introns located at the edge of nucleosomes display higher splicing efficiency than those at the center. However, multiple regression analysis indicates that the low GC content of introns, rather than nucleosome positioning, is associated with high splicing efficiency. Our data reveal a complex link between GC content, nucleosome positioning, and intron evolution in Paramecium.


Assuntos
Nucleossomos , Paramecium , Composição de Bases , Éxons , Íntrons/genética , Nucleossomos/genética , Paramecium/genética , Splicing de RNA/genética
2.
Nucleic Acids Res ; 51(2): 517-535, 2023 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35934316

RESUMO

N6-Methyladenosine (m6A), one of the most abundant internal modification of eukaryotic mRNAs, participates in the post-transcriptional control of gene expression through recruitment of specific m6A readers. In Saccharomyces cerevisiae, the m6A methyltransferase Ime4 is expressed only during meiosis and its deletion impairs this process. To elucidate how m6A control gene expression, we investigated the function of the budding yeast m6A reader Pho92. We show that Pho92 is an early meiotic factor that promotes timely meiotic progression. High-throughput RNA sequencing and mapping of Pho92-binding sites following UV-crosslinking reveal that Pho92 is recruited to specific mRNAs in an m6A-dependent manner during the meiotic prophase, preceding their down-regulation. Strikingly, point mutations altering m6A sites in mRNAs targeted by Pho92 are sufficient to delay their down-regulation and, in one case, to slow down meiotic progression. Altogether, our results indicate that Pho92 facilitate the meiotic progression by accelerating the down-regulation of timely-regulated mRNAs during meiotic recombination.


mRNAs molecules carry information contained in genes to direct the formation of proteins. In specific circumstances, the cellular machinery modifies some mRNAs through the formation of m6A residues. To understand the function of these m6A marks, the authors used the yeast Saccharomyces cerevisiae in which their formation only occurs during meiosis that leads to spore formation. Characterization of the Pho92 protein that specifically recognizes m6A residues revealed its importance for meiosis. m6A sites bound by Pho92 were identified and shown to be biologically functional. Unexpectedly, Pho92 was found to regulate an early step of meiosis by controlling DNA recombination. Overall, this study provides important clues on the role of m6A residues in mRNAs.


Assuntos
Proteínas de Ligação a RNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Recombinação Homóloga , Meiose , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a RNA/metabolismo , Metilação
3.
Bioinformatics ; 37(17): 2738-2740, 2021 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-33471071

RESUMO

MOTIVATION: Long-read sequencing technologies can be employed to detect and map DNA modifications at the nucleotide resolution on a genome-wide scale. However, published software packages neglect the integration of genomic annotation and comprehensive filtering when analyzing patterns of modified bases detected using Pacific Biosciences (PacBio) or Oxford Nanopore Technologies (ONT) data. Here, we present DNA Modification Annotation (DNAModAnnot), a R package designed for the global analysis of DNA modification patterns using adapted filtering and visualization tools. RESULTS: We tested our package using PacBio sequencing data to analyze patterns of the 6-methyladenine (6mA) in the ciliate Paramecium tetraurelia, in which high 6mA amounts were previously reported. We found P. tetraurelia 6mA genome-wide distribution to be similar to other ciliates. We also performed 5-methylcytosine (5mC) analysis in human lymphoblastoid cells using ONT data and confirmed previously known patterns of 5mC. DNAModAnnot provides a toolbox for the genome-wide analysis of different DNA modifications using PacBio and ONT long-read sequencing data. AVAILABILITY AND IMPLEMENTATION: DNAModAnnot is distributed as a R package available via GitHub (https://github.com/AlexisHardy/DNAModAnnot). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

4.
PLoS Genet ; 10(9): e1004665, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25254958

RESUMO

In the ciliate Paramecium tetraurelia, differentiation of the somatic nucleus from the zygotic nucleus is characterized by massive and reproducible deletion of transposable elements and of 45,000 short, dispersed, single-copy sequences. A specific class of small RNAs produced by the germline during meiosis, the scnRNAs, are involved in the epigenetic regulation of DNA deletion but the underlying mechanisms are poorly understood. Here, we show that trimethylation of histone H3 (H3K27me3 and H3K9me3) displays a dynamic nuclear localization that is altered when the endonuclease required for DNA elimination is depleted. We identified the putative histone methyltransferase Ezl1 necessary for H3K27me3 and H3K9me3 establishment and show that it is required for correct genome rearrangements. Genome-wide analyses show that scnRNA-mediated H3 trimethylation is necessary for the elimination of long, repeated germline DNA, while single copy sequences display differential sensitivity to depletion of proteins involved in the scnRNA pathway, Ezl1- a putative histone methyltransferase and Dcl5- a protein required for iesRNA biogenesis. Our study reveals cis-acting determinants, such as DNA length, also contribute to the definition of germline sequences to delete. We further show that precise excision of single copy DNA elements, as short as 26 bp, requires Ezl1, suggesting that development specific H3K27me3 and H3K9me3 ensure specific demarcation of very short germline sequences from the adjacent somatic sequences.


Assuntos
Epigênese Genética , Rearranjo Gênico , Genoma de Protozoário , Paramecium tetraurellia/genética , Zigoto/metabolismo , Elementos de DNA Transponíveis , Histonas/metabolismo , Macronúcleo , Metilação , Paramecium tetraurellia/classificação , Paramecium tetraurellia/metabolismo , Filogenia , Poliploidia , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Deleção de Sequência
5.
Am J Physiol Endocrinol Metab ; 305(7): E845-52, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23921141

RESUMO

Transgenic female mice overexpressing the hCGß subunit (hCGß(+)) and producing elevated levels of luteinizing hormone (LH)/hCG bioactivity present as young adults with enhanced ovarian steroidogenesis, precocious puberty, and infertility. They subsequently develop pituitary prolactinomas, high circulating prolactin (PRL) levels, and marked mammary gland lobuloalveolar development followed by adenocarcinomas. None of these phenotypes appear in gonadectomized mice, indicating that the hCG-induced aberrations of ovarian function are responsible for the extragonadal phenotypes. PRL receptor-deficient (PRLR(-/-)) female mice are sterile, despite ovulating, due to a failure of embryo implantation, as a consequence of decreased ovarian LH receptor (Lhcgr) expression and inadequate corpus luteum formation and progesterone production. To study further the presumed permissive role of PRL in the maintenance of gonadal responsiveness to LH/hCG stimulation, we crossed the hCGß(+) and PRLR(-/-) mice. The double-mutant hCGß(+)/PRLR(-/-) females remained sterile with an ovarian phenotype similar to PRLR(-/-) mice, indicating that LH action, Lhcgr expression, and consequent luteinization are not possible without simultaneous PRL signaling. The high frequency of pituitary prolactinomas in PRLR(-/-) mice was not affected by transgenic hCGß expression. In contrast, none of the hCGß(+)/PRLR(-/-) females showed either mammary gland lobuloalveolar development or tumors, and the increased mammary gland Wnt-5b expression, possibly responsible for the tumorigenesis in hCGß(+) mice, was absent in double-mutant mice. Hence, high LH/hCG stimulation is unable to compensate for missing PRL signaling in the maintenance of luteal function. PRL thus appears to be a major permissive regulator of LH action in the ovary and of its secondary extragonadal effects.


Assuntos
Carcinogênese/metabolismo , Hormônio Luteinizante/metabolismo , Ovário/metabolismo , Prolactina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Carcinogênese/patologia , Gonadotropina Coriônica Humana Subunidade beta/genética , Gonadotropina Coriônica Humana Subunidade beta/metabolismo , Corpo Lúteo/metabolismo , Feminino , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Transgênicos , Progesterona/metabolismo , Prolactina/sangue , Receptores da Prolactina/genética , Receptores da Prolactina/metabolismo
6.
Genome Biol ; 20(1): 272, 2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31831055

RESUMO

BACKGROUND: Genomic imprinting is essential for mammalian development and provides a unique paradigm to explore intra-cellular differences in chromatin configuration. So far, the detailed allele-specific chromatin organization of imprinted gene domains has mostly been lacking. Here, we explored the chromatin structure of the two conserved imprinted domains controlled by paternal DNA methylation imprints-the Igf2-H19 and Dlk1-Dio3 domains-and assessed the involvement of the insulator protein CTCF in mouse cells. RESULTS: Both imprinted domains are located within overarching topologically associating domains (TADs) that are similar on both parental chromosomes. At each domain, a single differentially methylated region is bound by CTCF on the maternal chromosome only, in addition to multiple instances of bi-allelic CTCF binding. Combinations of allelic 4C-seq and DNA-FISH revealed that bi-allelic CTCF binding alone, on the paternal chromosome, correlates with a first level of sub-TAD structure. On the maternal chromosome, additional CTCF binding at the differentially methylated region adds a further layer of sub-TAD organization, which essentially hijacks the existing paternal-specific sub-TAD organization. Perturbation of maternal-specific CTCF binding site at the Dlk1-Dio3 locus, using genome editing, results in perturbed sub-TAD organization and bi-allelic Dlk1 activation during differentiation. CONCLUSIONS: Maternal allele-specific CTCF binding at the imprinted Igf2-H19 and the Dlk1-Dio3 domains adds an additional layer of sub-TAD organization, on top of an existing three-dimensional configuration and prior to imprinted activation of protein-coding genes. We speculate that this allele-specific sub-TAD organization provides an instructive or permissive context for imprinted gene activation during development.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Impressão Genômica , Animais , Proteínas de Ligação ao Cálcio/genética , Fator de Crescimento Insulin-Like II/genética , Iodeto Peroxidase/genética , Camundongos , RNA Longo não Codificante/genética
7.
Methods Mol Biol ; 1480: 223-41, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27659989

RESUMO

3D chromatin organization is essential for many aspects of transcriptional regulation. Circular Chromosome Conformation Capture followed by Illumina sequencing (4C-seq) is among the most powerful techniques to determine 3D chromatin organization. 4C-seq, like other modifications of the original 3C technique, uses the principle of "proximity ligation" to identify and quantify ten thousands of genomic interactions at a kilobase scale in a single experiment for predefined loci in the genome.In this chapter we focus on the experimental steps in the 4C-seq protocol, providing detailed descriptions on the preparation of cells, the construction of the circularized 3C library and the generation of the Illumina high throughput sequencing library. This protocol is particularly suited for the use of mammalian tissue samples, but can be used with minimal changes on circulating cells and cell lines from other sources as well. In the final section of this chapter, we provide a brief overview of data analysis approaches, accompanied by links to publicly available analysis tools.


Assuntos
Cromatina/isolamento & purificação , Mapeamento Cromossômico/métodos , Cromossomos/genética , Genômica/métodos , Cromatina/genética , Cromossomos/química , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Conformação de Ácido Nucleico
8.
PLoS One ; 10(8): e0134892, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26244508

RESUMO

In all organisms several enzymes that are needed upon replication impediment are targeted to replication forks by interaction with a replication protein. In most cases these proteins interact with the polymerase clamp or with single-stranded DNA binding proteins (SSB). In Escherichia coli an accessory replicative helicase was also shown to interact with the DnaB replicative helicase. Here we have used cytological observation of Venus fluorescent fusion proteins expressed from their endogenous loci in live E. coli cells to determine whether DNA repair and replication restart proteins that interact with a replication protein travel with replication forks. A custom-made microscope that detects active replisome molecules provided that they are present in at least three copies was used. Neither the recombination proteins RecO and RecG, nor the replication accessory helicase Rep are detected specifically in replicating cells in our assay, indicating that either they are not present at progressing replication forks or they are present in less than three copies. The Venus-PriA fusion protein formed foci even in the absence of replication forks, which prevented us from reaching a conclusion.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Helicases/genética , Reparo do DNA , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , DnaB Helicases/genética , DnaB Helicases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Conformação de Ácido Nucleico , Ligação Proteica
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