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1.
Mol Cell ; 83(18): 3268-3282.e7, 2023 09 21.
Article in English | MEDLINE | ID: mdl-37689068

ABSTRACT

Heritable non-genetic information can regulate a variety of complex phenotypes. However, what specific non-genetic cues are transmitted from parents to their descendants are poorly understood. Here, we perform metabolic methyl-labeling experiments to track the heritable transmission of methylation from ancestors to their descendants in the nematode Caenorhabditis elegans (C. elegans). We find heritable methylation in DNA, RNA, proteins, and lipids. We find that parental starvation elicits reduced fertility, increased heat stress resistance, and extended longevity in fed, naïve progeny. This intergenerational hormesis is accompanied by a heritable increase in N6'-dimethyl adenosine (m6,2A) on the 18S ribosomal RNA at adenosines 1735 and 1736. We identified DIMT-1/DIMT1 as the m6,2A and BUD-23/BUD23 as the m7G methyltransferases in C. elegans that are both required for intergenerational hormesis, while other rRNA methyltransferases are dispensable. This study labels and tracks heritable non-genetic material across generations and demonstrates the importance of rRNA methylation for regulating epigenetic inheritance.


Subject(s)
Caenorhabditis elegans , Hormesis , Animals , RNA, Ribosomal, 18S , Caenorhabditis elegans/genetics , Methyltransferases/genetics , Adenosine
2.
Nat Metab ; 3(6): 729-731, 2021 Jun.
Article in English | MEDLINE | ID: mdl-34140693
3.
Proc Natl Acad Sci U S A ; 117(23): 13033-13043, 2020 06 09.
Article in English | MEDLINE | ID: mdl-32461362

ABSTRACT

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract causing infections that range from asymptomatic to highly inflammatory. Recent works have highlighted the importance of histone modifications in the regulation of transcription and parasite pathogenesis. However, the nature of DNA methylation in the parasite remains unexplored. Using a combination of immunological techniques and ultrahigh-performance liquid chromatography (UHPLC), we analyzed the abundance of DNA methylation in strains with differential pathogenicity demonstrating that N6-methyladenine (6mA), and not 5-methylcytosine (5mC), is the main DNA methylation mark in T. vaginalis Genome-wide distribution of 6mA reveals that this mark is enriched at intergenic regions, with a preference for certain superfamilies of DNA transposable elements. We show that 6mA in T. vaginalis is associated with silencing when present on genes. Interestingly, bioinformatics analysis revealed the presence of transcriptionally active or repressive intervals flanked by 6mA-enriched regions, and results from chromatin conformation capture (3C) experiments suggest these 6mA flanked regions are in close spatial proximity. These associations were disrupted when parasites were treated with the demethylation activator ascorbic acid. This finding revealed a role for 6mA in modulating three-dimensional (3D) chromatin structure and gene expression in this divergent member of the Excavata.


Subject(s)
Adenine/metabolism , Chromatin/chemistry , DNA Methylation/genetics , Trichomonas vaginalis/genetics , Ascorbic Acid/pharmacology , Cell Culture Techniques , Chromatin/genetics , Chromatin/metabolism , Computational Biology , DNA Methylation/drug effects , DNA Transposable Elements/genetics , Gene Expression Regulation , High-Throughput Nucleotide Sequencing , Molecular Conformation , Sequence Analysis, DNA
4.
Sci Rep ; 6: 36916, 2016 11 15.
Article in English | MEDLINE | ID: mdl-27845378

ABSTRACT

The mechanisms by which sex differences in the mammalian brain arise are poorly understood, but are influenced by a combination of underlying genetic differences and gonadal hormone exposure. Using a mouse embryonic neural stem cell (eNSC) model to understand early events contributing to sexually dimorphic brain development, we identified novel interactions between chromosomal sex and hormonal exposure that are instrumental to early brain sex differences. RNA-sequencing identified 103 transcripts that were differentially expressed between XX and XY eNSCs at baseline (FDR = 0.10). Treatment with testosterone-propionate (TP) reveals sex-specific gene expression changes, causing 2854 and 792 transcripts to become differentially expressed on XX and XY genetic backgrounds respectively. Within the TP responsive transcripts, there was enrichment for genes which function as epigenetic regulators that affect both histone modifications and DNA methylation patterning. We observed that TP caused a global decrease in 5-methylcytosine abundance in both sexes, a transmissible effect that was maintained in cellular progeny. Additionally, we determined that TP was associated with residue-specific alterations in acetylation of histone tails. These findings highlight an unknown component of androgen action on cells within the developmental CNS, and contribute to a novel mechanism of action by which early hormonal organization is initiated and maintained.


Subject(s)
Epigenesis, Genetic/drug effects , Testosterone/pharmacology , Transcriptome/drug effects , Acetylation/drug effects , Animals , Cell Lineage/drug effects , DNA Methylation/drug effects , Embryonic Stem Cells/cytology , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Female , Histones/metabolism , Male , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Neural Stem Cells/cytology , Neural Stem Cells/drug effects , Neural Stem Cells/metabolism , RNA/chemistry , RNA/isolation & purification , RNA/metabolism , Sequence Analysis, RNA , Sex Characteristics , Sex Chromosomes
5.
Hum Reprod ; 31(4): 905-14, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26911863

ABSTRACT

STUDY QUESTION: Can whole exome sequencing (WES) and in vitro validation studies be used to find the causative genetic etiology in a patient with primary ovarian failure and infertility? SUMMARY ANSWER: A novel follicle-stimulating hormone receptor (FSHR) mutation was found by WES and shown, via in vitro flow cytometry studies, to affect membrane trafficking. WHAT IS KNOWN ALREADY: WES may diagnose up to 25-35% of patients with suspected disorders of sex development (DSD). FSHR mutations are an extremely rare cause of 46, XX gonadal dysgenesis with primary amenorrhea due to hypergonadotropic ovarian failure. STUDY DESIGN, SIZE, DURATION: A WES study was followed by flow cytometry studies of mutant protein function. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study subjects were two Turkish sisters with hypergonadotropic primary amenorrhea, their parents and two unaffected sisters. The affected siblings and both parents were sequenced (trio-WES). Transient transfection of HEK 293T cells was performed with a vector containing wild-type FSHR as well as the novel FSHR variant that was discovered by WES. Cellular localization of FSHR protein as well as FSH-stimulated cyclic AMP (cAMP) production was evaluated using flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: Both affected sisters were homozygous for a previously unreported missense mutation (c.1222G>T, p.Asp408Tyr) in the second transmembrane domain of FSHR. Modeling predicted disrupted secondary structure. Flow cytometry demonstrated an average of 48% reduction in cell-surface signal detection (P < 0.01). The mean fluorescent signal for cAMP (second messenger of FSHR), stimulated by FSH, was reduced by 50% in the mutant-transfected cells (P < 0.01). LIMITATIONS, REASONS FOR CAUTION: This is an in vitro validation. All novel purported genetic variants can be clinically reported only as 'variants of uncertain significance' until more patients with a similar phenotype are discovered with the same variant. WIDER IMPLICATIONS OF THE FINDINGS: We report the first WES-discovered FSHR mutation, validated by quantitative flow cytometry. WES is a valuable tool for diagnosis of rare genetic diseases, and flow cytometry allows for quantitative characterization of purported variants. WES-assisted diagnosis allows for treatments aimed at the underlying molecular etiology of disease. Future studies should focus on pharmacological and assisted reproductive treatments aimed at the disrupted FSHR, so that patients with FSH resistance can be treated by personalized medicine. STUDY FUNDING/COMPETING INTERESTS: E.V. is partially funded by the DSD Translational Research Network (NICHD 1R01HD068138). M.S.B. is funded by the Neuroendocrinology, Sex Differences and Reproduction training grant (NICHD 5T32HD007228). The authors have no competing interests to disclose.


Subject(s)
Models, Molecular , Mutation, Missense , Primary Ovarian Insufficiency/genetics , Receptors, FSH/genetics , Adult , Consanguinity , Exome , Female , Genome-Wide Association Study , HEK293 Cells , Homozygote , Humans , Primary Ovarian Insufficiency/metabolism , Protein Structure, Secondary , Protein Transport , Receptors, FSH/chemistry , Receptors, FSH/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Siblings , Turkey , Young Adult
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