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1.
Sci Rep ; 11(1): 19156, 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34580381

RESUMO

Various bioactive food compounds may confer health and longevity benefits, possibly through altering or preserving the epigenome. While bioactive food compounds are widely being marketed for human consumption as 'improving health and longevity' by counteracting harmful effects of poor nutrition and lifestyle, claimed effects are often not adequately documented. Using the honey bee (Apis mellifera) as a model species, we here employed a multi-step screening approach to investigate seven compounds for effects on lifespan and DNA methylation using ELISA and whole genome bisulfite sequencing (WGBS). A positive longevity effect was detected for valproic acid, isovaleric acid, and cyanocobalamin. For curcumin, we found that lifespan shortening caused by ethanol intake, was restored when curcumin and ethanol were co-administered. Furthermore, we identified region specific DNA methylation changes as a result of ethanol intake. Ethanol specific changes in DNA methylation were fully or partially blocked in honey bees receiving ethanol and curcumin together. Ethanol-affected and curcumin-blocked differentially methylated regions covered genes involved in fertility, temperature regulation and tubulin transport. Our results demonstrate fundamental negative effects of low dose ethanol consumption on lifespan and associated DNA methylation changes and present a proof-of-principle on how longevity and DNA methylation changes can be negated by the bioactive food component curcumin. Our findings provide a fundament for further studies of curcumin in invertebrates.

2.
PLoS One ; 16(8): e0256142, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34437579

RESUMO

Long-COVID-19 is a proposed syndrome negatively affecting the health of COVID-19 patients. We present data on self-rated health three to eight months after laboratory confirmed COVID-19 disease compared to a control group of SARS-CoV-2 negative patients. We followed a cohort of 8786 non-hospitalized patients who were invited after SARS-CoV-2 testing between February 1 and April 15, 2020 (794 positive, 7229 negative). Participants answered online surveys at baseline and follow-up including questions on demographics, symptoms, risk factors for SARS-CoV-2, and self-rated health compared to one year ago. Determinants for a worsening of self-rated health as compared to one year ago among the SARS-CoV-2 positive group were analyzed using multivariate logistic regression and also compared to the population norm. The follow-up questionnaire was completed by 85% of the SARS-CoV-2 positive and 75% of the SARS-CoV-2 negative participants on average 132 days after the SARS-CoV-2 test. At follow-up, 36% of the SARS-CoV-2 positive participants rated their health "somewhat" or "much" worse than one year ago. In contrast, 18% of the SARS-CoV-2 negative participants reported a similar deterioration of health while the population norm is 12%. Sore throat and cough were more frequently reported by the control group at follow-up. Neither gender nor follow-up time was associated with the multivariate odds of worsening of self-reported health compared to one year ago. Age had an inverted-U formed association with a worsening of health while being fit and being a health professional were associated with lower multivariate odds. A significant proportion of non-hospitalized COVID-19 patients, regardless of age, have not returned to their usual health three to eight months after infection.


Assuntos
COVID-19/complicações , COVID-19/patologia , Adolescente , Adulto , Idoso , COVID-19/etiologia , COVID-19/virologia , Fadiga/etiologia , Feminino , Febre/etiologia , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , RNA Viral/análise , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Autorrelato , Inquéritos e Questionários , Fatores de Tempo , Adulto Jovem
3.
PLoS Pathog ; 17(3): e1009476, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33788902

RESUMO

Infectious and inflammatory diseases in the intestine remain a serious threat for patients world-wide. Reprogramming of the intestinal epithelium towards a protective effector state is important to manage inflammation and immunity and can be therapeutically targeted. The role of epigenetic regulatory enzymes within these processes is not yet defined. Here, we use a mouse model that has an intestinal-epithelial specific deletion of the histone demethylase Lsd1 (cKO mice), which maintains the epithelium in a fixed reparative state. Challenge of cKO mice with bacteria-induced colitis or a helminth infection model both resulted in increased pathogenesis. Mechanistically, we discovered that LSD1 is important for goblet cell maturation and goblet-cell effector molecules such as RELMß. We propose that this may be in part mediated by directly controlling genes that facilitate cytoskeletal organization, which is important in goblet cell biology. This study therefore identifies intestinal-epithelial epigenetic regulation by LSD1 as a critical element in host protection from infection.


Assuntos
Infecções por Enterobacteriaceae/imunologia , Células Caliciformes/imunologia , Histona Desmetilases/imunologia , Mucosa Intestinal/metabolismo , Tricuríase/imunologia , Animais , Citrobacter rodentium , Células Caliciformes/metabolismo , Histona Desmetilases/metabolismo , Mucosa Intestinal/imunologia , Camundongos , Camundongos Knockout , Trichuris
4.
Nat Cell Biol ; 22(4): 380-388, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231309

RESUMO

The importance of germline-inherited post-translational histone modifications on priming early mammalian development is just emerging1-4. Histone H3 lysine 9 (H3K9) trimethylation is associated with heterochromatin and gene repression during cell-fate change5, whereas histone H3 lysine 4 (H3K4) trimethylation marks active gene promoters6. Mature oocytes are transcriptionally quiescent and possess remarkably broad domains of H3K4me3 (bdH3K4me3)1,2. It is unknown which factors contribute to the maintenance of the bdH3K4me3 landscape. Lysine-specific demethylase 4A (KDM4A) demethylates H3K9me3 at promoters marked by H3K4me3 in actively transcribing somatic cells7. Here, we report that KDM4A-mediated H3K9me3 demethylation at bdH3K4me3 in oocytes is crucial for normal pre-implantation development and zygotic genome activation after fertilization. The loss of KDM4A in oocytes causes aberrant H3K9me3 spreading over bdH3K4me3, resulting in insufficient transcriptional activation of genes, endogenous retroviral elements and chimeric transcripts initiated from long terminal repeats during zygotic genome activation. The catalytic activity of KDM4A is essential for normal epigenetic reprogramming and pre-implantation development. Hence, KDM4A plays a crucial role in preserving the maternal epigenome integrity required for proper zygotic genome activation and transfer of developmental control to the embryo.


Assuntos
Histona Desmetilases/metabolismo , Histonas/metabolismo , Oócitos/metabolismo , Processamento de Proteína Pós-Traducional , Zigoto/metabolismo , Animais , Implantação do Embrião , Embrião de Mamíferos , Feminino , Fertilização/genética , Heterocromatina/química , Heterocromatina/metabolismo , Histona Desmetilases/genética , Histonas/genética , Masculino , Metáfase , Metilação , Camundongos , Camundongos Knockout , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Transcrição Genética , Zigoto/citologia , Zigoto/crescimento & desenvolvimento
5.
Mol Neurobiol ; 57(2): 997-1008, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31654318

RESUMO

Neural stem/progenitor cells (NSPCs) persist in the mammalian brain throughout life and can be activated in response to the physiological and pathophysiological stimuli. Epigenetic reprogramming of NPSC represents a novel strategy for enhancing the intrinsic potential of the brain to regenerate after brain injury. Therefore, defining the epigenetic features of NSPCs is important for developing epigenetic therapies for targeted reprogramming of NSPCs to rescue neurologic function after injury. In this study, we aimed at defining different subtypes of NSPCs by individual histone methylations. We found the three histone marks, histone H3 lysine 4 trimethylation (H3K4me3), histone H3 lysine 27 trimethylation (H3K27me3), and histone H3 lysine 36 trimethylation (H3K36me3), to nicely and dynamically portray individual cell types during neurodevelopment. First, we found all three marks co-stained with NSPC marker SOX2 in mouse subventricular zone. Then, CD133, Id1, Mash1, and DCX immunostaining were used to define NSPC subtypes. Type E/B, B/C, and C/A cells showed high levels of H3K27me3, H3K36me3, and H3K4me3, respectively. Our results reveal defined histone methylations of NSPC subtypes supporting that epigenetic regulation is critical for neurogenesis and for maintaining NSPCs.


Assuntos
Histonas/metabolismo , Ventrículos Laterais/metabolismo , Metilação , Células-Tronco Neurais/metabolismo , Células-Tronco/citologia , Animais , Epigênese Genética/genética , Lisina/metabolismo , Camundongos Endogâmicos C57BL , Neurogênese/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Regeneração/fisiologia
6.
Wiley Interdiscip Rev Syst Biol Med ; 12(1): e1465, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31478357

RESUMO

Chromatin immunoprecipitation (ChIP) enables mapping of specific histone modifications or chromatin-associated factors in the genome and represents a powerful tool in the study of chromatin and genome regulation. Importantly, recent technological advances that couple ChIP with whole-genome high-throughput sequencing (ChIP-seq) now allow the mapping of chromatin factors throughout the genome. However, the requirement for large amounts of ChIP-seq input material has long made it challenging to assess chromatin profiles of cell types only available in limited numbers. For many cell types, it is not feasible to reach high numbers when collecting them as homogeneous cell populations in vivo. Nonetheless, it is an advantage to work with pure cell populations to reach robust biological conclusions. Here, we review (a) how ChIP protocols have been scaled down for use with as little as a few hundred cells; (b) which considerations to be aware of when preparing small-scale ChIP-seq and analyzing data; and (c) the potential of small-scale ChIP-seq datasets for elucidating chromatin dynamics in various biological systems, including some examples such as oocyte maturation and preimplantation embryo development. This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods Developmental Biology > Developmental Processes in Health and Disease Biological Mechanisms > Cell Fates.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Animais , Linhagem Celular Tumoral , Células Cultivadas , Genoma/genética , Genômica , Histonas/genética , Histonas/metabolismo , Camundongos , Técnicas Analíticas Microfluídicas , Oócitos/metabolismo
7.
Sci Rep ; 9(1): 11065, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363131

RESUMO

In most mammalian cells, DNA replication occurs once, and only once between cell divisions. Replication initiation is a highly regulated process with redundant mechanisms that prevent errant initiation events. In lower eukaryotes, replication is initiated from a defined consensus sequence, whereas a consensus sequence delineating mammalian origin of replication has not been identified. Here we show that 5-hydroxymethylcytosine (5hmC) is present at mammalian replication origins. Our data support the hypothesis that 5hmC has a role in cell cycle regulation. We show that 5hmC level is inversely proportional to proliferation; indeed, 5hmC negatively influences cell division by increasing the time a cell resides in G1. Our data suggest that 5hmC recruits replication-licensing factors, then is removed prior to or during origin firing. Later we propose that TET2, the enzyme catalyzing 5mC to 5hmC conversion, acts as barrier to rereplication. In a broader context, our results significantly advance the understating of 5hmC involvement in cell proliferation and disease states.


Assuntos
5-Metilcitosina/análogos & derivados , Ciclo Celular/genética , Divisão Celular/fisiologia , Proliferação de Células/fisiologia , Replicação do DNA/fisiologia , 5-Metilcitosina/metabolismo , Células HeLa , Humanos , Origem de Replicação
8.
Aging Cell ; 18(2): e12897, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30712319

RESUMO

The age of tissues and cells can be accurately estimated by DNA methylation analysis. The multitissue DNA methylation (DNAm) age predictor combines the DNAm levels of 353 CpG dinucleotides to arrive at an age estimate referred to as DNAm age. Recent studies based on short-term observations showed that the DNAm age of reconstituted blood following allogeneic hematopoietic stem cell transplantation (HSCT) reflects the age of the donor. However, it is not known whether the DNAm age of donor blood remains independent of the recipient's age over the long term. Importantly, long-term studies including child recipients have the potential to clearly reveal whether DNAm age is cell-intrinsic or whether it is modulated by extracellular cues in vivo. Here, we address this question by analyzing blood methylation data from HSCT donor and recipient pairs who greatly differed in chronological age (age differences between 1 and 49 years). We found that the DNAm age of the reconstituted blood was not influenced by the recipient's age, even 17 years after HSCT, in individuals without relapse of their hematologic disorder. However, the DNAm age of recipients with relapse of leukemia was unstable. These data are consistent with our previous findings concerning the abnormal DNAm age of cancer cells, and it can potentially be exploited to monitor the health of HSCT recipients. Our data demonstrate that transplanted human hematopoietic stem cells have an intrinsic DNAm age that is unaffected by the environment in a recipient of a different age.


Assuntos
Senescência Celular/genética , DNA de Neoplasias/genética , Epigênese Genética/genética , Transplante de Células-Tronco Hematopoéticas , Leucemia/patologia , Adolescente , Adulto , Criança , Pré-Escolar , Metilação de DNA , Humanos , Lactente , Leucemia/sangue , Leucemia/genética , Pessoa de Meia-Idade , Transplante Homólogo , Adulto Jovem
9.
RNA Biol ; 15(6): 829-831, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29671387

RESUMO

The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20 years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery that development, ageing, and stem-cell dependent regeneration but also several diseases including cancer are largely controlled by the epigenetic state of cells. Consequently, this research has already led to the first FDA approved drugs that exploit the gained knowledge to combat disease. In recent years, the ~150 modifications found in RNA have come to the focus of intense research. Here we provide a perspective on necessary and expected developments in the fast expanding area of RNA modifications, termed epitranscriptomics.


Assuntos
DNA de Neoplasias , Epigênese Genética , Epigenômica/normas , Perfilação da Expressão Gênica/normas , Regulação Neoplásica da Expressão Gênica , Neoplasias , RNA Neoplásico , Transcriptoma , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Europa (Continente) , Perfilação da Expressão Gênica/métodos , Humanos , Neoplasias/genética , Neoplasias/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo
10.
Sci Rep ; 8(1): 3055, 2018 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-29445184

RESUMO

Micronutrient status of parents can affect long term health of their progeny. Around 2 billion humans are affected by chronic micronutrient deficiency. In this study we use zebrafish as a model system to examine morphological, molecular and epigenetic changes in mature offspring of parents that experienced a one-carbon (1-C) micronutrient deficiency. Zebrafish were fed a diet sufficient, or marginally deficient in 1-C nutrients (folate, vitamin B12, vitamin B6, methionine, choline), and then mated. Offspring livers underwent histological examination, RNA sequencing and genome-wide DNA methylation analysis. Parental 1-C micronutrient deficiency resulted in increased lipid inclusion and we identified 686 differentially expressed genes in offspring liver, the majority of which were downregulated. Downregulated genes were enriched for functional categories related to sterol, steroid and lipid biosynthesis, as well as mitochondrial protein synthesis. Differential DNA methylation was found at 2869 CpG sites, enriched in promoter regions and permutation analyses confirmed the association with parental feed. Our data indicate that parental 1-C nutrient status can persist as locus specific DNA methylation marks in descendants and suggest an effect on lipid utilization and mitochondrial protein translation in F1 livers. This points toward parental micronutrients status as an important factor for offspring health and welfare.


Assuntos
Micronutrientes/deficiência , Micronutrientes/metabolismo , Animais , Animais Recém-Nascidos , Metilação de DNA , Dieta/métodos , Suplementos Nutricionais , Epigênese Genética , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Feminino , Ácido Fólico/metabolismo , Expressão Gênica , Metabolismo dos Lipídeos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Metionina/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Vitamina B 12/metabolismo , Vitamina B 6/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
11.
Brief Funct Genomics ; 17(2): 89-95, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29087438

RESUMO

In the past decade, chromatin immunoprecipitation sequencing (ChIP-seq) has emerged as the dominant technique for those wishing to perform genome-wide protein:DNA profiling. Owing to the tissue- and cell-type-specific nature of epigenetic marks, the field has been driven towards obtaining data from ever-lower cell numbers. In this review, we focus on the methodological developments that have lowered input requirements and the biological findings they have enabled, as we strive towards the ultimate goal of robust single-cell ChIP-seq.


Assuntos
Imunoprecipitação da Cromatina/métodos , Animais , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Microfluídica
12.
Cell Discov ; 3: 17013, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28529766

RESUMO

Sertoli cells have dual roles during the cells' lifetime. In the juvenile mammal, Sertoli cells proliferate and create the structure of the testis, and during puberty they cease to proliferate and take on the adult role of supporting germ cells through spermatogenesis. Accordingly, many genes expressed in Sertoli cells during testis formation are repressed during spermatogenesis. 5-Hydroxymethylcytosine (5hmC) is a DNA modification enzymatically generated from 5mC and present in all investigated mammalian tissues at varying levels. Using mass spectrometry and immunofluorescence staining we identified a substantial Sertoli cell-specific global 5hmC increase during rat puberty. Chemical labeling, pull-down and sequencing of 5hmC-containing genomic DNA from juvenile and adult rat Sertoli cells revealed that genes that lose or gain 5hmC belong to different functional pathways and mirror the functions of the cells in the two different states. Loss of 5hmC is associated with genes involved in development and cell structure, whereas gain of 5hmC is associated with genes involved in cellular pathways pertaining to the function of the adult Sertoli cells. This redistribution during maturation shows that 5hmC is a dynamic nucleotide modification, correlated to gene expression.

13.
Nat Methods ; 14(1): 18-22, 2016 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-28032624

RESUMO

Post-transcriptional RNA modifications were discovered several decades ago, but the reversible nature of RNA modifications has only recently been discovered. Owing to technological advances, knowledge of epitranscriptomic marks and their writers, readers and erasers has recently advanced tremendously. Here we focus on the roles of the dynamic methylation and demethylation of internal adenosines in mRNA in germ cells and pluripotent stem cells.


Assuntos
Epigênese Genética/genética , Meiose/genética , Células-Tronco Pluripotentes/metabolismo , RNA/química , RNA/genética , Animais , Humanos , Células-Tronco Pluripotentes/citologia
14.
Sci Rep ; 6: 34535, 2016 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-27731423

RESUMO

World Health Organization is concerned for parental vitamin deficiency and its effect on offspring health. This study examines the effect of a marginally dietary-induced parental one carbon (1-C) micronutrient deficiency on embryonic gene expression using zebrafish. Metabolic profiling revealed a reduced 1-C cycle efficiency in F0 generation. Parental deficiency reduced the fecundity and a total of 364 genes were differentially expressed in the F1 embryos. The upregulated genes (53%) in the deficient group were enriched in biological processes such as immune response and blood coagulation. Several genes encoding enzymes essential for the 1-C cycle and for lipid transport (especially apolipoproteins) were aberrantly expressed. We show that a parental diet deficient in micronutrients disturbs the expression in descendant embryos of genes associated with overall health, and result in inherited aberrations in the 1-C cycle and lipid metabolism. This emphasises the importance of parental micronutrient status for the health of the offspring.


Assuntos
Apolipoproteínas , Regulação da Expressão Gênica no Desenvolvimento , Metabolismo dos Lipídeos/imunologia , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Apolipoproteínas/biossíntese , Apolipoproteínas/imunologia , Deficiência de Vitaminas/embriologia , Deficiência de Vitaminas/imunologia , Feminino , Masculino , Peixe-Zebra/embriologia , Peixe-Zebra/imunologia , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/imunologia
15.
Nature ; 537(7621): 548-552, 2016 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-27626377

RESUMO

Maternal-to-zygotic transition (MZT) is essential for the formation of a new individual, but is still poorly understood despite recent progress in analysis of gene expression and DNA methylation in early embryogenesis. Dynamic histone modifications may have important roles in MZT, but direct measurements of chromatin states have been hindered by technical difficulties in profiling histone modifications from small quantities of cells. Recent improvements allow for 500 cell-equivalents of chromatin per reaction, but require 10,000 cells for initial steps or require a highly specialized microfluidics device that is not readily available. We developed a micro-scale chromatin immunoprecipitation and sequencing (µChIP-seq) method, which we used to profile genome-wide histone H3 lysine methylation (H3K4me3) and acetylation (H3K27ac) in mouse immature and metaphase II oocytes and in 2-cell and 8-cell embryos. Notably, we show that ~22% of the oocyte genome is associated with broad H3K4me3 domains that are anti-correlated with DNA methylation. The H3K4me3 signal becomes confined to transcriptional-start-site regions in 2-cell embryos, concomitant with the onset of major zygotic genome activation. Active removal of broad H3K4me3 domains by the lysine demethylases KDM5A and KDM5B is required for normal zygotic genome activation and is essential for early embryo development. Our results provide insight into the onset of the developmental program in mouse embryos and demonstrate a role for broad H3K4me3 domains in MZT.


Assuntos
Cromatina/metabolismo , Metilação de DNA , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Lisina/metabolismo , Oócitos/metabolismo , Zigoto/metabolismo , Acetilação , Animais , Linhagem Celular Tumoral , Cromatina/genética , Imunoprecipitação da Cromatina , Desenvolvimento Embrionário/genética , Feminino , Genoma/genética , Histonas/química , Humanos , Masculino , Metilação , Camundongos , Análise de Sequência de DNA , Sítio de Iniciação de Transcrição , Zigoto/citologia
16.
Fertil Steril ; 105(5): 1170-1179.e5, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26820768

RESUMO

OBJECTIVE: To assess whether men with reduced semen quality exhibit genetic variants in the genes coding for the messenger RNA methylation erasers FTO and ALKBH5. DESIGN: DNA of men undergoing infertility work-up was extracted and the FTO and ALKBH5 genes were sequenced. Statistical analysis was used to study the correlation between the identified ALKBH5 and FTO variants and sperm quality. SETTING: University hospital infertility clinic. PATIENT(S): Semen samples from 77 unselected men that had been referred to Oslo University Hospital for routine semen analysis as part of infertility work-up. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Immunohistochemistry and Western blot were used to confirm the presence of ALKBH5 and FTO in human testis. DNA extraction from samples was followed by Illumina MiSeq amplicon high throughput sequencing and sequence alignment. Variant calling was carried out using GATK's UnifiedGenotyper. Standard semen parameter analysis was performed according to World Health Organization guidelines. RESULT(S): We found an FTO genetic variant to be associated with reduced semen quality. We also identified two FTO missense variants, one mutation (p.Cys326Ser) was located in the important linker between the two protein domains; the other mutation (p.Ser256Asn) was situated in a flexible loop able to interact with other molecules. CONCLUSION(S): The discovery of two missense mutations with potentially detrimental effect on the functionality of the methylation eraser protein FTO, as well as a genetic variant of the same protein that is associated with altered semen quality could suggest that aberrant demethylation of messenger RNA is a factor involved in reduced male fertility.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Variação Genética/genética , Infertilidade Masculina/genética , Mutação de Sentido Incorreto/genética , Análise de Sequência de DNA/métodos , Humanos , Infertilidade Masculina/diagnóstico , Masculino , Estrutura Secundária de Proteína , Sêmen/fisiologia , Análise do Sêmen/métodos , Espermatozoides/fisiologia
17.
Biochem Biophys Rep ; 6: 9-15, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28955859

RESUMO

Well-known epigenetic DNA modifications in mammals include the addition of a methyl group and a hydroxyl group to cytosine, resulting in 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) respectively. In contrast, the abundance and the functional implications of these modifications in invertebrate model organisms such as the honey bee (Apis mellifera) and the fruit fly (Drosophila melanogaster) are not well understood. Here we show that both adult honey bees and fruit flies contain 5mC and also 5hmC. Using a highly sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) technique, we quantified 5mC and 5hmC in different tissues of adult honey bee worker castes and in adult fruit flies. A comparison of our data with reports from human and mouse shed light on notable differences in 5mC and 5hmC levels between tissues and species.

18.
Methods Mol Biol ; 1222: 227-45, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25287350

RESUMO

Chromatin immunoprecipitation (ChIP) is a powerful method for mapping protein-DNA interactions in vivo. Genomic localization of histone modifications, transcription factors, and other regulatory proteins can be revealed by ChIP. However, conventional ChIP protocols require the use of large numbers of cells, which prevents the application of ChIP to rare cell types. We have developed ChIP assays suited for the immunoprecipitation of histone proteins or transcription factors from small cell numbers. Here we describe a rapid, yet sensitive micro (µ)ChIP protocol producing high signal to noise ratio output, suitable for as few as 100 cells. This chapter provides a detailed protocol for µChIP from early mammalian embryos, also suitable for any sample of limited numbers of cells. Minor modifications of this optimized high signal to noise ChIP protocol make it a reliable tool for the use with any cell number (100-10(7)).


Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/metabolismo , Embrião de Mamíferos/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Bovinos , Reagentes para Ligações Cruzadas/química , DNA/isolamento & purificação , Embrião de Mamíferos/citologia , Histonas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fatores de Transcrição/genética
19.
Curr Opin Genet Dev ; 26: 47-52, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25005745

RESUMO

While the presence of 6-methyladenosine (m6A) modifications in mRNA was noted several decades ago, the first enzyme reversing this modification was identified very recently. Today we know of two methyltransferases introducing m6A in mRNA--METTL3 and METTL14--and two demethylases that remove it have been identified-FTO (ALKBH9) and ALKBH5. The conserved role of m6A seems to relate to meiosis, and mice lacking ALKBH5 are infertile. While loss-of-function mutation in FTO causes a recessive lethal syndrome, sequence variants in introns of the FTO gene are associated with obesity and type 2 diabetes.


Assuntos
Adenosina/análogos & derivados , Diabetes Mellitus Tipo 2/metabolismo , Obesidade/metabolismo , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Homólogo AlkB 5 da RNA Desmetilase , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Diabetes Mellitus Tipo 2/genética , Dioxigenases/genética , Dioxigenases/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Mutação , Obesidade/genética , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/genética
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