RESUMO
N 6-Methyladenosine (m6A) is a prevalent and highly regulated RNA modification essential for RNA metabolism and normal brain function. It is particularly important in the hippocampus, where m6A is implicated in neurogenesis and learning. Although extensively studied, its presence in specific cell types remains poorly understood. We investigated m6A in the hippocampus at a single-cell resolution, revealing a comprehensive landscape of m6A modifications within individual cells. Through our analysis, we uncovered transcripts exhibiting a dense m6A profile, notably linked to neurological disorders such as Alzheimer's disease. Our findings suggest a pivotal role of m6A-containing transcripts, particularly in the context of CAMK2A neurons. Overall, this work provides new insights into the molecular mechanisms underlying hippocampal physiology and lays the foundation for future studies investigating the dynamic nature of m6A RNA methylation in the healthy and diseased brain.
Assuntos
Adenosina , Hipocampo , Análise de Célula Única , Hipocampo/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Análise de Célula Única/métodos , Camundongos , Neurônios/metabolismo , Processamento Pós-Transcricional do RNA , Metilação , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , RNA/metabolismo , RNA/genética , Humanos , Metilação de RNARESUMO
Recently, more than 1000 large intergenic noncoding RNAs (lincRNAs) have been reported. These RNAs are evolutionarily conserved in mammalian genomes and thus presumably function in diverse biological processes. Here, we report the identification of lincRNAs that are regulated by p53. One of these lincRNAs (lincRNA-p21) serves as a repressor in p53-dependent transcriptional responses. Inhibition of lincRNA-p21 affects the expression of hundreds of gene targets enriched for genes normally repressed by p53. The observed transcriptional repression by lincRNA-p21 is mediated through the physical association with hnRNP-K. This interaction is required for proper genomic localization of hnRNP-K at repressed genes and regulation of p53 mediates apoptosis. We propose a model whereby transcription factors activate lincRNAs that serve as key repressors by physically associating with repressive complexes and modulate their localization to sets of previously active genes.
Assuntos
Regulação para Baixo , RNA não Traduzido/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Transcrição GênicaRESUMO
BACKGROUND: Salvage autologous hematopoietic stem cell transplantation (autoHSCT) may be used to treat relapse of multiple myeloma occurring after previous autoHSCT. When insufficient number of hematopoietic stem cells was stored from the initial harvest, remobilization of stem cells is necessary. PURPOSE: The analysis of stem cell remobilization after previous autoHSCT. PATIENTS AND METHODS: Fifty-eight patients, 60% males, median 59 years, were included. Median time interval between autoHSCT and remobilization was 42 months. The first remobilization was performed mostly after chemotherapy: cyclophosphamide (33%), cytarabine (43%), and etoposide (19%). RESULTS: The first remobilization was successful in 67% patients. About 19% patients required plerixafor rescue, among whom it allowed for successful harvesting in 14%. Use of cyclophosphamide, cytarabine, and etoposide allowed for successful remobilization in 53%, 84%, and 55% patients, respectively. Patients treated with cytarabine had the highest yield of CD34+ cells (median 7.5 × 106 /kg vs 5.8 and 2.4 for etoposide and cyclophosphamide, P = .001). Higher percentage of patients was able to collect ≥2 × 106 CD34+ cells/kg during one leukapheresis after cytarabine (76% vs 21% for cyclophosphamide vs 36% for etoposide, P = .001). Cytarabine use was associated with lower risk of remobilization failure OR = 0.217, P = .02. Toxicity comprised mostly hematological toxicity (thrombocytopenia and neutropenia). One patient succumbed to septic shock. CONCLUSION: Remobilization after previous autoHSCT is feasible only in a proportion of patients. Cytarabine is associated with the highest rate of successful mobilization and the highest yield of mobilized CD34+ cells. The toxicity requires careful surveillance of these patients.
Assuntos
Mobilização de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas , Mieloma Múltiplo/terapia , Adulto , Idoso , Ciclofosfamida/uso terapêutico , Feminino , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Mobilização de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva , Estudos Retrospectivos , Transplante AutólogoRESUMO
Large intergenic noncoding RNAs (lincRNAs) are emerging as key regulators of diverse cellular processes. Determining the function of individual lincRNAs remains a challenge. Recent advances in RNA sequencing (RNA-seq) and computational methods allow for an unprecedented analysis of such transcripts. Here, we present an integrative approach to define a reference catalog of >8000 human lincRNAs. Our catalog unifies previously existing annotation sources with transcripts we assembled from RNA-seq data collected from â¼4 billion RNA-seq reads across 24 tissues and cell types. We characterize each lincRNA by a panorama of >30 properties, including sequence, structural, transcriptional, and orthology features. We found that lincRNA expression is strikingly tissue-specific compared with coding genes, and that lincRNAs are typically coexpressed with their neighboring genes, albeit to an extent similar to that of pairs of neighboring protein-coding genes. We distinguish an additional subset of transcripts that have high evolutionary conservation but may include short ORFs and may serve as either lincRNAs or small peptides. Our integrated, comprehensive, yet conservative reference catalog of human lincRNAs reveals the global properties of lincRNAs and will facilitate experimental studies and further functional classification of these genes.
Assuntos
Anotação de Sequência Molecular/métodos , RNA não Traduzido/genética , Processamento Alternativo , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Homologia de Genes , Humanos , RNA não Traduzido/classificação , Homologia de Sequência do Ácido NucleicoRESUMO
The ultimate aim of nuclear reprogramming is to provide stem cells or differentiated cells from unrelated cell types as a cell source for regenerative medicine. A popular route towards this is transcription factor induction, and an alternative way is an original procedure of transplanting a single somatic cell nucleus to an unfertilized egg. A third route is to transplant hundreds of cell nuclei into the germinal vesicle (GV) of a non-dividing Amphibian meiotic oocyte, which leads to the activation of silent genes in 24â h and robustly induces a totipotency-like state in almost all transplanted cells. We apply this third route for potential therapeutic use and describe a procedure by which the differentiated states of cells can be reversed so that totipotency and pluripotency gene expression are regained. Differentiated cells are exposed to GV extracts and are reprogrammed to form embryoid bodies, which shows the maintenance of stemness and could be induced to follow new directions of differentiation. We conclude that much of the reprogramming effect of eggs is already present in meiotic oocytes and does not require cell division or selection of dividing cells. Reprogrammed cells by oocytes could serve as replacements for defective adult cells in humans.
Assuntos
Oócitos , Transplante de Células-Tronco , Adulto , Animais , Humanos , Núcleo Celular , Anfíbios , Reprogramação Celular , MamíferosRESUMO
Salvage autologous hematopoietic cell transplantation (auto-HCT) may be used to treat relapse of plasma cell myeloma occurring after previous auto-HCT. When an insufficient number of hematopoietic stem cells have been stored from the initial harvest, remobilization is necessary. Here, we aimed to analyze the efficacy and safety of different doses of cytarabine (total 800 vs. 1600 vs. 2400 mg/m2) for remobilization. Sixty-five patients, 55% male, with a median age at remobilization 63 years, were included. Remobilization was performed with cytarabine_800 in 7, cytarabine_1600 in 36, and cytarabine_2400 in 22 patients. Plerixafor rescue was used in 25% of patients receiving cytarabine_1600 and 27% of those receiving cytarabine_2400. Patients administered cytarabine_800 were not rescued with plerixafor. Remobilization was successful in 80% of patients (57% cytarabine_800; 86% cytarabine_1600; 77% cytarabine_2400; p = 0.199). The yield of collected CD34+ cells did not differ between the different cytarabine doses (p = 0.495). Patients receiving cytarabine_2400 were at the highest risk of developing severe cytopenias, requiring blood product support, or having blood-stream infections. One patient died of septic shock after cytarabine_2400. In summary, remobilization with cytarabine is feasible in most patients. All doses of cytarabine allow for successful remobilization. Cytarabine_2400 is associated with higher toxicity; therefore, lower doses (800 or 1600 mg/m2) seem to be preferable.
RESUMO
Regeneration is the regrowth of damaged tissues or organs, a vital process in response to damages from primitive organisms to higher mammals. Planarian possesses active whole-body regenerative capability owing to its vast reservoir of adult stem cells, neoblasts, providing an ideal model to delineate the underlying mechanisms for regeneration. RNA N6 -methyladenosine (m6 A) modification participates in many biological processes, including stem cell self-renewal and differentiation, in particular the regeneration of haematopoietic stem cells and axons. However, how m6 A controls regeneration at the whole-organism level remains largely unknown. Here, we demonstrate that the depletion of m6 A methyltransferase regulatory subunit wtap abolishes planarian regeneration, potentially through regulating genes related to cell-cell communication and cell cycle. Single-cell RNA-seq (scRNA-seq) analysis unveils that the wtap knockdown induces a unique type of neural progenitor-like cells (NP-like cells), characterized by specific expression of the cell-cell communication ligand grn. Intriguingly, the depletion of m6 A-modified transcripts grn, cdk9 or cdk7 partially rescues the defective regeneration of planarian caused by wtap knockdown. Overall, our study reveals an indispensable role of m6 A modification in regulating whole-organism regeneration.
Assuntos
Células-Tronco Adultas , Planárias , Animais , Planárias/genética , Planárias/metabolismo , Interferência de RNA , Diferenciação Celular/genética , Divisão Celular , MamíferosRESUMO
Breast cancer is the most common neoplasm among women. The risk of disease increases with age, particularly in postmenopausal period. The method of treatment depends on the stage of disease, state of receptors and molecular subtype. Depending on indications there are few options: surgical treatment, radiotherapy, hormonotherapy, chemotherapy and targeted therapy. Systemic treatment of metastatic triple-negative breast cancer is not satisfying. Hopes are laid upon poly(ADP-ribose) polymerase inhibitors. Studies over these substances in both: in connection with the standard chemotherapy and as monotherapy bring promising results. The article presents the molecular basics of polymerase poly(ADP-ribose) function as well as the possibilities of using its inhibitors in treatment of triple-negative breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Metástase Neoplásica , Estadiamento de Neoplasias , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Pós-Menopausa , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismoRESUMO
Vertebrate embryogenesis involves a conserved and fundamental process, called the maternal-to-zygotic transition (MZT), which marks the switch from a maternal factors-dominated state to a zygotic factors-driven state. Yet the precise mechanism underlying MZT remains largely unknown. Here we report that the RNA helicase Ddx3xb in zebrafish undergoes liquid-liquid phase separation (LLPS) via its N-terminal intrinsically disordered region (IDR), and an increase in ATP content promotes the condensation of Ddx3xb during MZT. Mutant form of Ddx3xb losing LLPS ability fails to rescue the developmental defect of Ddx3xb-deficient embryos. Interestingly, the IDR of either FUS or hnRNPA1 can functionally replace the N-terminal IDR in Ddx3xb. Phase separation of Ddx3xb facilitates the unwinding of 5' UTR structures of maternal mRNAs to enhance their translation. Our study reveals an unprecedent mechanism whereby the Ddx3xb phase separation regulates MZT by promoting maternal mRNA translation.
Assuntos
Peixe-Zebra , Zigoto , Animais , DNA Helicases , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , RNA Mensageiro Estocado/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Zigoto/metabolismoRESUMO
An 85-year-old male with a tumour in his right lung was admitted to Internal Diseases Ward to continue treatment after suffering a sudden cardiac arrest. An empiric antibiotic therapy with amoxycillin was introduced due to increased inflammation markers. Blood and sputum were collected. An abundant growth of AmpC ß-lactamase-producing Citrobacter freundii was observed in culture grown from the sputum. The antibiogram showed retained sensitivity to fluoroquinolones. The therapy was modified by replacing ß-lactam with ciprofloxacin. Neither clinical nor laboratory improvement were observed. Blood culture indicated sepsis of Acinetobacter baumannii etiology. The strain was suspected of producing OXA carbapenemase (CARBA test positive), KPC (-), MBL (-). Antibiogram illustrated retained sensitivity to gentamicin and colistin with complete resistance to ciprofloxacin. Another modification in treatment was implemented and ciprofloxacin was replaced with colistin.
Assuntos
Acinetobacter baumannii , Pneumopatias , Neoplasias , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Proteínas de Bactérias , Citrobacter freundii , Humanos , Pulmão , Masculino , Testes de Sensibilidade Microbiana , beta-LactamasesRESUMO
Innate lymphoid cells (ILCs) are a recently identified family of lymphocyte-like cells lacking a specific antigen receptor. They are part of the innate immune system. They play a key role in tissue homeostasis and also control inflammatory and neoplastic processes. In response to environmental stimuli, ILCs change their phenotype and functions, and influence the activity of other cells in the microenvironment. ILC dysfunction can lead to a wide variety of diseases, including cancer. ILC can be divided into three subgroups: ILC Group 1, comprising NK cells and ILC1; Group 2, including ILC2 alone; and Group 3, containing Lymphoid Tissue inducers (LTi) and ILC3 cells. While Group 1 ILCs mainly exert antitumour activity, Group 2 and Group 3 ILCs are protumorigenic in nature. A growing body of preclinical and clinical data support the role of ILCs in the pathogenesis of multiple myeloma (MM). Therefore, targeting ILCs may be of clinical benefit. In this manuscript, we review the available data on the role of ILCs in MM immunology and therapy.
RESUMO
Myelodysplastic syndromes (MDS) are clonal haematopoetic stem cells disorders, characterized by bone marrow dysplasia, ineffecitive haematopoesis and cytopenias. Due to neutropenia, infections are common. A case is presented of a patient with high-risk myelodysplastic syndrome (MDS) complicated by hidradenitis suppurativa that developed in both axillae. Abscesses required multiple incisions and drainage. After five cycles of treatment with azacitidine, the patient underwent allogenic bone marrow transplantation. Unfortunately, six months after the procedure, the patient lost post-transplant chimerism.Treatment with azacitidine was re-started. After the subsequent ten months, blast transformation was observed. Skin lesions in the course of hidradenitis suppurative persisted and were still considerably active.
Assuntos
Anemia Refratária com Excesso de Blastos , Síndromes Mielodisplásicas , Abscesso/tratamento farmacológico , Abscesso/etiologia , Azacitidina , Humanos , Síndromes Mielodisplásicas/complicaçõesRESUMO
Acquired haemophilia (AH) is a suddenly occurring severe blood diathesis that affects both males and females and is caused by autoantibodies which inhibit coagulation factor VIII. The report describes an unusual case of acquired haemophilia in which an epileptic seizure and haemorrhage into the ventricular system of the brain were the first manifestations of the disease. In addition, APTT was prolonged to 94.6 seconds and the factor VIII level was as low as 1.5%. The level of anti-FVIII antibody was extremely high - 272BU/ml. The patient did not undergo invasive diagnostic procedure or an operation. Recombinant factor VIIa was used to control the bleeding. In order to eradicate the inhibitor, the patient received prednisone and cyclophosphamide. Complete remission was achieved after 5.5 weeks of treatment.
Assuntos
Ventrículos Cerebrais/irrigação sanguínea , Hemofilia A/complicações , Convulsões/etiologia , Autoanticorpos/sangue , Ventrículos Cerebrais/diagnóstico por imagem , Fator VIII/metabolismo , Hemofilia A/diagnóstico por imagem , Hemofilia A/metabolismo , Hemofilia A/patologia , Hemorragia/diagnóstico por imagem , Hemorragia/etiologia , Hemorragia/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Convulsões/sangue , Convulsões/patologiaRESUMO
Salvage autologous hematopoietic stem cell transplantation (auto-HSCT) constitutes a therapeutic option for a group of well-selected patients with relapsed multiple myeloma (MM). However, if an insufficient number of stem cells were harvested and stored before the first auto-HSCT, stem cells need to be remobilized. Patients diagnosed with MM who following relapse after auto-HSCT, had remobilization and afterward, auto-HSCT with remobilized cells were included in this retrospective analysis. Thirty-three patients, 61% males, the median age 61 years, were included. With a median follow-up of 1.8 years, 2-year progression-free survival was 56.2%, non-relapse mortality 4.8%. The 2-year cumulative incidence of t-MDS was 4.9%. Factors important for the outcome were: the quality of response, previous radiotherapy, the time between the first and salvage auto-HSCT. To conclude, salvage auto-HSCT performed with cells procured after the previous auto-HSCT can be efficacious in relapsed MM, especially if a sufficiently long response had been obtained to the first auto-HSCT(s).
Assuntos
Transplante de Células-Tronco Hematopoéticas , Mieloma Múltiplo , Feminino , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/terapia , Polônia , Estudos Retrospectivos , Transplante Autólogo , Resultado do TratamentoRESUMO
Nuclear transfer to eggs or oocytes provides a potential route for cell-replacement therapies because oocytes directly reprogram transplanted mammalian somatic-cell nuclei such that they have an embryo-like pattern of gene expression. This includes a large increase in the mRNA level of the stem-cell marker gene oct4. We have developed a novel procedure to identify new proteins that greatly increase the level of oct4 mRNA upon nuclear transfer. We have isolated Xenopus oocyte proteins that bind to the regulatory region of the mouse oct4 gene and identified these by mass spectrometry. The proteins include the retinoic-acid-receptor gamma, a known repressor of oct4 transcription, and Tpt1, a cancer-associated factor. The depletion of transcripts of retinoic-acid receptor gamma from oocytes increases oct4 and nanog transcription as expected, and depletion of tpt1 transcripts in oocytes reduces oct4 and nanog transcription in injected HeLa nuclei. An elevation of tpt1 transcripts in oocytes results in an earlier activation of oct4 transcription. Therefore, we identify a novel role for tpt1 in activating pluripotency genes upon nuclear transfer. Our results help to elucidate the mechanism by which somatic-cell nuclei are reprogrammed to have an embryo-like pattern of gene expression.
Assuntos
Biomarcadores Tumorais/metabolismo , Núcleo Celular/metabolismo , Regulação da Expressão Gênica/fisiologia , Fator 3 de Transcrição de Octâmero/metabolismo , Oócitos/metabolismo , RNA Mensageiro/metabolismo , Animais , Proteínas de Ligação a DNA , Ensaio de Desvio de Mobilidade Eletroforética , Células HeLa , Proteínas de Homeodomínio , Humanos , Espectrometria de Massas , Camundongos , Proteína Homeobox Nanog , Técnicas de Transferência Nuclear , Receptores do Ácido Retinoico/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteína Tumoral 1 Controlada por Tradução , Xenopus laevis , Receptor gama de Ácido RetinoicoRESUMO
The aim of the present experiment was to investigate the influence of silver nanoparticles on Fusarium culmorum (W.G. Smith) Sacc. (FC) spores. The silver nanoparticles were produced by the high-voltage arc discharge method. To test the effect of silver nanoparticles on FC spores, 3 parameters were tested. One of these parameters was the vegetative mycelial growth in 2 experiments. The first involved the growth of FC spores on potato dextrose agar (PDA) medium after contact with 0.12-10 ppm of silver nanoparticles, and the second the growth of spores after contact with 0.12-2.5 ppm solutions of silver, but with culturing on 3 types of media (PDA, nutrient-poor PDA, and agar) instead. The next parameter was the formation of spores after the mycelia were cultured. The last parameter was spore germination in a 2.5 ppm solution of silver nanoparticles. A significant reduction in mycelial growth was observed for spores incubated with silver nanoparticles. This relationship was dependent on the incubation time and type of growth medium, but did not depend significantly on the concentration of silver nanoparticles up to 2.5 ppm. The sporulation test showed that, relative to control samples, the number of spores formed by mycelia increased in the culture after contact with silver nanoparticles, especially on the nutrient-poor PDA medium. The 24 h incubation of FC spores with a 2.5 ppm solution of silver nanoparticles greatly reduced the number of germinating fragments and sprout length relative to the control.
Assuntos
Antifúngicos/farmacologia , Fusarium/efeitos dos fármacos , Nanopartículas Metálicas , Prata/farmacologia , Esporos Fúngicos/efeitos dos fármacos , Ágar/farmacologia , Meios de Cultura/farmacologia , Fusarium/crescimento & desenvolvimento , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimentoRESUMO
A novel DNA modification, N-6 methylated deoxyadenosine (m6dA), has recently been discovered in eukaryotic genomes. Despite its low abundance in eukaryotes, m6dA is implicated in human diseases such as cancer. It is therefore important to precisely identify and characterize m6dA in the human genome. Here, we identify m6dA sites at nucleotide level, in different human cells, genome wide. We compare m6dA features between distinct human cells and identify m6dA characteristics in human genomes. Our data demonstrates for the first time that despite low m6dA abundance, the m6dA mark does often occur consistently at the same genomic location within a given human cell type, demonstrating m6dA homogeneity. We further show, for the first time, higher levels of m6dA homogeneity within one chromosome. Most m6dA are found on a single chromosome from a diploid sample, suggesting inheritance. Our transcriptome analysis not only indicates that human genes with m6dA are associated with higher RNA transcript levels but identifies allele-specific gene transcripts showing haplotype-specific m6dA methylation, which are implicated in different biological functions. Our analyses demonstrate the precision and consistency by which the m6dA mark occurs within the human genome, suggesting that m6dA marks are precisely inherited in humans.
Assuntos
Metilação de DNA/genética , Desoxiadenosinas/metabolismo , Genoma Humano , Linhagem Celular , Cromossomos Humanos/metabolismo , Humanos , Transcrição GênicaRESUMO
It is widely known that epigenetic modifications are important in regulating transcription, but several have also been reported in alternative splicing. The regulation of pre-mRNA splicing is important to explain proteomic diversity and the misregulation of splicing has been implicated in many diseases. Here, we give a brief overview of the role of epigenetics in alternative splicing and disease. We then discuss the bioinformatics methods that can be used to model interactions between epigenetic marks and regulators of splicing. These models can be used to identify alternative splicing and epigenetic changes across different phenotypes.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.
Assuntos
Processamento Alternativo , Biologia Computacional , Epigênese Genética/genética , Humanos , ProteomaRESUMO
dA6m DNA immunoprecipitation followed by deep sequencing (DIP-Seq) is a key tool in identifying and studying the genome-wide distribution of N6-methyldeoxyadenosine (dA6m). The precise function of this novel DNA modification remains to be fully elucidated, but it is known to be absent from transcriptional start sites and excluded from exons, suggesting a role in transcriptional regulation (Koziol et al., 2015). Importantly, its existence suggests that DNA might be more diverse than previously believed, as further DNA modifications might exist in eukaryotic DNA (Koziol et al., 2015). This protocol describes the method to perform dA6m DNA immunoprecipitation (DIP), as was applied to characterize the first dA6m methylome analysis in higher eukaryotes (Koziol et al., 2015). In this protocol, we describe how genomic DNA is isolated, fragmented and then DNA containing dA6m is pulled down with an antibody that recognizes dA6m in genomic DNA. After subsequent washes, DNA fragments that do not contain dA6m are eliminated, and the dA6m containing fragments are eluted from the antibody in order to be processed further for subsequent analyses. BACKGROUND: This protocol was developed in order to identify regions in the genome that contain dA6m. It can be used to detect dA6m in different genomes. As a guideline, this protocol was established from existing approaches used to detect adenosine methylation in RNA (Dominissini et al., 2013). We developed this protocol and adapted it for the detection of dA6m in DNA, rather than detecting adenosine methylation RNA. This was required, as no protocol was available at that time to allow the genome-wide identification of dA6m in eukaryotic DNA.
RESUMO
Methylation of cytosine deoxynucleotides generates 5-methylcytosine (m(5)dC), a well-established epigenetic mark. However, in higher eukaryotes much less is known about modifications affecting other deoxynucleotides. Here, we report the detection of N(6)-methyldeoxyadenosine (m(6)dA) in vertebrate DNA, specifically in Xenopus laevis but also in other species including mouse and human. Our methylome analysis reveals that m(6)dA is widely distributed across the eukaryotic genome and is present in different cell types but is commonly depleted from gene exons. Thus, direct DNA modifications might be more widespread than previously thought.