Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 34
Filtrar
1.
Nat Rev Genet ; 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38918545

RESUMO

DNA damage is a threat to genome integrity and can be a cause of many human diseases, owing to either changes in the chemical structure of DNA or conversion of the damage into a mutation, that is, a permanent change in DNA sequence. Determining the exact positions of DNA damage and ensuing mutations in the genome are important for identifying mechanisms of disease aetiology when characteristic mutations are prevalent and probably causative in a particular disease. However, this approach is challenging particularly when levels of DNA damage are low, for example, as a result of chronic exposure to environmental agents or certain endogenous processes, such as the generation of reactive oxygen species. Over the past few years, a comprehensive toolbox of genome-wide methods has been developed for the detection of DNA damage and rare mutations at single-nucleotide resolution in mammalian cells. Here, we review and compare these methods, describe their current applications and discuss future research questions that can now be addressed.

2.
Nature ; 477(7366): 606-10, 2011 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-21892189

RESUMO

Sperm and eggs carry distinctive epigenetic modifications that are adjusted by reprogramming after fertilization. The paternal genome in a zygote undergoes active DNA demethylation before the first mitosis. The biological significance and mechanisms of this paternal epigenome remodelling have remained unclear. Here we report that, within mouse zygotes, oxidation of 5-methylcytosine (5mC) occurs on the paternal genome, changing 5mC into 5-hydroxymethylcytosine (5hmC). Furthermore, we demonstrate that the dioxygenase Tet3 (ref. 5) is enriched specifically in the male pronucleus. In Tet3-deficient zygotes from conditional knockout mice, paternal-genome conversion of 5mC into 5hmC fails to occur and the level of 5mC remains constant. Deficiency of Tet3 also impedes the demethylation process of the paternal Oct4 and Nanog genes and delays the subsequent activation of a paternally derived Oct4 transgene in early embryos. Female mice depleted of Tet3 in the germ line show severely reduced fecundity and their heterozygous mutant offspring lacking maternal Tet3 suffer an increased incidence of developmental failure. Oocytes lacking Tet3 also seem to have a reduced ability to reprogram the injected nuclei from somatic cells. Therefore, Tet3-mediated DNA hydroxylation is involved in epigenetic reprogramming of the zygotic paternal DNA following natural fertilization and may also contribute to somatic cell nuclear reprogramming during animal cloning.


Assuntos
Reprogramação Celular , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Epigênese Genética , Oócitos/enzimologia , Oócitos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/metabolismo , Alelos , Animais , Citosina/análogos & derivados , Citosina/metabolismo , DNA/química , DNA/genética , DNA/metabolismo , Metilação de DNA/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Feminino , Fertilidade/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Masculino , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Oócitos/citologia , Oxirredução , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Zigoto/citologia , Zigoto/metabolismo
3.
Nucleic Acids Res ; 43(15): e100, 2015 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-25977295

RESUMO

Data on biological mechanisms of aging are mostly obtained from cross-sectional study designs. An inherent disadvantage of this design is that inter-individual differences can mask small but biologically significant age-dependent changes. A serially sampled design (same individual at different time points) would overcome this problem but is often limited by the relatively small numbers of available paired samples and the statistics being used. To overcome these limitations, we have developed a new vector-based approach, termed three-component analysis, which incorporates temporal distance, signal intensity and variance into one single score for gene ranking and is combined with gene set enrichment analysis. We tested our method on a unique age-based sample set of human skin fibroblasts and combined genome-wide transcription, DNA methylation and histone methylation (H3K4me3 and H3K27me3) data. Importantly, our method can now for the first time demonstrate a clear age-dependent decrease in expression of genes coding for proteins involved in translation and ribosome function. Using analogies with data from lower organisms, we propose a model where age-dependent down-regulation of protein translation-related components contributes to extend human lifespan.


Assuntos
Envelhecimento/genética , Epigênese Genética , Perfilação da Expressão Gênica , Biossíntese de Proteínas , Adulto , Idoso , Algoritmos , Células Cultivadas , Análise por Conglomerados , Metilação de DNA , Regulação para Baixo , Fatores de Transcrição Forkhead/metabolismo , Histonas/metabolismo , Humanos , Masculino , Metilação , Pessoa de Meia-Idade , Pele/metabolismo , Estatísticas não Paramétricas
4.
Genomics ; 106(6): 322-30, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26384656

RESUMO

Using MIRA-seq, we have characterized the DNA methylome of metastatic melanoma and normal melanocytes. Individual tumors contained several thousand hypermethylated regions. We discovered 179 tumor-specific methylation peaks present in all (27/27) melanomas that may be effective disease biomarkers, and 3113 methylation peaks were seen in >40% of the tumors. We found that 150 of the approximately 1200 tumor-associated methylation peaks near transcription start sites (TSSs) were marked by H3K27me3 in melanocytes. DNA methylation in melanoma was specific for distinct H3K27me3 peaks rather than for broadly covered regions. However, numerous H3K27me3 peak-associated TSS regions remained devoid of DNA methylation in tumors. There was no relationship between BRAF mutations and the number of methylation peaks. Gene expression analysis showed upregulated immune response genes in melanomas presumably as a result of lymphocyte infiltration. Down-regulated genes were enriched for melanocyte differentiation factors; e.g., KIT, PAX3 and SOX10 became methylated and downregulated in melanoma.


Assuntos
Metilação de DNA , Histonas/metabolismo , Melanócitos/metabolismo , Melanoma/genética , Sítio de Iniciação de Transcrição , Diferenciação Celular/genética , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunidade/genética , Lisina/metabolismo , Melanoma/metabolismo , Metilação
5.
J Am Chem Soc ; 136(33): 11582-5, 2014 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-25073028

RESUMO

Oxidation of 5-methylcytosine in DNA by ten-eleven translocation (Tet) family of enzymes has been demonstrated to play a significant role in epigenetic regulation in mammals. We found that Tet enzymes also possess the activity of catalyzing the formation of 5-hydroxymethylcytidine (5-hmrC) in RNA in vitro. In addition, the catalytic domains of all three Tet enzymes as well as full-length Tet3 could induce the formation of 5-hmrC in human cells. Moreover, 5-hmrC was present at appreciable levels (∼1 per 5000 5-methylcytidine) in RNA of mammalian cells and tissues. Our results suggest the involvement of this oxidation in RNA biology.


Assuntos
Citosina/análogos & derivados , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA/metabolismo , 5-Metilcitosina/análogos & derivados , Animais , Citosina/biossíntese , Citosina/química , Citosina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/deficiência , Dioxigenases/química , Dioxigenases/deficiência , Células-Tronco Embrionárias/metabolismo , Células HEK293 , Humanos , Camundongos , Oxigenases de Função Mista , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/deficiência , RNA/química
6.
Cell Tissue Res ; 356(3): 631-41, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24816989

RESUMO

The patterns of DNA methylation in human cancer cells are highly abnormal and often involve the acquisition of DNA hypermethylation at hundreds or thousands of CpG islands that are usually unmethylated in normal tissues. The recent discovery of 5-hydroxymethylcytosine (5hmC) as an enzymatic oxidation product of 5-methylcytosine (5mC) has led to models and experimental data in which the hypermethylation and 5mC oxidation pathways seem to be connected. Key discoveries in this setting include the findings that several genes coding for proteins involved in the 5mC oxidation reaction are mutated in human tumors, and that a broad loss of 5hmC occurs across many types of cancer. In this review, we will summarize current knowledge and discuss models of the potential roles of 5hmC in human cancer biology.


Assuntos
Citosina/análogos & derivados , Metilação de DNA , DNA de Neoplasias/metabolismo , Neoplasias/metabolismo , 5-Metilcitosina/análogos & derivados , Citosina/metabolismo , DNA de Neoplasias/genética , Humanos , Neoplasias/genética , Neoplasias/patologia , Oxirredução
7.
Proc Natl Acad Sci U S A ; 108(9): 3642-7, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21321204

RESUMO

Genome-wide erasure of DNA cytosine-5 methylation has been reported to occur along the paternal pronucleus in fertilized oocytes in an apparently replication-independent manner, but the mechanism of this reprogramming process has remained enigmatic. Recently, considerable amounts of 5-hydroxymethylcytosine (5hmC), most likely derived from enzymatic oxidation of 5-methylcytosine (5mC) by TET proteins, have been detected in certain mammalian tissues. 5hmC has been proposed as a potential intermediate in active DNA demethylation. Here, we show that in advanced pronuclear-stage zygotes the paternal pronucleus contains substantial amounts of 5hmC but lacks 5mC. The converse is true for the maternal pronucleus, which retains 5mC but shows little or no 5hmC signal. Importantly, 5hmC persists into mitotic one-cell, two-cell, and later cleavage-stage embryos, suggesting that 5mC oxidation is not followed immediately by genome-wide removal of 5hmC through excision repair pathways or other mechanisms. This conclusion is supported by bisulfite sequencing data, which shows only limited conversion of modified cytosines to cytosines at several gene loci. It is likely that 5mC oxidation is carried out by the Tet3 oxidase. Tet3, but not Tet1 or Tet2, was expressed at high levels in oocytes and zygotes, with rapidly declining levels at the two-cell stage. Our results show that 5mC oxidation is part of the early life cycle of mammals.


Assuntos
5-Metilcitosina/metabolismo , Reprogramação Celular/genética , Fertilização/genética , Genoma/genética , Animais , Proteínas Cromossômicas não Histona , Fase de Clivagem do Zigoto/citologia , Fase de Clivagem do Zigoto/metabolismo , Citosina/análogos & derivados , Citosina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Oócitos/citologia , Oócitos/metabolismo , Oxirredução , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Análise de Sequência de DNA , Sulfitos , Zigoto/citologia , Zigoto/metabolismo
8.
Photochem Photobiol Sci ; 12(8): 1409-15, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23677065

RESUMO

Much of the cancer-causing effects of ultraviolet radiation from the sun have been linked to the formation of dimerized DNA bases. These dimeric DNA photoproducts include the cyclobutane pyrimidine dimers (CPDs) and the pyrimidine(6-4)pyrimidone photoproducts [(6-4)PPs]. CPDs are highly mutagenic and are produced in substantial quantities by UVB radiation. These dimers can form between any two adjacent pyrimidines and can involve thymine, cytosine, or 5-methylcytosine. Very recently, a sixth DNA base, 5-hydroxymethylcytosine (5hmC) has been identified and characterized as a normal component of mammalian DNA. Here, we investigated the formation of CPDs at different DNA sequences containing 5hmC following irradiation with UVA, UVB, or UVC light sources. We show that the formation of CPDs at dipyrimidines containing 5hmC occurs at different DNA sequences but is not enhanced relative to cytosine or 5-methylcytosines at the same sequence positions. In fact, in some sequence contexts, CPDs containing 5hmC are formed at very low levels. Nonetheless, CPD formation at 5hmC pyrimidines is expected to be biologically relevant since three types of human skin-derived cells, fibroblasts, keratinocytes and melanocytes, all contain detectable levels of this modified base.


Assuntos
Citosina/análogos & derivados , DNA/química , Dímeros de Pirimidina/análise , 5-Metilcitosina/análogos & derivados , Sequência de Bases/efeitos da radiação , Células Cultivadas , Citosina/análise , Fibroblastos/química , Fibroblastos/efeitos da radiação , Humanos , Queratinócitos/química , Queratinócitos/efeitos da radiação , Melanócitos/química , Melanócitos/efeitos da radiação , Raios Ultravioleta
9.
Nucleic Acids Res ; 39(12): 5015-24, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21378125

RESUMO

Methylation at the 5-position of cytosine is a well-studied epigenetic pathway. In addition to 5-methylcytosine (5mC), substantial amounts of 5-hydroxymethylcytosine (5hmC) also referred to as the sixth DNA base have been detected in certain tissues, most notably the brain. However, the genomic distribution of this cytosine modification is unknown. Here, we have used an immunoprecipitation technique (5hmC-IP) to examine the occurrence of 5hmC in DNA from human brain frontal lobe tissue. The distribution of 5hmC was compared to that of 5mC. We show that 5hmC is more selectively targeted to genes than is 5mC. 5hmC is particularly enriched at promoters and in intragenic regions (gene bodies) but is largely absent from non-gene regions. 5hmC peaks at transcription start sites did not correlate with gene expression levels for promoters with intermediate or high CpG content. However, the presence of 5hmC in gene bodies was more positively correlated with gene expression levels than was the presence of 5mC. Promoters of testis-specific genes showed strong 5mC peaks in brain DNA but were almost completely devoid of 5hmC. Our data provide an overview of the genomic distribution of 5hmC in human brain and will set the stage for further functional characterization of this novel DNA modification.


Assuntos
Química Encefálica , Citosina/análogos & derivados , DNA/química , 5-Metilcitosina/análise , Animais , Citosina/análise , Expressão Gênica , Genômica , Humanos , Camundongos , Regiões Promotoras Genéticas
10.
Methods Mol Biol ; 2660: 247-262, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37191802

RESUMO

There are many sources of endogenous and exogenous DNA damage. Damaged bases represent a threat to genome integrity and may interfere with normal cellular processes such as replication and transcription. To understand the specificity and biological consequences of DNA damage, it is essential to employ methods that are sensitive enough to detect damaged DNA bases at the level of single nucleotide resolution and genome-wide. Here we describe in detail a method we developed for this purpose, circle damage sequencing (CD-seq). This method is based on the circularization of genomic DNA that contains damaged bases and conversion of the damaged sites into double-strand breaks using specific DNA repair enzymes. Library sequencing of the opened circles yields the precise positions of the DNA lesions that are present. CD-seq can be adopted to various types of DNA damage as long as a specific cleavage scheme can be designed.


Assuntos
Dano ao DNA , Genoma , DNA/genética , Reparo do DNA , Replicação do DNA
11.
Cancer Res ; 83(15): 2480-2495, 2023 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-37272752

RESUMO

Hypermethylation of CpG islands (CGI) is a common feature of cancer cells and predominantly affects Polycomb-associated genomic regions. Elucidating the underlying mechanisms leading to DNA hypermethylation in human cancer could help identify chemoprevention strategies. Here, we evaluated the role of Polycomb complexes and 5-methylcytosine (5mC) oxidases in protecting CGIs from DNA methylation and observed that four genes coding for components of Polycomb repressive complex 1 (PRC1) are downregulated in tumors. Inactivation of RYBP, a key activator of variant PRC1 complexes, in combination with all three 5mC oxidases (TET proteins) in nontumorigenic bronchial epithelial cells led to widespread hypermethylation of Polycomb-marked CGIs affecting almost 4,000 target genes, which closely resembled the DNA hypermethylation landscape observed in human squamous cell lung tumors. The RYBP- and TET-deficient cells showed methylation-associated aberrant regulation of cancer-relevant pathways, including defects in the Hippo tumor suppressor network. Notably, the quadruple knockout cells acquired a transformed phenotype, including anchorage-independent growth and formation of squamous cell carcinomas in mice. This work provides a mechanism promoting hypermethylation of CGIs and shows that such hypermethylation can lead to cell transformation. The breakdown of a two-pronged protection mechanism can be a route towards genome-wide hypermethylation of CGIs in tumors. SIGNIFICANCE: Dysfunction of the Polycomb component RYBP in combination with loss of 5-methylcytosine oxidases promotes widespread hypermethylation of CpG islands in bronchial cells and induces tumorigenesis, resembling changes seen in human lung tumors.


Assuntos
Carcinoma de Células Escamosas , Neoplasias Pulmonares , Humanos , Animais , Camundongos , Ilhas de CpG/genética , Oxirredutases/genética , 5-Metilcitosina/metabolismo , Metilação de DNA , Transformação Celular Neoplásica/genética , Carcinoma de Células Escamosas/genética , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Neoplasias Pulmonares/genética , DNA/metabolismo , Proteínas Repressoras/genética
12.
Cell Rep ; 42(1): 112012, 2023 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-36680774

RESUMO

Long bones are generated by mesoderm-derived skeletal progenitor/stem cells (SSCs) through endochondral ossification, a process of sequential chondrogenic and osteogenic differentiation tightly controlled by the synergy between intrinsic and microenvironment cues. Here, we report that loss of TRIM28, a transcriptional corepressor, in mesoderm-derived cells expands the SSC pool, weakens SSC osteochondrogenic potential, and endows SSCs with properties of ectoderm-derived neural crest cells (NCCs), leading to severe defects of skeletogenesis. TRIM28 preferentially enhances H3K9 trimethylation and DNA methylation on chromatin regions more accessible in NCCs; loss of this silencing upregulates neural gene expression and enhances neurogenic potential. Moreover, TRIM28 loss causes hyperexpression of GREM1, which is an extracellular signaling factor promoting SSC self-renewal and SSC neurogenic potential by activating AKT/mTORC1 signaling. Our results suggest that TRIM28-mediated chromatin silencing establishes a barrier for maintaining the SSC lineage trajectory and preventing a transition to ectodermal fate by regulating both intrinsic and microenvironment cues.


Assuntos
Osteogênese , Proteína 28 com Motivo Tripartido , Diferenciação Celular/genética , Cromatina , Expressão Gênica , Proteínas Proto-Oncogênicas c-akt/genética , Células-Tronco , Serina-Treonina Quinases TOR/genética , Animais , Camundongos , Proteína 28 com Motivo Tripartido/metabolismo , Transdução de Sinais
13.
Nucleic Acids Res ; 38(11): e125, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20371518

RESUMO

DNA cytosine-5 methylation is a well-studied epigenetic pathway implicated in gene expression control and disease pathogenesis. Different technologies have been developed to examine the distribution of 5-methylcytosine (5mC) in specific sequences of the genome. Recently, substantial amounts of 5-hydroxymethylcytosine (5hmC), most likely derived from enzymatic oxidation of 5mC by TET1, have been detected in certain mammalian tissues. Here, we have examined the ability of several commonly used DNA methylation profiling methods to distinguish between 5mC and 5hmC. We show that techniques based on sodium bisulfite treatment of DNA are incapable of distinguishing between the two modified bases. In contrast, techniques based on immunoprecipitation with anti-5mC antibody (methylated DNA immunoprecipitation, MeDIP) or those based on proteins that bind to methylated CpG sequences (e.g. methylated-CpG island recovery assay, MIRA) do not detect 5hmC and are specific for 5mC unless both modified bases occur in the same DNA fragment. We also report that several methyl-CpG binding proteins including MBD1, MBD2 and MBD4 do not bind to sequences containing 5hmC. Selective mapping of 5hmC will require the development of unique tools for the detection of this modified base.


Assuntos
5-Metilcitosina/análise , Citosina/análogos & derivados , Metilação de DNA , 5-Metilcitosina/imunologia , Anticorpos , Citosina/análise , Citosina/metabolismo , DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Imunoprecipitação , Oligonucleotídeos/química , Análise de Sequência de DNA , Sulfitos
14.
ACS Omega ; 7(37): 32936-32948, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36157735

RESUMO

Melanoma is a lethal type of skin tumor that has been linked with sunlight exposure chiefly in fair-skinned human populations. Wavelengths from the sun that can reach the earth's surface include UVA radiation (320-400 nm) and UVB radiation (280-320 nm). UVB effectively induces the formation of dimeric DNA photoproducts, preferentially the cyclobutane pyrimidine dimers (CPDs). The characteristic UVB signature mutations in the form of C to T mutations at dipyrimidine sequences are prevalent in melanoma tumor genomes and have been ascribed to deamination of cytosines within CPDs before DNA polymerase bypass. However, evidence from epidemiological, animal, and other experimental studies also suggest that UVA radiation may participate in melanoma formation. The DNA damage relevant for UVA includes specific types of CPDs at TT sequences and perhaps oxidative DNA damage to guanine, both induced by direct or indirect, photosensitization-mediated chemical and biophysical processes. We summarize the evidence for a potential role of UVA in melanoma and discuss some of the mechanistic pathways of how UVA may induce mutagenesis in melanocytes.

15.
Sci Adv ; 8(22): eabn3815, 2022 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-35658030

RESUMO

Oxidative DNA damage has been linked to inflammation, cancer, and aging. Here, we have mapped two types of oxidative DNA damage, oxidized guanines produced by hydrogen peroxide and oxidized thymines created by potassium permanganate, at a single-base resolution. 8-Oxo-guanine occurs strictly dependent on the G/C sequence context and shows a pronounced peak at transcription start sites (TSSs). We determined the trinucleotide sequence pattern of guanine oxidation. This pattern shows high similarity to the cancer-associated single-base substitution signatures SBS18 and SBS36. SBS36 is found in colorectal cancers that carry mutations in MUTYH, encoding a repair enzyme that operates on 8-oxo-guanine mispairs. SBS18 is common in inflammation-associated upper gastrointestinal tract tumors including esophageal and gastric adenocarcinomas. Oxidized thymines induced by permanganate occur with a distinct dinucleotide specificity, 5'T-A/C, and are depleted at the TSS. Our data suggest that two cancer mutational signatures, SBS18 and SBS36, are caused by reactive oxygen species.


Assuntos
Neoplasias , Trato Gastrointestinal Superior , Dano ao DNA , Guanina , Humanos , Peróxido de Hidrogênio/farmacologia , Inflamação , Mutação , Oxirredução
16.
Nat Cell Biol ; 24(7): 1141-1153, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35787683

RESUMO

Mutagenic purine-pyrimidine repeats can adopt the left-handed Z-DNA conformation. DNA breaks at potential Z-DNA sites can lead to somatic mutations in cancer or to germline mutations that are transmitted to the next generation. It is not known whether any mechanism exists in the germ line to control Z-DNA structure and DNA breaks at purine-pyrimidine repeats. Here we provide genetic, epigenomic and biochemical evidence for the existence of a biological process that erases Z-DNA specifically in germ cells of the mouse male foetus. We show that a previously uncharacterized zinc finger protein, ZBTB43, binds to and removes Z-DNA, preventing the formation of DNA double-strand breaks. By removing Z-DNA, ZBTB43 also promotes de novo DNA methylation at CG-containing purine-pyrimidine repeats in prospermatogonia. Therefore, the genomic and epigenomic integrity of the species is safeguarded by remodelling DNA structure in the mammalian germ line during a critical window of germline epigenome reprogramming.


Assuntos
DNA Forma Z , Animais , DNA/metabolismo , Metilação de DNA , DNA Forma Z/metabolismo , Epigenoma , Células Germinativas/metabolismo , Masculino , Mamíferos/metabolismo , Camundongos , Conformação de Ácido Nucleico , Purinas/metabolismo , Pirimidinas
17.
PLoS Genet ; 4(3): e1000013, 2008 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-18369439

RESUMO

Although DNA methylation patterns in somatic cells are thought to be relatively stable, they undergo dramatic changes during embryonic development, gametogenesis, and during malignant transformation. The enzymology of DNA methyltransferases is well understood, but the mechanism that removes methylated cytosines from DNA (active DNA demethylation) has remained enigmatic. Recently, a role of the growth arrest and DNA damage inducible protein GADD45A in DNA demethylation has been reported [1]. We have investigated the function of GADD45A in DNA demethylation in more detail using gene reactivation and DNA methylation assays. Contrary to the previous report, we were unable to substantiate a functional role of GADD45A in DNA demethylation. The mechanism of active DNA demethylation in mammalian cells remains unknown.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Metilação de DNA , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animais , Sequência de Bases , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Endonucleases/genética , Endonucleases/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Células NIH 3T3 , Oócitos/metabolismo , Plasmídeos/genética , Gravidez , Proteínas Recombinantes/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Zigoto/metabolismo
18.
Sci Adv ; 7(31)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34330711

RESUMO

Sunlight-associated melanomas carry a unique C-to-T mutation signature. UVB radiation induces cyclobutane pyrimidine dimers (CPDs) as the major form of DNA damage, but the mechanism of how CPDs cause mutations is unclear. To map CPDs at single-base resolution genome wide, we developed the circle damage sequencing (circle-damage-seq) method. In human cells, CPDs form preferentially in a tetranucleotide sequence context (5'-Py-T<>Py-T/A), but this alone does not explain the tumor mutation patterns. To test whether mutations arise at CPDs by cytosine deamination, we specifically mapped UVB-induced cytosine-deaminated CPDs. Transcription start sites (TSSs) were protected from CPDs and deaminated CPDs, but both lesions were enriched immediately upstream of the TSS, suggesting a mutation-promoting role of bound transcription factors. Most importantly, the genomic dinucleotide and trinucleotide sequence specificity of deaminated CPDs matched the prominent mutation signature of melanomas. Our data identify the cytosine-deaminated CPD as the leading premutagenic lesion responsible for mutations in melanomas.


Assuntos
Melanoma , Dímeros de Pirimidina , Citosina/metabolismo , Dano ao DNA , Desaminação , Humanos , Melanoma/genética , Mutação , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/metabolismo , Raios Ultravioleta
19.
Methods Mol Biol ; 2272: 225-237, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009617

RESUMO

The 5-methylcytosine (5mC) oxidation pathway mediated by TET proteins involves step-wise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC can be removed from DNA by base excision repair and the completion of this pathway results in "demethylation" of 5mC by converting the modified base back into cytosine. In vitro studies with TET proteins aimed at analyzing their DNA substrate specificities and their activity within defined chromatin templates are relatively limited. Here we describe purification methods for mammalian TET proteins based on expression in insect cells or in 293T cells. We also briefly summarize a method that can be used to monitor 5-methylcytosine oxidase activity of the purified TET proteins in vitro.


Assuntos
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , Metilação de DNA , DNA/análise , Oxigenases de Função Mista/isolamento & purificação , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas/metabolismo , DNA/química , DNA/genética , Humanos , Oxigenases de Função Mista/genética , Oxirredução , Proteínas Proto-Oncogênicas/genética , Especificidade por Substrato
20.
Sci Adv ; 5(10): eaax0080, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31681843

RESUMO

The characteristics of DNA methylation changes that occur during neurogenesis in vivo remain unknown. We used whole-genome bisulfite sequencing to quantitate DNA cytosine modifications in differentiating neurons and their progenitors isolated from mouse brain at the peak of embryonic neurogenesis. Localized DNA hypomethylation was much more common than hypermethylation and often occurred at putative enhancers within genes that were upregulated in neurons and encoded proteins crucial for neuronal differentiation. The hypomethylated regions strongly overlapped with mapped binding sites of the key neuronal transcription factor NEUROD2. The 5-methylcytosine oxidase ten-eleven translocation 2 (TET2) interacted with NEUROD2, and its reaction product 5-hydroxymethylcytosine accumulated at the demethylated regions. NEUROD2-targeted differentially methylated regions retained higher methylation levels in Neurod2 knockout mice, and inducible expression of NEUROD2 caused TET2-associated demethylation at its in vivo binding sites. The data suggest that the reorganization of DNA methylation in developing neurons involves NEUROD2 and TET2-mediated DNA demethylation.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Córtex Cerebral/citologia , Metilação de DNA , Neurônios/citologia , Neuropeptídeos/metabolismo , 5-Metilcitosina/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Elementos Facilitadores Genéticos/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurogênese , Motivos de Nucleotídeos/genética , Oxirredução , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA