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1.
Int J Mol Sci ; 22(6)2021 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-33801130

RESUMO

It was suggested that the epigenetic alterations of the placenta are associated with obesity, as well as the delivery mode. This study aimed to assess the effect of maternal outcome and delivery procedure on global placental DNA methylation status, as well as selected 5'-Cytosine-phosphate-Guanine-3' (CpG) sites in ADIPOQ and LEP genes. Global DNA methylation profile in the placenta was assessed using the 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) ratio evaluated with the ELISA, followed by target gene methylation patterns at selected gene regions which were determined using methylation-specific qPCR in 70 placentas from healthy, pregnant women with single pregnancy. We found no statistically significant differences in 5-mC/5-hmC ratio between intrapartum cesarean sections (CS) and vaginal deliveries (p = 0.214), as well as between elective cesarean sections and vaginal deliveries (p = 0.221). In intrapartum cesarean sections, the ADIPOQ demethylation index was significantly higher (the average: 1.75) compared to elective cesarean section (the average: 1.23, p = 0.010) and vaginal deliveries (the average: 1.23, p = 0.011). The LEP demethylation index did not significantly differ among elective CS, intrapartum CS, and vaginal delivery groups. The demethylation index of ADIPOQ correlated negatively with LEP in the placenta in the vaginal delivery group (r = -0.456, p = 0.017), but not with the global methylation. The methylation of a singular locus might be different depending on the mode of delivery and uterine contractions. Further studies should be conducted with locus-specific analysis of the whole genome to detect the methylation index of specific genes involved in metabolism.


Assuntos
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Adiponectina/genética , Metilação de DNA , Leptina/genética , Placenta/metabolismo , Adiponectina/metabolismo , Parto Obstétrico , Epigênese Genética , Feminino , Regulação da Expressão Gênica , Loci Gênicos , Humanos , Leptina/metabolismo , Gravidez
2.
Nat Commun ; 12(1): 795, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542217

RESUMO

Epigenetic modifications of DNA play important roles in many biological processes. Identifying readers of these epigenetic marks is a critical step towards understanding the underlying mechanisms. Here, we present an all-to-all approach, dubbed digital affinity profiling via proximity ligation (DAPPL), to simultaneously profile human TF-DNA interactions using mixtures of random DNA libraries carrying different epigenetic modifications (i.e., 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine) on CpG dinucleotides. Many proteins that recognize consensus sequences carrying these modifications in symmetric and/or hemi-modified forms are identified. We further demonstrate that the modifications in different sequence contexts could either enhance or suppress TF binding activity. Moreover, many modifications can affect TF binding specificity. Furthermore, symmetric modifications show a stronger effect in either enhancing or suppressing TF-DNA interactions than hemi-modifications. Finally, in vivo evidence suggests that USF1 and USF2 might regulate transcription via hydroxymethylcytosine-binding activity in weak enhancers in human embryonic stem cells.


Assuntos
5-Metilcitosina/análogos & derivados , DNA/metabolismo , Epigenômica/métodos , 5-Metilcitosina/metabolismo , Linhagem Celular , Ilhas de CpG/genética , DNA/genética , Elementos Facilitadores Genéticos , Epigênese Genética , Biblioteca Gênica , Células-Tronco Embrionárias Humanas , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fatores Estimuladores Upstream/genética , Fatores Estimuladores Upstream/isolamento & purificação , Fatores Estimuladores Upstream/metabolismo
3.
Nat Commun ; 12(1): 618, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33504799

RESUMO

Although various methods have been developed for sequencing cytosine modifications, it is still challenging for specific and quantitative sequencing of individual modification at base-resolution. For example, to obtain both true 5-methylcytosine (5mC) and true 5-hydroxymethylcytosine (5hmC) information, the two major epigenetic modifications, it usually requires subtraction of two methods, which increases noise and requires high sequencing depth. Recently, we developed TET-assisted pyridine borane sequencing (TAPS) for bisulfite-free direct sequencing of 5mC and 5hmC. Here we demonstrate that two sister methods, TAPSß and chemical-assisted pyridine borane sequencing (CAPS), can be effectively used for subtraction-free and specific whole-genome sequencing of 5mC and 5hmC, respectively. We also demonstrate pyridine borane sequencing (PS) for whole-genome profiling of 5-formylcytosine and 5-carboxylcytosine, the further oxidized derivatives of 5mC and 5hmC. This work completes the set of versatile borane reduction chemistry-based methods as a comprehensive toolkit for direct and quantitative sequencing of all four cytosine epigenetic modifications.


Assuntos
5-Metilcitosina/metabolismo , Análise de Sequência de DNA , Sulfitos/metabolismo , 5-Metilcitosina/análogos & derivados , Animais , Sequência de Bases , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Oxirredução , Piridinas/metabolismo
4.
Nat Commun ; 12(1): 73, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397987

RESUMO

In the male germ cells of placental mammals, 26-30-nt-long PIWI-interacting RNAs (piRNAs) emerge when spermatocytes enter the pachytene phase of meiosis. In mice, pachytene piRNAs derive from ~100 discrete autosomal loci that produce canonical RNA polymerase II transcripts. These piRNA clusters bear 5' caps and 3' poly(A) tails, and often contain introns that are removed before nuclear export and processing into piRNAs. What marks pachytene piRNA clusters to produce piRNAs, and what confines their expression to the germline? We report that an unusually long first exon (≥ 10 kb) or a long, unspliced transcript correlates with germline-specific transcription and piRNA production. Our integrative analysis of transcriptome, piRNA, and epigenome datasets across multiple species reveals that a long first exon is an evolutionarily conserved feature of pachytene piRNA clusters. Furthermore, a highly methylated promoter, often containing a low or intermediate level of CG dinucleotides, correlates with germline expression and somatic silencing of pachytene piRNA clusters. Pachytene piRNA precursor transcripts bind THOC1 and THOC2, THO complex subunits known to promote transcriptional elongation and mRNA nuclear export. Together, these features may explain why the major sources of pachytene piRNA clusters specifically generate these unique small RNAs in the male germline of placental mammals.


Assuntos
Epigênese Genética , Éxons/genética , Mamíferos/genética , Estágio Paquíteno/genética , RNA Interferente Pequeno/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Acetilação , Animais , Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Histonas/metabolismo , Íntrons/genética , Masculino , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas/genética , Processamento de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/genética , Testículo/metabolismo , Transcrição Genética
5.
Mol Cell ; 81(4): 859-869.e8, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33352108

RESUMO

Active DNA demethylation via ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of the role of TETs in chromatin reprogramming. Here, we describe the first application of biochemically engineered TET mutants that unlink 5mC oxidation steps, examining their effects on somatic cell reprogramming. We show that only TET enzymes proficient for oxidation to 5fC/5caC can rescue the reprogramming potential of Tet2-deficient mouse embryonic fibroblasts. This effect correlated with rapid DNA demethylation at reprogramming enhancers and increased chromatin accessibility later in reprogramming. These experiments demonstrate that DNA demethylation through 5fC/5caC has roles distinct from 5hmC in somatic reprogramming to pluripotency.


Assuntos
5-Metilcitosina/metabolismo , Reprogramação Celular , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/metabolismo , Elementos Facilitadores Genéticos , Epigênese Genética , Fibroblastos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Mutação , Células NIH 3T3 , Proteínas Proto-Oncogênicas/genética
6.
J Virol ; 95(6)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33361417

RESUMO

Hepatitis B virus (HBV) transcription and replication increase progressively throughout postnatal liver development with maximal viral biosynthesis occurring at around 4 weeks of age in the HBV transgenic mouse model of chronic infection. Increasing viral biosynthesis is associated with a corresponding progressive loss of DNA methylation. The loss of DNA methylation is associated with increasing levels of 5-hydroxymethylcytosine (5hmC) residues which correlate with increased liver-enriched pioneer transcription factor Forkhead box protein A (FoxA) RNA levels, a rapid decline in postnatal liver DNA methyltransferase (Dnmt) transcripts, and a very modest reduction in ten-eleven translocation (Tet) methylcytosine dioxygenase expression. These observations are consistent with the suggestion that the balance between active HBV DNA methylation and demethylation is regulated by FoxA recruitment of Tet in the presence of declining Dnmt activity. These changes lead to demethylation of the viral genome during hepatocyte maturation with associated increases in viral biosynthesis. Consequently, manipulation of the relative activities of these two counterbalancing processes might permit the specific silencing of HBV gene expression with the loss of viral biosynthesis and the resolution of chronic HBV infections.IMPORTANCE HBV biosynthesis begins at birth and increases during early postnatal liver development in the HBV transgenic mouse model of chronic infection. The levels of viral RNA and DNA synthesis correlate with pioneer transcription factor FoxA transcript plus Tet methylcytosine dioxygenase-generated 5hmC abundance but inversely with Dnmt transcript levels and HBV DNA methylation. Together, these findings suggest that HBV DNA methylation during neonatal liver development is actively modulated by the relative contributions of FoxA-recruited Tet-mediated DNA demethylation and Dnmt-mediated DNA methylation activities. This mode of gene regulation, mediated by the loss of DNA methylation at hepatocyte-specific viral and cellular promoters, likely contributes to hepatocyte maturation during liver development in addition to the postnatal activation of HBV transcription and replication.


Assuntos
DNA Viral/metabolismo , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/virologia , Fígado/crescimento & desenvolvimento , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Animais Recém-Nascidos , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Replicação do DNA , DNA Viral/biossíntese , Desmetilação , Dioxigenases/genética , Dioxigenases/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Regulação Viral da Expressão Gênica , Hepatite B Crônica/metabolismo , Hepatite B Crônica/patologia , Fatores Nucleares de Hepatócito/genética , Fatores Nucleares de Hepatócito/metabolismo , Fígado/metabolismo , Fígado/virologia , Camundongos , Camundongos Transgênicos , RNA Viral/biossíntese , Replicação Viral
7.
Cell Mol Life Sci ; 78(6): 3045-3055, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33313982

RESUMO

Excess maternal fat intake and obesity increase offspring susceptibility to conditions such as chronic anxiety and substance abuse. We hypothesised that environmentally modulated DNA methylation changes (5mC/5hmC) in regulatory regions of the genome that modulate mood and consumptive behaviours could contribute to susceptibility to these conditions. We explored the effects of environmental factors on 5mC/5hmC levels within the GAL5.1 enhancer that controls anxiety-related behaviours and alcohol intake. We first observed that 5mC/5hmC levels within the GAL5.1 enhancer differed significantly in different parts of the brain. Moreover, we noted that early life stress had no significant effect of 5mC/5hmC levels within GAL5.1. In contrast, we identified that allowing access of pregnant mothers to high-fat diet (> 60% calories from fat) had a significant effect on 5mC/5hmC levels within GAL5.1 in hypothalamus and amygdala of resulting male offspring. Cell transfection-based studies using GAL5.1 reporter plasmids showed that 5mC has a significant repressive effect on GAL5.1 activity and its response to known stimuli, such as EGR1 transcription factor expression and PKC agonism. Intriguingly, CRISPR-driven disruption of GAL5.1 from the mouse genome, although having negligible effects on metabolism or general appetite, significantly decreased intake of high-fat diet suggesting that GAL5.1, in addition to being epigenetically modulated by high-fat diet, also actively contributes to the consumption of high-fat diet suggesting its involvement in an environmentally influenced regulatory loop. Furthermore, considering that GAL5.1 also controls alcohol preference and anxiety these studies may provide a first glimpse into an epigenetically controlled mechanism that links maternal high-fat diet with transgenerational susceptibility to alcohol abuse and anxiety.


Assuntos
Alcoolismo/patologia , Ansiedade/patologia , Dieta Hiperlipídica , Elementos Facilitadores Genéticos/genética , 5-Metilcitosina/metabolismo , Alcoolismo/genética , Tonsila do Cerebelo/metabolismo , Animais , Ansiedade/genética , Linhagem Celular Tumoral , Metilação de DNA , Modelos Animais de Doenças , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Epigênese Genética , Feminino , Humanos , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase C/química , Proteína Quinase C/metabolismo
8.
Methods Mol Biol ; 2198: 147-157, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32822029

RESUMO

DNA methylation undergoes dynamic changes at the genome-wide scale during the early steps of mammalian embryo development. Immunochemical detection of 5-methylcytosine (5mC) in the zygote has led to the discovery that a global loss of DNA methylation takes place soon after fertilization, occurring rapidly in the paternal pronucleus. Using the same method employed above, which detects modified bases in the denatured single stranded DNA, we showed that this active DNA "demethylation" in the paternal pronucleus involves oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) by the TET3 enzyme. By immunostaining of genetically altered zygotes we revealed that the maternal pronucleus is protected from TET3-mediated oxidation by histone H3K9 methyltransferase enzymes, EHMT2 and SETDB1. The same assays are also applicable for visualizing the temporal and spatial distribution of the modified cytosine residues in preimplantation embryos. Here, we provide a detailed protocol for detecting 5mC, 5hmC, 5fC, and 5caC in mouse zygotes and preimplantation-stage embryos using antibodies raised against modified cytosine species.


Assuntos
Blastocisto/metabolismo , Citosina/metabolismo , Metilação de DNA , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , 5-Metilcitosina/metabolismo , Animais , Citosina/análogos & derivados , Embrião de Mamíferos/embriologia , Humanos , Imunoquímica/métodos , Mamíferos , Microscopia Confocal , Zigoto/metabolismo
9.
Methods Mol Biol ; 2198: 169-181, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32822031

RESUMO

Immunocytochemistry can be instrumental in assessing the spatial distribution and relative levels of epigenetic modifications. Although conventional immunostaining has been utilized for the detection of 5-methylcytosine (5mC) in animal cells and tissues for several decades, the sensitivity of techniques based on the use of fluorophore-conjugated secondary antibodies is not always sufficient for studying DNA modifications that are less abundant in DNA compared with 5mC. Here we describe a protocol for sensitive immunocytochemistry that utilizes peroxidase-conjugated secondary antibodies coupled with catalyzed reporter deposition and allows for detection of low-abundance noncanonical bases (e.g., 5-carboxylcytosine, 5caC, 5-formylcytosine, 5fC, 5-hydroxymethyluracil, 5hmU) in mammalian DNA. This method can be employed for evaluation of the levels and nuclear distribution of DNA modifications and permits their colocalization with protein markers in animal cells.


Assuntos
DNA/imunologia , Imuno-Histoquímica/métodos , 5-Metilcitosina/metabolismo , Animais , Anticorpos/metabolismo , Núcleo Celular/metabolismo , Citosina/análogos & derivados , Citosina/análise , DNA/genética , Metilação de DNA/imunologia , Epigênese Genética/genética , Humanos , Pentoxil (Uracila)/análogos & derivados , Pentoxil (Uracila)/análise , Peroxidase/química
10.
Methods Mol Biol ; 2198: 183-191, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32822032

RESUMO

The modified cytosine base 5-hydroxymethylcytosine (5hmC) is abundantly present in the central nervous system (CNS), and visualization of global 5hmC levels is possible through use of immunohistochemistry. In this chapter we describe an adaptable method of brain tissue collection and immunohistochemical staining that allows for detection of 5hmC in mouse or rat brain, meaning that the method can be applied to many rodent models of CNS diseases and disorders.


Assuntos
5-Metilcitosina/análogos & derivados , DNA/imunologia , Imuno-Histoquímica/métodos , 5-Metilcitosina/química , 5-Metilcitosina/metabolismo , Animais , Anticorpos/metabolismo , Encéfalo , Núcleo Celular/metabolismo , Citosina , DNA/genética , Metilação de DNA/imunologia , Epigênese Genética/genética , Camundongos , Peroxidase/química , Ratos , Roedores
11.
Methods Mol Biol ; 2198: 193-208, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32822033

RESUMO

5-methylcytosine (5mC) is an epigenetic modification to DNA which modulates transcription. 5mC can be sequentially oxidized to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Collectively, these marks are referred to as the oxidized derivatives of 5mC (i.e., oxi-mCs). Their formation is catalyzed by the ten-eleven translocation methylcytosine dioxygenases (TETs 1, 2 and 3). Various techniques have been developed for the detection of oxi-mCs. The following chapter describes an immunochemical protocol for the simultaneous detection of 5hmC and 5caC in embryonic zebrafish tissue sections. The embryos are fixed, permeabilized and embedded in paraffin blocks. The blocks are cut into sections that are mounted onto slides. Depurination of the DNA is performed to allow immunodetection of the oxi-mCs. The 5hmC is detected with the help of a mouse anti-5hmC monoclonal primary antibody and a goat anti-mouse Alexa Fluor 633-conjugated secondary antibody. The weak 5caC signal requires enzymatic amplification. Its detection involves a rabbit anti-5caC polyclonal primary antibody and a goat anti-rabbit secondary antibody that is conjugated to horseradish peroxidase (HRP). HRP amplifies the 5caC signal by catalyzing the deposition of large quantities of fluorescein-labeled tyramide. Sections immunostained for 5hmC and 5caC are analyzed by fluorescent light or confocal laser scanning microscopy. This immunochemical method allows for highly sensitive detection of 5hmC and 5caC in zebrafish tissues.


Assuntos
5-Metilcitosina/análogos & derivados , DNA/imunologia , Imuno-Histoquímica/métodos , 5-Metilcitosina/química , 5-Metilcitosina/metabolismo , Animais , Anticorpos/metabolismo , Núcleo Celular/metabolismo , Citosina/análogos & derivados , DNA/genética , Metilação de DNA , Dioxigenases , Embrião não Mamífero , Peixe-Zebra
12.
Methods Mol Biol ; 2198: 209-216, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32822034

RESUMO

Methylated cytosine (5-methylcytosine) is the most studied epigenetic mark involved in the regulation of gene expression. Although it displays highly variable dynamics during plant ontogenesis, it is possible to gain a fine spatial perspective with immunohistochemistry techniques that use specific antibodies and fluorochromes. Besides, there are other cytosine modifications described in plants, although their biological significance is still unknown (i.e., 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine). Here we present a standardized protocol to detect cytosine modifications in plant tissues.


Assuntos
5-Metilcitosina/análogos & derivados , DNA/imunologia , Imuno-Histoquímica/métodos , 5-Metilcitosina/química , 5-Metilcitosina/metabolismo , Anticorpos/metabolismo , Núcleo Celular/metabolismo , Citosina/análogos & derivados , DNA/genética , Metilação de DNA , Dioxigenases , Epigênese Genética , Corantes Fluorescentes , Plantas/genética , Plantas/imunologia , Plantas/metabolismo
13.
Methods Mol Biol ; 2198: 311-319, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32822041

RESUMO

DNA methylation (5-methylcytosine, 5mC) is involved in regulation of a wide range of biological processes. TET proteins can oxidize 5mC to 5-hydroxymethylcytosine, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Although both 5fC and 5caC serve as intermediates in active demethylation pathway, growing body of experimental evidence indicate that these DNA modifications may also interact with specific sets of reader proteins and therefore may represent bona fide epigenetic marks. Despite a number of single-base resolution techniques have recently been proposed for 5fC/5caC mapping, antibody-based approaches still represent a relatively simple and plausible alternative for the analysis of genomic distribution of these DNA modifications. Here, we describe a protocol for 5caC DNA immunoprecipitation (5caC DIP) that can be used for both locus-specific and genome-wide assessment of 5caC distribution. In combination with mass spectrometry-based techniques and single base resolution mapping methods, this approach may contribute to elucidating the role of 5caC in development, differentiation, and tumorigenesis.


Assuntos
Imunoprecipitação da Cromatina/métodos , Citosina/análogos & derivados , Metilação de DNA/imunologia , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Citosina/análise , Citosina/metabolismo , DNA/imunologia , DNA/metabolismo , Humanos , Imunoprecipitação/métodos
14.
Int J Biol Macromol ; 170: 514-522, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33359809

RESUMO

Non-CpG DNA methylation (non-mCpG) is enriched in the genome of brain neurons and germline cells in mammals. Accumulation of non-mCpG during postnatal brain development correlates with gene regulation and inactivation of distal regulatory elements. Recently, UHRF1 has been found to contribute to de novo non-CpG methylation, however, whether UHRF1 could recognize non-mCpG is unknown. Here, we have demonstrated through calorimetric measurements that the UHRF1 SRA can recognize mCpH and fully-mCpHpG, types of non-mCpG. Our ITC binding studies endorse the preferential reading of hemi-mCpG by UHRF1 SRA and also show 6-fold weaker binding for fully-mCpG than hemi-mCpG. Despite presence of symmetrical (5-methyl cytosine) 5mCs, stoichiometry of 1:1 for UHRF1 SRA binding to fully-mCpG indicates that UHRF1 SRA may not form a stable complex with fully-mCpG DNA. Contrarily, UHRF1 SRA recognizes fully-mCpHpG with a stoichiometry of 2:1 protein to DNA duplex with binding affinity higher than fully-mCpG. Our crystal structure of UHRF1 SRA bound to fully-mCpHpG DNA reveals dual flip-out mechanism of 5mC recognition. Metadynamics studies corroborates with ITC data that UHRF1 SRA could not form a stable complex with fully-mCpG DNA. Altogether, this study demonstrates that UHRF1 SRA recognizes non-mCpG DNA and exhibits contrasting mechanisms for hemi-mCpG and fully-mCpHpG DNA recognition.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ilhas de CpG/fisiologia , Citosina/metabolismo , Metilação de DNA/fisiologia , DNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , 5-Metilcitosina/metabolismo , Sequência de Aminoácidos , Animais , DNA Complementar/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Camundongos , Ligação Proteica/fisiologia , Alinhamento de Sequência
15.
Int J Mol Sci ; 22(1)2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33374812

RESUMO

5-Hydroxymethylcytosine (5hmC) is a functionally active epigenetic modification. We analyzed whether changes in DNA 5-hydroxymethylation are an element of age-related epigenetic drift. We tested primary fibroblast cultures originating from individuals aged 22-35 years and 74-94 years. Global quantities of methylation-related DNA modifications were estimated by the dot blot and colorimetric methods. Regions of the genome differentially hydroxymethylated with age (DHMRs) were identified by hMeDIP-seq and the MEDIPS and DiffBind algorithms. Global levels of DNA modifications were not associated with age. We identified numerous DHMRs that were enriched within introns and intergenic regions and most commonly associated with the H3K4me1 histone mark, promoter-flanking regions, and CCCTC-binding factor (CTCF) binding sites. However, only seven DHMRs were identified by both algorithms and all of their settings. Among them, hypo-hydroxymethylated DHMR in the intron of Rab Escort Protein 1 (CHM) coexisted with increased expression in old cells, while increased 5-hydroxymethylation in the bodies of Arginine and Serine Rich Protein 1 (RSRP1) and Mitochondrial Poly(A) Polymerase (MTPAP) did not change their expression. These age-related differences were not associated with changes in the expression of any of the ten-eleven translocation (TET) enzymes or their activity. In conclusion, the distribution of 5hmC in DNA of in vivo aged human fibroblasts underwent age-associated modifications. The identified DHMRs are, likely, marker changes.


Assuntos
5-Metilcitosina/análogos & derivados , Metilação de DNA , Envelhecimento da Pele/genética , 5-Metilcitosina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas
16.
Int J Mol Sci ; 21(24)2020 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-33353068

RESUMO

Breast cancer is one of the most frequently diagnosed malignancies and common causes of cancer death in women. Recent studies suggest that environmental exposures to certain chemicals, such as 7,12-Dimethylbenzanthracene (DMBA), a chemical present in tobacco, may increase the risk of developing breast cancer later in life. The first-line treatments for breast cancer (surgery, chemotherapy or a combination of both) are generally invasive and frequently associated with severe side effects and high comorbidity. Consequently, novel approaches are strongly required to find more natural-like experimental models that better reflect the tumors' etiology, physiopathology and response to treatments, as well as to find more targeted, efficient and minimally invasive treatments. This study proposes the development and an in deep biological characterization of an experimental model using DMBA-tumor-induction in Sprague-Dawley female rats. Moreover, a photothermal therapy approach using a near-infrared laser coupled with gold nanoparticles was preliminarily assessed. The gold nanoparticles were functionalized with Epidermal Growth Factor, and their physicochemical properties and in vitro effects were characterized. DMBA proved to be a very good and selective inductor of breast cancer, with 100% incidence and inducing an average of 4.7 tumors per animal. Epigenetic analysis showed that tumors classified with worst prognosis were hypomethylated. The tumor-induced rats were then subjected to a preliminary treatment using functionalized gold nanoparticles and its activation by laser (650-900 nm). The treatment outcomes presented very promising alterations in terms of tumor histology, confirming the presence of necrosis in most of the cases. Although this study revealed encouraging results as a breast cancer therapy, it is important to define tumor eligibility and specific efficiency criteria to further assess its application in breast cancer treatment on other species.


Assuntos
5-Metilcitosina/metabolismo , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Hipertermia Induzida , Neoplasias Mamárias Experimentais/terapia , Nanopartículas Metálicas/administração & dosagem , Modelos Teóricos , Animais , Peso Corporal , Feminino , Ouro/química , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/patologia , Nanopartículas Metálicas/química , Ratos , Ratos Sprague-Dawley
17.
Nat Commun ; 11(1): 4956, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009383

RESUMO

Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), show decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation is found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment.


Assuntos
5-Metilcitosina/análogos & derivados , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA/metabolismo , 5-Metilcitosina/metabolismo , Animais , Especificidade de Anticorpos/imunologia , Sequência de Bases , Corpos Embrioides/metabolismo , Camundongos , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismo , Ligação Proteica , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genética
18.
Nat Commun ; 11(1): 5270, 2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-33077732

RESUMO

Pancreatic cancer is often detected late, when curative therapies are no longer possible. Here, we present non-invasive detection of pancreatic ductal adenocarcinoma (PDAC) by 5-hydroxymethylcytosine (5hmC) changes in circulating cell free DNA from a PDAC cohort (n = 64) in comparison with a non-cancer cohort (n = 243). Differential hydroxymethylation is found in thousands of genes, most significantly in genes related to pancreas development or function (GATA4, GATA6, PROX1, ONECUT1, MEIS2), and cancer pathogenesis (YAP1, TEAD1, PROX1, IGF1). cfDNA hydroxymethylome in PDAC cohort is differentially enriched for genes that are commonly de-regulated in PDAC tumors upon activation of KRAS and inactivation of TP53. Regularized regression models built using 5hmC densities in genes perform with AUC of 0.92 (discovery dataset, n = 79) and 0.92-0.94 (two independent test sets, n = 228). Furthermore, tissue-derived 5hmC features can be used to classify PDAC cfDNA (AUC = 0.88). These findings suggest that 5hmC changes enable classification of PDAC even during early stage disease.


Assuntos
5-Metilcitosina/análogos & derivados , Ácidos Nucleicos Livres/metabolismo , Neoplasias Pancreáticas/genética , 5-Metilcitosina/metabolismo , Adulto , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Estudos de Coortes , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
19.
BMC Bioinformatics ; 21(1): 489, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-33126851

RESUMO

BACKGROUND: As one of the most common post-transcriptional modifications (PTCM) in RNA, 5-cytosine-methylation plays important roles in many biological functions such as RNA metabolism and cell fate decision. Through accurate identification of 5-methylcytosine (m5C) sites on RNA, researchers can better understand the exact role of 5-cytosine-methylation in these biological functions. In recent years, computational methods of predicting m5C sites have attracted lots of interests because of its efficiency and low-cost. However, both the accuracy and efficiency of these methods are not satisfactory yet and need further improvement. RESULTS: In this work, we have developed a new computational method, m5CPred-SVM, to identify m5C sites in three species, H. sapiens, M. musculus and A. thaliana. To build this model, we first collected benchmark datasets following three recently published methods. Then, six types of sequence-based features were generated based on RNA segments and the sequential forward feature selection strategy was used to obtain the optimal feature subset. After that, the performance of models based on different learning algorithms were compared, and the model based on the support vector machine provided the highest prediction accuracy. Finally, our proposed method, m5CPred-SVM was compared with several existing methods, and the result showed that m5CPred-SVM offered substantially higher prediction accuracy than previously published methods. It is expected that our method, m5CPred-SVM, can become a useful tool for accurate identification of m5C sites. CONCLUSION: In this study, by introducing position-specific propensity related features, we built a new model, m5CPred-SVM, to predict RNA m5C sites of three different species. The result shows that our model outperformed the existing state-of-art models. Our model is available for users through a web server at https://zhulab.ahu.edu.cn/m5CPred-SVM .


Assuntos
5-Metilcitosina/metabolismo , RNA/genética , Máquina de Vetores de Suporte , Animais , Arabidopsis/genética , Sequência de Bases , Humanos , Internet , Camundongos , Curva ROC
20.
Mol Immunol ; 126: 136-142, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32823238

RESUMO

Interleukin (IL)-1ß produced by macrophages plays an important role in inflammation development. However, the underlying mechanism in epigenetic regulation of IL-1ß production is not fully addressed. Though DNA methylcytosine dioxygenase ten-eleven translocation 2 (TET2) is known to be involved in the regulation of inflammatory factors by oxidizing 5-methylcytosine (5mC), the underlying molecular mechanism is largely unknown. In this study, we found that the expression of both IL-1ß and TET2 is upregulated by lipopolysaccharide (LPS)-stimulated mononuclear macrophage. We then knocked down TET2 in mouse macrophagelike cell line (J774.1) and found that LPS-induced IL-1ß is also downregulated. In addition, LPS-stimulated phosphorylation of the mitogen-activated protein kinase (MAPK) signaling pathway and intracellular effectors of the toll-like receptor 4 (TLR4) signaling pathway were also suppressed in TET2-knockdown cells. The methylation status in the promoter regions of myeloid differentiation primary response gene (MyD)88 and TAK1 binding protein 2 (TAB2) were estimated by bisulfite polymerase chain reaction. Compared with that of the control, the 5mC level on the TAB2 promoter is downregulated in the LPS-stimulated cells which can be reversed by TET2-knockdown. These findings altogether suggest that LPS-upregulated TET2 enhances IL-1ß expression through demethylating the promoter region of TAB2, the key member of the TLR4/MAPK signaling pathway, a previously unreported molecular mechanism in TET2-regulated expression of inflammatory factors.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética/imunologia , Interleucina-1beta/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/metabolismo , Animais , Linhagem Celular , Desmetilação do DNA , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Lipopolissacarídeos/imunologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos , Camundongos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/metabolismo , Receptor 4 Toll-Like/metabolismo , Regulação para Cima/imunologia
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