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1.
Cell Rep ; 34(8): 108776, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33626359

RESUMO

Estrogen receptor-α (ER) drives tumor development in ER-positive (ER+) breast cancer. The transcription factor GATA3 has been closely linked to ER function, but its precise role in this setting remains unclear. Quantitative proteomics was used to assess changes to the ER complex in response to GATA3 depletion. Unexpectedly, few proteins were lost from the ER complex in the absence of GATA3, with the only major change being depletion of the dioxygenase TET2. TET2 binding constituted a near-total subset of ER binding in multiple breast cancer models, with loss of TET2 associated with reduced activation of proliferative pathways. TET2 knockdown did not appear to change global methylated cytosine (5mC) levels; however, oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) was significantly reduced, and these events occurred at ER enhancers. These findings implicate TET2 in the maintenance of 5hmC at ER sites, providing a potential mechanism for TET2-mediated regulation of ER target genes.


Assuntos
5-Metilcitosina/análogos & derivados , Neoplasias da Mama/enzimologia , Montagem e Desmontagem da Cromatina , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Elementos Facilitadores Genéticos , Receptor alfa de Estrogênio/metabolismo , 5-Metilcitosina/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/genética , Bases de Dados Genéticas , Dioxigenases/genética , Antagonistas do Receptor de Estrogênio/farmacologia , Receptor alfa de Estrogênio/genética , Feminino , Fulvestranto/farmacologia , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos Endogâmicos NOD , Camundongos SCID , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Biochemistry ; 59(27): 2541-2550, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32543182

RESUMO

Cytosine methylation is an important epigenetic mark, but how the distinctive patterns of DNA methylation arise remains elusive. For the first time, we systematically investigated how these patterns can be imparted by the inherent enzymatic preferences of mammalian de novo DNA methyltransferases in vitro and the extent to which this applies in cells. In a biochemical experiment, we subjected a wide variety of DNA sequences to methylation by DNMT3A or DNMT3B and then applied deep bisulfite sequencing to quantitatively determine the sequence preferences for methylation. The data show that DNMT3A prefers CpG and non-CpG sites followed by a 3'-pyrimidine, whereas DNMT3B favors a 3'-purine. Overall, we show that DNMT3A has a sequence preference for a TNC[G/A]CC context, while DNMT3B prefers TAC[G/A]GC. We extended our finding using publicly available data from mouse Dnmt1/3a/3b triple-knockout cells in which reintroduction of either DNMT3A or DNMT3B expression results in the acquisition of the same enzyme specific signature sequences observed in vitro. Furthermore, loss of DNMT3A or DNMT3B in human embryonic stem cells leads to a loss of methylation at the corresponding enzyme specific signatures. Therefore, the global DNA methylation landscape of the mammalian genome can be fundamentally determined by the inherent sequence preference of de novo methyltransferases.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Células-Tronco Embrionárias/fisiologia , Animais , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Genoma , Humanos , Motivos de Nucleotídeos , Especificidade por Substrato , DNA Metiltransferase 3B
3.
Nat Struct Mol Biol ; 25(10): 951-957, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30275516

RESUMO

Control of DNA methylation level is critical for gene regulation, and the factors that govern hypomethylation at CpG islands (CGIs) are still being uncovered. Here, we provide evidence that G-quadruplex (G4) DNA secondary structures are genomic features that influence methylation at CGIs. We show that the presence of G4 structure is tightly associated with CGI hypomethylation in the human genome. Surprisingly, we find that these G4 sites are enriched for DNA methyltransferase 1 (DNMT1) occupancy, which is consistent with our biophysical observations that DNMT1 exhibits higher binding affinity for G4s as compared to duplex, hemi-methylated, or single-stranded DNA. The biochemical assays also show that the G4 structure itself, rather than sequence, inhibits DNMT1 enzymatic activity. Based on these data, we propose that G4 formation sequesters DNMT1 thereby protecting certain CGIs from methylation and inhibiting local methylation.


Assuntos
Metilação de DNA , Quadruplex G , Ilhas de CpG , DNA/metabolismo , Epigenômica , Regulação da Expressão Gênica , Genoma Humano , Humanos , Células K562 , Método de Monte Carlo , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas
4.
Stem Cell Res ; 15(2): 435-43, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26397890

RESUMO

TET proteins have been found to play an important role in active demethylation at CpG sites in mammals. There are some reports implicating their functions in removal of DNA methylation imprint at the imprinted regions in the germline. However, it is not well established whether TET proteins can also be involved in demethylation of DNA methylation imprint in embryonic stem (ES) cells. Here we report that loss of TET proteins caused a significant increase in DNA methylation at the Igf2-H19 imprinted region in ES cells. We also observed a variable increase in DNA methylation at the Peg1 imprinted region in the ES clones devoid of TET proteins, in particular in the differentiated ES cells. By contrast, we did not observe a significant increase of DNA methylation imprint at the Peg3, Snrpn and Dlk1-Dio3 imprinted regions in ES cells lacking TET proteins. Interestingly, loss of TET proteins did not result in a significant increase of DNA methylation imprint at the Igf2-H19 and Peg1 imprinted regions in the embryoid bodies (EB). Therefore, TET proteins seem to be differentially involved in maintaining DNA methylation imprint at a subset of imprinted regions in ES cells and EBs.


Assuntos
Células-Tronco Embrionárias/metabolismo , Impressão Genômica , Animais , Proteínas de Ligação ao Cálcio , Ilhas de CpG , Metilação de DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Dioxigenases , Células-Tronco Embrionárias/citologia , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Iodeto Peroxidase/genética , Camundongos , Proteínas/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas Centrais de snRNP/genética
5.
Mol Cell ; 56(2): 286-297, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25263596

RESUMO

In mammals, cytosine methylation (5mC) is widely distributed throughout the genome but is notably depleted from active promoters and enhancers. While the role of DNA methylation in promoter silencing has been well documented, the function of this epigenetic mark at enhancers remains unclear. Recent experiments have demonstrated that enhancers are enriched for 5-hydroxymethylcytosine (5hmC), an oxidization product of the Tet family of 5mC dioxygenases and an intermediate of DNA demethylation. These results support the involvement of Tet proteins in the regulation of dynamic DNA methylation at enhancers. By mapping DNA methylation and hydroxymethylation at base resolution, we find that deletion of Tet2 causes extensive loss of 5hmC at enhancers, accompanied by enhancer hypermethylation, reduction of enhancer activity, and delayed gene induction in the early steps of differentiation. Our results reveal that DNA demethylation modulates enhancer activity, and its disruption influences the timing of transcriptome reprogramming during cellular differentiation.


Assuntos
Diferenciação Celular/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Citosina/análogos & derivados , Citosina/metabolismo , Proteínas de Ligação a DNA/genética , Dioxigenases , Camundongos , Camundongos Knockout , Oxirredução , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Análise de Sequência de DNA , Transcriptoma/genética , Dedos de Zinco/genética
6.
Cell Stem Cell ; 14(4): 512-22, 2014 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-24529596

RESUMO

Tet-mediated DNA oxidation is a recently identified mammalian epigenetic modification, and its functional role in cell-fate transitions remains poorly understood. Here, we derive mouse embryonic fibroblasts (MEFs) deleted in all three Tet genes and examine their capacity for reprogramming into induced pluripotent stem cells (iPSCs). We show that Tet-deficient MEFs cannot be reprogrammed because of a block in the mesenchymal-to-epithelial transition (MET) step. Reprogramming of MEFs deficient in TDG is similarly impaired. The block in reprogramming is caused at least in part by defective activation of key miRNAs, which depends on oxidative demethylation promoted by Tet and TDG. Reintroduction of either the affected miRNAs or catalytically active Tet and TDG restores reprogramming in the knockout MEFs. Thus, oxidative demethylation to promote gene activation appears to be functionally required for reprogramming of fibroblasts to pluripotency. These findings provide mechanistic insight into the role of epigenetic barriers in cell-lineage conversion.


Assuntos
Reprogramação Celular , DNA Glicosilases/fisiologia , Metilação de DNA , Proteínas de Ligação a DNA/fisiologia , Células-Tronco Embrionárias/citologia , Transição Epitelial-Mesenquimal , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Western Blotting , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Dioxigenases , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica , Técnicas Imunoenzimáticas , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs/fisiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
Nat Genet ; 45(12): 1504-9, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24162740

RESUMO

Vitamin C, a micronutrient known for its anti-scurvy activity in humans, promotes the generation of induced pluripotent stem cells (iPSCs) through the activity of histone demethylating dioxygenases. TET hydroxylases are also dioxygenases implicated in active DNA demethylation. Here we report that TET1 either positively or negatively regulates somatic cell reprogramming depending on the absence or presence of vitamin C. TET1 deficiency enhances reprogramming, and its overexpression impairs reprogramming in the context of vitamin C by modulating the obligatory mesenchymal-to-epithelial transition (MET). In the absence of vitamin C, TET1 promotes somatic cell reprogramming independent of MET. Consistently, TET1 regulates 5-hydroxymethylcytosine (5hmC) formation at loci critical for MET in a vitamin C-dependent fashion. Our findings suggest that vitamin C has a vital role in determining the biological outcome of TET1 function at the cellular level. Given its benefit to human health, vitamin C should be investigated further for its role in epigenetic regulation.


Assuntos
Ácido Ascórbico/farmacologia , Reprogramação Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Células Cultivadas , Embrião de Mamíferos , Epigênese Genética/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout
8.
J Am Chem Soc ; 135(28): 10396-403, 2013 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-23768208

RESUMO

DNA hydroxymethylation and its mediated DNA demethylation are critical for multiple cellular processes, for example, nuclear reprogramming, embryonic development, and many diseases. Here, we demonstrate that a vital nutrient ascorbic acid (AA), or vitamin C (Vc), can directly enhance the catalytic activity of Tet dioxygenases for the oxidation of 5-methylcytosine (5mC). As evidenced by changes in intrinsic fluorescence and catalytic activity of Tet2 protein caused by AA and its oxidation-resistant derivatives, we further show that AA can uniquely interact with the C-terminal catalytic domain of Tet enzymes, which probably promotes their folding and/or recycling of the cofactor Fe(2+). Other strong reducing chemicals do not have a similar effect. These results suggest that AA also acts as a cofactor of Tet enzymes. In mouse embryonic stem cells, AA significantly increases the levels of all 5mC oxidation products, particularly 5-formylcytosine and 5-carboxylcytosine (by more than an order of magnitude), leading to a global loss of 5mC (∼40%). In cells deleted of the Tet1 and Tet2 genes, AA alters neither 5mC oxidation nor the overall level of 5mC. The AA effects are however restored when Tet2 is re-expressed in the Tet-deficient cells. The enhancing effects of AA on 5mC oxidation and DNA demethylation are also observed in a mouse model deficient in AA synthesis. Our data establish a direct link among AA, Tet, and DNA methylation, thus revealing a role of AA in the regulation of DNA modifications.


Assuntos
5-Metilcitosina/metabolismo , Ácido Ascórbico/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/química , Animais , Ácido Ascórbico/química , DNA/química , Metilação de DNA , Dioxigenases , Camundongos , Oxirredução
9.
Cell ; 153(3): 678-91, 2013 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-23602153

RESUMO

TET proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are excised by mammalian DNA glycosylase TDG, implicating 5mC oxidation in DNA demethylation. Here, we show that the genomic locations of 5fC can be determined by coupling chemical reduction with biotin tagging. Genome-wide mapping of 5fC in mouse embryonic stem cells (mESCs) reveals that 5fC preferentially occurs at poised enhancers among other gene regulatory elements. Application to Tdg null mESCs further suggests that 5fC production coordinates with p300 in remodeling epigenetic states of enhancers. This process, which is not influenced by 5hmC, appears to be associated with further oxidation of 5hmC and commitment to demethylation through 5fC. Finally, we resolved 5fC at base resolution by hydroxylamine-based protection from bisulfite-mediated deamination, thereby confirming sites of 5fC accumulation. Our results reveal roles of active 5mC/5hmC oxidation and TDG-mediated demethylation in epigenetic tuning at regulatory elements.


Assuntos
Citosina/análogos & derivados , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Técnicas Genéticas , Estudo de Associação Genômica Ampla , 5-Metilcitosina/metabolismo , Animais , Citosina/metabolismo , Camundongos , Elementos Reguladores de Transcrição , Fatores de Transcrição de p300-CBP/metabolismo
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