RESUMO
TET proteins oxidize 5-methylcytosine in DNA to 5-hydroxymethylcytosine and other oxidation products. We found that simultaneous deletion of Tet2 and Tet3 in mouse CD4+CD8+ double-positive thymocytes resulted in dysregulated development and proliferation of invariant natural killer T cells (iNKT cells). Tet2-Tet3 double-knockout (DKO) iNKT cells displayed pronounced skewing toward the NKT17 lineage, with increased DNA methylation and impaired expression of genes encoding the key lineage-specifying factors T-bet and ThPOK. Transfer of purified Tet2-Tet3 DKO iNKT cells into immunocompetent recipient mice resulted in an uncontrolled expansion that was dependent on the nonclassical major histocompatibility complex (MHC) protein CD1d, which presents lipid antigens to iNKT cells. Our data indicate that TET proteins regulate iNKT cell fate by ensuring their proper development and maturation and by suppressing aberrant proliferation mediated by the T cell antigen receptor (TCR).
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Células T Matadoras Naturais/fisiologia , Células Precursoras de Linfócitos T/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Animais , Antígenos CD1d/genética , Antígenos CD1d/metabolismo , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Dioxigenases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
TET proteins are DNA demethylases that can oxidize 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC) and other oxidized mC bases (oxi-mCs). Importantly, TET proteins govern cell fate decisions during development of various cell types by activating a cell-specific gene expression program. In this review, we focus on the role of TET proteins in T-cell lineage specification. We explore the multifaceted roles of TET proteins in regulating gene expression in the contexts of T-cell development, lineage specification, function, and disease. Finally, we discuss the future directions and experimental strategies required to decipher the precise mechanisms employed by TET proteins to fine-tune gene expression and safeguard cell identity.
Assuntos
Metilação de DNA , Linfócitos T , 5-Metilcitosina , Diferenciação Celular , Linhagem da Célula , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Linfócitos T/metabolismoRESUMO
Cancer genomes are characterized by focal increases in DNA methylation, co-occurring with widespread hypomethylation. Here, we show that TET loss of function results in a similar genomic footprint. Both 5hmC in wild-type (WT) genomes and DNA hypermethylation in TET-deficient genomes are largely confined to the active euchromatic compartment, consistent with the known functions of TET proteins in DNA demethylation and the known distribution of 5hmC at transcribed genes and active enhancers. In contrast, an unexpected DNA hypomethylation noted in multiple TET-deficient genomes is primarily observed in the heterochromatin compartment. In a mouse model of T cell lymphoma driven by TET deficiency (Tet2/3 DKO T cells), genomic analysis of malignant T cells revealed DNA hypomethylation in the heterochromatic genomic compartment, as well as reactivation of repeat elements and enrichment for single-nucleotide alterations, primarily in heterochromatic regions of the genome. Moreover, hematopoietic stem/precursor cells (HSPCs) doubly deficient for Tet2 and Dnmt3a displayed greater losses of DNA methylation than HSPCs singly deficient for Tet2 or Dnmt3a alone, potentially explaining the unexpected synergy between DNMT3A and TET2 mutations in myeloid and lymphoid malignancies. Tet1-deficient cells showed decreased localization of DNMT3A in the heterochromatin compartment compared with WT cells, pointing to a functional interaction between TET and DNMT proteins and providing a potential explanation for the hypomethylation observed in TET-deficient genomes. Our data suggest that TET loss of function may at least partially underlie the characteristic pattern of global hypomethylation coupled to regional hypermethylation observed in diverse cancer genomes, and highlight the potential contribution of heterochromatin hypomethylation to oncogenesis.
Assuntos
Metilação de DNA , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/deficiência , Células-Tronco Hematopoéticas/metabolismo , Linfoma de Células T/metabolismo , Neoplasias Experimentais/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas/deficiência , Animais , DNA (Citosina-5-)-Metiltransferases/deficiência , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Estudo de Associação Genômica Ampla , Células-Tronco Hematopoéticas/patologia , Heterocromatina/genética , Heterocromatina/metabolismo , Heterocromatina/patologia , Humanos , Linfoma de Células T/genética , Linfoma de Células T/patologia , Camundongos , Camundongos Knockout , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Dioxygenases of the TET (Ten-Eleven Translocation) family produce oxidized methylcytosines, intermediates in DNA demethylation, as well as new epigenetic marks. Here we show data suggesting that TET proteins maintain the consistency of gene transcription. Embryos lacking Tet1 and Tet3 (Tet1/3 DKO) displayed a strong loss of 5-hydroxymethylcytosine (5hmC) and a concurrent increase in 5-methylcytosine (5mC) at the eight-cell stage. Single cells from eight-cell embryos and individual embryonic day 3.5 blastocysts showed unexpectedly variable gene expression compared with controls, and this variability correlated in blastocysts with variably increased 5mC/5hmC in gene bodies and repetitive elements. Despite the variability, genes encoding regulators of cholesterol biosynthesis were reproducibly down-regulated in Tet1/3 DKO blastocysts, resulting in a characteristic phenotype of holoprosencephaly in the few embryos that survived to later stages. Thus, TET enzymes and DNA cytosine modifications could directly or indirectly modulate transcriptional noise, resulting in the selective susceptibility of certain intracellular pathways to regulation by TET proteins.
Assuntos
5-Metilcitosina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Proto-Oncogênicas/metabolismo , Transcriptoma/genética , Animais , Biomarcadores/metabolismo , Blastocisto/metabolismo , Blastômeros/metabolismo , Blastômeros/patologia , Linhagem da Célula , Colesterol/biossíntese , DNA/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Dioxigenases , Regulação para Baixo/genética , Perda do Embrião/metabolismo , Perda do Embrião/patologia , Embrião de Mamíferos/patologia , Impressão Genômica , Proteínas Hedgehog/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de RNA , Transdução de Sinais/genéticaRESUMO
The discovery of Ten Eleven Translocation proteins, enzymes that oxidize 5-methylcytosine (5mC) in DNA, has revealed novel mechanisms for the regulation of DNA methylation. We have mapped 5-hydroxymethylcytosine (5hmC) at different stages of T-cell development in the thymus and T-cell differentiation in the periphery. We show that 5hmC is enriched in the gene body of highly expressed genes at all developmental stages and that its presence correlates positively with gene expression. Further emphasizing the connection with gene expression, we find that 5hmC is enriched in active thymus-specific enhancers and that genes encoding key transcriptional regulators display high intragenic 5hmC levels in precursor cells at those developmental stages where they exert a positive effect. Our data constitute a valuable resource that will facilitate detailed analysis of the role of 5hmC in T-cell development and differentiation.
Assuntos
Citosina/análogos & derivados , Linfócitos T/citologia , Linfócitos T/metabolismo , 5-Metilcitosina/análogos & derivados , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem da Célula/genética , Linhagem da Célula/fisiologia , Citosina/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismoRESUMO
DNA demethylases TET2 and TET3 play a fundamental role in thymic invariant natural killer T (iNKT) cell differentiation by mediating DNA demethylation of genes encoding for lineage specifying factors. Paradoxically, differential gene expression analysis revealed that significant number of genes were upregulated upon TET2 and TET3 loss in iNKT cells. This unexpected finding could be potentially explained if loss of TET proteins was reducing the expression of proteins that suppress gene expression. In this study, we discover that TET2 and TET3 synergistically regulate Drosha expression, by generating 5hmC across the gene body and by impacting chromatin accessibility. As DROSHA is involved in microRNA biogenesis, we proceed to investigate the impact of TET2/3 loss on microRNAs in iNKT cells. We report that among the downregulated microRNAs are members of the Let-7 family that downregulate in vivo the expression of the iNKT cell lineage specifying factor PLZF. Our data link TET proteins with microRNA expression and reveal an additional layer of TET mediated regulation of gene expression.
RESUMO
Ten-eleven translocation (TET) proteins are DNA dioxygenases that mediate active DNA demethylation. TET3 is the most highly expressed TET protein in thymic developing T cells. TET3, either independently or in cooperation with TET1 or TET2, has been implicated in T cell lineage specification by regulating DNA demethylation. However, TET-deficient mice exhibit complex phenotypes, suggesting that TET3 exerts multifaceted roles, potentially by interacting with other proteins. We performed liquid chromatography with tandem mass spectrometry in primary developing T cells to identify TET3 interacting partners in endogenous, in vivo conditions. We discover TET3 interacting partners. Our data establish that TET3 participates in a plethora of fundamental biological processes, such as transcriptional regulation, RNA polymerase elongation, splicing, DNA repair, and DNA replication. This resource brings in the spotlight emerging functions of TET3 and sets the stage for systematic studies to dissect the precise mechanistic contributions of TET3 in shaping T cell biology.
RESUMO
Ten-eleven translocation (TET) proteins are dioxygenases that oxidize 5-methylcytosine to form 5-hydroxymethylcytosine and downstream oxidized modified cytosines. In the past decade, intensive research established that TET-mediated DNA demethylation is critical for immune cell development and function. In this study, we discuss major advances regarding the role of TET proteins in regulating gene expression in the context of T cell lineage specification, function, and proliferation. Then, we focus on open questions in the field. We discuss recent findings regarding the diverse roles of TET proteins in other systems, and we ask how these findings might relate to T cell biology. Finally, we ask how this tremendous progress on understanding the multifaceted roles of TET proteins in shaping T cell identity and function can be translated to improve outcomes of human disease, such as hematological malignancies and immune response to cancer.
Assuntos
Epigênese Genética , Neoplasias , Humanos , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Linfócitos T/metabolismo , Neoplasias/genética , Neoplasias/metabolismoRESUMO
This article discusses methods to assess invariant natural killer T (iNKT) cell subsets isolated from the thymus, as well as the spleen, the liver, and the lung. iNKT cells can be subdivided in distinct, functional subsets based on the transcription factors they express and the cytokines they produce to regulate the immune response. Basic Protocol 1 focuses on characterizing murine iNKT subsets ex vivo by flow cytometry by evaluating the expression of lineage-specifying transcription factors such as PLZF and RORγt. The Alternate Protocol describes a detailed approach to define subsets based on expression of surface markers. This approach can be very useful for maintaining the subsets alive, without fixing them, in order to isolate them for downstream molecular assays such as DNA/RNA isolation, genome-wide analysis to assess gene expression (such as RNA-seq), assessment of chromatin accessibility (for instance, by ATAC-seq), and assessment of DNA methylation by whole-genome bisulfite sequencing. Basic Protocol 2 describes the functional characterization of iNKT cells, which are activated in vitro with PMA and ionomycin for a short period of time and subsequently stained and characterized for production of cytokines, such as IFNγ and IL-4, by flow cytometry. Basic Protocol 3 describes the process of activating iNKT cells in vivo using α-galactosyl-ceramide, a lipid that can be recognized specifically by iNKT cells, allowing assessment of their functionality in vivo. Cells are then isolated and directly stained for cytokine secretion. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Identifying iNKT cell subsets based on transcription factor expression by flow cytometry Alternate Protocol: Identifying iNKT cell subsets based on surface marker expression by flow cytometry Basic Protocol 2: iNKT cell functional characterization based on in vitro activation and assessment of cytokine secretion Basic Protocol 3: iNKT cell in vivo activation and assessment of cytokine secretion by flow cytometry.
Assuntos
Células T Matadoras Naturais , Animais , Camundongos , Citometria de Fluxo/métodos , Células T Matadoras Naturais/metabolismo , Citocinas/metabolismo , Expressão Gênica , Fatores de Transcrição/metabolismoRESUMO
The cylindromatosis tumor suppressor gene (Cyld) encodes an enzyme (CYLD) with deubiquitinating activity that has been implicated in the regulation of thymocyte selection in an NF-κB-essential-modulator (NEMO)-dependent manner. The main known molecular defects in thymocytes with inactive CYLD (LckCre-Cyld(flx9/flx9) ) are the aberrant hyperactivation of NF-κB and JNK pathways. In order to dissect further the molecular mechanism of CYLD-dependent thymocyte selection and address the role of NF-κB specifically, we generated double mutant mice (LckCre-Cyld(flx9/flx9) -Ikk2(flx/flx) ) in which CYLD was inactivated concomitantly with IKK2 (IκB-kinase 2) in thymocytes. Interestingly, thymic development and NF-κB activity in double mutant mice were fully restored, indicating that an IKK2-dependent function of CYLD that leads to the hyperactivation of the NF-κB pathway is primarily responsible for the defective selection of thymocytes. Intriguingly, we observed a greater reduction of CD4(+) and CD8(+) T cells in the periphery of LckCre-Cyld(flx9/flx9) -Ikk2(flx/flx) mice compared with LckCre-Ikk2(flx/flx) mice. Collectively, our data establish CYLD as a critical regulator of thymocyte selection in a manner that depends on IKK2 and NF-κB activation. In addition, our data uncover an IKK2-independent role for CYLD in the establishment of physiological T-cell populations in the periphery.
Assuntos
Cisteína Endopeptidases/metabolismo , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Cisteína Endopeptidases/deficiência , Cisteína Endopeptidases/genética , Enzima Desubiquitinante CYLD , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Quinase I-kappa B/deficiência , Quinase I-kappa B/genética , Immunoblotting , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Timo/imunologiaRESUMO
The cylindromatosis tumor suppressor gene (Cyld) encodes a deubiquitinating enzyme (CYLD) with immunoregulatory function. In this study, we evaluated the role of Cyld in T cell ontogeny by generating a mouse (Cyld(Delta9)) with a thymocyte-restricted Cyld mutation that causes a C-terminal truncation of the protein and reciprocates catalytically inactive human mutations. Mutant mice had dramatically reduced single positive thymocytes and a substantial loss of peripheral T cells. The analyses of polyclonal and TCR-restricted thymocyte populations possessing the mutation revealed a significant block in positive selection and an increased occurrence of apoptosis at the double-positive stage. Interestingly, in the context of MHC class I and II restricted TCR transgenes, lack of functional CYLD caused massive deletion of thymocytes that would have been positively selected, which is consistent with an impairment of positive selection. Biochemical analysis revealed that Cyld(Delta9) thymocytes exhibit abnormally elevated basal activity of NF-kappaB and JNK. Most importantly, inactivation of NF-kappaB essential modulator fully restored the NF-kappaB activity of Cyld(Delta9) thymocytes to physiologic levels and rescued their developmental and survival defect. This study identifies a fundamental role for functional CYLD in establishing the proper threshold of activation for thymocyte selection by a mechanism dependent on NF-kappaB essential modulator.
Assuntos
Cisteína Endopeptidases/metabolismo , NF-kappa B/metabolismo , Timo/imunologia , Timo/metabolismo , Animais , Sítios de Ligação/genética , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Cisteína Endopeptidases/genética , Enzima Desubiquitinante CYLD , Feminino , Citometria de Fluxo , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Timo/citologia , UbiquitinaçãoRESUMO
Here, we describe steps to isolate mature thymic T cell subsets, namely CD4 single positive (SP), CD8 SP, and invariant natural killer T (iNKT) cells starting from murine total thymocytes using fluorescence-activated cell sorting. We detail protocols to study gene expression by RNA-seq and assess binding of transcription factors across the genome using CUT&RUN. This approach deciphers the molecular principles that govern T cell lineage specification and function. This protocol works well with limited starting material. For complete details on the use and execution of this protocol, please refer to Äijö et al. (2022).1.
Assuntos
Subpopulações de Linfócitos T , Fatores de Transcrição , Camundongos , Animais , Citometria de Fluxo , RNA-Seq , Fatores de Transcrição/genética , Expressão GênicaRESUMO
TET proteins mediate DNA demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and other oxidative derivatives. We have previously demonstrated a dynamic enrichment of 5hmC during T and invariant natural killer T cell lineage specification. Here, we investigate shared signatures in gene expression of Tet2/3 DKO CD4 single positive (SP) and iNKT cells in the thymus. We discover that TET proteins exert a fundamental role in regulating the expression of the lineage specifying factor Th-POK, which is encoded by Zbtb7b. We demonstrate that TET proteins mediate DNA demethylation - surrounding a proximal enhancer, critical for the intensity of Th-POK expression. In addition, TET proteins drive the DNA demethylation of site A at the Zbtb7b locus to facilitate GATA3 binding. GATA3 induces Th-POK expression in CD4 SP cells. Finally, by introducing a novel mouse model that lacks TET3 and expresses full length, catalytically inactive TET2, we establish a causal link between TET2 catalytic activity and lineage specification of both conventional and unconventional T cells.
Assuntos
Dioxigenases , Células T Matadoras Naturais , Animais , Linhagem da Célula , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/genética , Camundongos , Células T Matadoras Naturais/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
TET proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation products in DNA. The oxidized methylcytosines (oxi-mCs) facilitate DNA demethylation and are also novel epigenetic marks. TET loss-of-function is strongly associated with cancer; TET2 loss-of-function mutations are frequently observed in hematological malignancies that are resistant to conventional therapies. Importantly, TET proteins govern cell fate decisions during development of various cell types by activating a cell-specific gene expression program. In this review, we seek to provide a conceptual framework of the mechanisms that fine tune TET activity. Then, we specifically focus on the multifaceted roles of TET proteins in regulating gene expression in immune cell development, function, and disease.
RESUMO
Invariant natural killer T cells (iNKTs) are distinct from conventional T cells. iNKT cells express a semi-invariant T cell receptor (TCR) that can specifically recognize lipid antigens presented by CD1d, an MHC class I-like antigen-presenting molecule. Currently, iNKT cells are distinguished in three functionally distinct subsets. Each subset is defined by lineage-specifying factors: T-bet shapes the fate of NKT1 subset that mainly secretes IFNγ, Gata3 specifies the NKT2 subset that produces robustly IL-4 whereas RORγt seals the differentiation of NKT17 subset that secretes IL-17. In the present review, the focus is placed on the regulation of NKT17 specification and their function.
Assuntos
Antígenos CD1d/imunologia , Diferenciação Celular/imunologia , Interleucina-17/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Fator de Transcrição GATA3/imunologia , Humanos , Interferon gama/imunologia , Interleucina-4/imunologia , Células T Matadoras Naturais/citologiaRESUMO
During the last years, intensive research has shed light in the transcriptional networks that shape the invariant NKT (iNKT) cell lineage and guide the choices towards functionally distinct iNKT cell subsets (Constantinides and Bendelac, 2013; Engel and Kronenberg, 2014; Gapin, 2016; Kim et al., 2015). However, the epigenetic players that regulate gene expression and orchestrate the iNKT cell lineage choices remain poorly understood. Here, we summarize recent advances in our understanding of epigenetic regulation of iNKT cell development and lineage choice. Particular emphasis is placed on DNA modifications and the Ten Eleven Translocation (TET) family of DNA demethylases.
Assuntos
Linhagem da Célula , Epigênese Genética , Células T Matadoras Naturais/citologia , Proteínas Proto-Oncogênicas/metabolismo , Animais , Metilação de DNA/genética , Reparo do DNA , HumanosRESUMO
DNA methylation is established by DNA methyltransferases and is a key epigenetic mark. Ten-eleven translocation (TET) proteins are enzymes that oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidization products (oxi-mCs), which indirectly promote DNA demethylation. Here, we provide an overview of the effect of TET proteins and altered DNA modification status in T and B cell development and function. We summarize current advances in our understanding of the role of TET proteins and 5hmC in T and B cells in both physiological and pathological contexts. We describe how TET proteins and 5hmC regulate DNA modification, chromatin accessibility, gene expression, and transcriptional networks and discuss potential underlying mechanisms and open questions in the field.
RESUMO
We present a generative model, Lux, to quantify DNA methylation modifications from any combination of bisulfite sequencing approaches, including reduced, oxidative, TET-assisted, chemical-modification assisted, and methylase-assisted bisulfite sequencing data. Lux models all cytosine modifications (C, 5mC, 5hmC, 5fC, and 5caC) simultaneously together with experimental parameters, including bisulfite conversion and oxidation efficiencies, as well as various chemical labeling and protection steps. We show that Lux improves the quantification and comparison of cytosine modification levels and that Lux can process any oxidized methylcytosine sequencing data sets to quantify all cytosine modifications. Analysis of targeted data from Tet2-knockdown embryonic stem cells and T cells during development demonstrates DNA modification quantification at unprecedented detail, quantifies active demethylation pathways and reveals 5hmC localization in putative regulatory regions.
Assuntos
5-Metilcitosina/metabolismo , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Teorema de Bayes , Citosina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Humanos , Oxirredução , Análise de Sequência de DNA/métodosRESUMO
Ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine and other oxidized methylcytosines, intermediates in DNA demethylation. In this study, we examine the role of TET proteins in regulating Foxp3, a transcription factor essential for the development and function of regulatory T cells (T reg cells), a distinct lineage of CD4(+) T cells that prevent autoimmunity and maintain immune homeostasis. We show that during T reg cell development in the thymus, TET proteins mediate the loss of 5mC in T reg cell-specific hypomethylated regions, including CNS1 and CNS2, intronic cis-regulatory elements in the Foxp3 locus. Similar to CNS2-deficient T reg cells, the stability of Foxp3 expression is markedly compromised in T reg cells from Tet2/Tet3 double-deficient mice. Vitamin C potentiates TET activity and acts through Tet2/Tet3 to increase the stability of Foxp3 expression in TGF-ß-induced T reg cells. Our data suggest that targeting TET enzymes with small molecule activators such as vitamin C might increase induced T reg cell efficacy.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/metabolismo , Animais , Ácido Ascórbico/farmacologia , Diferenciação Celular/efeitos dos fármacos , Citosina/metabolismo , Metilação de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/deficiência , Dioxigenases , Células HEK293 , Humanos , Masculino , Camundongos , Estabilidade Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas/deficiência , Linfócitos T Reguladores/imunologiaRESUMO
Jarid2 is a reported component of three lysine methyltransferase complexes, polycomb repressive complex 2 (PRC2) that methylates histone 3 lysine 27 (H3K27), and GLP-G9a and SETDB1 complexes that methylate H3K9. Here we show that Jarid2 is upregulated upon TCR stimulation and during positive selection in the thymus. Mice lacking Jarid2 in T cells display an increase in the frequency of IL-4-producing promyelocytic leukemia zinc finger (PLZF)(hi) immature invariant natural killer T (iNKT) cells and innate-like CD8(+) cells; Itk-deficient mice, which have a similar increase of innate-like CD8(+) cells, show blunted upregulation of Jarid2 during positive selection. Jarid2 binds to the Zbtb16 locus, which encodes PLZF, and thymocytes lacking Jarid2 show increased PLZF and decreased H3K9me3 levels. Jarid2-deficient iNKT cells perturb Th17 differentiation, leading to reduced Th17-driven autoimmune pathology. Our results establish Jarid2 as a novel player in iNKT cell maturation that regulates PLZF expression by modulating H3K9 methylation.