TET proteins regulate expression and impact microRNAs in iNKT cells.

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.

CAR-redirected natural killer T cells demonstrate superior antitumor activity to CAR-T cells through multimodal CD1d-dependent mechanisms.

Human natural killer T (NKT) cells have been proposed as a promising cell platform for chimeric antigen receptor (CAR) therapy in solid tumors. Here we generated murine CAR-NKT cells and compared them with CAR-T cells in immune-competent mice. Both CAR-NKT cells and CAR-T cells showed similar antitumor effects in vitro, but CAR-NKT cells showed superior antitumor activity in vivo via CD1d-dependent immune responses in the tumor microenvironment.

TET proteins regulate Drosha expression and impact microRNAs in iNKT cells.

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.

Deciphering the TET3 interactome in primary thymic developing T cells.

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.

TET Proteins in the Spotlight: Emerging Concepts of Epigenetic Regulation in T Cell Biology.

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.

Protocol to isolate mature thymic T cell subsets using fluorescence-activated cell sorting for assessing gene expression by RNA-seq and transcription factor binding across the genome by CUT&RUN.

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.

TET proteins regulate T cell and iNKT cell lineage specification in a TET2 catalytic dependent manner.

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 DKO CD4 single positive (SP) and iNKT cells in the thymus.

TET-Mediated Epigenetic Regulation in Immune Cell Development and Disease.

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; loss-of-function mutations are frequently observed in hematological malignancies that are resistant to conventional therapies.

Deciphering the multifaceted roles of TET proteins in T-cell lineage specification and malignant transformation.

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.

Paradoxical association of TET loss of function with genome-wide DNA hypomethylation.

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 -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.

Unveiling the regulation of NKT17 cell differentiation and function.

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.

TET mediated epigenetic regulation of iNKT cell lineage fate choice and function.

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.

TET Methylcytosine Oxidases in T Cell and B Cell Development and Function.

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.

TET proteins regulate the lineage specification and TCR-mediated expansion of iNKT cells.

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 CD4CD8 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.

A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways.

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.

Control of Foxp3 stability through modulation of TET activity.

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.

Simultaneous deletion of the methylcytosine oxidases Tet1 and Tet3 increases transcriptome variability in early embryogenesis.

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.

Jarid2 is induced by TCR signalling and controls iNKT cell maturation.

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.

Dissecting the dynamic changes of 5-hydroxymethylcytosine in T-cell development and differentiation.

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.

TET proteins and 5-methylcytosine oxidation in the immune system.

DNA methylation in the form of 5-methylcytosine (5mC) is essential for normal development in mammals and influences a variety of biological processes, including transcriptional regulation, imprinting, and the maintenance of genomic stability. The recent discovery of TET proteins, which oxidize 5mC to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine, has changed our understanding of the process of DNA demethylation. Here, we summarize our current knowledge of the roles of DNA methylation and TET proteins in cell differentiation and function.

Inactivation of the deubiquitinase CYLD in hepatocytes causes apoptosis, inflammation, fibrosis, and cancer.

The tumor suppressor cylindromatosis (CYLD) inhibits the NFκB and mitogen-activated protein kinase (MAPK) activation pathways by deubiquitinating upstream regulatory factors. Here we show that liver-specific disruption of CYLD triggers hepatocyte cell death in the periportal area via spontaneous and chronic activation of TGF-β activated kinase 1 (TAK1) and c-Jun N-terminal kinase (JNK). This is followed by hepatic stellate cell and Kupffer cell activation, which promotes progressive fibrosis, inflammation, tumor necrosis factor (TNF) production, and expansion of hepatocyte apoptosis toward the central veins.

Differential requirement of IKK2 for CYLD-dependent representation of thymic and peripheral T-cell populations.

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.

Truncation of the deubiquitinating domain of CYLD in myelomonocytic cells attenuates inflammatory responses.

The cylindromatosis tumor suppressor (CYLD) is a deubiquitinating enzyme that has been implicated in various aspects of adaptive and innate immune responses. Nevertheless, the role of CYLD in the function of specific types of immune cells remains elusive. In this report we have used conditional gene targeting in mice to address the role of the deubiquitinating activity of CYLD in the myelomonocytic lineage.