DNA cytosine methylation is crucial for retrotransposon silencing and mammalian development. In a computational search for enzymes that could modify 5-methylcytosine (5mC), we identified TET proteins as mammalian homologs of the trypanosome proteins JBP1 and JBP2, which have been proposed to oxidize the 5-methyl group of thymine. We show here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro. hmC is present in the genome of mouse embryonic stem cells, and hmC levels decrease upon RNA interference–mediated depletion of TET1. Thus, TET proteins have potential roles in epigenetic regulation through modification of 5mC to hmC.

TET2 is a close relative of TET1, an enzyme that converts 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) in DNA1,2. The gene encoding TET2 resides at chromosome 4q24, in a region showing recurrent microdeletions and copy-neutral loss of heterozygosity (CN-LOH) in patients with diverse myeloid malignancies3. Somatic TET2 mutations are frequently observed in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), MDS/MPN overlap syndromes including chronic myelomonocytic leukemia (CMML), acute myeloid leukemias (AML) and secondary AML (sAML)4–12. We show here that TET2 mutations associated with myeloid malignancies compromise TET2 catalytic activity. Bone marrow samples from patients with TET2 mutations displayed uniformly low levels of 5-hmC in genomic DNA compared to bone marrow samples from healthy controls. Moreover, small hairpin RNA (shRNA)-mediated depletion of Tet2 in mouse haematopoietic precursors skewed their differentiation towards monocyte/macrophage lineages in culture. There was no significant difference in DNA methylation between bone marrow samples from patients with high 5-hmC versus healthy controls, but samples from patients with low 5-hmC showed hypomethylation relative to controls at the majority of differentially-methylated CpG sites. Our results demonstrate that TET2 is important for normal myelopoiesis, and suggest that disruption of TET2 enzymatic activity favours myeloid tumorigenesis. Measurement of 5-hmC levels in myeloid malignancies may prove valuable as a diagnostic and prognostic tool, to tailor therapies and assess responses to anti-cancer drugs.

The Ten-Eleven-Translocation 2 (TET2) gene encodes a member of TET family enzymes that alters the epigenetic status of DNA by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Somatic loss-of-function mutations of TET2 are frequently observed in patients with diverse myeloid malignancies, including myelodysplastic syndromes, myeloproliferative neoplasms, and chronic myelomonocytic leukemia. By analyzing mice with targeted disruption of the Tet2 catalytic domain, we show here that Tet2 is a critical regulator of self-renewal and differentiation of hematopoietic stem cells (HSCs). Tet2 deficiency led to decreased genomic levels of 5hmC and augmented the size of the hematopoietic stem/ progenitor cell pool in a cell-autonomous manner. In competitive transplantation assays, Tet2-deficient HSCs were capable of multilineage reconstitution and possessed a competitive advantage over wild-type HSCs, resulting in enhanced hematopoiesis into both lymphoid and myeloid lineages. In vitro, Tet2 deficiency delayed HSC differentiation and skewed development toward the monocyte/macrophage lineage. Our data indicate that Tet2 has a critical role in regulating the expansion and function of HSCs, presumably by controlling 5hmC levels at genes important for the self-renewal, proliferation, and differentiation of HSCs.

Atopic asthma is an inflammatory pulmonary disease associated with Th2 adaptive immune responses triggered by innocuous antigens. While dendritic cells (DCs) are known to shape the adaptive immune response, the mechanisms by which DCs promote Th2 differentiation remain elusive. Herein we demonstrate that Th2-promoting stimuli induce DC expression of IRF4. Mice with conditional deletion of Irf4 in DCs show a dramatic defect in Th2-type lung inflammation, yet retain the ability to elicit pulmonary Th1 anti-viral responses. Using loss- and gain-of-function analysis, we demonstrate that Th2 differentiation is dependent on IRF4 expression in DCs. Finally, IRF4 directly targets and activates the Il10 and Il33 genes in DCs. Reconstitution with exogenous IL-10 and IL-33 recovers the ability of Irf4 deficient DCs to promote Th2 differentiation. These findings reveal a regulatory module in DCs by which IRF4 modulates IL-10 and IL-33 cytokine production to specifically promote Th2 differentiation and inflammation.

TET (Ten-Eleven-Translocation) proteins are Fe(II) and α-ketoglutarate-dependent dioxygenases1-3 that modify the methylation status of DNA by successively oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine and 5-carboxycytosine1,3-5, potential intermediates in the active erasure of DNA methylation marks5,6. We show here that IDAX/ CXXC4, a player in the Wnt signaling pathway7 that has been implicated in malignant renal cell carcinoma8 and colonic villous adenoma9, functions as a negative regulator of TET2 protein expression. IDAX/ CXXC4 was originally encoded within an ancestral TET2 gene that underwent a chromosomal gene inversion during evolution, thus separating the TET2 CXXC domain from the catalytic domain. The Idax CXXC domain binds DNA sequences containing unmethylated CpGs, localises to promoters and CpG islands in genomic DNA, and interacts directly with the catalytic domain of Tet2. Unexpectedly, Idax expression resulted in caspase activation and Tet2 protein downregulation, in a manner that depended on DNA-binding through the Idax CXXC domain. Idax depletion prevented Tet2 downregulation in differentiating mouse embryonic stem (ES) cells, and shRNA against IDAX increased TET2 protein expression in the human monocytic cell line U937. Notably, we find that the expression and activity of TET3 are also regulated through its CXXC domain. Taken together, these results establish the separate and linked CXXC domains of TET2 and TET3 respectively as novel regulators of caspase activation and TET enzymatic activity.

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Bromodomain-containing proteins bind acetylated lysine residues on histone tails and are involved in the recruitment of additional factors that mediate histone modifications and enable transcription. A compound, I-BET-762, that inhibits binding of an acetylated histone peptide to proteins of the bromodomain and extra-terminal domain (BET) family, was previously shown to suppress the production of proinflammatory proteins by macrophages and block acute inflammation in mice. Here, we investigated the effect of short-term treatment with I-BET-762 on T-cell function. Treatment of naïve CD4 + T cells with I-BET-762 during the first 2 d of differentiation had long-lasting effects on subsequent gene expression and cytokine production. Gene expression analysis revealed up-regulated expression of several antiinflammatory gene products, including IL-10, Lag3, and Egr2, and down-regulated expression of several proinflammatory cytokines including GM-CSF and IL-17. The short 2-d treatment with I-BET-762 inhibited the ability of antigen-specific T cells, differentiated under Th1 but not Th17 conditions in vitro, to induce pathogenesis in an adoptive transfer model of experimental autoimmune encephalomyelitis. The suppressive effects of I-BET-762 on T-cell mediated inflammation in vivo were accompanied by decreased recruitment of macrophages, consistent with decreased GM-CSF production by CNS-infiltrating T cells. These effects were mimicked by an inhibitor of c-myc function, implicating reduced expression of c-myc and GM-CSF as one avenue by which I-BET-762 suppresses the inflammatory functions of T cells. Our study demonstrates that inhibiting the functions of BET-family proteins during early T-cell differentiation causes long-lasting suppression of the proinflammatory functions of Th1 cells.A promising approach for limiting production of proinflammatory molecules by T cells for treatment of autoimmune disorders has been to target enzymes that facilitate the addition or removal of epigenetic modifications. An additional level of gene regulation derives from proteins that "read" histone and DNA modifications, such as bromodomain-containing proteins that bind acetylated histones. Specifically, BRD2, BRD3, and BRD4-members of the bromodomain and extra-terminal domain (BET) family-contain two tandem N-terminal bromodomains and an extraterminal domain that has been demonstrated to bind a number of chromatin-modifying proteins. The BET family member, BRD4 has a unique C-terminal domain that binds to the positive transcription elongation factor b (P-TEFb; composed of the cyclin-dependent kinase CDK9 and its partner, cyclin T1) complex. BRD4 recruits P-TEFb to acetylated histones, promoting phosphorylation of paused RNA polymerase II (Pol II) and the repressive complexes DSIF and NELF by CDK9, thereby allowing productive mRNA elongation (reviewed in refs. 1 and 2).Given the pivotal role of BET proteins in transcriptional regulation, small molecule compounds that inhibit binding of acetylated histones to bromodomains of BET proteins ...

The transcription factor FOXP3 is essential for the suppressive function of regulatory T cells that are required for maintaining self-tolerance. We have solved the crystal structure of the FOXP3 forkhead domain, as a ternary complex with the DNA-binding domain of nuclear factor of activated T cells-1 (NFAT1) and a DNA oligonucleotide from the interleukin-2 promoter. A striking feature of this structure is that FOXP3 forms a domain-swapped dimer that bridges two molecules of DNA. Structure-guided or autoimmune disease (IPEX)-associated mutations in the domain-swap interface diminished dimer formation by the FOXP3 forkhead domain without compromising FOXP3 DNA binding. These mutations also eliminated T cell suppressive activity conferred by FOXP3, both in vitro and in a murine model of autoimmune diabetes in vivo. We conclude that FOXP3-mediated suppressor function requires dimerization through the forkhead domain, and that mutations in the dimer interface can lead to the systemic autoimmunity observed in IPEX patients.