TET/JBP enzymes oxidize 5-methylpyrimidines in DNA. In mammals, the oxidized methylcytosines (oxi-mCs) function as epigenetic marks and likely intermediates in DNA demethylation. Here we present a method based on diglucosylation of 5-hydroxymethylcytosine (5hmC) to simultaneously map 5hmC, 5-formylcytosine, and 5-carboxylcytosine at near-base-pair resolution. We have used the method to map the distribution of oxi-mC across the genome of Coprinopsis cinerea, a basidiomycete that encodes 47 TET/JBP paralogs in a previously unidentified class of DNA transposons. Like 5-methylcytosine residues from which they are derived, oxi-mC modifications are enriched at centromeres, TET/JBP transposons, and multicopy paralogous genes that are not expressed, but rarely mark genes whose expression changes between two developmental stages. Our study provides evidence for the emergence of an epigenetic regulatory system through recruitment of selfish elements in a eukaryotic lineage, and describes a method to map all three different species of oxi-mCs simultaneously.T he discovery that oxidative modifications of DNA bases are catalyzed by the TET/JBP family of 2-oxoglutarate and irondependent dioxygenases (1-4) opened a major area of research into the epigenetics of various eukaryotic lineages (reviewed in refs. 5-7). In metazoans, the TET/JBP family is represented by TET proteins, which are present in all animals that are known to possess DNA cytosine methylation (3,8,9). The three TET paralogs of vertebrates have been shown to catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) (2), which is progressively oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (10-12). The discovery of these oxidized methylcytosine (oxi-mC) modifications triggered a flurry of studies on the mammalian TET paralogs; the reports increasingly point toward important roles for these oxidative modifications as intermediates in the enigmatic process of DNA demethylation, and also as epigenetic marks in their own right (13, 14) (reviewed in refs. 5-7). TET proteins have roles in diverse biological processes, including epigenetic regulation of gene transcription, embryonic development, stem cell function, and cancer (5), but the mechanisms underlying their biological activities are still poorly defined.Several methods have been developed to profile individual oxi-mC species at base resolution in genomic DNA (reviewed in ref. 5). However, none of these methods can simultaneously map all three oxi-mC species-5hmC, 5fC, and 5caC-at the same time; rather, they rely on chemical or enzymatic conversion of individual oxi-mCs followed by bisulfite sequencing. Two recent sequencing technologies, single-molecule, real-time (SMRT) sequencing and protein nanopore sequencing, are capable of recognizing modified bases in unamplified genomic DNA (15-18). In SMRT sequencing, 5mC and 5hmC are barely detectable in unmodified DNA, whereas 5fC and 5caC yield a robust kinetic signature (16). As 5hmC is an abundant oxi-mC modification in ...

BackgroundSevere autoinflammatory diseases are associated with mutations in the Foxp3 locus in both mice and humans. Foxp3 is required for the development, function, and maintenance of regulatory T cells (Tregs), a subset of CD4 cells that suppress T cell activation and inflammatory processes. Siva is a pro-apoptotic gene that is expressed across a range of tissues, including CD4 T cells. Siva interacts with three tumor necrosis factor receptor (TNFR) family members that are constitutively expressed on Treg cells: CD27, GITR, and OX40.ResultsHere we report a biophysical interaction between FOXP3 and Siva. We mapped the interaction domains to Siva's C-terminus and to a central region of FOXP3. We showed that Siva repressed IL-2 induction by suppressing IL-2 promoter activity during T cell activation. Siva-1's repressive effect on IL-2 gene expression appears to be mediated by inhibition of NFkappaB, whereas FOXP3 repressed both NFkappaB and NFAT activity.ConclusionsIn summary, our data suggest that both FOXP3 and Siva function as negative regulators of IL-2 gene expression in Treg cells, via suppression of NFAT by FOXP3 and of NFkappaB by both FOXP3 and Siva. Our work contributes evidence for Siva's role as a T cell signalling mediator in addition to its known pro-apoptotic function. Though further investigations are needed, evidence for the biophysical interaction between FOXP3 and Siva invites the possibility that Siva may be important for proper Treg cell function.

Regulatory agencies around the world have committed to reducing or eliminating animal testing for establishing chemical safety. Adverse outcome pathways can facilitate replacement by providing a mechanistic framework for identifying the appropriate non-animal methods and connecting them to apical adverse outcomes. This study separated 11,992 chemicals with curated rat oral acute toxicity information into clusters of structurally similar compounds. Each cluster was then assigned one or more ToxCast/Tox21 assays by looking for the minimum number of assays required to record at least one positive hit call below cytotoxicity for all acutely toxic chemicals in the cluster. When structural information is used to select assays for testing, none of the chemicals required more than four assays and 98% required two assays or less. Both the structure-based clusters and activity from the associated assays were significantly associated with the GHS toxicity classification of the chemicals, which suggests that a combination of bioactivity and structural information could be as reproducible as traditional in vivo studies. Predictivity is improved when the in vitro assay directly corresponds to the mechanism of toxicity, but many indirect assays showed promise as well. Given the lower cost of in vitro testing, a small assay battery including both general cytotoxicity assays and two or more orthogonal assays targeting the toxicological mechanism could be used to improve performance further. This approach illustrates the promise of combining existing in silico approaches, such as the Collaborative Acute Toxicity Modeling Suite (CATMoS), with structure-based bioactivity information as part of an efficient tiered testing strategy that can reduce or eliminate animal testing for acute oral toxicity.

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