Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by the MLL Partner TET1

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.Chemistry and Chemical Biolog

Inducible Costimulator Expression Regulates the Magnitude of Th2-Mediated Airway Inflammation by Regulating the Number of Th2 Cells

Inducible Costimulator (ICOS) is an important regulator of Th2 lymphocyte function and a potential immunotherapeutic target for allergy and asthma. A SNP in the ICOS 5' promoter in humans is associated with increased atopy and serum IgE in a founder population and increased ICOS surface expression and Th2 cytokine production from peripheral blood mononuclear cells. However, it is unknown if increased ICOS expression contributes to disease progression or is a result of disease pathology.We developed a mouse model in which ICOS surface expression levels are genetically predetermined to test our hypothesis that genetic regulation of ICOS expression controls the severity of Th2 responses in vivo. Using ICOS+/+ and ICOS+/- mice in a Th2 model of airway inflammation, we found that T cells from the ICOS+/- mice had reduced ICOS expression and decreased Th2-mediated inflammation in vivo. Although the activation status of the T cells did not differ, T cells isolated from the lungs and draining lymph nodes of ICOS+/- mice at the peak of inflammation produced less Th2 cytokines upon stimulation ex vivo. Using 4get mice, which express GFP upon IL-4 transcription, we determined that the decreased Th2 cytokines in ICOS+/- is due to reduced percentage of Th2 cells and not a defect in their ability to produce IL-4.These data suggest that in both mice and humans, the level of ICOS surface expression regulates the magnitude of the in vivo Th2 response, perhaps by influencing Th2 differentiation

Inducible Costimulator Expression Regulates the Magnitude of Th2-Mediated Airway Inflammation by Regulating the Number of Th2 Cells

Background: Inducible Costimulator (ICOS) is an important regulator of Th2 lymphocyte function and a potential immunotherapeutic target for allergy and asthma. A SNP in the ICOS 5′ promoter in humans is associated with increased atopy and serum IgE in a founder population and increased ICOS surface expression and Th2 cytokine production from peripheral blood mononuclear cells. However, it is unknown if increased ICOS expression contributes to disease progression or is a result of disease pathology.Methodology/Principal Findings: We developed a mouse model in which ICOS surface expression levels are genetically predetermined to test our hypothesis that genetic regulation of ICOS expression controls the severity of Th2 responses in vivo. Using ICOS+/+ and ICOS+/− mice in a Th2 model of airway inflammation, we found that T cells from the ICOS+/− mice had reduced ICOS expression and decreased Th2-mediated inflammation in vivo. Although the activation status of the T cells did not differ, T cells isolated from the lungs and draining lymph nodes of ICOS+/− mice at the peak of inflammation produced less Th2 cytokines upon stimulation ex vivo. Using 4get mice, which express GFP upon IL-4 transcription, we determined that the decreased Th2 cytokines in ICOS+/− is due to reduced percentage of Th2 cells and not a defect in their ability to produce IL-4.Conclusion: These data suggest that in both mice and humans, the level of ICOS surface expression regulates the magnitude of the in vivo Th2 response, perhaps by influencing Th2 differentiation.</p

ICOS^+/− T cells have decreased ICOS expression but equal activation status.

<p>ICOS^+/+ (black lines) and ICOS^+/− (grey line) T cells were isolated and stimulated with anti-CD3 and anti-CD28 antibodies in media alone or in the presence of IL-4 or anti-IL-4 antibodies. After 48 hours the cells were removed and expression of the indicated surface markers were analyzed via flow cytometry. (A) ICOS expression on CD4⁺ T cells stimulated under the indicated conditions. (B) Expression of the indicated cell-surface markers after stimulation for 48 hours in media alone cultures.</p

ICOS cell-surface expression regulates the magnitude of a Th2 response <i>in vivo</i>.

<p>ICOS^+/+ (black bars), ICOS^+/− (grey bars) and ICOS^−/− (white bars) mice were activated as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007525#s2" target="_blank">Materials and Methods</a>. (A) Total cells in BAL fluid were counted and percent CCR3⁺ eosinophils, CD4⁺ T cells and CD8⁺ T cells were analyzed via flow cytometry and used to calculate total cell numbers. (B) ICOS expression on BAL CD4⁺ T cells from ICOS^+/+ (black line), ICOS^+/− (grey line), ICOS^−/− (dotted line) is shown. (C) Serum IgE levels were analyzed via ELISA.</p

Decreased Th2 response in B7RP-1^+/− mice.

<p>Splenocytes from Wild-type and B7RP-1^+/− mice were removed and single-cell suspensions were made. The cells were stained with B7RP-1 and either CD19 or CD11c. Black = WT Grey = B7RP-1^+/−. (B) B6. B7RP-1^+/+ (black bars), B6. B7RP-1^+/− (grey bars), and B6. B7RP-1^−/− (white bars) mice were sensitized and challenged as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007525#s2" target="_blank">Materials and Methods</a>. Total cells in BAL fluid were counted and percent CCR3⁺ eosinophils, CD4⁺ T cells and CD8⁺ T cells were analyzed via flow cytometry and used to calculate total cell numbers and normalized to the average of the wild-type value. ICOS expression on BAL CD4⁺ T cells isolated from the mediastinal lymph nodes of mice is shown. Serum IgE levels were analyzed via ELISA.</p