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The nuclear orphan receptor Nr4a2 induces Foxp3 and regulates differentiation of CD4+ T cells.

Sekiya T, Kashiwagi I, Inoue N, Morita R, Hori S, Waldmann H, Rudensky AY, Ichinose H, Metzger D, Chambon P, Yoshimura A - Nat Commun (2011)

Bottom Line: Regulatory T cells (Tregs) have a central role in maintaining immune homoeostasis through various mechanisms.Nr4a2 binds to regulatory regions of Foxp3, where it mediates permissive histone modifications.Nr4a2-deficeint Tregs are prone to lose Foxp3 expression and have attenuated suppressive ability both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo 160-8582, Japan.

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Nr4a2 induces Treg-like active histone modifications in Foxp3 regulatory regions.(a–c) Native ChIP assay of Foxp3 promoter (left), CNS1 (middle) and CNS2 (right) enhancers was performed with antibodies against modified histones (a: anti-acetylated H4, open bars; b: anti-tri-methylated H3K4, dotted bars; c: anti-tri-methylated H3K27, striped bars) and control IgG (filled bars). Naïve T cells, Tregs, mock-transduced cultured cells and sorted GFP+ cells from the population of eMIGR1 or eMIGR1-Nr4a2 retrovirus-transduced cells were analysed. Values are depicted as the percentage of corresponding input. Data are representative of two independent experiments (mean and s.d. of triplicate). (d) Bisulphite sequencing revealed that Nr4a2 can not mediate the demethylation of the CpG island in the CNS2 region of the foxp3 locus. Sequencing was performed against the bisulphite-treated genomic DNA from naïve T cells, Tregs, sorted GFP+ cells from the eMIGR1 retrovirus-transduced cells and sorted GFP+Foxp3+ cells from the eMIGR1-Nr4a2 retrovirus-transduced cells. Ten CG sites in the CNS2 locus, located at the indicated positions, were analysed. Top: closed and open circles indicate methylated and unmethylated CG sites, respectively. Bottom: data shown in top panels were quantitated, and shown as percentages of methylation at each position. Data are pooled from two independent experiments. Foxp3+ cells were sorted with APC-conjugated anti-hCD2 antibody unless otherwise indicated.
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f2: Nr4a2 induces Treg-like active histone modifications in Foxp3 regulatory regions.(a–c) Native ChIP assay of Foxp3 promoter (left), CNS1 (middle) and CNS2 (right) enhancers was performed with antibodies against modified histones (a: anti-acetylated H4, open bars; b: anti-tri-methylated H3K4, dotted bars; c: anti-tri-methylated H3K27, striped bars) and control IgG (filled bars). Naïve T cells, Tregs, mock-transduced cultured cells and sorted GFP+ cells from the population of eMIGR1 or eMIGR1-Nr4a2 retrovirus-transduced cells were analysed. Values are depicted as the percentage of corresponding input. Data are representative of two independent experiments (mean and s.d. of triplicate). (d) Bisulphite sequencing revealed that Nr4a2 can not mediate the demethylation of the CpG island in the CNS2 region of the foxp3 locus. Sequencing was performed against the bisulphite-treated genomic DNA from naïve T cells, Tregs, sorted GFP+ cells from the eMIGR1 retrovirus-transduced cells and sorted GFP+Foxp3+ cells from the eMIGR1-Nr4a2 retrovirus-transduced cells. Ten CG sites in the CNS2 locus, located at the indicated positions, were analysed. Top: closed and open circles indicate methylated and unmethylated CG sites, respectively. Bottom: data shown in top panels were quantitated, and shown as percentages of methylation at each position. Data are pooled from two independent experiments. Foxp3+ cells were sorted with APC-conjugated anti-hCD2 antibody unless otherwise indicated.

Mentions: We investigated the effect of Nr4a2 on the epigenetic status of the foxp3 promoter/enhancer. First, we isolated the total green fluorescence protein (GFP)-positive fractions from Nr4a2-virus- or control-virus-transduced CD4-SP cells, and performed native ChIP to compare their histone modification status with those of Tregs and naïve CD4+ T cells. As depicted in Figure 2a–c, Nr4a2 induced active histone modifications, histone H4-acetylation and histone H3K4-trimethylation, similar to the patterns in Tregs. H3K27-trimethylation, a repressive histone modification was not detected in both Tregs and Nr4a2-transduced T cells.


The nuclear orphan receptor Nr4a2 induces Foxp3 and regulates differentiation of CD4+ T cells.

Sekiya T, Kashiwagi I, Inoue N, Morita R, Hori S, Waldmann H, Rudensky AY, Ichinose H, Metzger D, Chambon P, Yoshimura A - Nat Commun (2011)

Nr4a2 induces Treg-like active histone modifications in Foxp3 regulatory regions.(a–c) Native ChIP assay of Foxp3 promoter (left), CNS1 (middle) and CNS2 (right) enhancers was performed with antibodies against modified histones (a: anti-acetylated H4, open bars; b: anti-tri-methylated H3K4, dotted bars; c: anti-tri-methylated H3K27, striped bars) and control IgG (filled bars). Naïve T cells, Tregs, mock-transduced cultured cells and sorted GFP+ cells from the population of eMIGR1 or eMIGR1-Nr4a2 retrovirus-transduced cells were analysed. Values are depicted as the percentage of corresponding input. Data are representative of two independent experiments (mean and s.d. of triplicate). (d) Bisulphite sequencing revealed that Nr4a2 can not mediate the demethylation of the CpG island in the CNS2 region of the foxp3 locus. Sequencing was performed against the bisulphite-treated genomic DNA from naïve T cells, Tregs, sorted GFP+ cells from the eMIGR1 retrovirus-transduced cells and sorted GFP+Foxp3+ cells from the eMIGR1-Nr4a2 retrovirus-transduced cells. Ten CG sites in the CNS2 locus, located at the indicated positions, were analysed. Top: closed and open circles indicate methylated and unmethylated CG sites, respectively. Bottom: data shown in top panels were quantitated, and shown as percentages of methylation at each position. Data are pooled from two independent experiments. Foxp3+ cells were sorted with APC-conjugated anti-hCD2 antibody unless otherwise indicated.
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Related In: Results  -  Collection

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f2: Nr4a2 induces Treg-like active histone modifications in Foxp3 regulatory regions.(a–c) Native ChIP assay of Foxp3 promoter (left), CNS1 (middle) and CNS2 (right) enhancers was performed with antibodies against modified histones (a: anti-acetylated H4, open bars; b: anti-tri-methylated H3K4, dotted bars; c: anti-tri-methylated H3K27, striped bars) and control IgG (filled bars). Naïve T cells, Tregs, mock-transduced cultured cells and sorted GFP+ cells from the population of eMIGR1 or eMIGR1-Nr4a2 retrovirus-transduced cells were analysed. Values are depicted as the percentage of corresponding input. Data are representative of two independent experiments (mean and s.d. of triplicate). (d) Bisulphite sequencing revealed that Nr4a2 can not mediate the demethylation of the CpG island in the CNS2 region of the foxp3 locus. Sequencing was performed against the bisulphite-treated genomic DNA from naïve T cells, Tregs, sorted GFP+ cells from the eMIGR1 retrovirus-transduced cells and sorted GFP+Foxp3+ cells from the eMIGR1-Nr4a2 retrovirus-transduced cells. Ten CG sites in the CNS2 locus, located at the indicated positions, were analysed. Top: closed and open circles indicate methylated and unmethylated CG sites, respectively. Bottom: data shown in top panels were quantitated, and shown as percentages of methylation at each position. Data are pooled from two independent experiments. Foxp3+ cells were sorted with APC-conjugated anti-hCD2 antibody unless otherwise indicated.
Mentions: We investigated the effect of Nr4a2 on the epigenetic status of the foxp3 promoter/enhancer. First, we isolated the total green fluorescence protein (GFP)-positive fractions from Nr4a2-virus- or control-virus-transduced CD4-SP cells, and performed native ChIP to compare their histone modification status with those of Tregs and naïve CD4+ T cells. As depicted in Figure 2a–c, Nr4a2 induced active histone modifications, histone H4-acetylation and histone H3K4-trimethylation, similar to the patterns in Tregs. H3K27-trimethylation, a repressive histone modification was not detected in both Tregs and Nr4a2-transduced T cells.

Bottom Line: Regulatory T cells (Tregs) have a central role in maintaining immune homoeostasis through various mechanisms.Nr4a2 binds to regulatory regions of Foxp3, where it mediates permissive histone modifications.Nr4a2-deficeint Tregs are prone to lose Foxp3 expression and have attenuated suppressive ability both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo 160-8582, Japan.

Show MeSH
Related in: MedlinePlus