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Akt1-mediated Gata3 phosphorylation controls the repression of IFNγ in memory-type Th2 cells.

Hosokawa H, Tanaka T, Endo Y, Kato M, Shinoda K, Suzuki A, Motohashi S, Matsumoto M, Nakayama KI, Nakayama T - Nat Commun (2016)

Bottom Line: We also identify Akt1 as a Gata3-phosphorylating kinase, and the activation of Akt1 induces derepression of Tbx21 and Ifng expression in Th2 cells.Moreover, T-bet-dependent IFNγ expression in IFNγ-producing memory Th2 cells appears to be controlled by the phosphorylation status of Gata3 in human and murine systems.Thus, this study highlights the molecular basis for posttranslational modifications of Gata3 that control the regulation of IFNγ expression in memory Th2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan.

ABSTRACT
Th2 cells produce Th2 cytokines such as IL-4, IL-5 and IL-13, but repress Th1 cytokine IFNγ. Recent studies have revealed various distinct memory-type Th2 cell subsets, one of which produces a substantial amount of IFNγ in addition to Th2 cytokines, however it remains unclear precisely how these Th2 cells produce IFNγ. We herein show that phosphorylation of Gata3 at Ser308, Thr315 and Ser316 induces dissociation of a histone deacetylase Hdac2 from the Gata3/Chd4 repressive complex in Th2 cells. We also identify Akt1 as a Gata3-phosphorylating kinase, and the activation of Akt1 induces derepression of Tbx21 and Ifng expression in Th2 cells. Moreover, T-bet-dependent IFNγ expression in IFNγ-producing memory Th2 cells appears to be controlled by the phosphorylation status of Gata3 in human and murine systems. Thus, this study highlights the molecular basis for posttranslational modifications of Gata3 that control the regulation of IFNγ expression in memory Th2 cells.

No MeSH data available.


Related in: MedlinePlus

Identification of Gata3 phosphorylation in the C-terminal zinc finger.(a) Schematic representations of the Flag-tagged Gata3 WT or deletion mutants are shown (top panel). Flag-tagged Gata3 WT, dNF, dCF or dNCF plasmid constructs were transfected with Myc-tagged Chd4 into 293T cells. Two days later, the amount of Myc-tagged Chd4 associated with the Flag-tagged WT or mutant Gata3 was assessed by immunoprecipitation (IP) followed by immunoblotting (IB) (middle panel). Total lysates were also subjected to IB in parallel (lower panel). (b) Naive CD4 T cells were stimulated under Th1 conditions and then infected with a retrovirus vector carrying WT or mutant Gata3 cDNA. Three days later, the retrovirus-infected GFP-expressing cells were purified and the levels of mRNA of Tbx21 and Ifng were measured by RT-qPCR. The relative expression (/Hprt) is shown with s.d.'s. (c) The amount of Myc-tagged Hdac2 associated with Flag-tagged Gata3 mutants were assessed as in Fig. 1a. (d) D10G4.1 cells were infected with a lentivirus encoding Flag-Gata3 and then the immunopurified Gata3 was subjected to a LC-MS/MS analysis to assess posttranslational modifications. All Gata3 peptides including Thr315 and Ser316 detected by our mass spectrometry analysis are shown. Blue characters indicate phosphorylated amino acids. (e) The phosphorylated residues of Gata3 in the linker region of tandem zinc fingers are highly conserved from Drosophila to human. (f) The 3D structure of Gata3 zinc fingers bound to DNA, including the novel phosphorylation sites (Ser308, Thr315 and Ser316) determined using the Molecular Modeling Database (MMDB ID; 105495)30, was drawn using the Cn3D software programme. The phosphorylated Ser/Thr residues are highlighted in yellow. Four (b) and three (a,c) independent experiments were performed with similar results.
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f1: Identification of Gata3 phosphorylation in the C-terminal zinc finger.(a) Schematic representations of the Flag-tagged Gata3 WT or deletion mutants are shown (top panel). Flag-tagged Gata3 WT, dNF, dCF or dNCF plasmid constructs were transfected with Myc-tagged Chd4 into 293T cells. Two days later, the amount of Myc-tagged Chd4 associated with the Flag-tagged WT or mutant Gata3 was assessed by immunoprecipitation (IP) followed by immunoblotting (IB) (middle panel). Total lysates were also subjected to IB in parallel (lower panel). (b) Naive CD4 T cells were stimulated under Th1 conditions and then infected with a retrovirus vector carrying WT or mutant Gata3 cDNA. Three days later, the retrovirus-infected GFP-expressing cells were purified and the levels of mRNA of Tbx21 and Ifng were measured by RT-qPCR. The relative expression (/Hprt) is shown with s.d.'s. (c) The amount of Myc-tagged Hdac2 associated with Flag-tagged Gata3 mutants were assessed as in Fig. 1a. (d) D10G4.1 cells were infected with a lentivirus encoding Flag-Gata3 and then the immunopurified Gata3 was subjected to a LC-MS/MS analysis to assess posttranslational modifications. All Gata3 peptides including Thr315 and Ser316 detected by our mass spectrometry analysis are shown. Blue characters indicate phosphorylated amino acids. (e) The phosphorylated residues of Gata3 in the linker region of tandem zinc fingers are highly conserved from Drosophila to human. (f) The 3D structure of Gata3 zinc fingers bound to DNA, including the novel phosphorylation sites (Ser308, Thr315 and Ser316) determined using the Molecular Modeling Database (MMDB ID; 105495)30, was drawn using the Cn3D software programme. The phosphorylated Ser/Thr residues are highlighted in yellow. Four (b) and three (a,c) independent experiments were performed with similar results.

Mentions: We wished to identify the mechanisms by which the molecular switch for organizing activating and repressive Gata3 complexes occurs in Th2 cells. When Gata3 associates with the Chd4-NuRD repressive complex, the Gata3/Chd4-NuRD complex binds to the Tbx21 locus and represses its expression in Th2 cells4. First, to determine which domains of Gata3 are important for binding to Chd4, Myc-tagged Chd4 and Flag-tagged wild type (WT) or deletion mutants of Gata3 (Fig. 1a, upper) were co-transfected into 293T cells and then pull-down assays were performed. The association with Chd4 was almost completely abrogated by the deletion of the two zinc finger domains of Gata3 (Fig. 1a), suggesting that the tandem zinc finger motifs of Gata3 are important for binding to Chd4.


Akt1-mediated Gata3 phosphorylation controls the repression of IFNγ in memory-type Th2 cells.

Hosokawa H, Tanaka T, Endo Y, Kato M, Shinoda K, Suzuki A, Motohashi S, Matsumoto M, Nakayama KI, Nakayama T - Nat Commun (2016)

Identification of Gata3 phosphorylation in the C-terminal zinc finger.(a) Schematic representations of the Flag-tagged Gata3 WT or deletion mutants are shown (top panel). Flag-tagged Gata3 WT, dNF, dCF or dNCF plasmid constructs were transfected with Myc-tagged Chd4 into 293T cells. Two days later, the amount of Myc-tagged Chd4 associated with the Flag-tagged WT or mutant Gata3 was assessed by immunoprecipitation (IP) followed by immunoblotting (IB) (middle panel). Total lysates were also subjected to IB in parallel (lower panel). (b) Naive CD4 T cells were stimulated under Th1 conditions and then infected with a retrovirus vector carrying WT or mutant Gata3 cDNA. Three days later, the retrovirus-infected GFP-expressing cells were purified and the levels of mRNA of Tbx21 and Ifng were measured by RT-qPCR. The relative expression (/Hprt) is shown with s.d.'s. (c) The amount of Myc-tagged Hdac2 associated with Flag-tagged Gata3 mutants were assessed as in Fig. 1a. (d) D10G4.1 cells were infected with a lentivirus encoding Flag-Gata3 and then the immunopurified Gata3 was subjected to a LC-MS/MS analysis to assess posttranslational modifications. All Gata3 peptides including Thr315 and Ser316 detected by our mass spectrometry analysis are shown. Blue characters indicate phosphorylated amino acids. (e) The phosphorylated residues of Gata3 in the linker region of tandem zinc fingers are highly conserved from Drosophila to human. (f) The 3D structure of Gata3 zinc fingers bound to DNA, including the novel phosphorylation sites (Ser308, Thr315 and Ser316) determined using the Molecular Modeling Database (MMDB ID; 105495)30, was drawn using the Cn3D software programme. The phosphorylated Ser/Thr residues are highlighted in yellow. Four (b) and three (a,c) independent experiments were performed with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829694&req=5

f1: Identification of Gata3 phosphorylation in the C-terminal zinc finger.(a) Schematic representations of the Flag-tagged Gata3 WT or deletion mutants are shown (top panel). Flag-tagged Gata3 WT, dNF, dCF or dNCF plasmid constructs were transfected with Myc-tagged Chd4 into 293T cells. Two days later, the amount of Myc-tagged Chd4 associated with the Flag-tagged WT or mutant Gata3 was assessed by immunoprecipitation (IP) followed by immunoblotting (IB) (middle panel). Total lysates were also subjected to IB in parallel (lower panel). (b) Naive CD4 T cells were stimulated under Th1 conditions and then infected with a retrovirus vector carrying WT or mutant Gata3 cDNA. Three days later, the retrovirus-infected GFP-expressing cells were purified and the levels of mRNA of Tbx21 and Ifng were measured by RT-qPCR. The relative expression (/Hprt) is shown with s.d.'s. (c) The amount of Myc-tagged Hdac2 associated with Flag-tagged Gata3 mutants were assessed as in Fig. 1a. (d) D10G4.1 cells were infected with a lentivirus encoding Flag-Gata3 and then the immunopurified Gata3 was subjected to a LC-MS/MS analysis to assess posttranslational modifications. All Gata3 peptides including Thr315 and Ser316 detected by our mass spectrometry analysis are shown. Blue characters indicate phosphorylated amino acids. (e) The phosphorylated residues of Gata3 in the linker region of tandem zinc fingers are highly conserved from Drosophila to human. (f) The 3D structure of Gata3 zinc fingers bound to DNA, including the novel phosphorylation sites (Ser308, Thr315 and Ser316) determined using the Molecular Modeling Database (MMDB ID; 105495)30, was drawn using the Cn3D software programme. The phosphorylated Ser/Thr residues are highlighted in yellow. Four (b) and three (a,c) independent experiments were performed with similar results.
Mentions: We wished to identify the mechanisms by which the molecular switch for organizing activating and repressive Gata3 complexes occurs in Th2 cells. When Gata3 associates with the Chd4-NuRD repressive complex, the Gata3/Chd4-NuRD complex binds to the Tbx21 locus and represses its expression in Th2 cells4. First, to determine which domains of Gata3 are important for binding to Chd4, Myc-tagged Chd4 and Flag-tagged wild type (WT) or deletion mutants of Gata3 (Fig. 1a, upper) were co-transfected into 293T cells and then pull-down assays were performed. The association with Chd4 was almost completely abrogated by the deletion of the two zinc finger domains of Gata3 (Fig. 1a), suggesting that the tandem zinc finger motifs of Gata3 are important for binding to Chd4.

Bottom Line: We also identify Akt1 as a Gata3-phosphorylating kinase, and the activation of Akt1 induces derepression of Tbx21 and Ifng expression in Th2 cells.Moreover, T-bet-dependent IFNγ expression in IFNγ-producing memory Th2 cells appears to be controlled by the phosphorylation status of Gata3 in human and murine systems.Thus, this study highlights the molecular basis for posttranslational modifications of Gata3 that control the regulation of IFNγ expression in memory Th2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan.

ABSTRACT
Th2 cells produce Th2 cytokines such as IL-4, IL-5 and IL-13, but repress Th1 cytokine IFNγ. Recent studies have revealed various distinct memory-type Th2 cell subsets, one of which produces a substantial amount of IFNγ in addition to Th2 cytokines, however it remains unclear precisely how these Th2 cells produce IFNγ. We herein show that phosphorylation of Gata3 at Ser308, Thr315 and Ser316 induces dissociation of a histone deacetylase Hdac2 from the Gata3/Chd4 repressive complex in Th2 cells. We also identify Akt1 as a Gata3-phosphorylating kinase, and the activation of Akt1 induces derepression of Tbx21 and Ifng expression in Th2 cells. Moreover, T-bet-dependent IFNγ expression in IFNγ-producing memory Th2 cells appears to be controlled by the phosphorylation status of Gata3 in human and murine systems. Thus, this study highlights the molecular basis for posttranslational modifications of Gata3 that control the regulation of IFNγ expression in memory Th2 cells.

No MeSH data available.


Related in: MedlinePlus