Critical Role of STAT5 Transcription Factor Tetramerization for Cytokine Responses and Normal Immune Function

IL-21 is a type I cytokine essential for immune cell differentiation and function. Although IL-21 can activate several STAT family transcription factors, previous studies focused mainly on the role of STAT3 in IL-21 signaling. Here, we investigated the role of STAT1 and show that STAT1 and STAT3 have at least partially opposing roles in IL-21 signaling in CD4 + T cells. IL-21 induced STAT1 phosphorylation, and this was augmented in Stat3-deficient CD4 + T cells. RNA-Seq analysis of CD4 + T cells from Stat1-and Stat3-deficient mice revealed that both STAT1 and STAT3 are critical for IL-21-mediated gene regulation. Expression of some genes, including Tbx21 and Ifng, was differentially regulated by STAT1 and STAT3. Moreover, opposing actions of STAT1 and STAT3 on IFN-γ expression in CD4 + T cells were demonstrated in vivo during chronic lymphocytic choriomeningitis infection. Finally, IL-21-mediated induction of STAT1 phosphorylation, as well as IFNG and TBX21 expression, were higher in CD4 + T cells from patients with autosomal dominant hyperIgE syndrome, which is caused by STAT3 deficiency, as well as in cells from STAT1 gain-of-function patients. These data indicate an interplay between STAT1 and STAT3 in fine-tuning IL-21 actions.) is a type I cytokine that signals via a receptor composed of IL-21R and the common cytokine receptor γ-chain, γ c (1). γ c is also shared by the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 and is mutated in humans with X-linked severe combined immunodeficiency (XSCID), a disease characterized by the absence of T and natural killer (NK) cells and the presence of nonfunctional B cells (2). IL-21 is primarily produced by CD4 + T cells and natural killer T (NKT) cells, but it has pleiotropic actions on both adaptive and innate immune cells, including T, B, NK, NKT, and dendritic cells (1). In T cells, IL-21 can act as a comitogen and cooperates with IL-7 and IL-15 to expand CD8 + T cells (3), promotes Th17 differentiation (4-6), and induces BCL6 expression (7) to promote T follicular helper cell development (8, 9). In B cells, IL-21 promotes plasma cell differentiation (10, 11), and in combination with IL-4, drives IgG1 and IgG3 class switch (11,12). Defective signaling by IL-21 appears to substantially explain the B-cell defect observed in patients with XSCID (11, 13). Furthermore, IL-21 can enhance the cytotoxic activity of NK and NKT cells (1) and induce the apoptosis of conventional dendritic cells (14).IL-21 activates multiple signaling pathways, including the JAK-STAT, PI 3-kinase (PI3K), and MAPK pathways (15). Of these, the JAK-STAT pathway has been most extensively studied. IL-21 induces phosphorylation of JAK1 and JAK3, which in turn leads to phosphorylation and nuclear translocation of STAT3, which then binds to IFN-γ-activated sequence (GAS) motifs and modulates expression of IL-21-responsive genes. IL-21 also activates STAT1, but the function of IL-21-activated STAT1 is largely unknown, although IL-21 was suggested to use STAT1 to promote CD8 + T-cell cytotoxicity and ap...

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“…RNA-Seq and ChIP-Seq were performed as reported (14,36). See SI Materials and Methods for more information.…”

Section: Materials Of Methodsmentioning

confidence: 99%

Opposing roles of STAT1 and STAT3 in IL-21 function in CD4 ⁺ T cells

Wan

Andraski

Spolski

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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108

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“…16 A recent study elucidated the differences in DNA binding between phosphorylated STAT5 dimers and tetramers by generating STAT5A-STAT5B double knockin (DKI) ND mutant mice that lack P-STAT5 tetramers. 17 By studying IL-2-induced gene expression in DKI T cells vs. wild-type (WT) T cells and using ChIP-seq methodology to identify genome-wide binding sites for P-STAT5A/B dimer and tetramer, Lin et al have been able to establish the consensus binding motifs for P-STAT5A/B, including the optimal spacing between two GAS motifs located within 30 nucleotides for tetramer binding. 17 We re-analyzed the data set for GAS distribution at longer distances, 17 and also performed similar analysis of LIF-induced STAT3 binding to DNA, 18 to determine the optimal spacing between neighboring GAS sites using the CisGenome program 19 that is widely used for tiling array, ChIP-seq and genome analysis.…”

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confidence: 99%

“…17 By studying IL-2-induced gene expression in DKI T cells vs. wild-type (WT) T cells and using ChIP-seq methodology to identify genome-wide binding sites for P-STAT5A/B dimer and tetramer, Lin et al have been able to establish the consensus binding motifs for P-STAT5A/B, including the optimal spacing between two GAS motifs located within 30 nucleotides for tetramer binding. 17 We re-analyzed the data set for GAS distribution at longer distances, 17 and also performed similar analysis of LIF-induced STAT3 binding to DNA, 18 to determine the optimal spacing between neighboring GAS sites using the CisGenome program 19 that is widely used for tiling array, ChIP-seq and genome analysis. Since the profiles of ChIP-seq or ChIP-chip data are often noisy and diffusive, spanning regions ranging from short DNA fragments wrapped around several nucleosomes to large DNA domains covering multiple genes, we first narrowed down the diffusive regions to ChIPenriched sequences and then mapped out location of the GAS-like motif (T/C) (T/A)(C/T)(T/C)N(A/G/T)G(A/T)A in the ChIP-enriched data.…”

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A new role for STAT3 as a regulator of chromatin topology

et al. 2013

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“…To date, cytokine-regulated genes, the expression of which requires STAT5 tetramers, have been identified using STAT5A-STAT5B double knock-in N-domain mutant mice with STAT5 proteins that form dimers and not tetramers. Of these, il-2r␣ was identified as one of the genes that was expressed through STAT5 tetramers (41). Among the STAT5 target genes analyzed, the expression pattern of il-2r␣ mRNA exhibited the closest relationship with the proliferation and tumorigenesis of cells expressing the JAK2 V617F mutant and EpoR mutants ( Fig.…”

Section: Discussionmentioning

confidence: 97%

Three Tyrosine Residues in the Erythropoietin Receptor Are Essential for Janus Kinase 2 V617F Mutant-induced Tumorigenesis

Ueda

Tago

Tamura

et al. 2017

Journal of Biological Chemistry

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Edited by Alex TokerThe erythropoietin receptor (EpoR) regulates development of blood cells, and its full activation normally requires the cytokine erythropoietin (Epo). In the case of myeloproliferative neoplasms (MPN), Epo-independent signaling through EpoR can be caused by a point mutation, V617F, in the EpoR-interacting tyrosine kinase Janus kinase 2 (JAK2). In cells expressing the JAK2 V617F mutant, eight tyrosine residues in the intracellular domain of EpoR are phosphorylated, but the functional role of these phosphorylations in oncogenic signaling is incompletely understood. Here, to evaluate the functional consequences of the phosphorylation of these tyrosine residues, we constructed an EpoR-8YF mutant in which we substituted all eight tyrosine residues with phenylalanine. Co-expression of EpoR-8YF with the JAK2 V617F mutant failed to induce cytokine-independent cell proliferation and tumorigenesis, indicating that JAK2-mediated EpoR phosphorylation is the reason for JAK2 V617F mutant-induced oncogenic signaling. An exhaustive mutational analysis of the eight EpoR tyrosine residues indicated that three of these residues, Tyr-343, Tyr-460, and Tyr-464, are required for the JAK2 V617F mutant to exhibit its oncogenic activity. We also showed that phosphorylation at these three residues was necessary for full activation of the transcription factor STAT5, which is a critical downstream factor of JAK2 V617F-induced oncogenic signaling. In contrast, Epo stimulation could moderately stimulate the proliferation of cells expressing wild type JAK2 and EpoR-8YF, suggesting that the requirement of the phosphorylation of these three tyrosine residues seems to be specific for the oncogenic proliferation provoked by V617F mutation. Collectively, these results have revealed that phosphorylation of Tyr-343, Tyr-460, and Tyr-464 in EpoR underlies JAK2 V617F mutant-induced tumorigenesis. We propose that the targeted disruption of this pathway has therapeutic utility for managing MPN.The proliferation and differentiation of hematopoietic cells are regulated by various cytokines that act through their specific receptors (1, 2). Erythropoietin (Epo) 2 is a pleiotropic cytokine that exhibits hematopoietic functions such as the development of erythrocytes through the erythropoietin receptor (EpoR) (3). Once Epo binds to EpoR, the non-receptor tyrosine kinase, Janus kinase 2 (JAK2), which interacts with EpoR, is activated. Activated JAK2 then immediately phosphorylates multiple tyrosine residues in the cytoplasmic domain of EpoR (4).EpoR contains the following eight tyrosine residues in its intracellular domain that are phosphorylated by JAK2: Tyr-343, Tyr-401, Tyr-429, Tyr-431, Tyr-443, Tyr-460, Tyr-464, and Tyr-479. The JAK2-induced phosphorylation of these tyrosine residues in EpoR provides a platform for the recruitment and activation of signaling mediators and is critical for Epo-induced cellular proliferation (3, 5, 6). Previous studies clarified that the transcription factor, signal transducer and activator of transcript...

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Critical Role of STAT5 Transcription Factor Tetramerization for Cytokine Responses and Normal Immune Function

Cited by 173 publications

References 42 publications

Opposing roles of STAT1 and STAT3 in IL-21 function in CD4 ⁺ T cells

Opposing roles of STAT1 and STAT3 in IL-21 function in CD4 ⁺ T cells

A new role for STAT3 as a regulator of chromatin topology

Three Tyrosine Residues in the Erythropoietin Receptor Are Essential for Janus Kinase 2 V617F Mutant-induced Tumorigenesis

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Critical Role of STAT5 Transcription Factor Tetramerization for Cytokine Responses and Normal Immune Function

Cited by 173 publications

References 42 publications

Opposing roles of STAT1 and STAT3 in IL-21 function in CD4 + T cells

Opposing roles of STAT1 and STAT3 in IL-21 function in CD4 + T cells

A new role for STAT3 as a regulator of chromatin topology

Three Tyrosine Residues in the Erythropoietin Receptor Are Essential for Janus Kinase 2 V617F Mutant-induced Tumorigenesis

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Opposing roles of STAT1 and STAT3 in IL-21 function in CD4 ⁺ T cells

Opposing roles of STAT1 and STAT3 in IL-21 function in CD4 ⁺ T cells