Deletion of a conserved Il4 silencer impairs T helper type 1–mediated immunity

Ansel, K. Mark; Greenwald, Rebecca J.; Agarwal, Suneet; Bassing, Craig H.; Monticelli, Silvia; Interlandi, Jeneen; Djuretic, Ivana; Lee, Dong U.; Sharpe, Arlene H.; Alt, Frederick W.; Rao, Anjana

doi:10.1038/ni1135

Cited by 104 publications

(128 citation statements)

References 48 publications

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“…T cell isolation, retroviral transduction, and differentiation were performed as described previously (43). A more detailed description is included in the SI Text.…”

Section: Methodsmentioning

confidence: 99%

Requirement for balanced Ca/NFAT signaling in hematopoietic and embryonic development

Müller

Sasaki

Stevanović

et al. 2009

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

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NFAT transcription factors are highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by the phosphatase calcineurin, NFAT proteins translocate to the nucleus, where they orchestrate developmental and activation programs in diverse cell types. NFAT is rephosphorylated and inactivated through the concerted action of at least 3 different kinases: CK1, GSK-3, and DYRK. The major docking sites for calcineurin and CK1 are strongly conserved throughout vertebrate evolution, and conversion of either the calcineurin docking site to a high-affinity version or the CK1 docking site to a low-affinity version results in generation of hyperactivable NFAT proteins that are still fully responsive to stimulation. In this study, we generated transgenic mice expressing hyperactivable versions of NFAT1 from the ROSA26 locus. We show that hyperactivable NFAT increases the expression of NFAT-dependent cytokines by differentiated T cells as expected, but exerts unexpected signal-dependent effects during T cell differentiation in the thymus, and is progressively deleterious for the development of B cells from hematopoietic stem cells. Moreover, progressively hyperactivable versions of NFAT1 are increasingly deleterious for embryonic development, particularly when normal embryos are also present in utero. Forced expression of hyperactivable NFAT1 in the developing embryo leads to mosaic expression in many tissues, and the hyperactivable proteins are barely tolerated in organs such as brain, and cardiac and skeletal muscle. Our results highlight the need for balanced Ca/NFAT signaling in hematopoietic stem cells and progenitor cells of the developing embryo, and emphasize the evolutionary importance of kinase and phosphatase docking sites in preventing inappropriate activation of NFAT.T he activities of many signaling proteins and transcription factors are tightly regulated by phosphorylation and dephosphorylation. Protein kinases and phosphatases bind to specific docking sites on these intracellular proteins to allow their activation or inactivation at the appropriate location and time. A well-studied example of a transcription factor regulated in this fashion is nuclear factor of activated T cells (NFAT) (1-3). In resting cells, NFAT proteins are highly phosphorylated and reside in the cytoplasm; upon cell activation, they are dephosphorylated by the calcium/calmodulindependent phosphatase calcineurin and translocate to the nucleus. NFAT transcription factors play a key role in orchestrating diverse developmental programs, including those of the immune, central nervous, cardiovascular, and musculoskeletal systems (4-11). NFAT also is implicated in maintaining the quiescent state of stem cells in the skin (12).NFAT activation is initiated by dephosphorylation of the NFAT regulatory domain, a conserved 300-amino acid region located N-terminal to the DNA-binding domain (Fig. 1A) (2, 13). The phosphorylated residues (serines) in this domain are distributed among several classes of conserved serine...

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“…T cell isolation, retroviral transduction, and differentiation were performed as described previously (43). A more detailed description is included in the SI Text.…”

Section: Methodsmentioning

confidence: 99%

Requirement for balanced Ca/NFAT signaling in hematopoietic and embryonic development

Müller

Sasaki

Stevanović

et al. 2009

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

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“…Chromatin remodeling and enhancement of transcription of a gene are regulated by trans-acting factors that bind to specific DNA regulatory elements. Many laboratories including ours have shown that the Th2 cytokine locus is regulated by a number of regulatory elements including enhancers [CNS-1/ HSS (9, 20-22), CNS-2/HSV (20, 23), IE/HSII (20)], a silencer (HSIV) (20,24), and a locus control region (LCR) (25).We have shown previously that the expression of Th2 cytokines is coordinately regulated by the Th2 LCR that is located in the 3′ region of the rad50 gene (25). The Th2 LCR is composed of four DNase I-hypersensitive sites, namely RHS4, RHS5, RHS6, and RHS7 (13, 26).…”

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confidence: 99%

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confidence: 99%

Th2 LCR is essential for regulation of Th2 cytokine genes and for pathogenesis of allergic asthma

Koh

Hwang

Kim

et al. 2010

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

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Previous studies have shown that Th2 cytokine genes on mouse chromosome 11 are coordinately regulated by the Th2 locus control region (LCR). To examine the in vivo function of Th2 LCR, we generated CD4-specific Th2 LCR-deficient (cLCR KO) mice using CreLoxP recombination. The number of CD4 T cells in the cLCR KO mouse was comparable to that in wild-type mice. The expression of Th2 cytokines was dramatically reduced in in vitro-stimulated naïve CD4 T cells. Deletion of the LCR led to a loss of general histone H3 acetylation and histone H3-K4 methylation, and demethylation of DNA in the Th2 cytokine locus. Upon ovalbumin challenge in the mouse model of allergic asthma, cLCR KO mice exhibited marked reduction in the recruitment of eosinophils and lymphocytes in the bronchoalveolar lavage fluid, serum IgE level, lung airway inflammation, mucus production in the airway walls, and airway hyperresponsiveness. These results directly demonstrate that the Th2 LCR is critically important in the regulation of Th2 cytokine genes, in chromatin remodeling of the Th2 cytokine locus, and in the pathogenesis of allergic asthma.chromatin remodeling | differentiation | locus control region C D4 T cells play an essential role in the activation and regulation of a variety of immune responses. Effector CD4 T cells are composed of several different subsets including Th1, Th2, and Th17 cells (1-5). The expression of subset-specific cytokines is critical for the differentiation and function of T helper cells (6, 7). The Th2 cytokine genes, il4, il5, and il13, are clustered on human chromosome 5 and mouse chromosome 11. The Th2 cytokine locus undergoes structural changes in its chromatin upon Th2 cell differentiation to accommodate the high level expression of Th2 cytokine genes. The changes include acquisition of DNase I hypersensitivity (8, 9), restriction enzyme accessibility (10), histone acetylation (11-16), histone methylation (17), and DNA demethylation (8,13,18,19). Chromatin remodeling and enhancement of transcription of a gene are regulated by trans-acting factors that bind to specific DNA regulatory elements. Many laboratories including ours have shown that the Th2 cytokine locus is regulated by a number of regulatory elements including enhancers [CNS-1/ HSS (9, 20-22), CNS-2/HSV (20, 23), IE/HSII (20)], a silencer (HSIV) (20,24), and a locus control region (LCR) (25).We have shown previously that the expression of Th2 cytokines is coordinately regulated by the Th2 LCR that is located in the 3′ region of the rad50 gene (25). The Th2 LCR is composed of four DNase I-hypersensitive sites, namely RHS4, RHS5, RHS6, and RHS7 (13, 26). We have shown that deletion of RHS7 causes marked reduction of Th2 cytokine genes under Th0 conditions and partial reduction under Th2 conditions (27), suggesting that RHS7 is important for Th2 cytokine expression. The Th2 LCR interacts with the promoters of Th2 cytokine genes through intrachromosomal associations (28). Deletion of RHS7 disrupts these (27), suggesting that RHS7 is critical for these int...

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“…Likewise, the mechanisms responsible for locking in the activity or silencing of cytokine genes during helper T cell maturation are still incompletely defined. We do not yet know whether the stability we observe is truly epigenetic, maintained by self-propagating chromatin marks, or is more accurately genetic, maintained by trans-activators other than T-bet, Hlx, or GATA-3, and conventional repressors acting at negative cis-elements (32). In either case, it is remarkable, although not fully explicable, that recruitment of an engrailed repression domain has such a minor effect on cytokine expression in mature cells.…”

Section: Discussionmentioning

confidence: 92%

Transcriptional Activators of Helper T Cell Fate Are Required for Establishment but Not Maintenance of Signature Cytokine Expression

Martins

Hutchins

Reiner

2005

The Journal of Immunology

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The stability of helper T cell fates is not well understood. Using conditional introduction of dominant-negative factors, we now show that T-bet and GATA-3 are far more critical in establishment than maintenance of IFN-γ and IL-4 activity during Th1 and Th2 maturation, respectively. We also show that a genetic interaction between T-bet and its target Hlx seems to be required for Th1 maturation, but that Hlx may also be dispensable for maintenance of a transcriptionally permissive ifng gene. In parallel to progressive activator independence in the permissive lineage, the ifng gene becomes more recalcitrant to switching as the forbidden lineage matures. T-bet plus Hlx can disrupt ifng silencing when introduced into developing Th2 cells, but they fail to perturb ifng silencing in mature Th2 cells. In contrast, a hypermorphic allele of T-bet can reverse silencing of the ifng gene in mature Th2 cells. These results suggest that signature gene activity of helper T cells is initially plastic but later becomes epigenetically fixed and offer an initial strategy for inducing mature cells to switch their fate.

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Deletion of a conserved Il4 silencer impairs T helper type 1–mediated immunity

Cited by 104 publications

References 48 publications

Requirement for balanced Ca/NFAT signaling in hematopoietic and embryonic development

Requirement for balanced Ca/NFAT signaling in hematopoietic and embryonic development

Th2 LCR is essential for regulation of Th2 cytokine genes and for pathogenesis of allergic asthma

Transcriptional Activators of Helper T Cell Fate Are Required for Establishment but Not Maintenance of Signature Cytokine Expression

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