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• W2140155690 abstract "Naturally arising regulatory T (Treg) cells express the transcription factor FoxP3, which critically controls the development and function of Treg cells. FoxP3 interacts with another transcription factor Runx1 (also known as AML1). Here, we showed that Treg cell-specific deficiency of Cbfβ, a cofactor for all Runx proteins, or that of Runx1, but not Runx3, induced lymphoproliferation, autoimmune disease, and hyperproduction of IgE. Cbfb-deleted Treg cells exhibited impaired suppressive function in vitro and in vivo, with altered gene expression profiles including attenuated expression of FoxP3 and high expression of interleukin-4. The Runx complex bound to more than 3000 gene loci in Treg cells, including the Foxp3 regulatory regions and the Il4 silencer. In addition, knockdown of RUNX1 showed that RUNX1 is required for the optimal regulation of FoxP3 expression in human T cells. Taken together, our results indicate that the Runx1-Cbfβ heterodimer is indispensable for in vivo Treg cell function, in particular, suppressive activity and optimal expression of FoxP3. Naturally arising regulatory T (Treg) cells express the transcription factor FoxP3, which critically controls the development and function of Treg cells. FoxP3 interacts with another transcription factor Runx1 (also known as AML1). Here, we showed that Treg cell-specific deficiency of Cbfβ, a cofactor for all Runx proteins, or that of Runx1, but not Runx3, induced lymphoproliferation, autoimmune disease, and hyperproduction of IgE. Cbfb-deleted Treg cells exhibited impaired suppressive function in vitro and in vivo, with altered gene expression profiles including attenuated expression of FoxP3 and high expression of interleukin-4. The Runx complex bound to more than 3000 gene loci in Treg cells, including the Foxp3 regulatory regions and the Il4 silencer. In addition, knockdown of RUNX1 showed that RUNX1 is required for the optimal regulation of FoxP3 expression in human T cells. Taken together, our results indicate that the Runx1-Cbfβ heterodimer is indispensable for in vivo Treg cell function, in particular, suppressive activity and optimal expression of FoxP3. CD4+CD25+FoxP3+ naturally occurring regulatory T (Treg) cells play essential roles for the maintenance of immunological self-tolerance and immune homeostasis by actively suppressing aberrant or excessive immune responses harmful to the host (Sakaguchi et al., 2006Sakaguchi S. Ono M. Setoguchi R. Yagi H. Hori S. Fehervari Z. Shimizu J. Takahashi T. Nomura T. Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease.Immunol. Rev. 2006; 212: 8-27Crossref PubMed Scopus (1231) Google Scholar). Natural Treg cells specifically express the transcription factor FoxP3, which critically controls the development and the function of Treg cells as illustrated by FOXP3 mutations (Ochs et al., 2005Ochs H.D. Ziegler S.F. Torgerson T.R. FOXP3 acts as a rheostat of the immune response.Immunol. Rev. 2005; 203: 156-164Crossref PubMed Scopus (170) Google Scholar). FoxP3 deficiency or dysfunction in humans results in the development of IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome, which is characterized by severe autoimmune disease, allergy, and inflammatory bowel disease (Sakaguchi et al., 2006Sakaguchi S. Ono M. Setoguchi R. Yagi H. Hori S. Fehervari Z. Shimizu J. Takahashi T. Nomura T. Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease.Immunol. Rev. 2006; 212: 8-27Crossref PubMed Scopus (1231) Google Scholar). FoxP3 expression can confer suppressive activity to Treg cells, suppress the production of cytokines such as interleukin-2 (IL-2) and interferon-gamma (IFN-γ), and upregulate the expression of Treg cell-associated molecules including CD25 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (Fontenot et al., 2003Fontenot J.D. Gavin M.A. Rudensky A.Y. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells.Nat. Immunol. 2003; 4: 330-336Crossref PubMed Scopus (5716) Google Scholar, Hori et al., 2003Hori S. Nomura T. Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3.Science. 2003; 299: 1057-1061Crossref PubMed Scopus (36) Google Scholar, Khattri et al., 2003Khattri R. Cox T. Yasayko S.A. Ramsdell F. An essential role for Scurfin in CD4+CD25+ T regulatory cells.Nat. Immunol. 2003; 4: 337-342Crossref PubMed Scopus (2286) Google Scholar). Recent studies have shown that the gene regulatory function of FoxP3 requires its association with other transcription factors, such as NFAT (nuclear factor of activated T cells), NF-κB (nuclear factor-κB), and Runx1 (runt-related transcription factor 1), also known as AML1 (acute myeloid leukemia 1), and with histone deacetylases and acetyltransferases (Bettelli et al., 2005Bettelli E. Dastrange M. Oukka M. Foxp3 interacts with nuclear factor of activated T cells and NF-kappa B to repress cytokine gene expression and effector functions of T helper cells.Proc. Natl. Acad. Sci. USA. 2005; 102: 5138-5143Crossref PubMed Scopus (436) Google Scholar, Li et al., 2007Li B. Samanta A. Song X. Iacono K.T. Bembas K. Tao R. Basu S. Riley J.L. Hancock W.W. Shen Y. et al.FOXP3 interactions with histone acetyltransferase and class II histone deacetylases are required for repression.Proc. Natl. Acad. Sci. USA. 2007; 104: 4571-4576Crossref PubMed Scopus (317) Google Scholar, Ono et al., 2007Ono M. Yaguchi H. Ohkura N. Kitabayashi I. Nagamura Y. Nomura T. Miyachi Y. Tsukada T. Sakaguchi S. Foxp3 controls regulatory T-cell function by interacting with AML1/Runx1.Nature. 2007; 446: 685-689Crossref PubMed Scopus (489) Google Scholar, Wu et al., 2006Wu Y. Borde M. Heissmeyer V. Feuerer M. Lapan A.D. Stroud J.C. Bates D.L. Guo L. Han A. Ziegler S.F. et al.FOXP3 controls regulatory T cell function through cooperation with NFAT.Cell. 2006; 126: 375-387Abstract Full Text Full Text PDF PubMed Scopus (864) Google Scholar). Yet, the precise molecular mechanisms by which FoxP3 controls Treg cell function remain to be elucidated. The Runx (AML) transcription factors consist of three members: Runx1 (AML1), Runx2 (AML3), and Runx3 (AML2) (van Wijnen et al., 2004van Wijnen A.J. Stein G.S. Gergen J.P. Groner Y. Hiebert S.W. Ito Y. Liu P. Neil J.C. Ohki M. Speck N. Nomenclature for Runt-related (RUNX) proteins.Oncogene. 2004; 23: 4209-4210Crossref PubMed Scopus (94) Google Scholar). All Runx proteins bind to the specific DNA consensus sequences (ACCACA) via a highly conserved DNA-binding runt domain. Runx binding is stabilized by the association with Cbfβ (core-binding factor β), a non-DNA-binding cofactor essential for the function of all Runx proteins (Speck, 2001Speck N.A. Core binding factor and its role in normal hematopoietic development.Curr. Opin. Hematol. 2001; 8: 192-196Crossref PubMed Scopus (62) Google Scholar). The Runx-Cbfβ heterodimeric complex interacts with other DNA-binding transcription factors, coactivators, or corepressors to either activate or repress expression of the target genes in a context-dependent manner (Durst and Hiebert, 2004Durst K.L. Hiebert S.W. Role of RUNX family members in transcriptional repression and gene silencing.Oncogene. 2004; 23: 4220-4224Crossref PubMed Scopus (147) Google Scholar, Taniuchi and Littman, 2004Taniuchi I. Littman D.R. Epigenetic gene silencing by Runx proteins.Oncogene. 2004; 23: 4341-4345Crossref PubMed Scopus (49) Google Scholar). In addition to the essential requirement of Runx proteins for definitive hematopoiesis (de Bruijn and Speck, 2004de Bruijn M.F. Speck N.A. Core-binding factors in hematopoiesis and immune function.Oncogene. 2004; 23: 4238-4248Crossref PubMed Scopus (188) Google Scholar), Runx1 and Runx3 are crucially involved in the differentiation and function of peripheral T cells (Djuretic et al., 2007Djuretic I.M. Levanon D. Negreanu V. Groner Y. Rao A. Ansel K.M. Transcription factors T-bet and Runx3 cooperate to activate Ifng and silence Il4 in T helper type 1 cells.Nat. Immunol. 2007; 8: 145-153Crossref PubMed Scopus (363) Google Scholar, Komine et al., 2003Komine O. Hayashi K. Natsume W. Watanabe T. Seki Y. Seki N. Yagi R. Sukzuki W. Tamauchi H. Hozumi K. et al.The Runx1 transcription factor inhibits the differentiation of naive CD4+ T cells into the Th2 lineage by repressing GATA3 expression.J. Exp. Med. 2003; 198: 51-61Crossref PubMed Scopus (101) Google Scholar, Naoe et al., 2007Naoe Y. Setoguchi R. Akiyama K. Muroi S. Kuroda M. Hatam F. Littman D.R. Taniuchi I. Repression of interleukin-4 in T helper type 1 cells by Runx/Cbf beta binding to the Il4 silencer.J. Exp. Med. 2007; 204: 1749-1755Crossref PubMed Scopus (185) Google Scholar, Zhang et al., 2008Zhang F. Meng G. Strober W. Interactions among the transcription factors Runx1, RORgammat and Foxp3 regulate the differentiation of interleukin 17-producing T cells.Nat. Immunol. 2008; 9: 1297-1306Crossref PubMed Scopus (363) Google Scholar) as well as thymic T cell development (Grueter et al., 2005Grueter B. Petter M. Egawa T. Laule-Kilian K. Aldrian C.J. Wuerch A. Ludwig Y. Fukuyama H. Wardemann H. Waldschuetz R. et al.Runx3 regulates integrin alpha E/CD103 and CD4 expression during development of CD4-/CD8+ T cells.J. Immunol. 2005; 175: 1694-1705Crossref PubMed Scopus (88) Google Scholar, Sato et al., 2005Sato T. Ohno S. Hayashi T. Sato C. Kohu K. Satake M. Habu S. Dual functions of Runx proteins for reactivating CD8 and silencing CD4 at the commitment process into CD8 thymocytes.Immunity. 2005; 22: 317-328Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, Setoguchi et al., 2008Setoguchi R. Tachibana M. Naoe Y. Muroi S. Akiyama K. Tezuka C. Okuda T. Taniuchi I. Repression of the transcription factor Th-POK by Runx complexes in cytotoxic T cell development.Science. 2008; 319: 822-825Crossref PubMed Scopus (177) Google Scholar, Taniuchi et al., 2002Taniuchi I. Osato M. Egawa T. Sunshine M.J. Bae S.C. Komori T. Ito Y. Littman D.R. Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development.Cell. 2002; 111: 621-633Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar, Woolf et al., 2003Woolf E. Xiao C. Fainaru O. Lotem J. Rosen D. Negreanu V. Bernstein Y. Goldenberg D. Brenner O. Berke G. et al.Runx3 and Runx1 are required for CD8 T cell development during thymopoiesis.Proc. Natl. Acad. Sci. USA. 2003; 100: 7731-7736Crossref PubMed Scopus (283) Google Scholar). We have previously shown that Runx1 binds to the promoter of the Il2 and Ifng genes and upregulates the production of IL-2 and IFN-γ, respectively. Further, FoxP3 binds to Runx1 in Treg cells, thereby repressing Il2 and Ifng and activating the genes encoding CD25 (Il2ra) and CTLA-4 (Ctla4) (Ono et al., 2007Ono M. Yaguchi H. Ohkura N. Kitabayashi I. Nagamura Y. Nomura T. Miyachi Y. Tsukada T. Sakaguchi S. Foxp3 controls regulatory T-cell function by interacting with AML1/Runx1.Nature. 2007; 446: 685-689Crossref PubMed Scopus (489) Google Scholar). We have also shown that, in vitro, FoxP3 can interact with the other members of the Runx family, Runx2 and Runx3, in addition to Runx1 (Ono et al., 2007Ono M. Yaguchi H. Ohkura N. Kitabayashi I. Nagamura Y. Nomura T. Miyachi Y. Tsukada T. Sakaguchi S. Foxp3 controls regulatory T-cell function by interacting with AML1/Runx1.Nature. 2007; 446: 685-689Crossref PubMed Scopus (489) Google Scholar). These findings collectively suggest that the Runx-dependent transcription program operating in conventional T cells could be modulated in Treg cells through interaction with FoxP3. Yet, it remains obscure whether the Runx-mediated gene regulation is indeed required for the in vivo function of Treg cells. In this report, we have generated mice with Treg cell-specific conditional deletion of Cbfb to analyze in vivo the possible contribution of Runx-dependent gene regulation to Treg cell function because all Runx proteins need to form a heterodimeric complex with Cbfβ for exerting transcriptinal activities and Cbfβ deficiency disrupts the function of the Runx complex (Speck, 2001Speck N.A. Core binding factor and its role in normal hematopoietic development.Curr. Opin. Hematol. 2001; 8: 192-196Crossref PubMed Scopus (62) Google Scholar). Here, we showed that Treg cell-specific Cbfβ-deficient mice spontaneously developed lymphoproliferation, autoimmune disease, and IgE hyperproduction and that Cbfb-deleted Treg cells exhibited impaired suppressive activity both in vitro and in vivo. In addition, Treg cell-specific conditional deletion of Runx1, but not Runx3, led to the development of immunological diseases similar to those observed in Treg cell-specific Cbfβ deficiency. Our findings thus indicate that the heterodimeric Runx1-Cbfβ complex is an indispensable transcription regulator for in vivo functions of Treg cells and that it is a potential therapeutic target for controlling physiological and pathological immune responses. To determine whether Runx proteins were required for in vivo function of FoxP3+ Treg cells, we generated Treg cell-specific Cbfb-deleted mice by crossing mice harboring LoxP-flanked Cbfb allele with Foxp3-Ires-Cre (FIC) knockin mice, which faithfully express Cre recombinase in FoxP3+ T cells (Naoe et al., 2007Naoe Y. Setoguchi R. Akiyama K. Muroi S. Kuroda M. Hatam F. Littman D.R. Taniuchi I. Repression of interleukin-4 in T helper type 1 cells by Runx/Cbf beta binding to the Il4 silencer.J. Exp. Med. 2007; 204: 1749-1755Crossref PubMed Scopus (185) Google Scholar, Wing et al., 2008Wing K. Onishi Y. Prieto-Martin P. Yamaguchi T. Miyara M. Fehervari Z. Nomura T. Sakaguchi S. CTLA-4 control over Foxp3+ regulatory T cell function.Science. 2008; 322: 271-275Crossref PubMed Scopus (1914) Google Scholar). FIC-mediated genomic deletion of LoxP-flanked region occurred in almost 100% CD4+FoxP3+ cells and a small population of CD8+ T cells (Wing et al., 2008Wing K. Onishi Y. Prieto-Martin P. Yamaguchi T. Miyara M. Fehervari Z. Nomura T. Sakaguchi S. CTLA-4 control over Foxp3+ regulatory T cell function.Science. 2008; 322: 271-275Crossref PubMed Scopus (1914) Google Scholar). With genomic DNA-PCR analyses of subpopulations of thymocytes and splenic T cells from CbfbF/F: FIC mice, inactivation of the Cbfb gene was initiated specifically in CD4-single positive (CD4SP) HSAloCD25hi mature thymocytes and was completed in CD4+CD25hi splenic T cells, indicating Treg cell-specific deletion of the Cbfb gene (Figure 1A, left). Some of the CD4+CD25− T cells also harbored the genomic Cbfb deletion (Figure 1A, left). This can be attributed to the presence of CD4+CD25-FoxP3+ Treg cells in this CD4+CD25− population (Figure S1 and Supplemental Data available online). As a consequence of the gene inactivation, the Cbfβ protein was undetectable in CD4+CD25hi splenocytes, although a substantial amount of the Cbfβ protein remained in CD4SP HSAloCD25hi thymocytes (Figure 1A, right). Thus, the Cbfβ protein is gradually decreased in FoxP3+ cells after Cbfb gene deletion in the thymus and almost completely lost in the periphery, which is consistent with a similar finding with conditional Cbfb deletion by Cd4-Cre transgene (Naoe et al., 2007Naoe Y. Setoguchi R. Akiyama K. Muroi S. Kuroda M. Hatam F. Littman D.R. Taniuchi I. Repression of interleukin-4 in T helper type 1 cells by Runx/Cbf beta binding to the Il4 silencer.J. Exp. Med. 2007; 204: 1749-1755Crossref PubMed Scopus (185) Google Scholar). Notably, CbfbF/F: FIC mice spontaneously developed severe lymphadenopathy and splenomegaly with significantly increased numbers of splenocytes by 14 weeks of age (Figure 1B). Various types of immune cells including CD4+ T cells, CD8+ T cells, B cells, macrophages, and dendritic cells increased in the enlarged spleens of CbfbF/F: FIC mice (Figure S2). Seventy percent of CbfbF/F: FIC mice developed histologically evident gastritis accompanying high titers of anti-gastric parietal cell autoantibodies in the sera, whereas control CbfbF/+: FIC littermates did not (Figures 1C and 1D). Flow cytometric analysis revealed that non-Treg T cells, i.e., CD4+FoxP3− conventional T cells and CD8+ T cells, in CbfbF/F: FIC mice showed an activated or memory phenotype; e.g., CD69+, CD122+, CD44hi, and CD62Llo (Figure 1E). CD4+ and CD8+ T cells abundantly produced cytokines such as IFN-γ, IL-2, IL-4, and IL-10, as revealed by intracellular cytokine staining after stimulation with PMA and ionomycin (Figure 1F). In addition, CbfbF/F: FIC mice showed 10-fold elevated concentrations of serum IgE and 1.5-fold increase in serum IgG (Figure 1G). Thus, Treg cell-specific Cbfβ deficiency produced autoimmune disease and led to hyperproduction of IgE. To analyze the mechanism of autoimmunity caused by Treg cell-specific Cbfb deficiency, we attempted to determine whether thymic generation and differentiation of Treg cells, their peripheral survival, or their suppressive function was affected by the deficiency. There was no significant difference in the number of total or FoxP3+ thymocytes between CbfbF/F: FIC and CbfbF/+: FIC mice (Figure 2A and Figure S3). HSAloCD25hiFoxP3+CD4SP thymic Treg cells normally developed in CbfbF/F: FIC mice as in control CbfbF/+: FIC mice (Figure 2B). Whereas Runx proteins were required for the differentiation of immature thymocytes to TCRβhiHSAloCD4SP mature thymocytes (Egawa et al., 2007Egawa T. Tillman R.E. Naoe Y. Taniuchi I. Littman D.R. The role of the Runx transcription factors in thymocyte differentiation and in homeostasis of naive T cells.J. Exp. Med. 2007; 204: 1945-1957Crossref PubMed Scopus (211) Google Scholar), the generation of FoxP3+TCRβhiHSAloCD4SP mature thymocytes was not markedly impaired in CbfbF/F: FIC mice (Figure S4). Residual Cbfβ protein might be sufficient to support differentiation and maturation of FoxP3+ cells in the thymus (Figure 1A). We next examined whether Treg cell homeostasis was impaired in the periphery of Cbfb-deleted mice. The proportion of CD4+FoxP3+ Treg cells to total CD4+ T cells and the absolute number of Treg cells were slightly higher in CbfbF/F: FIC mice than in CbfbF/+: FIC mice (Figures 2C and 2D). Notably, Treg cells in CbfbF/F: FIC mice showed substantially decreased expression of FoxP3, compared to those in control mice (Figure 2D). Expression of Ki-67, a cellular marker for proliferation, indicated that an equivalent or larger proportion of Treg cells were active in cell cycle in CbfbF/F: FIC mice compared with control mice (Figure 2E). In CbfbF/F: FIC mice, Ki-67− resting Treg cells showed reduced expression of FoxP3, whereas Ki-67hi proliferating Treg cells expressed FoxP3 at equivalent amounts as Ki-67hi Treg cells in control mice (Figure 2E). In vivo BrdU labeling also revealed that CD4+CD25hi splenocytes in CbfbF/F: FIC mice were more actively proliferating than those in CbfbF/+: FIC mice (Figure 2F). Treg cells in the former expressed only slightly lower amounts of CD127 (IL-7 receptor α chain) and were not apoptotic according to 7-AAD (7-amino-actinomycin D) and Annexin V staining, in accord with the previous finding that Runx1-deficient Treg cells in Runx1F/F: Cd4-Cre mice were apoptosis resistant (Egawa et al., 2007Egawa T. Tillman R.E. Naoe Y. Taniuchi I. Littman D.R. The role of the Runx transcription factors in thymocyte differentiation and in homeostasis of naive T cells.J. Exp. Med. 2007; 204: 1945-1957Crossref PubMed Scopus (211) Google Scholar) (Figures 2G and 2H). Phenotypically, Cbfb-deleted Treg cells expressed CD25 and glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) at higher amounts and CTLA-4 at equivalent amounts compared to control Treg cells, whereas they scarcely expressed CD103 in accord with the finding that Runx3 controls CD103 expression (Grueter et al., 2005Grueter B. Petter M. Egawa T. Laule-Kilian K. Aldrian C.J. Wuerch A. Ludwig Y. Fukuyama H. Wardemann H. Waldschuetz R. et al.Runx3 regulates integrin alpha E/CD103 and CD4 expression during development of CD4-/CD8+ T cells.J. Immunol. 2005; 175: 1694-1705Crossref PubMed Scopus (88) Google Scholar) (Figure 3A). Neither Cbfb-deleted nor control Treg cells proliferated in response to in vitro polyclonal TCR stimulation with anti-CD3 (Figure 3B). Yet, Cbfb-deleted Treg cells were less suppressive in vitro (Figure 3C). In addition, they failed to prevent the development of colitis and weight loss in SCID mice when cotransferred with BALB/c CD4+CD25−CD45RBhi T cells, in contrast to effective disease prevention by cotransfer of control Treg cells (Figures 3D–3F). Similarly, Cbfb-deleted Treg cells failed to suppress the development of gastritis (data not shown). Cbfb-deleted Treg cells survived when transferred to SCID mice (Figure S5A), indicating that the impaired in vivo suppressive activity of Cbfb-deleted Treg cells was not due to their shorter survival. In addition, the attenuated CD103 expression in Cbfb-deleted Treg cells (Figure S5B) would not be responsible for the impaired Treg cell function because others reported that Treg cell-mediated control of colitis did not require CD103 expression by Treg cells (Annacker et al., 2005Annacker O. Coombes J.L. Malmstrom V. Uhlig H.H. Bourne T. Johansson-Lindbom B. Agace W.W. Parker C.M. Powrie F. Essential role for CD103 in the T cell-mediated regulation of experimental colitis.J. Exp. Med. 2005; 202: 1051-1061Crossref PubMed Scopus (392) Google Scholar). Collectively, these findings indicate that failure in Treg cell-mediated self-tolerance in CbfbF/F: FIC mice is not due to numerical deficiency, reduced proliferation, or enhanced apoptosis of FoxP3+ Treg cells, but due to their impaired suppressive activity. Treg cells hardly produce cytokines such as IL-2, IFN-γ, and IL-4 (Sakaguchi et al., 2006Sakaguchi S. Ono M. Setoguchi R. Yagi H. Hori S. Fehervari Z. Shimizu J. Takahashi T. Nomura T. Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease.Immunol. Rev. 2006; 212: 8-27Crossref PubMed Scopus (1231) Google Scholar). Flow cytometric analysis revealed that a larger proportion of FoxP3+ Treg cells from CbfbF/F: FIC mice produced IL-4 and IL-10 compared to Treg cells from control mice, whereas there were no substantial differences in the percentage of IL-2- or IFN-γ-producing cells among FoxP3+ Treg cells (Figure 3G). Few IL-17-expressing FoxP3+ Treg cells were present in both groups of mice (Figure 3G). The amount of mRNA for each cytokine in CD4+CD25hi cells in CbfbF/F: FIC and control mice correlated with the protein expression. However, mRNA for IL-17, which was detectable in CbfbF/+: FIC mice, was substantially lower in CbfbF/F: FIC mice (Figure 3H). Next, the expression of transcription factors Foxp3, Tbx21, Gata3, and Rorgt, all of which are essential for Th or Treg cell lineage differentiation, were examined in Cbfb-deleted Treg cells. Foxp3 mRNA expression decreased in Cbfb-deleted Treg cells, which is consistent with decreased FoxP3 expression at the protein level (Figure S6). In contrast to the hyperproduction of Th2 cell cytokines IL-4 and IL-10, mRNA expression of Th2 cell-specific transcription factor Gata3 in Cbfb-deleted Treg cells was equivalent to that in control Treg cells, whereas Cbfb-deleted Treg cells showed higher expression of Th1 cell-specific transcription factor Tbx21. Thus, hyperproduction of Th2 cell cytokines by Cbfb-deleted Treg cells was not due to overexpression of Gata3, although it has been reported that Runx1 represses Gata3 expression in conventional CD4+ T cells (Komine et al., 2003Komine O. Hayashi K. Natsume W. Watanabe T. Seki Y. Seki N. Yagi R. Sukzuki W. Tamauchi H. Hozumi K. et al.The Runx1 transcription factor inhibits the differentiation of naive CD4+ T cells into the Th2 lineage by repressing GATA3 expression.J. Exp. Med. 2003; 198: 51-61Crossref PubMed Scopus (101) Google Scholar). The expression of Rorgt, which controls the differentiation of IL-17-producing Th17 cells, also decreased in Cbfb-deleted Treg cells compared with control Treg cells (Figure S6). Taken together, our findings show that Cbfβ-deficient Treg cells transcribed Il17a and Rorgt to lesser extents than control Treg cells, whereas Il4, Il10, and Tbx21 were more actively transcribed in the former. To elucidate the molecular mechanisms underlying the dysfunction of Cbfb-deleted Treg cells, we examined gene expression profiles of CD4+CD25hi cells from CbfbF/F: FIC and littermate CbfbF/+: FIC mice by expression microarray. We first focused on the previously described “Treg cell signature” genes, which are differentially expressed between Treg cells and conventional CD4+ T cells and therefore thought to be closely related to Treg cell-intrinsic properties including suppressive function (Hill et al., 2007Hill J.A. Feuerer M. Tash K. Haxhinasto S. Perez J. Melamed R. Mathis D. Benoist C. Foxp3 transcription-factor-dependent and -independent regulation of the regulatory T cell transcriptional signature.Immunity. 2007; 27: 786-800Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). Sixty-nine signature genes including Socs2 and Nrp1 were found to be differentially expressed in Cbfb-deleted Treg cells (unpaired t test, p < 0.05. see Table S1). Yet, there were not significant differences in the expression of many well-known Treg cell-associated genes, such as Ctla4, Tnfrsf18 (Gitr), Gzmb, Folr4, and Gpr83, between Cbfb-deleted and control Treg cells (Figure 4A). Decreased mRNA expression of Itgae (CD103) in Cbfb-deleted Treg cells was consistent with the aforementioned flow cytometry results (Figure 4A and Figure 3A). Using the false discovery rate (FDR)-controlling procedure (FDR < 0.2), we further attempted to determine other genes that were differentially expressed in Cbfb-deleted Treg cells. We found that 22 and 24 genes were significantly up- or downregulated, respectively, in Cbfb-deleted Treg cells (Figure 4B and Table S2). Differentially expressed molecules included IL-4, CCR5, leukotriene B4 receptor 1 (Ltb4r1, also called BLT1), and CD160, which are secreted or have extracellular regions possibly involved in cellular interactions. To further investigate Runx-dependent gene regulation in Treg cells, we attempted to identify target genes of the Runx complex. Genome-wide analysis with chromatin immunoprecipitation (ChIP) coupled with promoter tiling array showed that the Runx complex bound to the promoter regions of 3566 genes including Foxp3 (Figure 5A and Table S3). Similar analysis with the customized array covering the Foxp3 gene locus revealed that the Runx complex bound to several regions of the Foxp3 gene in Treg cells prepared from wild-type BALB/c mice (Figure 5B, left). It has been reported that the following three conserved noncoding sequences (CNSs) contribute to Foxp3 expression: one CNS located in 0.5 kb upstream of the transcription start site (CNS1) and two CNSs located in the first intron (CNS2 and CNS3) (Kim and Leonard, 2007Kim H.P. Leonard W.J. CREB/ATF-dependent T cell receptor-induced FoxP3 gene expression: A role for DNA methylation.J. Exp. Med. 2007; 204: 1543-1551Crossref PubMed Scopus (471) Google Scholar, Mantel et al., 2006Mantel P.Y. Ouaked N. Ruckert B. Karagiannidis C. Welz R. Blaser K. Schmidt-Weber C.B. Molecular mechanisms underlying FOXP3 induction in human T cells.J. Immunol. 2006; 176: 3593-3602Crossref PubMed Scopus (338) Google Scholar, Tone et al., 2008Tone Y. Furuuchi K. Kojima Y. Tykocinski M.L. Greene M.I. Tone M. Smad3 and NFAT cooperate to induce Foxp3 expression through its enhancer.Nat. Immunol. 2008; 9: 194-202Crossref PubMed Scopus (573) Google Scholar). The binding of the Runx complex to CNS1 and CNS3 of the Foxp3 gene in Treg cells was detected, although the binding to CNS2 was not examined because of unavailability of appropriate probes for this region (Figure 5B, left). Conventional ChIP assays revealed that the Runx complex bound to CNS2 as well as CNS1 and CNS3 of the Foxp3 gene in Treg cells, but not to the region at 1 kb upstream of Zbtb7b (Th-POK) exon Ia (UP1), which was used as a negative control locus (Setoguchi et al., 2008Setoguchi R. Tachibana M. Naoe Y. Muroi S. Akiyama K. Tezuka C. Okuda T. Taniuchi I. Repression of the transcription factor Th-POK by Runx complexes in cytotoxic T cell development.Science. 2008; 319: 822-825Crossref PubMed Scopus (177) Google Scholar) (Figure 5B, right). In CNS1 and CNS3, but not in CNS2, there are conserved Runx-binding consensus sites (ACCACA) (Figure S7 and S8), suggesting that the Runx complex might bind to CNS2 via associating with other molecules. Because Cbfb-deleted Treg cells showed IL-4 hyperproduction without overexpression of Gata3, we investigated how the Runx complex controlled Il4 expression in Treg cells. By promoter tiling array analysis, we could not detect the binding of the Runx complex to the Il4 promoter region in Treg cells. However, by coupling ChIP assay with custom tiling array for the Th2 cytokine locus (∼200 base intervals), we found that the Runx complex bound to the Il4 silencer in Treg cells (Figure 5C, left). We further confirmed this binding by conventional ChIP assays (Figure 5C, right). Thus, the Runx complex may repress Il4 expression in Treg cells via binding to the Il4 silencer as in naive CD4+ T cells and Th1 cells, as we and others have recently reported (Djuretic et al., 2007Djuretic I.M. Levanon D. Negreanu V. Groner Y. Rao A. Ansel K.M. Transcription factors T-bet and Runx3 cooperate to activate Ifng and silence Il4 in T helper type 1 cells.Nat. Immunol. 2007; 8: 145-153Crossref PubMed Scopus (363) Google Scholar, Naoe et al., 2007Naoe Y. Setoguchi R. Akiyama K. Muroi S. Kuroda M. Hatam F. Littman D.R. Taniuchi I. Repression of interleukin-4 in T helper type 1 cells by Runx/Cbf beta binding to the Il4 silencer.J." @default.
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• W2140155690 title "Indispensable Role of the Runx1-Cbfβ Transcription Complex for In Vivo-Suppressive Function of FoxP3+ Regulatory T Cells" @default.
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