- Email: [email protected]
Chromatin remodeling of the Th2 cytokine gene loci
International Congress Series 1285 (2005) 137 – 144
www.ics-elsevier.com
Chromatin remodeling of the Th2 cytokine gene loci T. Nakayama a,*, M. Yamashita a, M. Kimura a, A. Hasegawa a, M. Omori a, M. Inami a, S. Motohashi a, M. Kitajima a, K. Hashimoto b, H. Hosokawa a, R. Shinnakasu a a
Department of Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan Department of Life and Environmental Sciences and High Technology Research Center, Chiba Institute of Technology, Narashino, Tsudanuma, Chiba, Japan
b
Abstract. Differentiation of naive CD4 T cells into Th2 cells is accompanied by chromatin remodeling including hyperacetylation of histones H3 and H4 in the nucleosomes associated with the IL-4, IL-13 and IL-5 genes. A conserved GATA3 response element (CGRE) containing four GATA consensus sequences was identified 1.6 kbp upstream of the IL-13 gene, corresponding with the 5V border of the Th2-specific histone hyperacetylation region. The CGRE was shown to bind to GATA3, histone acetyl transferase complexes including CBP/p300, and RNA polymerase II. As for the IL-5 gene locus, CD28 costimulation selectively enhanced histone hyperacetylation through NFnB activation and subsequent upregulation of GATA3. Chromatin remodeling of type 2 cytokine gene loci occurs also in developing Tc2 cells. IL-4 production and histone hyperacetylation in IL-4associated nucleosomes in developing Tc2 cells were significantly lower than those of Th2 cells; however, cytokine production and histone hyperacetylation of IL-5 and IL-13 genes were equivalent. Developing Tc2 cells expressed lower GATA3 levels and dramatically increased levels of repressor of GATA (ROG). A ROG response element in the IL-13 gene exon 4 displayed Tc2-specific binding of ROG, HDAC1 and HDAC2. Thus, ROG may confer CD8 T cell-specific repression of histone hyperacetylation and activation of the IL-4 gene locus. D 2005 Elsevier B.V. All rights reserved. Keywords: Th2; Tc2; ROG; Intergenic transcript; HDAC
1. Introduction After antigen recognition by T cell receptor (TCR), naive CD4 T cells differentiate into two distinct helper T (Th) cell subsets, Th1 and Th2 cells. Th1 cells produce IFNg and * Corresponding author. E-mail address: [email protected] (T. Nakayama). 0531-5131/ D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2005.08.007
138
T. Nakayama et al. / International Congress Series 1285 (2005) 137–144
tumor necrosis factor (TNF)h, and initiate cell-mediated immunity against intracellular pathogens. Th2 cells produce IL-4, IL-5 and IL-13, and are involved in humoral immunity and allergic responses. The cytokine environment is crucial in controlling the direction of Th cell differentiation. For Th1 cell differentiation, IL-12-mediated activation of signal transducer and activator of transcription (STAT) 4 is required, while IL-4-mediated STAT6 activation is important for Th2 cell generation. In addition, TCR stimulation events upon encounter with antigens are also indispensable for both Th1 and Th2 cell differentiation. We reported that efficient TCR-mediated activation of the p56lck , calcineurin, and RasERK MAPK signaling cascade is crucial for Th2 cell differentiation [1–3]. Recent studies have identified several transcription factors that control Th1/Th2 cell differentiation. Among them, GATA3 appears to be a master transcription factor for Th2 cell differentiation. GATA3 is selectively induced in developing Th2 cells and the ectopic expression of GATA3 induced Th2 cell differentiation even in the absence of IL-4 or STAT6. For Th1 cell differentiation, T-bet was recently identified as a key transcription factor. CD28 costimulation enhances Th2 responses significantly [4,5]. Upon anti-CD28 mAb stimulation, PI-3 kinase (PI3K) is recruited to CD28 and is activated, and then subsequent activation of NF-nB is induced. It has been reported that GATA3 induction was an outcome of the CD28-induced NF-nB activation in T cells [6,7]. This may be a mechanism by which Th2 responses were enhanced by CD28 costimulation. It is also known that IL-5 production and IL-5-dependent airway inflammation are dependent on NF-nB family members. Chromatin remodeling of the Th2 cytokine gene loci (IL-4/IL-5/IL-13) occurs during Th2 cell differentiation. A highly conserved 400 bp noncoding sequence 1 (CNS1) was identified, and an important role in coordinate expression of Th2 cytokines was revealed. More recently, a 3V distal IL-4 enhancer (VA) containing an inducible DNase I hypersensitive site was identified. Reiner and colleagues reported that demethylation of intron 2 region of the IL-4 gene was associated with cell cycle progression and Th2 cell differentiation. We reported that demethylation of this region is regulated by polycomb group genes [8] that are known to regulate transcriptional memory in Drosophila. Hyperacetylation of histone H3 and H4 by histone acetyl transferases (HATs) was suggested to be associated with active chromatin. Recently, we and others have demonstrated that histone hyperacetylation of the Th2 cytokine gene loci occurs in developing Th2 cells in a Th2-specific and STAT6-dependent manner [9–11]. We demonstrated an essential role for GATA3 in the Th2-specific hyperacetylation [9]. We also generated a precise map of the Th2-specific histone hyperacetylation within the IL-13 and IL-4 gene loci, and identified a 71 bp conserved GATA3 response element (CGRE) at 1.6 kbp upstream of the IL-13 locus exon 1. This histone hyperacetylation remodeling process could be a major target for the Th2 master transcription factor GATA3 to induce differentiation towards Th2 cells. Histone hyperacetylation of another Th2 cytokine gene locus, IL-5, occurs in a Th2specific STAT6- and GATA3-dependent manner with significantly different kinetics compared to that of the IL-4 and IL-13 genes [9]. The direction of transcription of the IL-5 gene is opposite to that of IL-13 and IL-4. In addition, the RAD50 gene encoding a DNA
T. Nakayama et al. / International Congress Series 1285 (2005) 137–144
139
repair enzyme is located between the IL-5 and IL-13 gene loci. A differential role for GATA3 in the regulation of promoter activity of the IL-5 gene from IL-4 has been suggested. These results encouraged us to explore possible novel molecular mechanisms that would govern histone hyperacetylation of the IL-5 gene locus. CD8+ cytotoxic T cells (CTLs) secrete type 1 cytokines after TCR stimulation. However, in vitro TCR/CD3 stimulation of CD8+ T cells in the presence of certain cytokines induced CD8 T cells to produce type 2 cytokines. The Tc1/Tc2 terminology was proposed by Sad et al. [12] in analogy to the Th1/Th2 subsets of CD4 T cells. Differences in the profiles of type 2 cytokine production between CD4 T cells and CD8 T cells have been noted. Particularly, the amount of IL-4 produced by type 2 CD8 T cells was reported to be 100-fold less than that by type 2 CD4 T cells. However, a full explanation for the limited production of IL-4 in type 2 CD8 T cells is not yet available. The unique cytokine production profiles of type 2 CD8 T cells suggest a distinct immunoregulatory role for these cells. In fact, putative unique functions of Tc2 cells in various immune responses and related diseases have been demonstrated. However, the true roles of the type 2 CD8 T cells in the immune responses against microorganisms are still unclear. 2. Materials and methods 2.1. Cell cultures and in vitro T cell differentiation Splenic CD4 T cells were stained with anti-CD4-FITC, and then, purified using magnetic beads and an Auto-MACS sorter (Miltenyi Biotec), yielding a purity of N98%. Enriched CD4 T cells (1.5 106) were stimulated for 2 days with immobilized anti-TCR mAb (H57-597, 3 Ag/ml) and soluble anti-CD28 mAb (37.51, 3 Ag/ml) in the presence of IL-2 (25 U/ml), IL-12 (100 U/ml), and anti-IL-4 mAb (11B11, 25% culture supernatant) for Th1-skewed conditions. For Th2-skewed conditions, cells were stimulated with immobilized anti-TCR mAb as above but in the presence of IL-2 (25 U/ml), IL-4 (100 U/ ml) and anti-IFNg mAb (R4.6A2, 25% culture supernatant). The cells were then transferred to new dishes and were cultured for another 5 days in the presence of cytokines present in the initial culture. In order to enhance the generation of IL-5-producing cells, stimulation with anti-TCR and anti-CD28 mAbs was performed during the second culture for 5 days. This procedure is slightly different from that used in our previous report [4]. Where indicated Wortmannin (Calbiochem) was added to the culture at the doses of 30 or 300 nM for the first 2 days. In vitro differentiation was then assessed by intracellular cytokine staining with anti-IL-4, anti-IL-5, anti-IL-13 and anti-IFNg or by ELISA as described [13]. 2.2. Chromatin immunoprecipitation (ChIP) assay The ChIP assay was performed using histone H3 ChIP assay kits (#17-245: Upstate Biotechnology) as described [9]. Anti-GATA3 Ab (H-48: Santa Cruz Biotechnology) was used for precipitation. Where indicated, GFP-positive retrovirus-infected cells were sorted by flow cytometry, and subjected to ChIP assay. Several primer sequences for ChIP assay were described previously [9,13].
140
T. Nakayama et al. / International Congress Series 1285 (2005) 137–144
3. Results and discussion Fig. 1 shows the summary of the results of histone hyperacetylation of the IL-13 and IL-4 gene loci. In developing Th2 cells, the expression of GATA3 is induced and GATA3 bounds to the CGRE (conserved GATA3 response element) region. Then, HAT complex and RNAse polymerase II complex are recruited, and induced hyperacetylation of histones downstream of the CGRE region. A long-range histone hyperacetylation in the IL-4 and IL-13 gene loci is detected in developing Th2 cells. Intergenic transcription is also induced at the same region as that with Th2-specific histone hyperacetylation. The ChIP assay was performed on the IL-5 gene locus and the intergenic region of the IL-5 gene and the RAD50 gene. We detected long-range Th2 specific hyperacetylation in the intergenic region between the IL-5 and RAD50 gene loci, and the levels were increased if anti-CD28 mAb was included in the Th2 cell developing culture. Fig. 2 shows the schematic representation of the levels of histone H3 acetylation. The levels of histone hyperacetylation and intergenic transcription were not increased in the IL-13 and IL-4 gene loci in the presence of CD28 costimulation. However, the levels of histone hyperacetylation and intergenic transcription were significantly increased in IL-5 gene locus. It is not known the starting site of the histone hyperacetylation and intergenic transcription at the IL-5/ RAD50 gene loci. Fig. 3 shows the conserved region between mouse and human. There is a conserved region at 3V border of hyperacetylatin containing two GATA3 binding motifs. We are now currently investigating the actual levels of GATA3 binding and Pol II binding. In summary, a series of our investigation indicate that: (i) CD28-costimulation selectively enhanced histone hyperacetylation of the IL-5 gene locus. (ii) The histone hyperacetylation of the IL5 gene locus appeared to be controlled by NF-nB activation and subsequent upregulation of GATA3. (iii) CD28 costimulation-sensitive histone hyperacetylation and intergenic transcript were observed in the entire intergenic region between the IL-5 and RAD50 gene loci [14].
20 Kb
CGRE
IL-5
RAD50
Th2-specific acetylation
RAD50
IL-13
CNS1
IL-4 V A enhancer
IL-13 IL-4 KIF3A
IL-4
KIF3A
CGRE Transcription RNA pol HAT Ac
GATA-3
Ac
Ac
Ac
Acetylation
Ac
Fig. 1. Schematic representation of histone hyperacetylation of the IL-13 and IL-4 gene loci in developing Th2 cells.
T. Nakayama et al. / International Congress Series 1285 (2005) 137–144
20 Kb
CGRE
IL-5
RAD50
CNS1
141
IL-4 V A enhancer
IL-13 IL-4 KIF3A
IL-5
acetylation
RAD50
Anti-TCR + anti-CD28 Anti-TCR
Fig. 2. Schematic representation of histone hyperacetylation of the IL-5 and RAD50 gene loci in developing Th2 cells.
In developing Tc2 cells, the production of IL-4 is known to be selectively decreased with normal production of IL-13 and IL-5. Fig. 4 shows the IFNg/IL-4 cytokine profiles of the developing Th1, Th2, Tc1 and Tc2 cells. The levels of IL-4 producing cells were significantly reduced in Tc2 cells compared with those of Th2 cells. In addition, developing Tc2 cell population contains IFNg producing cells, that is not observed in developing Th2 cells. A schematic representation of possible molecular events underlying GATA3-dependent hyperacetylation within the IL-13 and IL-4 gene loci in developing Th2 and Tc2 cells is shown in Fig. 5. In developing Th2 cells, a GATA3-dependent long-range histone hyperacetylation spanning
: conserved region between human and mouse Two GATA3 Binding Motifs
IL-5
?
Th2-CD28specific acetylation
1kbp
RAD50
Fig. 3. Conserved regions within the intergenic region between the IL-5 and RAD50 gene loci.
4
T. Nakayama et al. / International Congress Series 1285 (2005) 137–144 10
142
Th1
3
10
0.5
22.4
Th2
10
2
10
1
IL-4
56.3
0
10
IL-4
Th2
10
3
10
2
0.1
Tc1
1.6 5.0
Tc2
10
1
10
0
Tc2
54.1
100 101
102
103
45.6
100
101
102
103
IL-4
104
IFNγγ Fig. 4. Limited IL-4 production in CD8 T cells developed under type-2 skewed culture conditions.
#A key molecule: ROG (repressor of GATA), ---CD8 T cell specific expression
IL-5 RAD50
CGRE IL-13 1 234
IL-4 12
KIF3A
3 4
5 kb p
IL-4
IL-13
Type 2-specific acetylation
400 bp
stop codon
CGRE
cROGRE
Th2
Tc2
Tc2 Transcription
Transcription
HDAC
Repressor complex
RNA pol
Acetylation
HAT Ac
GATA-3
Ac
Ac
Ac
Ac
Acetylation
ROG
cROGRE
CGRE
Initiation of histone hyperacetylation and tanscription at CGRE site
ROG-madiated inhibition of histone hyperacetylation and transcription
Fig. 5. Schematic representation of possible molecular events underlying GATA3-dependent hyperacetylation within the IL-13 and IL-4 gene loci in developing Th2 and Tc2 cells.
T. Nakayama et al. / International Congress Series 1285 (2005) 137–144
143
the IL-13 and IL-4 gene loci was demonstrated [9]. We identified a 71 bp conserved GATA response element (CGRE) at 1.6 kbp upstream of the IL-13 locus corresponding with the 5V border of the Th2specific histone hyperacetylation region. The CGRE was shown to bind GATA3 and also p300 and RNA polymerase II, and thus, we proposed a crucial role of the CGRE for GATA3-mediated targeting and downstream spreading of core histone hyperacetylation within the IL-13 and IL-4 gene loci [9]. We also detected intergenic transcripts at the region of Th2-specific histone hyperacetylation. It is most likely that histone hyperacetylation initiates at the CGRE site and spreads to a downstream region with transcription. In developing Tc2 cells, the CGRE appears to be also important as evidenced by the fact that the 5V border of the Th2-specific hyperacetylation region is the same. In addition to the GATA3/CGRE-dependent initiation of acetylation, CD8 T cells seem to possess an additional regulatory mechanism in the histone acetylation events. Activated CD8 T cells but not CD4 T cells expressed a large amount of ROG protein and cROGRE (conserved ROG response element) was able to associate with ROG, HDAC1 and HDAC2 molecules in a Tc2 cellspecific manner. Taken together, it is probable that ROG molecules recruit a putative HDAC complex either directly or by interacting with another molecule and target it to the cROGRE resulting in the repression of histone hyperacetylation that is initiated at the upstream CGRE site. This mechanism appears to be CD8 T cell-specific, and thus, may confer limited production of IL-4 on CD8 T cells to exert distinct immunoregulatory roles from CD4+ Th2 cells by producing a different set of cytokines [13].
Acknowledgements We thank Ms. Yoko Sawano, Kaoru Sugaya, and Aoi Nakazawa for their secretarial assistance. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology (Japan) (Grants-in-Aid for: Scientific Research in Priority Areas #17016010; Scientific Research B #17390139; Grant-in-Aid for Young Scientists #17790317 and Special Coordination Funds for Promoting Science and technology), the Ministry of Health, Labor and Welfare (Japan), the Program for Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (Japan), The Japan Health Science Foundation, Uehara Memorial Foundation, Mochida Foundation and Kanae Foundation. References [1] M. Yamashita, et al., Requirement for p56lck tyrosine kinase activation in Th subset differentiation, Int. Immunol. 10 (1998) 577 – 591. [2] M. Yamashita, et al., T cell antigen receptor-mediated activation of the Ras/mitogen-activated protein kinase pathway controls interleukin 4 receptor function and type-2 helper T cell differentiation, Proc. Natl. Acad. Sci. U. S. A. 96 (3) (1999) 1024 – 1029. [3] M. Yamashita, et al., T cell receptor-induced calcineurin activation regulates T helper type 2 cell development by modifying the interleukin 4 receptor signaling complex, J. Exp. Med. 191 (2000) 1869 – 1879. [4] M. Kubo, et al., CD28 costimulation accelerates IL-4 receptor sensitivity and IL-4-mediated Th2 differentiation, J. Immunol. 163 (5) (1999) 2432 – 2442. [5] I.C. Rulifson, et al., CD28 costimulation promotes the production of Th2 cytokines, J. Immunol. 158 (2) (1997) 658 – 665. [6] M. Rodriguez-Palmero, et al., Triggering of T cell proliferation through CD28 induces GATA-3 and promotes T helper type 2 differentiation in vitro and in vivo, Eur. J. Immunol. 29 (12) (1999) 3914 – 3924. [7] J. Das, et al., A critical role for NF-nB in GATA3 expression and TH2 differentiation in allergic airway inflammation, Nat. Immunol. 2 (1) (2001) 45 – 50.
144
T. Nakayama et al. / International Congress Series 1285 (2005) 137–144
[8] M. Kimura, et al., Regulation of Th2 cell differentiation by mel-18, a mammalian polycomb group gene, Immunity 15 (2) (2001) 275 – 287. [9] M. Yamashita, et al., Identification of a conserved GATA3 response element upstream proximal from the interleukin-13 gene locus, J. Biol. Chem. 277 (44) (2002) 42399 – 42408. [10] O. Avni, et al., TH cell differentiation is accompanied by dynamic changes in histone acetylation of cytokine genes, Nat. Immunol. 3 (7) (2002) 643 – 651. [11] P.E. Fields, S.T. Kim, R.A. Flavell, Cutting edge: changes in histone acetylation at the IL-4 and IFN-c loci accompany Th1/Th2 differentiation, J. Immunol. 169 (2) (2002) 647 – 650. [12] S. Sad, R. Marcotte, T.R. Mosmann, Cytokine-induced differentiation of precursor mouse CD8+ T cells into cytotoxic CD8+ T cells secreting Th1 or Th2 cytokines, Immunity 2 (1995) 271 – 279. [13] M. Omori, et al., CD8 T cell-specific downregulation of histone hyperacetylation and gene activation of the IL-4 gene locus by ROG, repressor of GATA, Immunity 19 (2) (2003) 281 – 294. [14] M. Inami, et al., CD28 costimulation controls histone hyperacetylation of the interleukin 5 gene locus in developing Th2 cells, J. Biol. Chem. 279 (22) (2004) 23123 – 23133.
www.ics-elsevier.com
Chromatin remodeling of the Th2 cytokine gene loci T. Nakayama a,*, M. Yamashita a, M. Kimura a, A. Hasegawa a, M. Omori a, M. Inami a, S. Motohashi a, M. Kitajima a, K. Hashimoto b, H. Hosokawa a, R. Shinnakasu a a
Department of Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan Department of Life and Environmental Sciences and High Technology Research Center, Chiba Institute of Technology, Narashino, Tsudanuma, Chiba, Japan
b
Abstract. Differentiation of naive CD4 T cells into Th2 cells is accompanied by chromatin remodeling including hyperacetylation of histones H3 and H4 in the nucleosomes associated with the IL-4, IL-13 and IL-5 genes. A conserved GATA3 response element (CGRE) containing four GATA consensus sequences was identified 1.6 kbp upstream of the IL-13 gene, corresponding with the 5V border of the Th2-specific histone hyperacetylation region. The CGRE was shown to bind to GATA3, histone acetyl transferase complexes including CBP/p300, and RNA polymerase II. As for the IL-5 gene locus, CD28 costimulation selectively enhanced histone hyperacetylation through NFnB activation and subsequent upregulation of GATA3. Chromatin remodeling of type 2 cytokine gene loci occurs also in developing Tc2 cells. IL-4 production and histone hyperacetylation in IL-4associated nucleosomes in developing Tc2 cells were significantly lower than those of Th2 cells; however, cytokine production and histone hyperacetylation of IL-5 and IL-13 genes were equivalent. Developing Tc2 cells expressed lower GATA3 levels and dramatically increased levels of repressor of GATA (ROG). A ROG response element in the IL-13 gene exon 4 displayed Tc2-specific binding of ROG, HDAC1 and HDAC2. Thus, ROG may confer CD8 T cell-specific repression of histone hyperacetylation and activation of the IL-4 gene locus. D 2005 Elsevier B.V. All rights reserved. Keywords: Th2; Tc2; ROG; Intergenic transcript; HDAC
1. Introduction After antigen recognition by T cell receptor (TCR), naive CD4 T cells differentiate into two distinct helper T (Th) cell subsets, Th1 and Th2 cells. Th1 cells produce IFNg and * Corresponding author. E-mail address: [email protected] (T. Nakayama). 0531-5131/ D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2005.08.007
138
T. Nakayama et al. / International Congress Series 1285 (2005) 137–144
tumor necrosis factor (TNF)h, and initiate cell-mediated immunity against intracellular pathogens. Th2 cells produce IL-4, IL-5 and IL-13, and are involved in humoral immunity and allergic responses. The cytokine environment is crucial in controlling the direction of Th cell differentiation. For Th1 cell differentiation, IL-12-mediated activation of signal transducer and activator of transcription (STAT) 4 is required, while IL-4-mediated STAT6 activation is important for Th2 cell generation. In addition, TCR stimulation events upon encounter with antigens are also indispensable for both Th1 and Th2 cell differentiation. We reported that efficient TCR-mediated activation of the p56lck , calcineurin, and RasERK MAPK signaling cascade is crucial for Th2 cell differentiation [1–3]. Recent studies have identified several transcription factors that control Th1/Th2 cell differentiation. Among them, GATA3 appears to be a master transcription factor for Th2 cell differentiation. GATA3 is selectively induced in developing Th2 cells and the ectopic expression of GATA3 induced Th2 cell differentiation even in the absence of IL-4 or STAT6. For Th1 cell differentiation, T-bet was recently identified as a key transcription factor. CD28 costimulation enhances Th2 responses significantly [4,5]. Upon anti-CD28 mAb stimulation, PI-3 kinase (PI3K) is recruited to CD28 and is activated, and then subsequent activation of NF-nB is induced. It has been reported that GATA3 induction was an outcome of the CD28-induced NF-nB activation in T cells [6,7]. This may be a mechanism by which Th2 responses were enhanced by CD28 costimulation. It is also known that IL-5 production and IL-5-dependent airway inflammation are dependent on NF-nB family members. Chromatin remodeling of the Th2 cytokine gene loci (IL-4/IL-5/IL-13) occurs during Th2 cell differentiation. A highly conserved 400 bp noncoding sequence 1 (CNS1) was identified, and an important role in coordinate expression of Th2 cytokines was revealed. More recently, a 3V distal IL-4 enhancer (VA) containing an inducible DNase I hypersensitive site was identified. Reiner and colleagues reported that demethylation of intron 2 region of the IL-4 gene was associated with cell cycle progression and Th2 cell differentiation. We reported that demethylation of this region is regulated by polycomb group genes [8] that are known to regulate transcriptional memory in Drosophila. Hyperacetylation of histone H3 and H4 by histone acetyl transferases (HATs) was suggested to be associated with active chromatin. Recently, we and others have demonstrated that histone hyperacetylation of the Th2 cytokine gene loci occurs in developing Th2 cells in a Th2-specific and STAT6-dependent manner [9–11]. We demonstrated an essential role for GATA3 in the Th2-specific hyperacetylation [9]. We also generated a precise map of the Th2-specific histone hyperacetylation within the IL-13 and IL-4 gene loci, and identified a 71 bp conserved GATA3 response element (CGRE) at 1.6 kbp upstream of the IL-13 locus exon 1. This histone hyperacetylation remodeling process could be a major target for the Th2 master transcription factor GATA3 to induce differentiation towards Th2 cells. Histone hyperacetylation of another Th2 cytokine gene locus, IL-5, occurs in a Th2specific STAT6- and GATA3-dependent manner with significantly different kinetics compared to that of the IL-4 and IL-13 genes [9]. The direction of transcription of the IL-5 gene is opposite to that of IL-13 and IL-4. In addition, the RAD50 gene encoding a DNA
T. Nakayama et al. / International Congress Series 1285 (2005) 137–144
139
repair enzyme is located between the IL-5 and IL-13 gene loci. A differential role for GATA3 in the regulation of promoter activity of the IL-5 gene from IL-4 has been suggested. These results encouraged us to explore possible novel molecular mechanisms that would govern histone hyperacetylation of the IL-5 gene locus. CD8+ cytotoxic T cells (CTLs) secrete type 1 cytokines after TCR stimulation. However, in vitro TCR/CD3 stimulation of CD8+ T cells in the presence of certain cytokines induced CD8 T cells to produce type 2 cytokines. The Tc1/Tc2 terminology was proposed by Sad et al. [12] in analogy to the Th1/Th2 subsets of CD4 T cells. Differences in the profiles of type 2 cytokine production between CD4 T cells and CD8 T cells have been noted. Particularly, the amount of IL-4 produced by type 2 CD8 T cells was reported to be 100-fold less than that by type 2 CD4 T cells. However, a full explanation for the limited production of IL-4 in type 2 CD8 T cells is not yet available. The unique cytokine production profiles of type 2 CD8 T cells suggest a distinct immunoregulatory role for these cells. In fact, putative unique functions of Tc2 cells in various immune responses and related diseases have been demonstrated. However, the true roles of the type 2 CD8 T cells in the immune responses against microorganisms are still unclear. 2. Materials and methods 2.1. Cell cultures and in vitro T cell differentiation Splenic CD4 T cells were stained with anti-CD4-FITC, and then, purified using magnetic beads and an Auto-MACS sorter (Miltenyi Biotec), yielding a purity of N98%. Enriched CD4 T cells (1.5 106) were stimulated for 2 days with immobilized anti-TCR mAb (H57-597, 3 Ag/ml) and soluble anti-CD28 mAb (37.51, 3 Ag/ml) in the presence of IL-2 (25 U/ml), IL-12 (100 U/ml), and anti-IL-4 mAb (11B11, 25% culture supernatant) for Th1-skewed conditions. For Th2-skewed conditions, cells were stimulated with immobilized anti-TCR mAb as above but in the presence of IL-2 (25 U/ml), IL-4 (100 U/ ml) and anti-IFNg mAb (R4.6A2, 25% culture supernatant). The cells were then transferred to new dishes and were cultured for another 5 days in the presence of cytokines present in the initial culture. In order to enhance the generation of IL-5-producing cells, stimulation with anti-TCR and anti-CD28 mAbs was performed during the second culture for 5 days. This procedure is slightly different from that used in our previous report [4]. Where indicated Wortmannin (Calbiochem) was added to the culture at the doses of 30 or 300 nM for the first 2 days. In vitro differentiation was then assessed by intracellular cytokine staining with anti-IL-4, anti-IL-5, anti-IL-13 and anti-IFNg or by ELISA as described [13]. 2.2. Chromatin immunoprecipitation (ChIP) assay The ChIP assay was performed using histone H3 ChIP assay kits (#17-245: Upstate Biotechnology) as described [9]. Anti-GATA3 Ab (H-48: Santa Cruz Biotechnology) was used for precipitation. Where indicated, GFP-positive retrovirus-infected cells were sorted by flow cytometry, and subjected to ChIP assay. Several primer sequences for ChIP assay were described previously [9,13].
140
T. Nakayama et al. / International Congress Series 1285 (2005) 137–144
3. Results and discussion Fig. 1 shows the summary of the results of histone hyperacetylation of the IL-13 and IL-4 gene loci. In developing Th2 cells, the expression of GATA3 is induced and GATA3 bounds to the CGRE (conserved GATA3 response element) region. Then, HAT complex and RNAse polymerase II complex are recruited, and induced hyperacetylation of histones downstream of the CGRE region. A long-range histone hyperacetylation in the IL-4 and IL-13 gene loci is detected in developing Th2 cells. Intergenic transcription is also induced at the same region as that with Th2-specific histone hyperacetylation. The ChIP assay was performed on the IL-5 gene locus and the intergenic region of the IL-5 gene and the RAD50 gene. We detected long-range Th2 specific hyperacetylation in the intergenic region between the IL-5 and RAD50 gene loci, and the levels were increased if anti-CD28 mAb was included in the Th2 cell developing culture. Fig. 2 shows the schematic representation of the levels of histone H3 acetylation. The levels of histone hyperacetylation and intergenic transcription were not increased in the IL-13 and IL-4 gene loci in the presence of CD28 costimulation. However, the levels of histone hyperacetylation and intergenic transcription were significantly increased in IL-5 gene locus. It is not known the starting site of the histone hyperacetylation and intergenic transcription at the IL-5/ RAD50 gene loci. Fig. 3 shows the conserved region between mouse and human. There is a conserved region at 3V border of hyperacetylatin containing two GATA3 binding motifs. We are now currently investigating the actual levels of GATA3 binding and Pol II binding. In summary, a series of our investigation indicate that: (i) CD28-costimulation selectively enhanced histone hyperacetylation of the IL-5 gene locus. (ii) The histone hyperacetylation of the IL5 gene locus appeared to be controlled by NF-nB activation and subsequent upregulation of GATA3. (iii) CD28 costimulation-sensitive histone hyperacetylation and intergenic transcript were observed in the entire intergenic region between the IL-5 and RAD50 gene loci [14].
20 Kb
CGRE
IL-5
RAD50
Th2-specific acetylation
RAD50
IL-13
CNS1
IL-4 V A enhancer
IL-13 IL-4 KIF3A
IL-4
KIF3A
CGRE Transcription RNA pol HAT Ac
GATA-3
Ac
Ac
Ac
Acetylation
Ac
Fig. 1. Schematic representation of histone hyperacetylation of the IL-13 and IL-4 gene loci in developing Th2 cells.
T. Nakayama et al. / International Congress Series 1285 (2005) 137–144
20 Kb
CGRE
IL-5
RAD50
CNS1
141
IL-4 V A enhancer
IL-13 IL-4 KIF3A
IL-5
acetylation
RAD50
Anti-TCR + anti-CD28 Anti-TCR
Fig. 2. Schematic representation of histone hyperacetylation of the IL-5 and RAD50 gene loci in developing Th2 cells.
In developing Tc2 cells, the production of IL-4 is known to be selectively decreased with normal production of IL-13 and IL-5. Fig. 4 shows the IFNg/IL-4 cytokine profiles of the developing Th1, Th2, Tc1 and Tc2 cells. The levels of IL-4 producing cells were significantly reduced in Tc2 cells compared with those of Th2 cells. In addition, developing Tc2 cell population contains IFNg producing cells, that is not observed in developing Th2 cells. A schematic representation of possible molecular events underlying GATA3-dependent hyperacetylation within the IL-13 and IL-4 gene loci in developing Th2 and Tc2 cells is shown in Fig. 5. In developing Th2 cells, a GATA3-dependent long-range histone hyperacetylation spanning
: conserved region between human and mouse Two GATA3 Binding Motifs
IL-5
?
Th2-CD28specific acetylation
1kbp
RAD50
Fig. 3. Conserved regions within the intergenic region between the IL-5 and RAD50 gene loci.
4
T. Nakayama et al. / International Congress Series 1285 (2005) 137–144 10
142
Th1
3
10
0.5
22.4
Th2
10
2
10
1
IL-4
56.3
0
10
IL-4
Th2
10
3
10
2
0.1
Tc1
1.6 5.0
Tc2
10
1
10
0
Tc2
54.1
100 101
102
103
45.6
100
101
102
103
IL-4
104
IFNγγ Fig. 4. Limited IL-4 production in CD8 T cells developed under type-2 skewed culture conditions.
#A key molecule: ROG (repressor of GATA), ---CD8 T cell specific expression
IL-5 RAD50
CGRE IL-13 1 234
IL-4 12
KIF3A
3 4
5 kb p
IL-4
IL-13
Type 2-specific acetylation
400 bp
stop codon
CGRE
cROGRE
Th2
Tc2
Tc2 Transcription
Transcription
HDAC
Repressor complex
RNA pol
Acetylation
HAT Ac
GATA-3
Ac
Ac
Ac
Ac
Acetylation
ROG
cROGRE
CGRE
Initiation of histone hyperacetylation and tanscription at CGRE site
ROG-madiated inhibition of histone hyperacetylation and transcription
Fig. 5. Schematic representation of possible molecular events underlying GATA3-dependent hyperacetylation within the IL-13 and IL-4 gene loci in developing Th2 and Tc2 cells.
T. Nakayama et al. / International Congress Series 1285 (2005) 137–144
143
the IL-13 and IL-4 gene loci was demonstrated [9]. We identified a 71 bp conserved GATA response element (CGRE) at 1.6 kbp upstream of the IL-13 locus corresponding with the 5V border of the Th2specific histone hyperacetylation region. The CGRE was shown to bind GATA3 and also p300 and RNA polymerase II, and thus, we proposed a crucial role of the CGRE for GATA3-mediated targeting and downstream spreading of core histone hyperacetylation within the IL-13 and IL-4 gene loci [9]. We also detected intergenic transcripts at the region of Th2-specific histone hyperacetylation. It is most likely that histone hyperacetylation initiates at the CGRE site and spreads to a downstream region with transcription. In developing Tc2 cells, the CGRE appears to be also important as evidenced by the fact that the 5V border of the Th2-specific hyperacetylation region is the same. In addition to the GATA3/CGRE-dependent initiation of acetylation, CD8 T cells seem to possess an additional regulatory mechanism in the histone acetylation events. Activated CD8 T cells but not CD4 T cells expressed a large amount of ROG protein and cROGRE (conserved ROG response element) was able to associate with ROG, HDAC1 and HDAC2 molecules in a Tc2 cellspecific manner. Taken together, it is probable that ROG molecules recruit a putative HDAC complex either directly or by interacting with another molecule and target it to the cROGRE resulting in the repression of histone hyperacetylation that is initiated at the upstream CGRE site. This mechanism appears to be CD8 T cell-specific, and thus, may confer limited production of IL-4 on CD8 T cells to exert distinct immunoregulatory roles from CD4+ Th2 cells by producing a different set of cytokines [13].
Acknowledgements We thank Ms. Yoko Sawano, Kaoru Sugaya, and Aoi Nakazawa for their secretarial assistance. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology (Japan) (Grants-in-Aid for: Scientific Research in Priority Areas #17016010; Scientific Research B #17390139; Grant-in-Aid for Young Scientists #17790317 and Special Coordination Funds for Promoting Science and technology), the Ministry of Health, Labor and Welfare (Japan), the Program for Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (Japan), The Japan Health Science Foundation, Uehara Memorial Foundation, Mochida Foundation and Kanae Foundation. References [1] M. Yamashita, et al., Requirement for p56lck tyrosine kinase activation in Th subset differentiation, Int. Immunol. 10 (1998) 577 – 591. [2] M. Yamashita, et al., T cell antigen receptor-mediated activation of the Ras/mitogen-activated protein kinase pathway controls interleukin 4 receptor function and type-2 helper T cell differentiation, Proc. Natl. Acad. Sci. U. S. A. 96 (3) (1999) 1024 – 1029. [3] M. Yamashita, et al., T cell receptor-induced calcineurin activation regulates T helper type 2 cell development by modifying the interleukin 4 receptor signaling complex, J. Exp. Med. 191 (2000) 1869 – 1879. [4] M. Kubo, et al., CD28 costimulation accelerates IL-4 receptor sensitivity and IL-4-mediated Th2 differentiation, J. Immunol. 163 (5) (1999) 2432 – 2442. [5] I.C. Rulifson, et al., CD28 costimulation promotes the production of Th2 cytokines, J. Immunol. 158 (2) (1997) 658 – 665. [6] M. Rodriguez-Palmero, et al., Triggering of T cell proliferation through CD28 induces GATA-3 and promotes T helper type 2 differentiation in vitro and in vivo, Eur. J. Immunol. 29 (12) (1999) 3914 – 3924. [7] J. Das, et al., A critical role for NF-nB in GATA3 expression and TH2 differentiation in allergic airway inflammation, Nat. Immunol. 2 (1) (2001) 45 – 50.
144
T. Nakayama et al. / International Congress Series 1285 (2005) 137–144
[8] M. Kimura, et al., Regulation of Th2 cell differentiation by mel-18, a mammalian polycomb group gene, Immunity 15 (2) (2001) 275 – 287. [9] M. Yamashita, et al., Identification of a conserved GATA3 response element upstream proximal from the interleukin-13 gene locus, J. Biol. Chem. 277 (44) (2002) 42399 – 42408. [10] O. Avni, et al., TH cell differentiation is accompanied by dynamic changes in histone acetylation of cytokine genes, Nat. Immunol. 3 (7) (2002) 643 – 651. [11] P.E. Fields, S.T. Kim, R.A. Flavell, Cutting edge: changes in histone acetylation at the IL-4 and IFN-c loci accompany Th1/Th2 differentiation, J. Immunol. 169 (2) (2002) 647 – 650. [12] S. Sad, R. Marcotte, T.R. Mosmann, Cytokine-induced differentiation of precursor mouse CD8+ T cells into cytotoxic CD8+ T cells secreting Th1 or Th2 cytokines, Immunity 2 (1995) 271 – 279. [13] M. Omori, et al., CD8 T cell-specific downregulation of histone hyperacetylation and gene activation of the IL-4 gene locus by ROG, repressor of GATA, Immunity 19 (2) (2003) 281 – 294. [14] M. Inami, et al., CD28 costimulation controls histone hyperacetylation of the interleukin 5 gene locus in developing Th2 cells, J. Biol. Chem. 279 (22) (2004) 23123 – 23133.