Friend of GATA Is Expressed in Naive Th
Cells and Functions As a Repressor of
GATA-3-Mediated Th2 Cell Development
This information is current as
of June 15, 2017.
Hirokazu Kurata, Hyun-Jun Lee, Terri McClanahan, Robert
L. Coffman, Anne O'Garra and Naoko Arai
J Immunol 2002; 168:4538-4545; ;
doi: 10.4049/jimmunol.168.9.4538
http://www.jimmunol.org/content/168/9/4538
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References
Friend of GATA Is Expressed in Naive Th Cells and Functions
As a Repressor of GATA-3-Mediated Th2 Cell Development1
Hirokazu Kurata,2* Hyun-Jun Lee,† Terri McClanahan,* Robert L. Coffman,* Anne O’Garra,*
and Naoko Arai*
C
larifying the mechanisms by which naive Th cells differentiate into effector cells is critical for understanding T
cell immune responses. CD4⫹ Th cells develop into at
least two distinct subsets (1). Th1 cells produce IFN-␥ and lymphotoxin and mediate delayed-type hypersensitivity responses and
protection against intracellular pathogens and viruses. In contrast, Th2 cells produce IL-4, IL-5, and IL-13, provide help to
B cells, and are implicated in atopic and allergic manifestations
(1). Th2 cell development has been considered to be essentially
an IL-4- (2) and Stat6-dependent process (3–5). The IL-4 signal
induces expression of GATA-3 and c-Maf in a Stat6-dependent
manner (6 –10).
GATA-3 is the key transcription factor of Th2 cell differentiation (6 – 8, 11): transgenic and retroviral expression of GATA-3
has induced the Th2 cytokine profile in Th1 cells (7, 8, 12), while
antisense and dominant-negative GATA-3 has reduced the Th2
cytokines in Th2 clones (7, 13). It is supposed that GATA-3 regulates the Th2 cell phenotype at multiple levels. First, GATA-3
strongly transactivates the IL-5 promoter and weakly activates the
IL-4 promoter (6, 11, 13–15). Second, it activates the enhancers
found within several regions surrounding the IL-4 gene (15).
Third, it appears to exert a more global influence by altering the
chromatin structure of Th2-specific gene loci (11, 16 –18). The
early stage of Th2 differentiation is accompanied by changes in the
*Department of Immunology, DNAX Research Institute of Molecular and Cellular
Biology, Palo Alto, CA 94304; and †Department of Molecular Genetics and Cell
Biology, University of Chicago, Chicago, IL 60634
Received for publication November 21, 2001. Accepted for publication February
20, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
DNAX Research Institute is supported by the Schering-Plough Corporation.
2
Address correspondence and reprint requests to Dr. Hirokazu Kurata, Department of
Immunology, DNAX Research Institute of Molecular and Cellular Biology, 901 California Avenue, Palo Alto, CA 94304. E-mail address: [email protected]
Copyright © 2002 by The American Association of Immunologists
DNase I hypersensitivity and DNA methylation patterns of the Th2
cytokine cluster loci (16, 19, 20). The changes render the loci more
accessible to GATA-3, as proven by the chromatin immunoprecipitation assay (21) and transgenic mice studies (22, 23).
The six vertebrate GATA factors regulate the development of
various tissues and are categorized into two subfamilies (GATA1/2/3 and GATA-4/5/6). The former are expressed primarily in the
hematopoietic system: GATA-1 regulates the differentiation of
erythroid and megakaryocytic cells, GATA-2 controls the proliferation of hematopoietic progenitors, and GATA-3 controls the
development of early T-lymphoid and effector Th2 cells (7, 24,
25). The latter are implicated in development of heart, intestine,
and endoderm (26 –29).
Of interest is how GATA factors function in transcriptional control. Each GATA factor contains an N-terminal activation domain
and two zinc fingers: the C-terminal finger (Cf)3 and the N-terminal finger (Nf) (30 –32). The Cf is essential for binding to the
consensus GATA sequence, whereas the Nf stabilizes the DNA
binding (33). Moreover, the activities of GATA factors are modulated through Cf-mediated interaction with several transcription
factors (p300/CBP, EKLF, Sp1, PU-1, Pit-1, Nkx2.5, and NFAT3) (29).
Recently, a yeast two-hybrid screening identified friend of
GATA (FOG) as a GATA-1 Nf-interacting molecule in the MEL
cell library (34). Subsequently, its homolog FOG-2 was cloned in
the embryonic brain (27) and heart (26, 28) libraries. In vitro reporter assays suggested that FOG and FOG-2 enhance or repress
the transcriptional activity of GATA factors depending on the cell
and the promoter context (26, 28).
FOG is highly expressed in erythroid and megakaryocytic cell
lines and in spleen, liver, and testis (34). FOG⫺/⫺ embryos
showed impaired primitive and definitive erythropoiesis, like
3
Abbreviations used in this paper: Cf, C-terminal finger; FOG, friend of GATA;
CtBp, C-terminal binding protein; DBD, DNA-binding domain; Nf, N-terminal finger; TAD, transactivation domain; RV, retrovirus; GFP, green fluorescent protein;
NLS, nuclear localization signal.
0022-1767/02/$02.00
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The commitment of naive T cells to polarized Th cells requires specific changes in their transcription factors. Retrovirally
overexpressed GATA-3 has been reported to induce the Th2 cytokine profile in developing Th1 cells. In this study, we examined
the role of the N-terminal finger (Nf) of GATA-3 in Th2 cell development. The Nf, as well as the C-terminal finger and the
transactivation domain, is critical for the induction of the Th2 phenotype. Using the GATA-3-Nf as a bait, our yeast two-hybrid
screening identified friend of GATA (FOG) in the Th2 cell-specific library. Naive T cells express significant levels of FOG mRNA,
which was rapidly down-regulated upon commitment to both Th1 and Th2 lineages. In reporter assays, FOG blocked the GATA3-mediated activation of several cytokine promoters. Finally, retroviral expression of FOG in developing Th2 cells suppressed both
IL-4 and IL-5 and allowed for IFN-␥ production, which was accompanied by a significant level of T-bet mRNA expression. Serial
deletion mutation analysis indicated that the N-terminal region, but not the consensus C-terminal binding protein-binding motif,
of FOG is critical for the effects. Our results clearly indicate that 1) FOG is a repressor of GATA-3 in naive T cells and 2) the
down-regulation of FOG induces Th2 cell differentiation by releasing GATA-3 from its repression. The Journal of Immunology,
2002, 168: 4538 – 4545.
The Journal of Immunology
GATA-1⫺/⫺ embryos, but a more extreme megakaryocytic defect
(35). In contrast, as FOG-2 is expressed in embryonic and adult
heart, brain, and urogenital tissues (26 –28), FOG-2⫺/⫺ embryos
are lethal due to the defects in heart morphogenesis (29, 36). These
results indicate the critical role of FOG family for the functions of
GATA family proteins. The role of the FOG proteins in T lymphocyte development becomes an interesting question, because
GATA-3 is the critical factor in early Th and effector Th2 cell
development (7).
In this work we report that FOG acts as a critical regulator of
GATA-3-mediated Th2 cell development.
Materials and Methods
Mice, cytokines, and Abs
DO11.10 TCR-transgenic mice, recombinant cytokines, and Abs were used
as previously described (9).
Yeast two-hybrid screening using Th2 cell-specific library
Transfected cells were stimulated with PMA (50 ng/ml) and ionomycin (1
␮M) 24 h after transfection, harvested 48 h after, and assayed for luciferase
and ␤-galactosidase activity using the Luciferase Assay System (Promega)
and Galacto-Light ␤-galactosidase detection kit (Tropix, Bedford, MA).
Luciferase activities were normalized to ␤-galactosidase activity.
Mammalian two-hybrid assay
GATA-3 Nf cDNA (aa 259 –309) was cloned into GAL4 DNA-binding
domain (GAL4-DBD) encoding plasmid pM (Clontech Laboratories) to
generate pM-G3-Nf. The GAL4 activation domain encoding plasmid
pVP16 (Clontech Laboratories) was used as the cloning vector for in-frame
FOG constructs. All FOG mutant constructs were prepared by inserting the
corresponding fragments in pVP16 in frame. 293T cells in 12-well plates
were transiently transfected by FuGene 6 (Roche), according to the manufacturer’s protocol, using 1 ␮g of plasmid DNAs (0.4 ␮g GAL4-DBD
vector, 0.4 ␮g GAL4-activation domain vector, 0.1 ␮g pGL5-luc vector
(Promega), and 0.1 ␮g pSV-␤-galactosidase control vector). Transfectants
were harvested 48 h later and assayed as above.
Retroviral constructs
Control retroviral plasmid (pMX-IRES-GFP) and GATA-3 expression retroviral plasmids (pMX-GATA3-wt) were described previously (11). The
pMX-GATA3-⌬CF, -⌬NF, -⌬TAD, and -V264G were prepared by inserting the corresponding fragments in pME18S vectors into pMX-IRES-GFP
(11). FOG retroviral plasmids were prepared by inserting full-length
(pMX-FOG) or mutant FOG cDNAs into the multicloning site in the pMXIRES-GFP vector.
RV infection
A retrovirus (RV) packaging cell line, PLAT-E (39), was transfected with
retroviral plasmids using FuGene 6 (Roche). Splenic naive T cells from
DO11.10 mice were prepared and activated weekly as previously described
(9). Cells were spin-infected with RV supernatants for 4 h at 3000 rpm at
35°C 1 and 2 days after activation and cultured under the Th1- and Th2priming conditions (9). Green fluorescent protein (GFP)-positive T cells
were harvested on day 7 with a purity of ⬎98% by FACScan (BD Biosciences, Mountain View, CA).
Cytokine ELISA
T cells were stimulated with PMA (50 ng/ml) and ionomycin (1 ␮M) for
48 h, and supernatants were measured as described (9).
Plasmid construction
pME18S-GATA-3 (pME-G3-wt) has been described previously (11).
pME-GATA-3-⌬Cf, -⌬Nf, -⌬TAD, and -V264G contain deletions of Cf
(aa 309 –345), Nf (aa 263–289), transactivation domain (TAD) (aa 29 –
168), and a single amino acid substitution of glycine for valine at aa 264.
The full-length FOG cDNA was constructed as below. A NotI/XhoIFOG fragment corresponding to aa 817–996 was obtained from a clone
(pACT-FOG) in the T cell lymphoma library. An EcoRI/KpnI-fragment
corresponding to aa 1–264 was PCR-amplified from day 15 embryo Marathon cDNA library using GC2-PCR kit (Clontech Laboratories); missense
mutations were corrected by PCR-based mutagenesis. A KpnI/NotI-fragment (aa 265– 816) was obtained from a clone in the Th2 library (pGD7FOG-402). The three fragments were cloned into BamHI/XhoI-sites of
pME18S to generate pME-FOG.
Reporter and expression plasmid construction
pGL3-mIL-4(⫺766) construct was made by inserting the KpnI/HindIII
fragment from pIL-4(⫺766)Luc (37) (⫺766 to ⫹63 of the transcription
start site) into the pGL3-basic luciferase vector (Promega, Madison, WI).
The pGL3-mIL-5 (1.2 kb) and pGL3-mIFN-␥ (⫺300 bp) constructs were
prepared by inserting a HindIII/XbaI fragment of pGL2-mIL-5 (⫺1.2 kb)
(from ⫺1174 to ⫹33 bp upstream of the translation start site) (14) and a
PCR-cloned fragment from pMCITK-IFN-␥ (⫺300 to ⫺5 bp) (38), into the
pGL3-basic luciferase vector (Promega), respectively.
Transcriptional reporter assays
Jurkat cells were transfected with 4 ␮g of plasmid DNAs using
XtremeGene Q2 transfection reagent (Roche, Indianapolis, IN) according
to the manufacturer’s instructions. All transfections were performed in duplicate and contained 0.1 ␮g of pSV-␤-galactosidase vector (Promega).
Quantification of mRNA expression
The expression profiles of the GATA and FOG family genes were detected
by real-time quantitative PCR (ABI PRISM 7700 Sequence Detection System; PE Applied Biosystems, Foster City, CA). Tissue RNAs were extracted from tissues of 8-wk-old BALB/c mice using STAT60 (Tel-Test,
Friendswood, TX). CD4⫹CD62L⫹ naive DO11.10 splenic T cells were
stimulated weekly with OVA and APC as described above, or with platebound anti-CD3 (10 ␮g/ml) and anti-CD28 (1 ␮g/ml). The cells were cultured under Th1- and Th2-priming conditions as described above, except
that 10 ␮g/ml anti-IFN-␥ (XMG1.2) was added in place of the anti-IL-12
Ab. The cells were activated with 5 ␮g/ml Con A on day 14. RNAs were
extracted from cultured T cells using an RNeasy kit (Qiagen, Valencia,
CA). cDNAs were synthesized from total RNA using an oligo(dT) primer
and random hexamers using Superscript II reverse transcriptase (Life Technologies). cDNA from 100 ng of total RNA was used per PCR. Real-time
PCR was performed with the PCR SYBR Green sequence detection system
(PE Applied Biosystems). The sequences of the primers are as follows:
retrovirally induced GATA-3, 5⬘-CCCTTCCAGCATGGTCACC-3⬘ and
5⬘-GGAATTTACGTAGCGGCCG-3⬘; endogenous GATA-3, 5⬘-GCCAT
GGGTTAGAGAGGCAG-3⬘ and 5⬘-TTGGAGACTCCTCACGCATGT3⬘; GATA-3 (total), 5⬘-CTGACGGAAGAGGTGGACGT-3⬘ and 5⬘-GTG
GTTGCCTTGACCATCG-3⬘; FOG, 5⬘-CAGCAGCCAAACTTCCTCCA-3⬘
and 5⬘-GCGAGTGCTGTTGAAAGCCT-3⬘ (a second set of primers was also
used: 5⬘-TGGCCGACTACCACGAGTG-3⬘ and 5⬘-CAGGTAAGCTTCGAG
GCTGTG-3⬘); FOG-2, 5⬘-GAACCTGCAAGCCCATTTGA-3⬘ and 5⬘-GCT
TCTCGTTGCCTCCCAC-3⬘; T-bet, 5⬘-CGGTACCAGAGCGGCAAGT-3⬘
and 5⬘-CATGCTGCCTTCTGCCTTTC-3⬘; IL12R␤2, 5⬘-CTTGGCACTGT
GACCGTCC-3⬘ and 5⬘-CAGCTGACCCAAGAGGAATCA-3⬘; and ubiquitin, 5⬘-TGGCTATTAATTATTCGGTCTGCAT-3⬘ and 5⬘-GCAAGTGGC
TAGAGTGCAGAGTAA-3⬘. All results were normalized with respect to the
expression of ubiquitin.
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The yeast two-hybrid bait vectors pY-G3-Nf and pY-G3-(N⫹C)f contain
the N-finger (aa residues 259 –308) and both zinc finger domains (aa 259 –
361) of mouse GATA-3, respectively, in pGBKT7 (Clontech Laboratories,
Palo Alto, CA). A Th2 cell-specific library was constructed. Briefly,
CD4⫹Mel-14⫹ naive T cells were stimulated weekly with 0.6 ␮M OVA
and 3000-rad irradiated spleen cells (APC) as described (9). Cells were
harvested with (days 7 and 14) or without (days 3, 5, 7, 10, 12, and 14)
stimulation by adding 5 ␮g/ml Con A (Sigma-Aldrich, St. Louis, MO) for
6 h. Poly(A)⫹ RNA was prepared from the pooled cells, and cDNA was
synthesized using an oligo(dT) primer by Superscript II reverse transcriptase (Life Technologies, Gaithersburg, MD). SalI/NotI-digested cDNA
fragments were cloned into the GAL4 activation domain-containing plasmid pGADT7 (Clontech Laboratories). Yeast two-hybrid screening was
performed using V13 Kaplan T cell lymphoma library (Clontech Laboratories) and the Th2 cDNA library, as described (Matchmaker Two-Hybrid
System 3 Protocol; Clontech Laboratories). Briefly, competent AH109
yeast cells were transformed sequentially with the bait and library plasmids, and colonies were selected on Ade-, His-, Leu-, and Trp-synthetic
dropout agar plates. The clones were confirmed to be truly positive by
Ade/His or ␤-galactosidase production when cotransformed with pY-G3
but not pGBKT7.
4539
4540
FOG REPRESSES Th2 CELL DEVELOPMENT
sixth zinc finger domains, and the latter two contained the fifth
through the sixth fingers, of which the first, fifth, and sixth fingers
are putative interacting domains with GATA factors.
To investigate the effects of GATA-3 domains on the induction of
Th2 cytokine profiles, we transduced RVs containing GATA-3 and
its mutants into developing Th1 cells. Compatible with previous
reports (11, 40), wild-type GATA-3 induced the expression of IL-4
and IL-5 and suppressed IFN-␥ production (Fig. 1A), whereas deletion of the Cf and the TAD resulted in a complete loss of the Th2
phenotype induction. Interestingly, deletion of the Nf led to a complete loss of IL-5 induction, but both the IL-4 induction and the
IFN-␥ suppression were partially conserved.
The induction of the Th2 profile was confirmed by quantitative
analysis of several Th type-specific genes (Fig. 1B). First, the levels of retrovirally expressed GATA-3 were similar among wildtype and mutant GATA-3-expressing Th1 cells (data of GATA-3⌬Cf and -⌬TAD are not shown). The induction of endogenous
GATA-3 was induced by wild-type- but not GATA-3-⌬Nf. Moreover, the suppression of T-bet and IL12R␤2 mRNAs was observed
by wild-type GATA-3. In contrast, GATA-3-⌬Nf partially suppressed T-bet but not IL12R␤2 mRNA. These results indicate the
critical but differential roles of three domains of GATA-3 for the
induction of the Th2 phenotype.
Tissue-restricted and developmentally regulated expression of
GATA-3 and FOG family proteins
GATA-3 induces Th2 cytokines and suppresses IFN-␥
production by developing Th1 cells in a zinc fingers- and
transactivation domain-dependent manner
Identification of FOG as a GATA-3-interacting molecule in Th2
cells
To identify proteins that interact with GATA-3, we prepared a
yeast two-hybrid library from mixtures of several developmental
stages of Th2 cells. We used an N-terminal-truncated GATA-3 (aa
169 – 422) that encompassed both zinc fingers as a bait to screen
the library. More than 800 clones of one million clones demonstrated histidine and adenine auxotrophy, and sequence analysis
revealed that 3 of 197 clones encoded FOG (aa 228 – 819, 443–
819, and 505– 819). The first one contained the first through the
Next, we examined the tissue- and developmental stage-specific
expression of FOG family proteins, including FOG and FOG-2,
and GATA-3 using a real-time quantitative RT-PCR (Fig. 2A, left
panels). Significantly, higher levels of GATA-3 mRNA were detected in lymph node and thymus, while much lower levels were
detected in spleen, liver, and brain stem. FOG mRNA is expressed
in many tissues, most prominently in spleen and lymph node and
moderately in thymus, kidney, heart, and brain stem. We confirmed the expression of FOG using two different sets of primers
(see sequences in Materials and Methods) and obtained similar
results (data not shown). In contrast, FOG-2 mRNA is expressed
very weakly in thymus and spleen but moderately in brain stem,
ovary, and lymph node, and strongly in heart. These data are compatible with previous reports (26 –28).
We further compared the expression of FOG and FOG-2
mRNAs in developing Th cells (Fig. 2A, right panels). Naive T
cells showed a lower but significant level of GATA-3 mRNA compared with thymic cells. Two weeks of Th1-driving culture with
OVA and APC led to the disappearance of GATA-3 and FOG
mRNAs. In contrast, 2 wk of Th2-driving culture led to prominent
up-regulation of GATA-3 and an almost complete disappearance
of FOG mRNA. FOG-2 mRNA was detected very weakly in naive
Th cells (⬍30 ⫻ 10⫺5/ubiquitin) but not in polarized Th1 or Th2
cells. This developmental stage-specific expression was further
confirmed by time-course analysis of in vitro cultured Th cells
(activated with anti-CD3 and anti-CD28 under Th1/Th2-driving
cultures) (Fig. 2B). As controls, the expression of both GATA-3
and T-bet mRNAs was examined (Fig. 2B, middle and lower panels). A slight level of GATA-3 mRNA was expressed in naive T
FIGURE 1. Retroviral expression of GATA-3 in developing Th1 cells induces Th2 cytokine patterns in a zinc fingers- and transactivation domaindependent manner. A, Induction of IL-4 and IL-5 and suppression of IFN-␥ by retrovirally expressed GATA-3 in developing Th1 cells. Naive Th cells from
DO11.10 spleens were infected with RV vectors containing GATA-3 wild-type and deletion mutants. Cells were sorted 7 days after the activation and
activated by PMA and ionomycin. Supernatants were collected after 48 h and assayed by ELISA. Uninfected cells cultured under Th1- and Th2-driving
conditions were analyzed as controls. Data bars represent the average of three independent samples, and error bars show the SD. B, GATA-3-mediated
induction of endogenous GATA-3 and suppression of T-bet and IL-12R␤2 mRNA expression. Total RNA was isolated from the above cells and analyzed
for expression of retroviral GATA-3, endogenous GATA-3, c-Maf, T-bet, and IL12R␤2 using the TaqMan quantitative PCR assay. mRNA levels are
normalized to ubiquitin mRNA (⫻10⫺5/ubiquitin).
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Results
The Journal of Immunology
4541
cells, rapidly up-regulated during Th2 cell development, but downregulated during Th1 cell development (6). In contrast, T-bet
mRNA was not expressed in naive T cells, but was rapidly upregulated in Th1 cells but not Th2 cells (41). Interestingly, FOG
mRNA was expressed significantly in naive Th cells but downregulated quickly during the development to both Th1 and Th2
lineages (Fig. 2B, upper panel). FOG-2 mRNA was not detected in
either of these polarized Th cells (detection limit, 1 ⫻ 10⫺5/ubiquitin) (data not shown). These results demonstrate that FOG and
GATA-3 are coexpressed in naive T cells but that the FOG mRNA
was rapidly down-regulated during Th cell differentiation.
FOG represses GATA-3-dependent transcription from cytokine
gene promoters
FIGURE 2. FOG is expressed in naive T cells and is down-regulated
during Th cell development. A, Quantitative RT-PCR analyses of GATA-3
and FOG family transcripts in mouse multiple tissue cDNA panels and in
vitro cultured Th1/Th2 cells from DO11.10-transgenic mouse spleens. LN,
Lymph node. B, Kinetics of induction of GATA-3 and T-bet mRNAs and
down-regulation of FOG mRNA during Th cell development. CD4⫹
CD62L⫹ naive T cells were activated with plate-bound anti-CD3 and soluble anti-CD28, and cultured under Th1-priming (IL-12, IL-2, and antiIL-4 Ab) and Th2-priming (IL-4, IL-2, and anti-IFN-␥ Ab) conditions for
indicated periods. The cells were activated with Con A on day 14. Quantitative RT-PCR was performed and analyzed as in Fig. 1B.
FIGURE 3. FOG represses the GATA-3-mediated activation of cytokine promoters in Jurkat cells. A, Activation of the IL-5, IL-4, and IFN-␥
promoters by GATA-3, and repression by FOG. pGL3-mIL-5-, mIL-4-, or
mIFN-␥ plasmid (1.3 ␮g) was cotransfected with pSV-␤-galactosidase (0.1
␮g) into Jurkat cells. Expression vectors for GATA-3 (pME-GATA-3-wt,
1.3 ␮g) and FOG (pME-FOG, 1.3 ␮g; or 0.3 or 0.65 ␮g for the IL-5
promoter), or equivalent amounts of empty pME18S vector, were added as
indicated (a total of 4 ␮g of plasmids). After 24 h, cells were stimulated
with PMA and ionomycin, and after 48 h luciferase and ␤-galactosidase
activities were measured. The basal promoter activity was arbitrarily assigned as a value of 1. Representative results from three independent experiments, each performed in duplicate, are shown. B, FOG represses the
IL-5 promoter through the Nf of GATA-3. The parallel experiments for
IL-5 promoter activation were done using expression vectors for GATA-3
(pME-GATA-3-wt, -⌬Cf, -⌬Nf, -⌬TAD, or -V264G) and FOG
(pME-FOG).
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FOG family proteins have been reported to function as both coactivators and repressors of GATA-dependent transcription depending on cellular context (34, 42). To study the effects of FOG
on GATA-3-dependent transcription in lymphocytes, Jurkat cells
were cotransfected with expression plasmids containing GATA-3
and FOG and luciferase reporter plasmids containing mouse IL-5,
IL-4, and IFN-␥ promoters (Fig. 1). Consistent with previous reports (11, 13, 15), GATA-3 alone transactivated the IL-5 and IL-4
4542
promoters by 5- and 3-fold, respectively (Fig. 3A). In contrast,
forced expression of FOG had little effect on the basal levels of
IL-5 promoter activation but significantly repressed the transactivation by GATA-3 in a dose-dependent manner (Fig. 3A, left panel). FOG also repressed GATA3-mediated activation of IL-4 and
IFN-␥ promoters. Next we examined the effect of interaction between GATA-3 and FOG on the transactivation (Fig. 3B). Interestingly, expression of FOG repressed the transactivation of the
IL-5 promoter by GATA-3-wild-type and -⌬TAD to the basal level
of activation but did not repress the transactivation by GATA-3⌬Nf and -V264G, which are supposed not to interact with FOG.
GATA-3-⌬Cf showed no transactivation. These results demonstrated that FOG specifically represses GATA-3-mediated transcription of several cytokine promoters through interaction with
the N-finger of GATA-3.
Retroviral gene transduction of FOG into primary T cells
results in decreased Th2 cytokine production and induced IFN-␥
production
Fig. 4 shows that FOG significantly suppressed the production
of IL-4 (78 vs 317 and 316 ng/ml in RV-GFP-infected and uninfected Th2 cells) and IL-5 (95 vs 205 and 222 ng/ml in RV-GFPinfected and uninfected Th2 cells) (Fig. 4A). In contrast, FOG
allowed for the production of IFN-␥ despite the Th2-priming culture condition (449 vs 38 and 107 ng/ml in RV-GFP-infected and
uninfected Th2 cells). The production of IL-10 was only slightly
reduced by FOG. Similar phenotypic changes were observed on
day 7 (data not shown).
The Th type-specific gene expression was examined on the same
samples by quantitative RT-PCR (Fig. 4B). Both RV-GFP- and
RV-FOG-infected Th2 cells expressed similar levels of GATA-3
mRNA to uninfected Th2 cells. In contrast, T-bet mRNA was
slightly induced in RV-FOG- but not RV-GFP-infected Th2 cells.
IL12R␤2 mRNA was not detected in either of the RV-infected Th2
cells.
The N-terminal region of FOG is critical for the repression of
GATA-3 in Th2 cells
To more precisely identify the domains of FOG required for
GATA-3-binding and transcriptional repression, we constructed a
series of N- and C-terminal truncations of FOG (Fig. 5A). The
binding of FOG to GATA factors is reported to be mediated by the
first, the sixth, the ninth, and, weakly, the fifth fingers of FOG and
N-finger of GATA factors (43). The putative repression domains
were reported to be the consensus C-terminal binding protein
(CtBP)-binding (Pro-Ile-Asp-Leu-Ser: PIDLS) motif between the
sixth and seventh fingers (44) or the N-terminal domain (45). The
nuclear localization signal (NLS) was supposed to be around the 3⬘
side of the PIDLS motif (Fig. 5A). FOG-⌬3⬘ mutant lacks the
C-terminal regions (aa 762–995) harboring the seventh to ninth
fingers, the PIDLS motif, and the NLS. FOG-⌬3⬘B lacks the three
fingers but contains the PIDLS and the NLS. FOG-⌬5⬘ lacks the
N-terminal putative repression domain (aa 1– 48). FOG-⌬(5⬘/3⬘)
lacks both the N-terminal (aa 1– 48) and the C-terminal (aa 817–
FIGURE 4. Retroviral gene transduction of FOG into naive T cells suppresses Th2 cytokines and induces IFN-␥ production in developing Th2 cells.
A, Retroviral transduction of primary CD4⫹CD62L⫹ T cells with control RV-GFP and RV-FOG. FACS-purified naive T cells from DO11.10 TCRtransgenic mouse spleens were infected with the indicated RVs 18 and 42 h after primary activation by OVA and APC and cultured in the Th2-priming
condition. Cells were sorted on day 7 for GFP expression, activated with OVA and APC, and cultured in the same condition for one more week. The cells
were stimulated on day 14 with PMA and ionomycin, and cytokine production was measured after 48 h. Control uninfected cells were cultured in Th1and Th2-priming conditions. Empty indicates RV-GFP-infected cells, and FOG indicates RV-FOG-infected cells. B, Expression of GATA-3, T-bet, and
IL12R␤2 mRNAs in RV-infected Th cells. Quantitative PCR of Th cell markers was performed as in Fig. 1B.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
The experiments described above argue for the repression by FOG
of the GATA-3-mediated transcription. The fact that FOG is expressed in naive T cells but down-regulated during Th cell development raises the possibility that the regulation of FOG level is
critical in the processes leading to Th2 cell development. To test
this, a retroviral transfer of FOG into naive T cells was performed.
We generated a RV vector expressing both FOG and GFP (RVFOG) and used a retroviral vector containing GFP alone (RVGFP) as a control. The CD4⫹CD62L⫹ T cells from DO11.10
TCR␣␤-transgenic mouse spleens were activated with OVA and
APC under Th2-priming culture and infected with RV 18 and 42 h
after primary activation. After 7 days, GFP-positive cells were
isolated by FACS and stimulated with OVA and APC for one more
week under Th2-priming culture. On day 14, the cells were stimulated with PMA/ionomycin for measurement of cytokine
production.
FOG REPRESSES Th2 CELL DEVELOPMENT
The Journal of Immunology
4543
995) regions, and FOG-290 –590 contains only the second to
fourth fingers (Fig. 5A).
Using a mammalian two-hybrid system, we confirmed the interaction of GATA-3 and FOG or FOG mutants in Jurkat cells
(Fig. 5B). The N-finger domain of GATA-3 fused to GAL4-DBD
was tested for its ability to interact with the FOG truncation mutants fused to the VP16-AD. Interaction of GATA-3-Nf and
pVP16-FOG-wild-type was demonstrated by an 3.4-fold activation
of luciferase activity compared with that of GATA-3-Nf and the
control pVP16 vector. Deletion of N-terminal domain (FOG-⌬5⬘),
C-terminal domains (FOG-⌬3⬘ and -⌬3⬘B), or both (FOG-⌬(5⬘/
3⬘)) did not significantly reduce the interaction of GATA-3-Nf and
FOG but increased the luciferase activity, most likely through the
deletion of repression domains. In contrast, deletion of all the putative interacting fingers (FOG-290 –590) completely abolished the
interaction (Fig. 5B). FOG aa 616 –995 corresponds to the clone
harvested from the yeast two-hybrid screening (pG7-FOG).
Retroviral transfer of FOG mutants into naive T cells was performed and the cells were cultured under Th2-priming conditions
as in Fig. 4. The IL-4 production was significantly reduced by
wild-type and C-terminal-deleted (⌬3⬘ and ⌬3⬘B) FOG mutants,
but the repression was abolished in N-terminal-deleted (⌬5⬘) FOG
and weakened by FOG ⌬(5⬘/3⬘) (Fig. 5C). Conversely, the production of IFN-␥ was induced by wild-type and C-terminal-deleted
(⌬3⬘ and ⌬3⬘B) FOG but not by N-terminal-deleted (⌬5⬘) or ⌬(5⬘/
3⬘)-FOG mutants (Fig. 5C).
Discussion
In this study, we have revealed that 1) FOG, as a GATA-3 Nfinteracting cofactor, is expressed in naive T cells and down-regulated during Th cell development, and 2) FOG functions as a repressor of GATA-3, as the introduction of FOG into developing
Th2 cells down-regulates IL-4 and IL-5 and allows for IFN-␥
production.
The requirement of functional domains is important for understanding the mechanism of GATA-3 functions. For GATA-1, TAD
is essential for the transactivation in nonhematopoietic cells but is
dispensable for the megakaryocytic conversion of mouse myeloid
cell line 416B (46) and terminal maturation of erythroid cells (47,
48). In contrast, although the Nf is dispensable for transactivation,
it is critical for the erythroid and megakaryocytic maturation (46 –
48). Our experiment with GATA-3 mutants indicated that the Nf is
critical for the induction of endogenous GATA-3 and IL-5 but
partially dispensable for the induction of IL-4 and suppression of
IFN-␥. This is partially in contrast to a previous report (40) showing that Nf-deleted GATA-3 mutants failed to induce IL-4 in developing Th1 cells, probably due to the differences in retroviral
expression levels and culture conditions. These results indicate the
functional significance of the Nf of GATA-3.
Using the Nf-containing region as a bait, we identified FOG in
the Th2 cell yeast two-hybrid library. Our quantitative PCR analysis revealed that FOG is expressed in a broad range of tissues,
including lymphoid tissues such as thymus, spleen, and lymph
nodes, while the expression of FOG-2 is restricted mainly to heart,
brain, and ovary (Fig. 2A) (26 –28). Although FOG mRNA expression was detected in several erythroid, megakaryocytic, and
progenitor cells (34), its expression in lymphoid cells was not examined in detail. We found that naive Th cells express significant
levels of FOG mRNA and that its expression is down-regulated
during development to both Th1 and Th2 cells, while FOG-2 is not
expressed significantly in Th cells (Fig. 2). The expression of FOG
protein in naive Th cells and its down-regulation was reported in
a recent paper (49).
It has been shown previously that FOG and GATA-1 can synergistically transactivate the NF-E2 p45 promoter (34). However,
the effects of FOG proteins depend on both cell types and promoter
contexts. Thus, other GATA-dependent promoters are strongly repressed by FOG (44), and the anti-MHC promoter is activated in
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FIGURE 5. The N-terminal region of FOG is
critical for the suppression of Th2 cytokines and
the induction of IFN-␥ in developing Th2 cells. A,
Structure of FOG mutants. The FOG truncation
mutants are shown schematically below the fulllength FOG protein. Numbers shown to the right
indicate the amino acids of FOG included in each
mutant. ⴱ, The putative GATA-interacting zinc
finger; 䉬, the CtBp-binding site and NLS. B, The
3⬘- and 5⬘-truncated mutants of FOG maintain the
ability to interact with GATA-3. The GAL4-(adenovirus major late promoter)-luciferase plasmid
pGL5-luc was cotransfected into 293T cells with
pM-GATA-3-Nf and pVP16 plasmids encoding
FOG fusion proteins. The mean fold activation
relative to that of pM-GATA-3-Nf and pVP16 is
shown. Representative results from three experiments, each in duplicate, are shown. C, Retroviral
transduction of primary CD4⫹CD62L⫹ T cells
with RV-FOG mutants. Naive DO11.10 transgenic naive T cells were stimulated with OVA
and APC and infected with RV-GFP, RV-FOG, or
RV-FOG mutants. On day 7, cells were sorted for
GFP expression and cultured, and cytokine production was measured as in Fig. 4. Representative
results from three independent experiments are
shown.
4544
vators during Th2 development, because GATA-3-⌬Nf has decreased Th2 cytokine production despite abolished FOG interaction (Fig. 1).
Although the mechanism of repression by FOG proteins remains
unsolved, previous studies suggest the role of a corepressor CtBP
(42, 44). Ectopically expressed FOG suppresses crystal cell production in a CtBP-dependent manner (42). In contrast, CtBP binding is not required for the repression of cardiac promoters in
NIH3T3 cells (45) or for the Drosophila eye and heart development (42), where the conserved N-terminal regions of FOG and
FOG-2 are necessary for the repression. We examined the serial Nand C-terminal deletion mutations for the repression on Th2 phenotype (Fig. 5A). Deletion of aa 1– 48 of FOG abolished the repression, while C-terminal deletions did not significantly reduce
the repression until all of the GATA-interacting zinc fingers were
removed (Fig. 5C). These results indicate that the N-terminal region, but not the CtBP-binding motif, of FOG is critical for the
repression of GATA-3-mediated Th2 cytokine gene activation.
These findings will be important for development of inhibitors of
GATA-3 and FOG by increasing our understanding of the transcriptional complexes in Th2 cell development.
In summary, FOG functions in naive Th cells as a repressor of
GATA-3, and the down-regulation of FOG is an essential step for
Th2 cell development.
Acknowledgments
We thank Drs. D. Rennick and J. Sedgwick for helpful support and suggestions, M. Sathe and C. Savkoor for technical support, Dr. D. Gorman for
gene analyses, Dr. J. Cupp for flow cytometry, Dr. T. Kitamura (Tokyo
University, Tokyo, Japan) for providing PLAT-E cells, and M. Angelopoulos for review of the manuscript.
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COS cells but suppressed in primary cardiomyocytes by FOG-2
(28). While many erythroid-specific genes required FOG for their
expression, the expression of GATA-2 and Myc is, conversely,
up-regulated by a GATA-1 mutation defective in FOG interaction
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We have shown that the expression of FOG was down-regulated
during Th cell development of naive T cells. Similar down-regulation of FOG was reported in other GATA-mediated differentiation systems (53, 54). FOG acts as a repressor of GATA-1-mediated induction of eosinophil-specific genes in multipotent
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FOG suppressed the erythroid differentiation in Xenopus embryos
(55). Therefore, these results, including ours, suggest a general
phenomenon that the down-regulation of the FOG family is a prerequisite for GATA-mediated differentiation. Although a previous
report (35) suggested that FOG is not required for the control of T
lymphocyte development by GATA-3, it is possible that the precise analysis of effector Th cell development was hindered by the
embryonic lethality of FOG⫺/⫺ mice.
Our data indicate that FOG inhibits Th2 cytokines and allows
for IFN-␥ production in developing Th2 cells (Figs. 4A and 5C).
Therefore, it appears that overexpression of FOG inhibits the
GATA-3-mediated process of Th2 cell development. The level of
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