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* Department of Immunology and Infectious Diseases, Harvard School of Public Health, and
Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine GATA-3 locus contains several T cell-specific
DNase I hypersensitivity sites. An 
2.5-kb genomic fragment
encompassing several of the DNase I hypersensitivity sites is
sufficient to support the expression of a reporter gene in a T
cell-specific manner in vitro (8, 9). A recent report
showed that the T cell specificity of the human GATA-3 gene
might be regulated by an upstream silencer and a positive
cis-acting element, which is located in the 3' end of the
first intron, however, neither of the regulatory elements was cell
type-specific (10, 11). Even more elusive are the
identities of the transcription factors that dictate the expression of
the GATA-3 gene. Mice rendered deficient in NF-
B
p50-/- or mel-18, a polycomb
protein, have defects in mounting Th2 immune responses, and their Th
cells express lower levels of GATA-3 (12, 13). It remains
to be determined, however, whether the subnormal GATA-3 level is the
cause or the result of impaired Th2 responses in these mice. Although
it was reported that E-box binding proteins could bind to the upstream
silencer of the human GATA-3 gene (10), the in
vivo functions of E-box binding proteins in regulating the expression
of GATA-3 remain unclear.
Fetal liver zinc finger protein 1
(Fliz1)3 is a recently
cloned CCCH-type zinc finger protein (14). Thus far, only
a handful of CCCH-type zinc finger proteins have been identified,
nearly all of which function as RNA binding proteins
(15, 16, 17). For example, TIS11/Nup475/TTP is a critical
protein that regulates the stability and levels of TNF-
transcripts
by specifically binding to an AU-rich element in the 3' untranslated
region of the transcripts (17, 18, 19). During embryogenesis,
Fliz1 is expressed nearly exclusively by hemopoietic progenitors in
fetal livers (14). Its unique tissue distribution suggests
that it might play a critical role in regulating the development and
maturation of hemopoietic cells.
In this study, we report that the 3' end of the first intron of the murine GATA-3 gene contains a very potent regulatory region, which is composed of both a negative and a positive cis-acting element. As a whole, the intronic regulatory region (IRR) serves as a T cell-specific enhancer and is essential for the activity of the GATA-3 promoter. By using yeast one-hybrid screening, we uncovered that Fliz1 could bind to the negative cis-acting element in a sequence-specific fashion. Overexpression of Fliz1 in vitro and in vivo substantially repressed the activities of exogenous and endogenous GATA-3 promoters. Taken together, our data demonstrate that the transcriptional regulation of GATA-3 might be partly regulated by IRR and Fliz1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A genomic fragment containing the upstream region (up to -1475
bp), the first exon, the first intron, and the untranslated part of the
second exon (up to +901 bp) of murine GATA-3 gene was
amplified from genomic DNA prepared from M12 cells. PCR amplification
was performed by using the Platinum Taq DNA Polymerase High
Fidelity (Invitrogen, Carlsbad, CA), and the sequences of the
genomic fragment thus obtained were confirmed by sequence analysis and
were found to be identical to the published sequences of the GATA-3
promoter. This genomic fragment was cloned into the BglII
site of a luciferase reporter plasmid, pBS-luc (a gift of
Dr. K. Murphy, Washington University School of Medicine, St. Louis,
MO), to form the parental construct 2.5-kb
GATA-3-luc. Subsequent deletion mutants as indicated in Fig. 1
A were generated by
restriction digestion. Subfragments of IRR were generated by
restriction digestion or PCR amplification and cloned into an enhancer
trap vector, thymidine kinase (TK)-luc (a gift of Dr. T.
Hoey, Tularik, South San Francisco, CA), upstream to a minimal
thymidine kinase promoter. The mutants of D-25-bp TK-luc
reporter gene were generated by site-directed mutagenesis according to
the manufacturer’s manual (Clontech Laboratories, Palo Alto,
CA).
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Murine T cell lines (EL4, WEHI7.1, and BW5147) and B cell lines
(M12, 18.81, and Bal 17) were maintained and transfected in RPMI 1640
supplemented with 10% FCS (Life Technologies, Grand Island, NY).
Transfection of lymphoid cells (5 x 106
cells/transfection) was performed by electroporation at 280 V/975 µF
with a total 20 µg of DNA. NIH3T3 and Cos7 cells were cultured in
DMEM supplemented with 10% FCS and transfected with Effectene reagent
according to the manufacturer’s protocol (Qiagen, Valencia, CA). Cell
extracts were prepared in reporter lysis buffer (Promega, Madison, WI)
and luciferase activity was assayed by using a luminometer. All
transfection experiments were performed at least four times and
included a pCMV
(Clontech Laboratories) reporter to serve as an
internal control.
EMSA
The truncated cDNA of murine Fliz1 (mFliz1) obtained from the
yeast one-hybrid screening was cloned in-frame into the pET29 vector
(Novagen, Madison, WI). The resulting plasmid was used to transform
BL21 (DE3). The transformed BL21 (DE3) cells were induced with 1 mM
isopropyl -D-thiogalactoside at 37°C for 2
h to produce recombinant truncated mFliz1. The recombinant protein was
enriched in inclusion bodies, solubilized with 6 M urea, and refolded
in PBS. One microgram of such prepared rmFliz1 was used in each EMSA
reaction. Double-stranded oligonucleotides corresponding to the D-25 bp
or SP1 binding sequences were labeled with T4 polynucleotide kinase
(New England Biolabs, Beverly, MA) in the presence of
[
-32P]ATP (New England Nuclear, Boston, MA),
and 10,000 cpm of labeled oligomer was incubated with rmFliz1 protein
in binding buffer (20 mM HEPES, pH 7.9, 1 mM EDTA, 20 mM KCl, 4 mM
MgCl2, 5 mM DTT, 1 µg poly(dI-dC)) at room
temperature for 20 min. Complexes were resolved on 4% polyacrylamide
gels and subjected to autoradiography. For competition assay, excessive
unlabeled D-25 bp or SP1 oligonucleotides were preincubated with
rmFliz1 protein before incubation with specific probes.
Immunoblot analysis
Whole cell extracts were prepared by NE-PER (Pierce, Rockford, IL), resolved on 10% SDS-PAGE gels, and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were incubated with mouse monoclonal anti-myc (9E10) Ab (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-GAL4 DB Ab (Santa Cruz Biotechnology) diluted 1/500 or 1/1000, respectively, in PBS containing Tween 20, followed by incubation with HRP-conjugated specific secondary Abs and detection with ECL kit according to the manufacturer’s instruction (Amersham, Piscataway, NJ).
Real-time PCR
Total RNA was prepared and 1 µg of total RNA was used for
reverse transcription (RT) reaction and amplification by using a
Superscript II RT kit according to the manufacturer’s protocol
(Invitrogen). A master mix of TaqMan reagents was prepared and 10 ng of
each RT product was used in TaqMan PCR (Applied Biosystems, Foster
City, CA). The standard curve method was used to quantitate the amounts
of each species of transcripts relative to the -actin in each
reaction. Reactions were conducted in 96-well plates by using the ABI
PRISM 7700 Sequence Detection System (Applied Biosystems). The
sequences of primers and probes are: GATA-3; 5'-AGAACCGGCCCCTTATCAA-3',
5'-AGTTCGCGCAGGATGTCC3', 6FAM-CCAAGCGAAGGCTGTCGGCAG-TAMRA, T
cell-specific factor 1 (TCF1); 5'-CGCTGCCATCAACCAGATC-3',
5'-ACTTGGCCTGCTCTTCTCGAG-3',
FAM-TGGGTCGCAGGTGGCATGCA-TAMRA,-actin;
5'-ACCAACTGGGACCGATATGGAGAAGA-3',
5'-TACGACCAGAGGCATACAGGGACAA-3',
FAM-CCCTCTGAACCCTA-TAMRA.The primers and probes were designed
with the use of Primer Expression Version 1.0 (Applied Biosystems).
Generation of inducible Fliz1 or Fliz1-
N double-transgenic mice
A full-length cDNA (Fliz1) or an N-terminal truncated mutant
(Fliz1-N), containing amino acid residues 103–291 of human Fliz1,
was tagged with six copies of Myc peptides and cloned into the
BamHI site of pTRE (Clontech Laboratories), which contains
the TetO and a minimal CMV promoter. The resulting constructs were
separately injected into ICR mouse zygotes. The injected embryos were
implanted in the oviducts of day 1 pseudopregnant foster females. Two
independent TetO/Fliz1 and one TetO/Fliz1-N-transgenic mouse lines
were thus generated. The TetO/Fliz1 and TetO/Fliz1-N-transgenic mice
were subsequently bred with CD2-rtTA-transgenic mice (20),
which expressed rtTA under the control of a human CD2 promoter and were
kindly provided by Dr. R. Zamoyska (National Institute of Medical
Research, London, U.K.), to create Fliz1 x rtTA and
Fliz1-N x rtTA double-transgenic mice. The expression of Fliz1
or Fliz1-N was induced by feeding 6–8 wk-old double-transgenic mice
with doxycycline (Sigma-Aldrich, St. Louis, MO)-containing water for
4 days.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously, it was reported that an 2.5-kb genomic fragment,
encompassing the murine GATA-3 promoter, its upstream region, the first
exon, the first intron, and the untranslated part of the second exon,
was sufficient to confer T cell specificity in vitro (8, 9). In addition, the 3' end of the first intron of the human
GATA-3 gene was found to be critical for the activity of a
human GATA-3 promoter (10). To identify the
cis-acting elements that are important for the activity of
the 2.5-kb murine GATA-3 promoter, we have cloned the 2.5-kb promoter
(-1475 to +901) upstream to a luciferase reporter. The resulting
construct was used to transfect various cell lines. In agreement with
previous reports, we found that the 2.5-kb murine GATA-3 promoter was
more active in T cells (EL4) than in B (M12) or NIH3T3 cells (Fig. 1
A). The T cell-specific activity was even more obvious in a
truncated 0.9-kb promoter (-308-+608) (Fig. 1, A and
B). Further deletion of the 3' end of the first intron by
100 bp (0.8 kb; -308 to +500) nearly completely abrogated the
promoter activity. A 0.3-kb minimal promoter, encompassing -308 to
+25, yielded almost no activity and was used as a background control.
These results indicate that the 2.5-kb murine GATA-3 promoter is
sufficient to confer T cell-specificity in vitro, and that the 3' end
of the first intron contains an essential regulatory region. We
henceforth called the 100-bp region in the 3' end of the first intron
IRR.
To further map the critical cis-acting element within IRR,
we generated series of truncation mutants of IRR and cloned them
upstream to a minimal TK promoter driving a luciferase reporter gene.
The resulting constructs were used to transfect EL4, M12, and NIH3T3
cells. In agreement with the result in Fig. 1A, IRR alone
augmented the luciferase activities by 150-fold (Fig. 1C,
100-bp TK-luc). In contrast, <30-fold induction was
observed in M12 or NIH3T3 cells. In addition, nearly 100% of IRR
activity and T cell-specificity were retained in a 25-bp fragment
located in the very 3'end of IRR (D-25 bp TK-luc). We
subsequently performed mutagenesis analysis by introducing a series of
point mutations into the D-25-bp fragment, and examined the activities
of the mutants in EL4 cells. As shown in Fig. 1D, M1 and M2
mutations did not affect the activity of the 25-bp fragment, whereas M3
mutation increased the activity by 3-fold. In contrast, mutations in M4
and M4-2 dramatically reduced the activity by 90%. Taken together,
these results indicate that IRR contains at least one negative and one
positive cis-acting element. Interestingly, the negative
cis-acting element overlaps with a sequence AGGTCTC, which
is conserved between murine and human GATA-3 genes (Fig. 1D).
Molecular cloning of Fliz1, which binds to IRR in a sequence-specific fashion
To identify trans-acting factors that might interact
with IRR, we performed yeast one-hybrid screening by using the D-25-bp
fragment as bait to screen a murine Th2 cDNA library, which was
described previously (21). cDNA clones thus obtained were
further examined by EMSA by using recombinant proteins derived from
each clone. One clone thus identified encodes a novel open reading
frame that contains three CCCH-type zinc finger domains, the sequences
of which are identical to those of the recently cloned mFliz1
(15). The truncated mFliz1 cDNA obtained from the yeast
one-hybrid screening lacks 90 amino acid residues in the N terminus
which were subsequently obtained by 5' RACE. In addition, we also
obtained human Fliz1 (hFliz1) cDNA from several expressed sequence
tag clones (GenBank accession numbers AL530577, AL563786, and
AW958306). Sequence analysis revealed that Fliz1 is highly conserved
between mouse and human. To determine whether Fliz1 can bind to the
D-25-bp fragment in a sequence-specific fashion, we repeated EMSA. As
shown in Fig. 2A, rmFliz1
proteins strongly bind to the D-25 bp, but not to a double-stranded
oligonucleotide containing a consensus SP1 binding site. In addition,
the formation of the Fliz1/D-25-bp complex was completely inhibited by
excessive amounts of unlabeled D-25 bp, but not by unlabeled SP1 probe
(Fig. 2B). To determine the binding sequences of Fliz1
within the D-25 bp, we introduced a series of single base pair
mutations into the D-25-bp probe. The resulting mutant probes were then
radiolabeled and used in EMSA reactions. Interestingly, any single base
pair mutation within the conserved AGGTCTC sequence dramatically
attenuated the binding of Fliz1 protein (Fig. 2C). In
contrast, mutations outside the AGGTCTC sequence did not affect the
binding of Fliz1. Taken together, these results demonstrate that Fliz1,
a CCCH-type zinc finger protein, can function as a DNA binding protein
and can specifically bind to the AGGTCTC sequence within IRR.
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The fact that Fliz1 specifically binds to the AGGTCTC, overlapping
with the negative cis-acting element within IRR, suggests
that Fliz1 might function as a transcriptional repressor (Fig. 1D). To test this hypothesis, we performed cotransfection
experiments. EL4 cells were cotransfected with the 2.5-kb GATA-3
reporter construct along with an expression vector of a
myc-tagged hFliz1. As shown in Fig. 3A, overexpression of Fliz1
inhibited the activity of the 2.5-kb GATA-3 promoter in a
dose-dependent manner (Fig. 3A). Similar results were
obtained when the D-25-bp TK-luc was used (Fig. 3B, left panel). In contrast, overexpression of
Fliz1 did not affect the activity of D-25-bp M3 TK-luc,
which partially ablates the AGGTCTC sequence (Fig. 1D; Fig. 3B, right panel), or the U-25-bp
TK-luc, which does not contain the AGGTCTC sequence (data
not shown). These results suggest that Fliz1 can function as a
transcriptional repressor of GATA-3 in vitro.
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To map the functional domains that are required for the repressor
activity of Fliz1, we performed structure-function analyses. We first
created several truncation mutants of myc-tagged hFliz1 and
examined their effects on the 2.5-kb GATA-3 promoter in EL4 cells by in
vitro transfection assays. The expression of the mutants was confirmed
by immunoblot analysis (Fig. 4A, lower panel).
We found that deletion of the N-terminal 102 amino acid residues
resulted in an appreciable reduction in repression (Fig. 4A,
Fliz1 103–291), whereas deletion of residues 192–291 (Fliz1 1–191),
containing the zinc finger domains, completely abrogated the repression
effects of Fliz1. These results indicate that residues 192–291 are
essential for the repressor activity. The N-terminal 102 residues
(Fliz1 1–102), while required for full repressor activity, are not
sufficient to repress the promoter. However, it is possible that Fliz1
1–102, lacking the zinc finger domains, does not contain a DNA binding
domain and cannot be recruited to the GATA-3 promoter. To test this
hypothesis and to further identify the repression domain in the C
terminus of Fliz1, we generated a series of constructs expressing
various truncation mutants of Fliz1, which were fused to a GAL4 DNA
binding domain. Each of the vectors yielded fusion proteins of expected
m.w. as examined by immunoblot analysis (Fig. 4B,
lower panel). The expression vectors and a luciferase
reporter construct containing GAL4-UAS were used to transfect Cos7
cells. As expected, overexpression of full-length Fliz1 significantly
reduced the luciferase activity to 23% (Fig. 4B, Fliz1
1–291), and a substantial repression effect was retained in the mutant
Fliz1 192–291. Further removal of residues 192 to 218, containing the
first zinc finger, resulted in a complete loss of repressor activity
(Fig. 4B, Fliz1 219–291). Interestingly, the N-terminal 102
residues, when fused with GAL4 DB, also conferred moderate repression
effects (Fig. 4B, Fliz1 1–102). These results demonstrate
that Fliz1 contains at least two independent repression domains. One is
located in the N-terminal 102 residues and the other between residues
192–218.
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To further examine whether Fliz1 can serve as a transcriptional
repressor of the GATA-3 gene in vivo, we have generated
transgenic mice overexpressing Fliz1 in a T cell-specific and
tetracycline-inducible fashion. The generation of the mice was
described in Materials and Methods. Mice were induced to
express a myc-tagged Fliz1 with doxycycline or left
uninduced before analyses, and the overexpression of the
myc-tagged Fliz1 in thymocytes was confirmed by immunoblot
analysis (Fig. 5A, top
panel). We found that overexpression of Fliz1 in vivo resulted in
a substantial reduction (50–75%) in the levels of GATA-3 transcripts
in thymocytes as examined by real-time PCR (Fig. 5A).
Comparable results were obtained from two independently generated
Fliz1 x rtTA transgenic mouse lines (Fig. 5B). The
effect of Fliz1 is specific to the GATA-3 gene because no
reduction was detected in the expression of other transcription factors
such as TCF-1, LEF-1, T-bet, and c-maf (Fig. 5C;
data not shown). In addition, the reduction in the levels of GATA-3
transcripts appears to be dependent on the N-terminal repression
domain, because in vivo overexpression of a truncated Fliz1
(Fliz1-N) lacking the N-terminal 102 amino acid residues did not
affect the expression of GATA-3 in thymocytes (Fig. 5D).
Taken together, these results demonstrate that Fliz1 can attenuate the
expression of GATA-3 in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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625-kb genomic fragment encompassing
the murine GATA-3 locus fails to support the expression of a
reporter gene in lymphoid organs in vivo (22). This
finding strongly indicates that cis-acting elements outside
the 625-kb region are also required for the expression of the
GATA-3 gene in T cells. Murine IRR and the upstream silencer
of the human GATA-3 gene, however, might contribute more to
T cell-specificity than to activity in regulating the expression
of the GATA-3 gene. In addition, IRR probably has a very
limited role in dictating the expression of GATA-3 in Th2 cells,
because IRR is comparably active in both Th1 and Th2 cells in in vitro
transfection experiments (data not shown). The real function of IRR can
be addressed by studying "knock-in" mice bearing deletion or
mutations of IRR via homologous recombination. It is still unclear what transcription factor, if any, binds to the positive cis-acting element within IRR. Various attempts of EMSA have failed to show any binding activity to IRR in endogenous T cell extracts. Repeated yeast one-hybrid screenings were also nonrevealing. Possibly, endogenous IRR-binding proteins, including Fliz1, are present in very low levels or are degraded during the process of preparation. Furthermore, alternative EMSA binding conditions might be needed to allow the formation of protein-DNA complexes.
Although our data firmly demonstrate that overexpression of Fliz1 can repress the activities of the GATA-3 promoter in vitro and in vivo, the real function of Fliz1 remains unclear. Of note, during embryogenesis, Fliz1 is exclusively expressed in fetal liver hemopoietic progenitors (14). It is tempting to postulate that Fliz1 might play a critical role in lineage determination by repressing GATA-3 expression and allowing the development of non-T cells. In adult animals, Fliz1 is expressed at low levels in limited numbers of organs, including thymus, and it has been shown that GATA-3 levels vary in thymocytes maturing from the double-negative to the single-positive stage (2, 23). Thus, it is possible that Fliz1 might contribute to the subtle variation of GATA-3 levels during thymic ontogeny. Alternatively, Fliz1 might regulate the expression of other genes, in addition to GATA-3, which have yet to be identified. In the inducible Fliz1-transgenic mice, a very low level of Fliz1 was induced in peripheral T cells and precluded analyses on its effects on peripheral T cells (data not shown). However, Fliz1 probably has a very limited role in regulating the Th2 cell-specificity of GATA-3, given the very low expression of endogenous Fliz1 in the peripheral T cells (14). Generation and study of Fliz1-deficient mice will be very informative.
The observation that Fliz1 can function as a sequence-specific DNA binding protein merits further discussion. Thus far, nearly all CCCH-type zinc finger proteins are RNA binding proteins (15, 16, 17, 18, 19). To the best of our knowledge, Fliz1 is the first sequence-specific DNA-binding CCCH-type zinc finger protein. It is still very likely, however, that Fliz1 can also bind to RNA with a yet-to-be-defined sequence. Thus, Fliz1 might regulate the levels of GATA-3 by altering the stability of GATA-3 transcripts in addition to repressing the transcription of the GATA-3 gene.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. I-Cheng Ho, Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women’s Hospital, Smith Building, Room 526D, One Jimmy Fund Way, Boston, MA 02115. E-mail address: iho{at}hsph.harvard.edu
3 Abbreviations used in this paper: Fliz1, fetal liver zinc finger protein 1; IRR, intronic regulatory region; mFliz1, murine Fliz1; RT, reverse transcription; TCF1, T cell-specific factor 1; TK, thymidine kinase; hFliz1, human Fliz1.
Received for publication February 11, 2002. Accepted for publication April 23, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene. EMBO J. 10:1187.[Medline]
12 gene enhancer. Mol. Cell. Biol. 11:2778.B in GATA3 expression and TH2 differentiation in allergic airway inflammation. Nat. Immunol. 2:45.[Medline]
mRNA. Mol. Cell. Biol. 19:4311.This article has been cited by other articles:
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