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Molecular and Cellular Biology, January 2001, p. 164-174, Vol. 21, No. 1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.1.164-174.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
A Novel GATA Factor Transcriptionally Represses Yolk Protein
Precursor Genes in the Mosquito Aedes aegypti via
Interaction with the CtBP Corepressor
David
Martín,1,
Maria-Dolors
Piulachs,2 and
Alexander S.
Raikhel1,*
Department of Entomology and Program in
Genetics, Michigan State University, East Lansing, Michigan
48824,1 and Department of Physiology
and Molecular Biodiversity, Institut de Biologia Molecular de
Barcelona, CID, CSIC, Barcelona, Spain2
Received 9 June 2000/Returned for modification 24 July
2000/Accepted 16 October 2000
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ABSTRACT |
In anautogenous mosquitoes, vitellogenesis, the key event in egg
maturation, requires a blood meal. Consequently, mosquitoes are vectors
of many devastating human diseases. An important adaptation for
anautogenicity is the previtellogenic arrest (the state of arrest)
preventing the activation of the yolk protein precursor (YPP) genes Vg and VCP prior to
blood feeding. A novel GATA factor (AaGATAr) that recognizes GATA
binding motifs (WGATAR) in the upstream region of the YPP
genes serves as a transcriptional repressor at the state of arrest.
Importantly, AaGATAr can override the 20-hydroxyecdysone
transactivation of YPP genes, and its transcriptional repression involves the recruitment of CtBP, one of the universal corepressors. AaGATAr transcript is present only in the adult female
fat body. Furthermore, in nuclear extracts of previtellogenic fat
bodies with transcriptionally repressed YPP genes, there is a GATA binding protein forming a band with mobility similar to that of
AaGATAr. The specific repression of YPP genes by AaGATAr in
the fat body of the female mosquito during the state of arrest represents an important molecular adaptation for anautogenicity.
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INTRODUCTION |
The recent resurgence of several of
the most threatening mosquito-borne diseases is due to the failure to
generate effective vaccines and to the rise of resistance in mosquitoes
and in pathogens to insecticides and preventative drugs, respectively.
Malaria is a particularly devastating mosquito-borne disease, taking a heavy toll on the human population in many parts of the world (4).
Mosquitoes serve as disease vectors because they require blood feeding
for their egg development. In anautogenous mosquitoes, vitellogenesis,
the cornerstone of egg maturation, is initiated only after a female
mosquito ingests vertebrate blood. This blood meal triggers a hormonal
cascade with 20-hydroxyecdysone (20E) as the terminal signal, which
activates yolk protein precursor (YPP) genes in the
metabolic tissue, called the fat body (11, 15, 29). An
important adaptation for anautogenicity is the developmental arrest
(the state of arrest), which prevents the activation of YPP
genes in previtellogenic females prior to blood feeding. Understanding
the molecular nature of the state of arrest and the mechanisms
underlying blood meal activation of YPP genes is of
paramount importance for current efforts that utilize molecular genetics to develop novel strategies for controlling mosquito-mediated disease transmission.
In the anautogenous mosquito Aedes aegypti, a key model
vector, the fat body produces several YPPs: vitellogenin, vitellogenic carboxypeptidase, and vitellogenic cathepsin B (5, 6, 8). Genes encoding vitellogenic carboxypeptidase and vitellogenin have been
cloned (10, 31). Analysis of the regulatory regions of
Vg and VCP genes by binding and expression assays
have suggested that both of these genes are under the synergistic
control of the hormone- and tissue-specific gene-regulatory hierarchies
(D. Martín, V. Kokoza, S. F. Wang, and A. S. Raikhel,
unpublished data).
At the state of arrest, the ecdysteroid receptor is the target of the
20E signaling modification in the mosquito fat body. The functional
ecdysteroid receptor is a heterodimer of the ecdysone receptor (EcR)
and the retinoid X receptor homolog, Ultraspiracle (37,
38). In A. aegypti, two EcR isoforms and two
Ultraspiracle isoforms have been cloned (7, 19, 36; Wang,
C. Li, and Raikhel, unpublished data). We have found that at the state
of arrest, AHR38, the mosquito homolog of DHR38 and vertebrate
NGFI-B/Nurr1 orphan receptors, is the key factor inhibiting the
ecdysone response in the A. aegypti fat body. At this stage,
AHR38 interacts strongly with the AaUSP protein, preventing the
formation of a functional ecdysteroid receptor (40).
In addition to the hormone-specific gene-regulatory hierarchy,
transcriptional activation of Vg and VCP genes is
under the control of a tissue-specific GATA factor (Martín and
Raikhel, unpublished data). In this paper, we report a mosquito homolog of the GATA family of transcription factors (AaGATAr) that recognizes GATA binding motifs in the upstream region of the YPP genes,
Vg and VCP. Significantly, AaGATAr acts as a
repressor of YPP genes in cell transfection assays. The
transcriptional repression by AaGATAr involves the corepressor CtBP
(23, 27). AaGATAr mRNA is only present in adult female fat
bodies and the binding activity corresponding to AaGATAr in the nuclei
of previtellogenic fat bodies.
Thus, we have identified a novel transcriptional repressor, belonging
to the GATA family of transcription factors, which recruits CtBP, one
of the universal corepressors. Our data further suggest the involvement
of AaGATAr in the specific repression of YPP genes in the
fat body of the A. aegypti female at the state of arrest.
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MATERIALS AND METHODS |
Animals.
Mosquitoes, A. aegypti, were reared
according to the method described by Hays and Raikhel
(16). Vitellogenesis was initiated by allowing females 3 to 5 days after eclosion to feed on an anesthetized white rat.
Cloning and sequencing of cDNA.
Two degenerated
oligonucleotides derived from highly conserved amino acid sequences of
the GATA finger region (from human, mouse, chicken, and fruit fly GATA
proteins) were designed: GATAS (a sense strand composed of 29 nucleotides corresponding to the amino acid residues -ECVNCG-) and
GATAR (an antisense 27-nucleotide strand corresponding to amino acid
sequence -GCANCV-). The sequences of the oligonucleotides were as
follows: GATAS,
5'-AGATCTAGAGARTGYGTNAAYTGYGGNGC-3'; and GATAR,
5'-AGAGAATTCNCCRCANGCRTTRCANAC-3', where R is A or G; Y is
C or T; and N is A, T, C, or G.
To facilitate cloning of the amplified product, anchor sequences
containing EcoRI and XbaI restriction sites were
added 5' of the forward and the reverse primers.
Total RNA prepared from previtellogenic female fat bodies was used to
generate randomly primed double-stranded cDNA using
reverse
transcription (RT) (Gibco BRL). The initial PCRs were
performed in a
final volume of 50 µl containing 0.5 µg of each
of the two
degenerate primers, 10 ng of cDNA, and 0.25 U of AmpliTaq
gold
polymerase (Perkin-Elmer). After a denaturation step of 2
min at
94°C, amplifications were carried out for 10 cycles at
94°C for
30 s, 45°C for 1 min, and 72°C for 2 min, followed by
30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min.
The amplified fragment was subcloned into vector pGEMT-easy
(Promega)
for sequencing. After confirmation of its sequence, the
243-bp
PCR fragment was used as a probe to screen a
ZAPII cDNA
library
generated from fat bodies of vitellogenic female mosquitoes
from
6 to 48 h post-blood meal (PBM) (
9). After four
rounds of screening,
one clone with an insert size of around 3.8 kb was
isolated and
sequenced from both strands. The clone did not contain the
initiation
sequence. The missing part of the 5' end sequence was
obtained
by 5' rapid amplification of cDNA ends-PCR (Gibco BRL) using
an
antisense primer located 397 bp downstream of the AaGATAr cDNA
start
(5'-ACAAACGAGGAGCATGTAAACACT-3'). Total RNA purified from
previtellogenic fat bodies was used as a template for RT. An 800-bp
PCR
fragment was subcloned into pGEMT-easy for sequencing and
contained the
initiation sequence, completing then the entire
open reading frame
(ORF).
Alignments of nucleotide and amino acid sequences.
The
software package of the Genetics Computer Group (GCG, version 9.1) of
the University of Wisconsin was used for sequence alignments, which
were carried out with Pileup and were not further hand refined;
alignment was displayed with the Box option. Phylogenetic analyses were
carried out using amino acid sequences, with the Phylogeny Inference
Package (Phylip, version 3.57c). We followed the method of neighbor
joining, and the distances between different sequences were estimated
with Kimura's formula and the application Protdist. Bootstrap analyses
were carried out with the application Seqboot in the Phylip package,
and the procedure was repeated 100 times.
Northern blot analysis.
Total RNA from mosquitoes of
different stages, taken from tissues as well as from whole males, was
prepared using the RNeasy Qiagen kit. Fifteen micrograms of total RNA
was separated in a 1.2% agarose-formaldehyde gel, blotted to a nylon
membrane (N+-Hybond; Amersham), hybridized, and washed
under stringent conditions. The probe used was a 2.5-kb
EcoRI fragment of the full-length AaGATAr cDNA clone
containing the DNA-binding domain, which was labeled by random priming
(32) with [32P]dATP (3,000 Ci/mmol) (New
England Nuclear). Northern blots were also hybridized with a labeled
0.8-kb EcoRI fragment from the VCP gene
(10) and with a 0.85-kb EcoRI fragment from the
A. aegypti actin gene (11).
In vitro transcription and translation.
The entire AaGATAr
cDNA was cloned into pBluescript SK(+). A coupled in vitro
transcription-translation TNT system (Promega), utilizing the T7
promoter, was used for expression of the AaGATAr cDNA. To monitor the
in vitro reaction, the synthesized protein was labeled with
[35S]methionine (1,200 Ci/mmol) from ICN Radiochemicals,
and the radiolabeled product was visualized by electrophoresis and autoradiography.
EMSA.
Preparation of nuclear extracts from A. aegypti fat bodies and the electrophoretic mobility shift assay
(EMSA) using the nuclear extracts were carried out according to the
method described by Miura et al. (22). DNA probes for EMSA
were made by annealing together complementary oligonucleotides. The
GATA binding site (box A) in the Drosophila mulleri alcohol
dehydrogenase gene (Adh) (1) was used as a positive
control for binding. The oligonucleotides (only sense strands are
shown) used to generate the different probes were D. mulleri
box A, 5'-AGTGGTATTGATAAGAC-3'; AaVgGATAa,
5'-TTAATGCTTATCATCGCG-3'; AaVgGATAb,
5'-TTTTGCTTATCTTACTATCTTCA-3'; AaVgGATAc, 5'-GAATTTCAACAATGATAGCCTTTCA-3'.
(Boldface letters correspond to the core GATA motif within the
primers.)
Plasmid construction and cell transient transfection.
The
pPac-ABF and Adh-1-(BoxA)6/CAT plasmids were provided by T. Abel (1). The pAc5-AaGATAr expression plasmid was
constructed by subcloning the entire AaGATAr cDNA into the expression
vector pAc5/V5/His(C) (Invitrogen), under the control of the actin 5C promoter. The reporter plasmid (AaVgGATAb)4/CAT was
constructed by ligating four copies of the AaVgGATAb oligonucleotide
5'-AGCTTTTTGCTTATCTTACTATCTTCAATT-3' (only one
strand is shown; the HindIII site is underlined) into the HindIII site of Adh-1/CAT (21) just
upstream of the Drosophila Adh promoter controlling the
expression of CAT. The 0.6Vg-Luc reporter plasmid was constructed by
cloning a 0.6-kb KpnI-SmaI fragment of the 5'
regulatory region of the A. aegypti Vg gene (31) into the promoterless plasmid pGL3basic (Promega).
This construct include sequences from 
600 to +115 bp of the
Vg gene. The pAc5-dCtBP expression vector was constructed by
excising the entire dCtBP cDNA from pGEX-3X-dCtBP (provided by S. Parkhurst) with BamHI, blunt ending the recessed termini
with Klenow, and cloning the cDNA into pAc5/V5/His(C) digested with
EcoRV. pAc5-AaGATAr
CtBP was obtained by PCR using two
primers flanking the CtBP binding site within the AaGATAr protein.
Expression vectors pAc5-AaEcR and pAc5-AaUSPb are described in
reference 36. All constructs were confirmed by restriction
analysis and sequencing.
Transfections were carried out using the Schneider
Drosophila cell line (S2; Invitrogen), as described by Wang
et al. (
36)
with minor modifications. Transfection was
conducted with LipofectACE
(Gibco BRL) with a DNA-to-lipid ratio of
1:10 (wt/wt).
GST pulldown assay.
AaGATAr, AaGATArCtBP, and
Drosophila ABF were 35S labeled with a coupled
in vitro transcription translation protocol (TNT; Promega). GST-dCtBP
was provided by S. Parkhurst and was prepared as described previously
(27). Binding assays were performed as described
previously (40). Generation of the mutated AaGATArCtBP protein was carried out by PCR using Pfu polymerase
(Promega) and a pair of primers flanking the PCDLSYK sequence.
Sequencing was performed to verify the deletion.
Nucleotide sequence accession number.
The AaGATAr sequence
has been submitted to the DDBJ/EMBL/GenBank database under
accession no. AJ400338.
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RESULTS |
Cloning and characterization of AaGATAr cDNA.
Two motifs were
selected from a highly conserved GATA DNA-binding domain for the design
and synthesis of degenerated primers, which were then used for the
initial PCR isolation of the cDNA encoding a mosquito GATAr (see
Materials and Methods). RT-PCR of the fat body total RNA from
previtellogenic females yielded a cDNA fragment of 256 bp. The sequence
of this fragment exhibited a high degree of identity with the GATA
DNA-binding domain. This cDNA fragment was then used to screen the
ZAP II vitellogenic fat body cDNA library. The clone with the
longest insert (3.8 kb) was sequenced from both strands. The missing 5'
portion of the cDNA was obtained by 5' rapid amplification of cDNA
ends-PCR using a specific antisense primer designed from the isolated clone.
The entire sequence of the putative AaGATAr cDNA was 4,275 bp long and
encoded a protein of 866 amino acids with a predicted
molecular mass of
93.83 kDa (Fig.
1). The
deduced amino acid sequence
started with a methionine at nucleotide 412 and ended with a stop
codon at nucleotide 3016. The initiation codon
conformed to the
Kozak consensus sequence (
20) (Fig.
1, double underlined). This
putative
methionine start codon was preceded by several in-frame
stop codons,
indicating that the AaGATAr sequence represented
a full-length ORF. At
the 3' end, the AaGATAr polyadenylation
signal AATAAA was
followed by an additional 9-bp sequence and
lacked a poly(A) tail,
indicating that this cDNA was not a full-length
transcript. To verify
that the cloned cDNA contained a translatable
ORF, it was expressed in
a coupled TNT system under the control
of the T7 promoter. Sodium
dodecyl sulfate-polyacrylamide gel
electrophoresis and fluorography
showed that the in vitro-synthesized
protein closely corresponded to
its expected molecular size (data
not shown).
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FIG. 1.
Nucleotide and deduced amino acid sequence of AaGATAr.
Boldface, stop codons; double underline, Kozak sequence; single
underline, polyadenylation signal; boldfaced italics, phosphorylation
signal; dotted underline, putative glycosylation sequences; striped
box, putative nuclear localization signal; grey box, CtBP interaction
motif. The DNA-binding domain is boxed.
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Analysis of the AaGATAr protein sequence revealed that it contained a
double C.X
2.C.X
17.C.X
2.C. zinc
finger (Fig.
1), characteristic
for the GATA DNA-binding transcription
factors (
25). Its DNA-binding
domain showed a high level
of conservation of the residues involved
in direct contact with the DNA
(
24) (Fig.
2). Outside the
DNA-binding
domain, however, the level of conservation between AaGATAr
and
other GATA family members was very low. Characteristic features
of
AaGATAr included a putative nuclear localization signal located
downstream of the second zinc finger (RKRKPK), several potential
N-linked glycosylation sites (NXS/T), and a putative PKA
phosphorylation
site (RRPS) (Fig.
1). Surprisingly, AaGATAr also
contained a motif
(PCDLSYK) which was similar to the consensus binding
motif PXDLSXK/H
for the corepressor protein CtBP (Fig.
1)
(
23).
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FIG. 2.
Similarities of AaGATAr to other members of the GATA
family. Shown is an alignment of different GATA amino acid sequences
belonging to different animal orders, displaying a high degree of
similarity in the area corresponding to the DNA-binding domain. The
alignment was made using a GCG PILEUP option, and results are displayed
by using the BOX option. Sequence accession numbers are chicken
(GATA4), P43691; Xenopus (GATA4), Q91677; human (GATA4),
P43694; zebra fish (GATA3), Q91428; Drosophila (dGATAa),
P52168; Aedes (AaGATAr), AJ400338; Bombyx
(bGATA 3), P52167.
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Next, we carried out phylogenetic analyses of amino acid sequences from
proteins of the GATA family using the neighbor-joining
and Kimura
distance methods. Two zinc finger GATA proteins were
clustered into two
separated branches. The first included GATA
factors of types 1, 2, and
3, while the second included types
4, 5, and 6 of vertebrate as well as
insect and worm GATAs (Fig.
3). AaGATAr
was classified as a member of the second group, being
phylogenetically
closest to
Bombyx mori GATA 1 and 2 (Fig.
3).
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FIG. 3.
Dendrogram generated from amino acid sequences
corresponding to DNA-binding domains of GATA transcription factors. The
method of neighbor joining, using the distance formula of Kimura, was
applied to generate the tree. Numbers at the nodes correspond to
bootstrap values in 100 replicates. The accession numbers for the GATA
sequences used are A. aegypti, AJ400338; B. mori
1, P52167; B. mori 2, P52167; Brachydanio rerio,
Q91428; Caenorhabditis elegans 1, P28515; Drosophila
melanogaster a, P52168; D. melanogaster c, D50542;
Gallus gallus 1, P17678; G. gallus 2, P23824;
G. gallus 3, P23825; G. gallus 4, P43691;
G. gallus 5, P43692; G. gallus 6, P43693;
Homo sapiens 1, P15976; H. sapiens 2, P23769;
H. sapiens 3, P23771; H. sapiens 4, P43694;
H. sapiens 6, Q92908; Mus musculus 1, P17679;
M. musculus 2, AB000096; M. musculus 3, P23772;
M. musculus 4, Q08369; M. musculus 5, P97489;
M. musculus 6, S82462; Rattus norvegicus 1, P43429; R. norvegicus 4, P46152; R. norvegicus 6, P46153; Xenopus laevis 1, P23767; X. laevis 2, P23770; X. laevis 3, P23773; X. laevis 4, Q91677;
X. laevis 5, P43695; X. laevis 6, Q91678.
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Tissue specificity and temporal pattern of AaGATAr mRNA
expression.
To determine the distribution of the AaGATAr mRNA in
the adult mosquito, samples of total RNA from whole body of males as well as fat body, ovary, gut, and thorax of vitellogenic females were
subjected to Northern blot analyses, using a 2.5-kb EcoRI AaGATAr cDNA fragment containing the DNA-binding domain as a probe. A
single RNA transcript of 4.3 kb, matching the size of the AaGATAr cDNA,
was found only in the female fat body (Fig.
4). No hybridization was detected in
total RNA collected from pupae at different developmental time points.
In contrast, the AaGATAr transcript was present in the fat bodies of
female mosquitoes after eclosion (Fig.
5A). The same transcript size was
detected when using a 1.7-kb cDNA fragment of the unique region of
AaGATAr as a probe (data not shown).
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FIG. 4.
Northern blot analysis of AaGATAr mRNA levels in males
and different tissues of female mosquitoes. Total RNA was extracted
from different female tissues as well as from whole males (15 µg per
lane). Northern blots were hybridized with a 32P-labeled
2.5-kb fragment from the protein-coding region (including the
DNA-binding domain) of the AaGATAr cDNA clone (upper panel). To control
the integrity of the isolated RNA, the same blot was hybridized to
A. aegypti actin cDNA (middle panel). Portions of the gel
containing rRNA were stained with ethidium bromide to control for
equivalent sample loading (lower panel).
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FIG. 5.
Northern blot analysis of AaGATAr mRNA levels during
pupal development and in the female mosquito fat body during the first
vitellogenic cycle. (A) Total RNA from various stages in A. aegypti development (15 µg per lane). Northern blots were
hybridized with a 32P-labeled 2.5-kb fragment from the
protein-coding region (including the DNA-binding domain) of the AaGATAr
cDNA clone (upper panel). (B) Total RNA was extracted from fat bodies
dissected from previtellogenic mosquitoes on indicated days
(Pre-vit.per.) after eclosion and from vitellogenic fat bodies at
indicated hours PBM (15 µg per lane). Northern blots were hybridized
with the same 32P-labeled 2.5-kb fragment from the AaGATAr
cDNA clone (upper panel) and with a 32P-labeled 0.8-kb
fragment from the AaVCP gene (middle panel). Portions of the gel
containing rRNA were stained with ethidium bromide to control for
equivalent sample loading (lower gels in panels A and B).
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Detailed analysis of the AaGATAr expression in the female fat bodies
showed that its transcript was present throughout the
previtellogenic,
vitellogenic, and postvitellogenic periods (Fig.
5B). The AaGATAr
transcript was more abundant in newly eclosed
females and newly fed
females (1 to 3 h PBM) as well as in those
at the postvitellogenic
stage (36 and 42 h PBM) (Fig.
5B). Thus,
the
AaGATAr
gene appears to be a fat body-specific gene expressed
only in adult
females.
AaGATAr protein recognizes GATA binding sites and binds to putative
GATA elements in 5' regions of mosquito YPP genes.
Next, we
elucidated whether the AaGATAr protein recognized the DNA-binding site
characteristic for the GATA family of transcription factors. The
TNT-expressed AaGATAr was tested in EMSAs using the box A sequence from
the D. mulleri alcohol dehydrogenase (Adh) gene
as a probe, which had been shown to bind a truncated version of the
Drosophila GATAb protein, ABF (1). In our
EMSAs, a binding complex was observed with AaGATAr and the box A
response element (Fig. 6A, lane 2). The
specificity of the complex was confirmed by its competition with a
50-fold molar excess of either the box A DNA (Fig. 6A, lane 3) or a
putative GATA binding site from the A. aegypti lysosomal
aspartic protease (LAP) gene (12) (Fig. 6A,
lane 4) but was not confirmed by nonspecific competitors like a
putative E75 binding site from the A. aegypti Vg gene (Fig. 6A, lane 5). Furthermore, the specificity of the complex was also established by a supershift, with the antibodies recognizing the first
zinc finger domain of B. mori GATA (13) (Fig.
6A, lane 6). Taken together, these results indicated that AaGATAr was
specifically bound to GATA DNA motifs in vitro.
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FIG. 6.
AaGATAr protein binds to a GATA binding site, and
identification is performed for a functional GATA protein in fat body
nuclear extracts of previtellogenic fat bodies. , absence of
substance; +, presence of substance. (A) EMSA using in
vitro-transcribed and -translated AaGATAr and 32P-labeled
Drosophila box A element as a probe. The GATA-specific
complex is indicated by an arrow (lane 2). Fiftyfold molar excesses of
cold probe (lane 3) or the AaLAP GATA binding site (lane 4) were
included as specific competitors. The same excess of cold
double-stranded oligonucleotide of an unrelated sequence (AaVgE75
binding site) was included in lane 5. The DNA-AaGATAr complex was
supershifted by a B. mori anti-GATA serum (lane 6). An
arrowhead indicates the position of the supershifted complex. In
vitro-expressed full-length AaGATAr showed a small product due to an
internal initiation site of transcription. (B) Nuclear extracts from
previtellogenic fat bodies were examined by EMSA with AaVgGATAb element
as a probe. The GATA complex was also supershifted by a B. mori anti-GATA serum (lane 1). An arrowhead indicates the position
of the supershifted complex. The GATA-specific complex is indicated by
an arrow. For competition analysis of this complex, 50-fold molar
excesses of cold probe (lane 4) or AaLAP GATA oligonucleotide (lane 5)
were included as specific competitors. The same amount of cold
double-stranded oligonucleotide of an unrelated sequence was included
in lane 3.
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Analysis of the 5' regulatory region of
Vg and
VCP genes showed the presence of several GATA binding sites.
As an example,
AaGATAr specifically bound to a putative binding site
from the
Vg gene designated AaVgGATAb (Fig.
7A, lanes 2 to
5).
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FIG. 7.
In vitro-synthesized AaGATAr protein binds to upstream
elements of the A. aegypti YPP genes. (A) AaGATAr
protein binds to a sequence in the Vg gene. An EMSA was done
with in vitro-produced AaGATAr and AaVgGATAb as probes. A complex due
to specific interaction between DNA and protein is clearly detected
(lane 2). Fiftyfold molar excesses of cold probe (lane 3) and AaLAP
GATA binding site (lane 4) were included as specific competition. The
same excess of a cold, unrelated oligonucleotide (AaVgE75) was included
in lane 5. (B) Relative affinity of different AaVg GATA binding sites
for AaGATAr protein. Binding of AaGATAr to Drosophila box A
element in the absence () or in the presence of increasing amounts of
cold competing probes (self-competition [DmBoxA.], AaVgGATAa,
AaVgGATAb, and AaVgGATAc) was performed to compare the binding affinity
of the different AaVg GATA elements. (C) GATA motifs in the upstream
regulatory regions of vitellogenin used in the competition analysis.
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To demonstrate the occurrence of different relative affinities of
AaGATAr binding to different GATA binding sites within the
5'
regulatory region of the
Vg gene, we carried out an EMSA
competition
analysis. The binding complex formed by the AaGATAr protein
and
the box A response element was competed with increasing
concentrations
of one of three different
AaVg GATA response
elements (designated
as AaVgGATAa, AaVgGATAb, and AaVgGATAc) (Fig.
7C) as well as with
the
Drosophila box A element as a
positive control. The competition
levels indicated that the DNA-binding
affinity of AaGATAr towards
the different AaVg GATA binding sites
followed the order AaVgGATAb
AaVgGATAa
AaVgGATAc
(Fig.
7B).
Previtellogenic fat body nuclear extracts contain a GATAr-like
factor bound to GATA elements in the YPP genes.
To identify the
occurrence of nuclear factors that bind specifically to these GATA
sequences in the Vg gene at the state of previtellogenic
arrest, we carried out EMSAs with nuclear extracts prepared from fat
bodies of previtellogenic female mosquitoes. A retardation complex was
observed when the radiolabeled AaVgGATAb response element was incubated
with the nuclear extract (Fig. 6B, lane 2). To determine the
specificity of this interaction, competition experiments were performed
using excess cold-specific or nonspecific oligonucleotides (Fig. 6B,
lanes 3 to 5). As done before, the specificity of the complex was also
established by a supershift with the anti-GATA serum (Fig. 6B, lane 1).
Developmental changes of this GATA binding complex in the fat body
nuclei showed that it was present during the stage of arrest (3 to 5 days posteclosion), as well as at 60 h PBM, when YPP synthesis was
completely halted (not shown).
AaGATAr protein acts as a transcriptional repressor.
To
establish whether AaGATAr was able to regulate transcription upon
binding to GATA binding sites, we used the transient transfection assay
in a Drosophila Schneider (S2) cell line. We employed an
adh-1-(BoxA)6-CAT reporter construct that contained six
copies of the box A site upstream of the adh-1 promoter with the CAT gene as a reporter. The pAc5-AaGATAr expression
vector contained AaGATAr cDNA under the control of the constitutive
actin 5C promoter. As a positive control, the pPac5-ABF vector
(Drosophila ABF controlled by the actin 5C promoter)
(1) was used. As shown in Fig.
8A, while ABF was able to activate the
reporter gene (up to 2.1-fold), AaGATAr repressed the reporter activity
(51% inhibition).
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|
FIG. 8.
AaGATAr represses gene transcription via GATA-specific
recognition sites. The Drosophila Schneider cell line was
transfected with the indicated plasmids by the lipid-mediated method.
, absence of substance; +, presence of substance. (A) Chloramphenicol
acetyltransferase (CAT) activity from cells transfected with 100 µg
of a reporter plasmid containing six copies of box A upstream of the
Adh promoter driving the expression of CAT, with either 0.5 µg of pPac5-ABF (Drosophila GATAb isoform controlled by
the actin promoter) or pAc5-AaGATAr (AaGATAr controlled by the actin
promoter). As a control, the reporter plasmid Adh-1/CAT (lacking any
GATA sequences) was used. (B) CAT activity from cells transfected with
100 ng of a reporter plasmid containing four copies of the AaVgGATAb
binding site upstream of the Adh promoter driving the expression of
CAT, with 0.5 µg of pPAc5-ABF or pAc5-AaGATAr. In all cases, cells
were transfected for 12 h and incubated for 48 h at 22°C.
The plasmid pAc5-LacZ was used in all assays for transfection
efficiency. Transfection was performed in two independent experiments
in triplicate. Significant differences (t test) between
treated and respective controls are represented by asterisks (*,
P  0.05; **, P 0.005). Each
value represents the mean ± standard deviation.
|
|
To verify that AaGATAr could exert similar repression via the GATA
binding sites present in the
Vg gene, we modified the
adh-1-(BoxA)
6-CAT
reporter plasmid, replacing six copies of
box A with four copies
of the AaVgGATAb binding site. This construct
was cotransfected
with either the ABF or AaGATAr expression
vectors; ABF was able
to activate the reporter up to 3.7-fold, whereas
AaGATAr repressed
the CAT activity about 50% (Fig.
8B). It is worth
noting that
in the absence of pPac-ABF or pAc5-AaGATAr, the reporter
construct
showed a high basal activity consistent with the presence of
Drosophila GATAb in S2 cells (
1).
Next, we carried out cell transfection experiments utilizing fragments
of the 5' regulatory regions of the mosquito
Vg gene
containing GATA binding sites in the context of their natural
flanking
sequences. The luciferase reporter was driven by the
Vg
promoter containing 0.6 kb of the 5' untranscribed region (
600
to
+115 bp) with both AaGATAb and AaGATAa binding sites (0.6Vg-Luc)
(Fig.
9). Cotransfection of the 0.6Vg-Luc
construct with the AaGATAr
expression plasmid resulted in a noteworthy
70% reduction of the
basal reporter activity (Fig.
9A). In addition to
GATA binding
sites, this region contained the ecdysone response element
sufficient
for a two- to threefold ecdysone-dependent activation after
cotransfection
with AaEcR and AaUSP expression plasmids
(Martín, Wang, and Raikhel,
unpublished data). Because of that,
cotransfection of the 0.6Vg-Luc
construct with the AaEcR and AaUSP
expression plasmids resulted
in about a 2.5-fold induction of
transcriptional activity in the
presence of 20E (Fig.
9B). Importantly,
this ecdysone-dependent
transactivation was abolished, a 65% total
reduction, as a result
of the cotransfection of AaGATAr (Fig.
9B). To
ensure that the
AaGATAr-dependent repression was not due to a
nonspecific squelching
of the general transcription machinery, cell
transfections were
carried out with Adh-1/CAT and pAc5-LacZ (reporters
without GATA
binding sites) as well as with 0.6Vg-Luc (reporter with
GATA binding
sites), along with pAc5-AaGATAr at the same time. Whereas
AaGATAr
inhibited the activity of 0.6Vg-Luc, it did not affect the
expression
of the other reporter plasmids which were driven by
promoters
independent of GATA (data not shown).
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FIG. 9.
AaGATAr inhibits basal and 20E-dependent activation of
the A. aegypti vitellogenin gene. Luciferase activity is
measured in arbitrary units. (A) Luciferase enzyme activity in
Drosophila Schneider cells transfected with 100 ng of a
reporter gene containing 0.6 kb of the 5' regulatory region of the
Vg gene, which controls the expression of luciferase and
pAc5-AaGATAr (500 ng). (B) The Vg gene-luciferase reporter and
pAc5-AaGATAr were cotransfected along with the expression vectors
pAc5-AaEcR (12.5 ng) and pAc5-AaUSPb (12.5 ng). Cells were treated,
where indicated, with 1 µM 20E for 36 h after transfection and
incubated at 22°C. The plasmid pAc5-LacZ was used in all assays for
transfection efficiency. Transfection was performed in two independent
experiments in triplicate. Significant differences (t test)
between treated and respective controls are represented by asterisks
(**, P 0.005). Each value represents the
mean ± standard deviation.
|
|
AaGATAr interacts with the corepressor CtBP.
We identified a
putative recognition site (PCDLSYK) for CtBP in AaGATAr (Fig. 1 and
10A). We used the glutathione
S-transferase (GST) pulldown in vitro binding assay to
examine the interaction between Drosophila CtBP (dCtBP) and
AaGATAr. Drosophila CtBP, fused to GST (GST-dCtBP), was
immobilized on glutathione-Sepharose beads and incubated with in
vitro-expressed 35S-labeled AaGATAr. This assay showed that
GST-dCtBP specifically interacted with AaGATAr (Fig. 10B, lanes 1 to
3). The mutant AaGATArCtBP with a deleted putative CtBP binding site
was expressed and radiolabeled in vitro (Fig. 10B, lane 4); however, it
failed to bind GST-dCtBP (Fig. 10B, lane 6). As a negative control, we
tested in the same assay the Drosophila ABF protein, a
transcriptional GATA activator. Drosophila ABF was prepared
in a way similar to that for AaGATAr (Fig. 10B, lane 7) and subjected
to binding with GST-dCtBP. No interaction between GST-dCtBP and ABF was
detected (Fig. 10B, lane 9).
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|
FIG. 10.
AaGATAr interacts with the corepressor dCtBP in vitro.
(A) Consensus interaction motif for CtBP. Shown are
Drosophila and mosquito repressors that have been
demonstrated to bind to the CtBP corepressor protein. (B)
35S-labeled full-length AaGATAr protein (lane 1) interacts
with GST-dCtBP (lane 3) but not with GST alone (lane 2). Mutated
AaGATAr protein lacking the CtBP recognition site (AaGATArCtBP)
(lane 4) is not able to retain GST-dCtBP (lane 6) or GST alone (lane
5). As a control, 35S-labeled ABF protein (lane 7) shows no
interaction with GST-dCtBP (lane 9) or GST alone (lane 8). In
vitro-expressed full-length AaGATAr showed a smaller product, due to an
internal initiation site of transcription.
|
|
We then cotransfected
Drosophila S2 cells with both
pAc5-AaGATAr and pAc5-dCtBP, along with the 0.6Vg-
Luc
plasmid as a reporter.
As shown, whereas AaGATAr was able to repress
the ecdysone-dependent
stimulation of the reporter by 53%, addition of
increasing amounts
of dCtBP resulted in an increase of the repression
in a dose-dependent
manner (up to a 70% inhibition with a 1:1
AaGATAr/dCtBP ratio)
(Fig.
11A). When
we transfected
Drosophila cells with the mutated
AaGATAr
CtBP protein, it displayed the same degree of repression
that
AaGATAr did alone, but this repression was not increased
by dCtBP
cotransfection (Fig.
11B).
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|
FIG. 11.
dCtBP cooperates with AaGATAr to repress transcription.
(A) Luciferase enzyme activity in Drosophila Schneider cells
transfected with 100 ng of the 0.6Vg-Luc reporter gene along with the
expression vectors pAc5-AaEcR (12.5 ng), pAc5-USPb (12.5 ng),
pAc5-AaGATAr (500 ng), and pAc5-dCtBP (10, 100, and 500 ng). (B)
Luciferase activity of Drosophila Schneider cells
transfected with 100 ng of the 0.6Vg-Luc reporter gene along with
expression vectors pAc5-AaEcR (12.5 ng), pAc5-USPb (12.5 ng),
pAc5-AaGATAr (250 ng), pAc5-dCtBP (250 ng), and pAc5-AaGATArCtBP
(250 ng). Cells were treated with 1 µM 20E for 36 h after
transfection. The plasmid pAc5-LacZ was used in each assay for
transfection efficiency. Transfections were performed in two
independent experiments in triplicate. Significant differences
(t test) between treated and respective controls are
represented by asterisks (*, P 0.05; **,
P 0.005). Each value represents the mean ± standard deviation.
|
|
| |
DISCUSSION |
AaGATAr is a novel member of the GATA family of transcription
factors that serves as a repressor.
In this work, we report the
characterization of a mosquito transcription factor belonging to the
GATA family. Analysis of the amino acid sequence of the AaGATAr protein
revealed that it contained a double
C.X2.C.X17.C.X2.C. zinc finger, a
signature motif of the GATA DNA-binding transcription factors
(25). Moreover, the Pileup analysis based on the alignment
of the DNA-binding domains of GATA family members confirmed that
AaGATAr belongs to the GATA family and is related to the subfamily that
includes isoforms 4 through 6 (Fig. 3). Furthermore, AaGATAr
protein recognizes the DNA-binding sites characteristic for the GATA
family of transcription factors and specifically binds to several
putative GATA elements in 5' regulatory regions of the mosquito
YPP genes Vg and VCP.
Previously, the large family of GATA-related transcription factors was
known for its transactivating properties (
3,
26).
The
novel aspect of our study is the finding that AaGATAr acts
as a
transcriptional repressor. AaGATAr was able to repress the
adh-1-(BoxA)
6-CAT reporter construct that contained six
copies
of box A GATA elements. Moreover, in the transient assay,
AaGATAr
could exert similar repression via the GATA binding sites
present
in the
Vg gene. Even more significant is the ability
of AaGATAr
to overcome and repress ecdysone-dependent activation of the
5'
regulatory regions of the mosquito
Vg gene containing
GATA binding
sites in the context of their natural flanking sequences,
as well
as the ecdysone response element. Recently, it has been shown
that transcriptional cofactors of the FOG (Friend of GATA) family
can
repress GATA-mediated activation (
14,
18). However, to
our
knowledge, this is the first report of a GATA transcription
factor that
serves as a specific transcriptional
repressor.
AaGATAr directly interacts with the corepressor CtBP.
AaGATAr is involved in transcriptional repression by recruiting the
corepressor protein CtBP. CtBP has been shown to interact with a highly
conserved PXDLSXK sequence within several zinc finger repressors which
are involved in the restricted patterns of gene expression during
Drosophila embryogenesis (such as krüppel, snail,
knirps, hairy, and zfh-1) (23, 27, 39) with the vertebrate proteins CtIP, ZEB, and BKLF (28, 33, 35) and the viral protein Ad2/5E1A (34). As in the case of these repressors,
the interaction with CtBP depends on the integrity of a highly
conserved motif, PCDLSYK, in AaGATAr. The CtBP recognition motif
has not been found in any other member of the GATA family of
transcription factors, which is consistent with the previously reported
role of GATA proteins as transcriptional activators (3,
26).
Cotransfection experiments validated the importance of CtBP in the
AaGATAr-mediated repression of the
Vg gene. Cotransfection
with increasing amounts of dCtBP resulted in an elevation of the
repression levels of the
Vg gene in a dose-dependent manner.
Although
CtBP significantly enhanced AaGATAr repression, AaGATAr
alone
or the mutated AaGATAr
CtBP was still able to repress up to
50%
of the ecdysone-dependent stimulation of the reporter, suggesting
the presence of an additional mode of repression associated with
the
AaGATAr action. The
Drosophila developmental transcriptional
repressor
hairy provides an example of such a dual
repression
mechanism. The action of this repressor is independently
mediated
by two corepressors, Groucho and dCtBP (
23,
27).
Previously, GATA activators have been shown to interact with cofactors,
such as FOG in vertebretes and u-
shaped in
Drosophila,
which can repress GATA-mediated activation.
Interestingly, FOG
and u-
shaped contain the CtBP recognition
motifs and subsequently
recruit CtBP to display repression activity
(
14,
17,
18,
35). Thus, CtBP appears to play a critical
role in either the
repression of GATA-mediated activation through
binding to a corepressor
or a direct interaction with the GATA
repressor, as reported in
this
paper.
AaGATAr serves the unique role of a specific repressor at the state
of vitellogenic arrest in the mosquito female.
The stage
specificity of gene expression can be regulated through different
mechanisms, which may include a permanent shutdown of temporally
expressed genes or the use of alternative promoters in order to shift
the regulation of a particular gene from one stage-specific regulatory
hierarchy to another. In Drosophila, transcriptional
repressors such as krüppel, snail, hairy, knirps, or zhf-1 are
responsible for the correct timing of the expression of genes involved
in development of the embryo through their sequential shutdown
(23, 27, 28, 39). The stage-specific expression of the
Drosophila alcohol dehydrogenase gene is achieved through the alternative activation of two tandem promoters (30).
The proximal promoter is active during embryonic development and early larval stages, while the distal promoter is active in late larval stages and adulthood. The switch to the late distal promoter is accomplished through a permanent shutdown of the early proximal promoter by a zinc finger repressor, AEF-1 (30).
Interestingly, AEF-1 has also been implicated in the permanent
repression of Drosophila fat body-specific YPP
genes in other female tissues (2).
Cyclic egg production in insects is activated either by environmental
factors such as food and mating or by internal developmental
and
endocrine signals. Unlike other patterns of temporal regulation
of gene
expression which involve permanent repression of stage-specific
genes,
expression of
YPP genes in insects with cyclic egg
production
suggests the need for a temporal alteration of
transcriptional
repression and activation of the same genes. However,
the precise
mechanism of such a cyclic regulation of
YPP
genes has not been
elucidated.
Analyses of tissue and temporal distribution of the AaGATAr
transcript have demonstrated that it is expressed only in the
fat
body of adult female mosquitoes. This specificity suggests
that AaGATAr
acts as a fat body-specific repressor regulating
the transcriptional
activity of genes expressed during the reproductive
phase of female
life. Furthermore, EMSAs of female fat body nuclear
extracts have shown
that a retardation band with mobility and
specificity of binding
similar to those of AaGATAr exists at the
state of arrest, as
well as during postvitellogenic stages when
YPP genes are
shut off. Taken together, our results from transcript
abundance and
binding activity suggest that AaGATAr represents
a specific factor
involved in temporal transcriptional repression
of
YPP genes
before and between vitellogenic
cycles.
In anautogenous mosquitoes, such as
A. aegypti, this
temporal alteration of transcriptional repression and activation
involved
in the regulation of
YPP genes is under the strict
control of
a hormonal cascade, initiation of which requires a blood
meal.
In this work, we have uncovered a significant element of the
underlying
molecular mechanism of the state of arrest, which involves a
unique
GATA repressor. Importantly, we showed that AaGATAr can not only
inhibit the basal activity presented by the natural
Vg
promoter
but can also overcome its ecdysone-dependent activation.
Impairing
ecdysone-dependent activation by AaGATAr during
previtellogenesis
is essential because at that period, detectable
amounts of 20E
as well as the components of the EcR, AaEcR and AaUSP
are present
(
15,
40). Without comparable studies on other
insects with
cyclic egg production, it is difficult to speculate
whether in
evolution, anautogenous mosquitoes have developed a unique
repression
mechanism utilizing GATAr. Alternatively, they may have
adapted
a more general mechanism for the cyclic regulation of
YPP genes
by developing its strict control via a blood
meal-activated hormonal
cascade.
In summary, the characterization of AaGATAr and its response elements
in the
Vg and
VCP genes allows us to better
understand
the complex regulatory program controlling the expression of
YPP genes in anautogenous mosquitoes, which serve as vectors
of pathogens
responsible for some major infectious diseases in
humans.
| |
ACKNOWLEDGMENTS |
We thank T. Abel for the kind gift of (BoxA)6/CAT plasmid and
pPac-ABF clones, S. Parkhurst for the GST-dCtBP clone, and K. Iatrou
for antibodies to B. mori GATA. We also thank A. Hays for his invaluable help in maintaining mosquitoes, M. Trail for editing the
manuscript, and D. Arnosti for helpful discussions of the manuscript.
This work was supported by grant AI-24716 from the National Institutes
of Health to A. S. Raikhel. D. Martín was a recipient of a
postdoctoral research grant from the Spanish Ministry of Education and
Culture. M.-D. Piulachs was supported in part by a travel grant from
the Spanish Ministry of Education and Culture.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Entomology and Program in Genetics, S-150 Plant Biology Building,
Michigan State University, East Lansing, MI 48824. Phone: (517)
353-7144. Fax: (517) 353-3396. E-mail:
araikhel{at}pilot.msu.edu.
Present address: Department of Physiology and Molecular
Biodiversity, Institut de Biologia Molecular de Barcelona, CID, CSIC, Barcelona, Spain.
| |
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Molecular and Cellular Biology, January 2001, p. 164-174, Vol. 21, No. 1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.1.164-174.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
