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Research Institute, Hospital for Sick Children, Departments
of Clinical Biochemistry and Biochemistry, University of Toronto
(D.L., Y.L.D., F.X., C.L.H.), Toronto, Ontario, Canada,
Loeb Institute for Medical Research, Ottawa Civic Hospital and
University of Ottawa (M.E.), Ottawa, Ontario, Canada
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1-fetoprotein gene in
rodent liver. However, similar to mouse steroidogenic factor 1, zFF1A
regulated chinook salmon gonadotropin IIß subunit gene expression. On
the contrary, zFF1B, which could bind a consensus gonadotrope-specific
element with an affinity similar to that of zFF1A, lacked both the
trans-activation function and synergistic interaction with
the estrogen receptor. Furthermore, cotransfection studies in HeLa
cells showed that zFF1B was a strong competitor for the action of zFF1A
on the chinook salmon gonadotropin IIß subunit gene promoter. Our
investigation suggests that 1) zFF1 represents an ancestor protein of
the vertebrate FTZ-F1 homologs; 2) the antagonistic relationship
between zFF1A and -B may dictate the expression of the FTZ-F1 target
genes in a variety of tissues, including the pituitary; and 3) the
naturally occurring zFF1B provides evidence that the C-terminal portion
of zFF1A (80 amino acid residues) contains a major
trans-activation function and a protein-protein interface. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(dFTZ-F1), a member of the nuclear receptor superfamily, is crucial
for activation of the homeobox segmentation gene fushi
tarazu in early embryogenesis (1, 2). Various dFTZ-F1 homologs
have been discovered in other insects and vertebrate species (3, 4, 5, 6, 7, 8),
and collectively, these FTZ-F1 homologs constitute a distinct subgroup
of the orphan receptors (9, 10). In particular, they share high
identities in their DNA-binding domains (DBD) and conserved FTZ-F1 box
(11), whereas other regions are either less conserved or quite
divergent. Recent evidence reveals that, in contrast to the majority of
nuclear receptors binding as dimer to direct or inverted repeat of
5'-AAGGTCA-3' (12), FTZ-F1 binds as monomer to its cognate
5'-PyCAAGGPyCPu-3' site. The FTZ-F1 box recognizes the first 3 bp (5')
of the DNA sequence and determines the specificity of the monomeric
binding (7, 9, 10, 11).
Mammalian steroidogenic factor 1 (SF-1) is a homolog of dFTZ-F1 (4).
SF-1 is a key regulator of the hypothalamus-pituitary-gonadal axis
(13, 14, 15, 16). It is expressed in all primary steroidogenic tissues and acts
as a crucial transcription factor of enzymes involved in steroid
production (including the sex hormones) (17, 18, 19, 20, 21, 22, 23). As demonstrated in
the SF-1 gene-disrupted mice, along with the disappearance of
steroidogenic organs, such as the adrenal cortex and gonads (13), the
loss of gonadotrope-specific markers, including LH ß-subunit (LHß)
and GnRH receptors, has been observed (15, 16). Furthermore, the
development of the ventromedial hypothalamus in FTZ-F1 null mice was
defective (14). In addition to its determinant role in the
steroidogenic events and gonadotrope function, SF-1 is strongly
suggested to be involved in sex determination events and gonadal
differentiation (24). Specifically, SF-1 is likely to be a regulator of
the anti-Mullerian hormone, as anti-Mullerian hormone gene promoter
activity in Sertoli cells is supported by a promoter region containing
the SF-1-binding site. Although SF-1 expression appears in the
urogenital ridge of both sexes at 9–9.5 days postconception, the
persistence of SF-1 is only found in males beyond 12.5 postconception,
at which stage the first differences in sexes develop. Furthermore, all
SF-1-disrupted mice are born with developed female internal genitalia
(13, 16, 25, 26).
Only recently, however, has a direct link between SF-1 and pituitary LHß gene been identified both in vitro and in vivo (27, 28, 29, 30). Our recent in vitro studies on the salmon gonadotropin gene showed that mouse SF-1 (mSF-1) is an essential transcription factor controlling salmon gonadotropin IIß subunit (sGTHIIß) gene expression (30). A dramatic enhancement of the sGTHIIß gene promoter activity was exhibited by the synergy of mSF-1 with ligand-activated rainbow trout estrogen receptor (rtER). Our data suggest that these two nuclear receptors ultimately lead to the GTHII surge in the later phase of salmon reproduction. Similarly, a synergistic effect is necessary for the mammalian LH surge, i.e. NGFI-A, a Cys-His zinc finger transcription factor. SF-1 could induce a dramatic increase in the activity of rodent LHß gene promoter in heterologous cell lines (31). In each case, two transcription factors of limited tissue specificity were needed to trigger gonadotropin ß-subunit (GTHß) gene expression, indicating a common mechanism of GTHß gene regulation.
To better understand reproductive regulation in teleost and explore the mechanisms of sex determination in fish, we cloned the zebrafish FTZ-F1 homolog (zFF1A) and a novel splicing variant (zFF1B) from a zebrafish embryonic complementary DNA (cDNA) library. We showed that zFF1B is a C-terminally truncated zFF1A, and zFF1 gene transcripts exhibit a wider tissue distribution than the mammalian SF-1. Cotransfection studies in HeLa cells indicate that zFF1A resembles mSF-1 functionally, whereas zFF1B lacks the trans-activation function. However, zFF1B is a strong competitor for the action of zFF1A on sGTHIIß promoter. Our results suggest a novel mechanism to control target gene(s) expression in salmon pituitary.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Two types of positive cDNA clones were identified (Fig. 1A
). The majority of them (zFF1A) were identical to each
other, with a 3126-bp insert, whereas the minor one (zFF1B) contained
an insert, 2286 bp in length, that was different from zFF1A at both its
5'- and 3'-untranslated regions (UTR). Except for an extra 118 bp at
the 5'-end of the zFF1B cDNA insert, both zFF1A and zFF1B cDNAs were
identical in their 5'-UTR, and the identity extended to the translation
start site and another 1292 bp. In their 3' UTRs, there was no
significant similarity between these two types of cDNAs (Fig. 1B).
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). Therefore, zFF1B represents a C-terminally truncated
zFF1A.
Both zFF1A and B Transcripts Are from the Same Gene Locus
Southern blot analysis of genomic DNA using a probe restricted
from zFF1B cDNA covering nucleotides 1–306 suggested that there was a
single gene copy responsible for both zFF1A and -B (data not shown).
When a primer based on the 5'-end of zFF1A cDNA was used in the inverse
PCR, a 500-bp DNA fragment was generated (Chung, B. C., personal
communication). In the 500-bp fragment, a 118-bp sequence unique to
zFF1B cDNA was found directly adjacent to the start site of zFF1A cDNA
(Fig. 1A), providing further evidence that both zFF1A and -B
transcripts are derived from a single genomic locus.
Polyadenylation signal and poly(A) tail have been found near or at the
3'-ends of all cloned cDNAs, arguing against the possibility that zFF1B
cDNA was a cloned artifact. It is likely that both zFF1A and -B
transcripts were derived from the same gene by differential splicing.
The divergence between them is reminiscent of the site where intron 6
was found in the mSF-1 genomic gene (Fig. 2B).
Furthermore, a nearly perfect intron donor site, GG/GTGAGT
(the vertebrate consensus is AG/GTRAGT), was present at the point of
divergence, strongly suggesting the involvement of an intron or
alternative exon during the zFF1 gene heterogeneous nuclear RNA process
(33). Therefore, alternative splicing results in the production of
zFF1A and -B messenger RNAs (mRNAs).
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Homologs).
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1-fetoprotein gene expression (32). To distinguish to
which category the cloned zFF1 gene belongs, a documentation of the
tissue distribution of zFF1A and -B messenger RNA is necessary. Except
for the absence of zFF1B mRNA in testis, both zFF1A and -B transcripts
were detected in brain, liver/pancreas, and gonads by RT-PCR, using
primers either common or specific to zFF1A and -B cDNAs (Fig. 4A).
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Cells in positions corresponding to that of the developing
pituitary expressed both the zFF1A and -B transcripts starting around
27 h (36), and the highest intensity of the in situ
hybridization signals in the developing pituitary cells was obtained
between 36–48 h (data not shown, or Fig. 4, bottom panel).
In addition to cells of the developing pituitary, a subset of cells in
the mandibular arch expressed both zFF1A and -B transcripts. Mandibular
arch expression of zFF1 persisted well into the third day of
development and diminished gradually (data not shown). RT-PCR using
primers specific to either zFF1A or -B cDNA confirmed the appearance of
both transcripts before 48 h (data not shown).
Recently, it has been proposed that mSF-1 and LRH-1 loci are the result of duplication of an ancestral FTZ-F1 gene in vertebrates (7). Our data suggest that the zebrafish gene might represent an ancestor for the mammalian FTZ-F1 homolog genes before gene duplication.
Although zFF1A Resembles mSF-1 Functionally, zFF1B Is Devoid of Its
Trans-Activation Function
Both full-length cDNAs and the ORF-A fragment were introduced into
the pCDNA3 expression vector, and the resulting constructs were used in
the cotransfection study in HeLa cells. The sGTHIIß -39/+42 promoter
was demonstrated as a strong basal promoter in HeLa cells (37). This
promoter, linked with a consensus GSE upstream (consGSE/CAT-39), was
chosen as the reporter construct. As shown in Fig. 5A, zFF1A, similarly to mSF-1, was sufficient to up-regulate the
GSE-containing reporter gene. Moreover, no significant variation was
found between zFF1A and ORF-A expression constructs in their
trans-activation abilities, suggesting that the UTR
sequences of zFF1A cDNA make a limited contribution to the expression
efficiency of the gene in the in vitro system. On the other
hand, with the truncation of 80 aa residues, including the AF-2 domain
in zFF1B, the reporter activity mediated by zFF1B is minimal. Neither
zFF1A nor zFF1-B affected sGTHIIß -39/+42 promoter in the absence of
consGSE, indicating that this DNA sequence was necessary to mediate the
trans-activation of the orphan receptor. Other non-GSE
sequences in front of the basal promoter could not be activated by
either zFF1A or -B (see Fig. 7B).
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shows that cell
extract from either zFF1A or B expression vector transfected cells were
able to retard the labeled consGSE with similar affinity. This binding
was comparable to the mSF-1 transfected cell extract. Competition
experiments further confirmed the specificity of the binding. The
shifted band was abolished with the addition of a 25-fold excess of
consGSE, while the same amount of unlabeled sGSE2 (the GSE sequence
found in sGTHIIß promoter) was less effective (30). When other probes
were used, such as the non-specific Homo oligo or the pERE (proximal
estrogen receptor element) palindrome (40), there was no significant
competition. Therefore, the failure in trans-activation
function of zFF1B is not due to its lack of direct GSE binding.
Another distinct feature of mSF-1 is its ability to synergize with rtER
to stimulate sGTHIIß promoter activity (30). To assess the functional
similarity between mSF-1 and zFF1, cotransfection studies were
performed in HeLa cells, with the sGTHIIß-289-CAT (chloramphenicol
acetyltransferase) promoter containing pERE and sGSE2 sites necessary
for the mSF-1/ER synergistic interaction. There was no significant
variation in the trans-activation of either zFF1A or mSF-1
alone, whereas zFF1B had no effect on the sGTHIIß -289/+42 promoter.
When rtER/estradiol (E2) was introduced, only zFF1A and
mSF-1 synergized with rtER to trigger a dramatic enhancement of the
promoter activity, whereas zFF1B failed to do so (Fig. 6). Similar results were obtained when longer promoters
were used (see Fig. 7). These data clearly demonstrate
that zFF1A resembles mSF-1 functionally in terms of its ability to
regulate sGTHIIß gene expression. However, zFF1B lost not only its
trans-activation function, but also its ability to interact
with ER.
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To verify this hypothesis, cotransfection studies were conducted by
including both zFF1A and -B with the reporters. As shown in Fig. 7A, trans-activation of sGTHIIß-1260-CAT by either zFF1A or
mSF-1 was indistinguishable, whereas zFF1B was ineffective. Similarly,
zFF1A could stimulate sGTHIIß-3358-CAT, but zFF1B could not (Fig. 7B). Interestingly, when an equal amount of zFF1B was added to the
cotransfection, the promoter activities activated by zFF1A decreased
(Fig. 7B). The inhibitory effect of zFF1B was GSE dependent. There was
no significant interference in the ER/E2-induced activity when the same
amount of zFF1B was incorporated into the pERE/sGTHIIß-39/ER
cotransfection. Such an antagonistic relationship between zFF1A and -B
was more dramatic when the sGTHIIß promoter was tested for FTZ-F1 and
ER synergy (Fig. 7A). The inhibitory effect is dependent on the
quantity of zFF1B present in cells. When the ratio of A to B was less
than 10:1, a significantly reduced CAT activity was found. Similarly,
stimulation of the target promoter triggered by mSF-1 and rtER was
impaired by the addition of an equal amount of zFF1B, confirming that
zFF1B is a strong competitor for either mSF-1 or zFF1A in sGTHIIß
gene regulation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The C-Terminal 80 aa Residues of zFF1A Contain a
Trans-Activation Domain and a Protein-Protein Interface
Domain
The nuclear receptor superfamily is characterized by the presence
of six regions (A–F) that provide the receptor with multiple functions
necessary for its DNA-binding ability and transcriptional activation.
Some recent studies revealed that in addition to the ligand-binding
domain, region E of the nuclear receptor contained a ligand-dependent
trans-activation function activating factor-2 (AF-2) (42, 43). The core of the AF-2 domain has been mapped in the C-terminal part
of the E region and shown to form an amphipathic -helix (43). This
-helical motif is well conserved among all known transcriptionally
active members of the superfamily. In the presence of its putative
ligand or coactivator, conformational changes in the E region could
reorientate the AF-2 surface to interface with the basal
transcriptional machinery.
The zFF1A and other transcriptionally active FTZ-F1 homologs in
vertebrate showed conserved AF-2 domain at their C-terminal ends (Fig. 3), whereas the complete loss of trans-activation of zFF1B
indicated that the AF-2 domain in zFF1A is solely responsible for its
transcriptional activity. Due to the loss of their DNA-binding
abilities, ELP and xFF1rAshort no long act as transcriptional
activators. In another case, complete removal of the AF-2 motif LLIEML
from the C-terminus of rat LRH-1 also caused the loss of its
trans-activation function (32). Taken together, these
results suggest that the transcriptional activities of the members of
the FTZ-F1 subfamily are mainly due to their C-terminal regions.
Two distinct dimerization interfaces have been found in nuclear hormone receptors. Besides the D box located in the second zinc finger of DBD, a helical segment (H9 and 10) of retinoic acid receptor (RAR) was implicated as part of the dimer interface. More recently, the helical segment has been confirmed as a transferable element critical for determining identity in the heterodimeric interaction and for high affinity DNA binding (44, 45). Both interfaces are suggested to differentially modulate target gene specificity of the receptors.
The search of the zFF1A identity box (I box) defined from E regions of
retinoid X receptor (RXR), RAR, thyroid hormone receptor (TR), and
chicken ovalbumin upstream promoter transcription factor (COUP-TF) (45)
revealed that a potential I box is localized at the N-terminus of the
extra 80-aa segment in zFF1A. Comparison of all vertebrate FTZ-F1
homologs within this 45-aa stretch indicated that this region is Leu
rich and highly conserved within the group. Moreover, the identity
among these FTZ-F homologs, hRXR, and COUP is significant (Fig. 8A). Given that only zFF1A was able to achieve the
synergistic effect on the sGTHIIß promoter with ER, it is tempting to
speculate that the I box may be directly related to the interaction of
the two nuclear receptors. On the other hand, although P box regions of
FTZ-F1 subfamily members are identical, D box regions are not well
conserved (Fig. 8B). The monomeric binding nature of this group of
receptors may explain the diversity of D box regions among species.
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proteins, all of the differentially splicing events generate
the C-terminal truncation in the E region compared with their wild type
proteins.
The ligand-binding domain of the E region, in the presence of the
receptor’s putative ligand or cofactor (32), is believed to govern
allosteric transitions in the receptor structure to facilitate the
binding of DBD to DNA. In many circumstances, conserved regions II and
III are involved directly in the DNA binding. For instance, a series of
deletion mutants with the truncated E region of xFF1rA, in which R-II,
R-III, or both are removed, showed impaired DNA-binding abilities (38).
Other examples come from TR2 and truncated Rev-ErbA (38). In both
cases, C-terminal truncation modulated their DNA-binding activities.
However, in all C-terminally truncated splicing variants of the FTZ-F1
subfamily, only zFF1B retained intact conserved R-II and R-III, whereas
all others have their truncations either in the region between R-II and
R-III or in R-II (Fig. 3). ELP has been indicative of a significantly
weaker DNA-binding ability compared with SF-1. Similarly, xFF1rAshort
has poor DNA-binding activity, as shown in the gel retardation assay
(38).
Therefore, zFF1B represents a novel splice variant in the FTZ-F1 subfamily. First, zFF1B is capable of binding to GSE, and the affinity is comparable to that of zFF1A. Secondly, due to the colocalization of both zFF1A and -B transcripts in the developing pituitaries of zebrafish embryos, zFF1B is a naturally occurring competitor to zFF1A in controlling their target genes in pituitary as well as in other tissues involved in reproduction.
Nature of the Transcriptional Competition by dFTZ-F1 Homolog
Splice Variants
We have clearly demonstrated that zFF1B is a strong competitor for
the action of zFF1A on the sGTHIIß gene promoter via direct binding
to the same DNA site recognized by mSF-1 and zFF1A. The functional
antagonism of differentially spliced variants of FTZ-F1 homolog genes
have been addressed in mouse and Xenopus. In both cases,
excessive amounts of truncated variants were needed in the in
vitro testing system despite the expression levels of ELP and
xFF1rAshort, which were significantly less than those of the
full-length isoforms (SF-1 and xFF1rA, respectively). In fact, ELP
exerted no significant inhibitory effect on the SF-1-related
trans-activation of the target gene promoter, whereas
xFF1rAshort was impaired in its trans-activation and ability
to bind to the target DNA sequence (23, 38). Nevertheless, it is
interesting to note that all splicing variants found in the FTZ-F1
subfamily are inevitably caused by the truncation of the presumed
trans-activation domains, either in the C-terminal AF-2 or
in the A/B AF-1 domain (23, 38, 46, 47). This common feature implies
that transcriptional antagonism is a shared mechanism in controlling
FTZ-F1 target gene expression.
Through RT-PCR experiments, the earliest detectable transcript of the zFF1 gene was at the 12 h stage (Liu, D., Y. Le Drean, and C. L. Hew, unpublished observation). Similarly, in X. laevis, both xFF1rA and xFF1rAshort transcripts could be detected in early embryos (38). ELP mRNA was not found in mouse embryo, but was located in undifferentiated embryonic carcinoma cells. However, the rationale for the early expression of these FTZ-F1 homologs (xFF1r and zFF1) in lower vertebrate embryos is presently unclear. In addition, determination of whether zFF1B is expressed only at particular developmental stages, to keep its target gene dormant or maintain it at a certain expression level, needs further investigation.
The coexpression of both zFF1A and -B transcripts in developing pituitaries would suggest that the expression of their target genes in the pituitary may have been initiated during embryogenesis. Like mammalian SF-1, zFF1 proteins may confer a variety of functions in endocrine tissues. The finding that the zFF1 gene is expressed in adult ovary and testis further proves its commitment in steroidogenesis. However, the lack of the zFF1B transcript in testis is in contrast to the fact that both SF-1 and ELP could be detected in rat gonads (23). Whether the absence of the inhibitory factor in testis is responsible for the different reproductive performances between the sexes will rely on understanding the nature of alternative splicing of zFF1A and zFF1B mRNAs. The antagonistic relationship between zFF1A and -B in the regulation of the sGTHIIß gene promoter provides a novel mechanism in controlling their target genes in the pituitary as well as in other tissues. In addition, the study of zFF1B should provide new insight into the structural and functional domains important for FTZ-F1 homologs’ actions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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For amplification of the probe used for the library screening, one set of primers, P (overlaps the P box) and A (covers the A box), was designed as follows: P, 5'-TGCAAGGGCTTCTTCAAGCGC-3'; and A, 5'-SCGRTCYCKYTTGTACATGGG-3'; S represents G/C, R = A/G, Y = C/T, and K = G/T. For amplification of the zFF1A and B transcripts, the upper strand primer was designed in the region shared by both zFF1A and B cDNAs, as I (5'-GGATCGCCGCCCTCCTTCCCT-3'); the lower strand primers were II (5'-GCACATCACGTAGTCCAGCAG-3') and III (5'-AGACGTGACTAGTCTGGCATC-3'), specific to zFF1A or -B, respectively. DNA amplified from the latter reaction was fractionated on 1.0% agarose gel and confirmed by Southern blotting with 32P-labeled zFF1A or -B cDNA. The exposure time to the x-ray film (Eastman Kodak, Rochester, NY) varied from 12 h (room temperature) to 48 h (-70 C).
Embryonic cDNA Library Screening and Identification of Zebrafish
FTZ-F1 Homolog cDNAs
The embryonic cDNA library was constructed as previously
described (49). A 273-bp fragment amplified from RT-PCR was excised
from low melting point agarose gel (Sigma), subcloned into the pT7Blue
T-vector (Novagen, Madison, WI), and sequenced. A search of the DNA
database confirmed the identity of the fragment as a partial sequence
of the zebrafish FTZ-F1 homolog.
The 273-bp fragment then served as the probe to screen 2 million phages, and a total 40 positive clones were obtained. After the first screening, all positive phages were eluted in sodium-magnesium buffer, and 2 µl of the elution were used to perform the PCR reaction by primers P and A. With this approach, 6 of 40 were identified as the real positives. The second screening was skipped by using massive in vivo excision and colony hybridization. Insertion of each selected bacterial colony was examined by PCR, and the plasmid DNA was restricted by a series of enzyme digestions. Two types of cDNA clone were identified based on their lengths and restriction patterns. Using a double stranded Nest deletion kit and a T7Sequencing kit (Pharmacia), both zFF1A and -B cDNAs were fully sequenced from their 3'-ends (T7 primer) and partially confirmed by SK and P primers. Further sequence confirmation was performed using internal primers deduced from the known sequences.
In Situ Hybridization
Zebrafish embryos were obtained from the natural cross, staged
accordingly (50), fixed in 4% paraformaldehyde in PBS, and stored in
100% methanol. Plasmids used for the in vitro transcription
labeling were constructed based on the pBluescript (Stratagene, La
Jolla, CA). Briefly, a fragment specific to zFF1A DNA (nucleotides
1582–3105) was subcloned into the PstI site of pBluescript,
while nucleotides 1144–2286 of zFF1B cDNA were inserted into
PstI and XhoI sites of the vector. Antisense RNA
probe generation and the whole mount in situ hybridization
procedure were essentially similar to those previously described
(51).
Plasmid Construction, Transfection, and CAT Assay
Both zFF1A and -B full-length cDNA fragments were released from
their original plasmids, as was a PCR fragment containing only ORF-A,
and were introduced into the eukaryotic expression vector pCDNA3
(Invitrogen, San Diego, CA) in the same fashion. The resulting
constructs were named, in sequence, z.FF1.A, z.FF1.B, and ORF-A. ER,
mSF-1, pCMV-lacZ, and (frgt pERE)-IIß-39 constructs were
previously described (30, 37, 40). The reporter consGSE/CAT-39 was
constructed as follows. Two oligo nucleotides
(5'-AGCTGCTGACCTTGACACT-3' and 5'-AGCTAGTGTCAAGGTCAGC-3') were annealed
and ligated with the HindIII-restricted -0.039-kb CAT. Only
the plasmid containing the single fragment with the same orientation as
that found in mammalian gonadotropin gene promoters was used in the
transfection studies.
Cell culture, transfection, hormone treatment, and complete CAT assay were performed as previously described (30, 37, 40).
Gel Shift Assay
Whole cell extract was obtained from HeLa cells individually
transfected with 5 µg z.FF1.A, z.FF1.B, pCMVmSF-1, and
pCMVß-lacZ. Cells were washed twice in PBS and harvested
in ice-cold PBS, swollen in cold 0.25 x PBS for 3 min, and
resuspended in buffer I (30). After two freeze-thaw cycles, the
supernatant was ready for the DNA binding assay. The consGSE fragment
was labeled, and the binding reaction was set up as previously
described (30). Similar amounts of proteins were used in each reaction.
In addition to the cold consGSE, other competitors included were: sGSE2
(5'-AAGTAGAGGTCAGGA-3') (30), pERE
(5'-ATTATGTCAATCTGACCCTAA-3'), and Homo
(5'-TCTATGACAATTATGTCAATCT-3'). The putative GSE and
palindromic sequences are italicized. The Homo palindrome
sequence overlaps pERE in the sGTHIIß gene promoter and serves as a
nonspecific competitor in the retardation assay.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by the Medical Research Council of Canada (to C.L.H.).
1 Recipient of a Restracom Trainee Fellowships from the Hospital for
Sick Children, Toronto. 
Received for publication January 23, 1997. Revision received March 19, 1997. Accepted for publication March 20, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-hydroxylase/c17–20 lysase). Mol Endocrinol 10:147–158-1 fetoprotein locus is
activated by a nuclear receptor of the Drosophila FTZ-F1
family. Mol Cell Biol 16:3853–3865[Abstract]
-subunit
produces hypogonadal and hypothyroid mice. Genes Dev 9:2007–2019
ligand-binding domain
bound to all-trans retinoic acid. Nature 378:681–689[CrossRef][Medline]
and FTZ-F1ß compete as monomers for
binding to a site in the fushi tarazu gene. Mol Cell Biol 14:3166–3175This article has been cited by other articles:
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 V. Giguère Orphan Nuclear Receptors: From Gene to Function Endocr. Rev., October 1, 1999; 20(5): 689 - 725. [Abstract] [Full Text]
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 M. Nitta, S. Ku, C. Brown, A. Y. Okamoto, and B. Shan CPF: An orphan nuclear receptor that regulates liver-specific expression of the human cholesterol 7alpha -hydroxylase gene PNAS, June 8, 1999; 96(12): 6660 - 6665. [Abstract] [Full Text] [PDF]
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 M. Li, Y.-H. Xie, Y.-Y. Kong, X. Wu, L. Zhu, and Y. Wang Cloning and Characterization of a Novel Human Hepatocyte Transcription Factor, hB1F, Which Binds and Activates Enhancer II of Hepatitis B Virus J. Biol. Chem., October 30, 1998; 273(44): 29022 - 29031. [Abstract] [Full Text] [PDF]
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D. Liu, M. Chandy, S.-K. Lee, Y. Le Drean, H. Ando, F. Xiong, J. Woon Lee, and C. L. Hew A Zebrafish Ftz-F1 (Fushi Tarazu Factor 1) Homologue Requires Multiple Subdomains in the D and E Regions for Its Transcriptional Activity J. Biol. Chem., May 26, 2000; 275(22): 16758 - 16766. [Abstract] [Full Text] [PDF]
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